Orphan GPCRs and their ligands

Associate editor:J.Wess

Orphan GPCRs and their ligands

Olivier Civelli *,Yumiko Saito,Zhiwei Wang,Hans-Peter Nothacker,Rainer K.Reinscheid

Department of Pharmacology and Department of Developmental and Cell Biology,University of California,Irvine,

Med Surge II Room 369,Irvine,CA 92697-4625,USA

Abstract

Due to their diversity,G-protein-coupled receptors (GPCRs)are major regulators of intercellular interactions.They exert their actions by being activated by a vast array of natural ligands,referred to in this article as ‘‘transmitters’’.Yet each GPCR is highly selective in its ligand recognition.Traditionally,the transmitters were found first and served to characterize the receptors pharmacologically.Since the end of the 1980s,however,it is the GPCRs that are first to be found because they are identified molecularly by homology screening approaches.But the GPCRs found this way suffer of one drawback,they lack their natural transmitters,they are ‘‘orphan’’GPCRs.Searching for transmitters of orphan GPCRs has given birth to the reverse pharmacology approach that uses orphan GPCRs as targets to identify their transmitters.The most salient successes of the reverse pharmacology approach were the discoveries of 9novel neuropeptide families.These have enriched our understanding of several important behavioral responses.But the application of reverse pharmacology has also led to some surprising results that question some basic pharmacological concepts.This review aims at describing the history of the orphan GPCRs and their impact on our understanding of biology.D 2005Elsevier Inc.All rights reserved.

Keywords:Orphan GPCR;Reverse pharmacology;Transmitters;Neuropeptides

Contents

1.

Introduction...........................................5251.1.The transmitters .....................................5261.2.The physiological importance of the G-protein-coupled receptors ...........5262.The orphan G-protein-coupled receptors .................

...........5262.1.Reverse pharmacology .................................5272.2.The search for novel transmitters ............................5272.3.Physiological roles of the orphan G-protein-coupled receptors .............5283.The surprises of the reverse pharmacology approach...........

...........5283.1.The unexpected transmitters...............................5293.2.The promiscuous G-protein-coupled receptors .....................5293.3.The non-selective G-protein-coupled receptors .....................5294.The impact of the orphan G-protein-coupled receptor research.................530Acknowledgments...........................................530References...............................................

530

1.Introduction

The seminal discovery that the h 2-adrenergic receptor and the opsins share a 7-transmembrane domains (TMs)topology (Dixon et al.,1986),created the concept that receptors that

0163-7258/$-see front matter D 2005Elsevier Inc.All rights reserved.doi:10.1016/j.pharmthera.2005.10.001

*Corresponding author.Tel.:9498242522;fax:9498244855.E-mail address: ocivelli@https://www.wendangku.net/doc/345242128.html, (O. Civelli).

Pharmacology &Therapeutics 110(2006)525–

532

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couple to G proteins form a homologous supergene family, thereafter called the G-protein-coupled receptors(GPCRs) (Hall,1987).The7TMs topology has since evolved to include receptors that do not couple to G proteins,and our under-standing of receptor activation has been shown to include proteins other than the G proteins(Bockaert&Pin,1999; Angers et al.,2002;Pierce et al.,2002;Kristiansen,2004).Yet, one tenet of the basic concept has remained constant over the last20years:all the small molecules that have evolved to direct intercellular interactions interact with GPCRs(7TMRs).This positions the GPCRs at the center of signal transmission and endows them with an extraordinary importance in the organism’s life and survival.

1.1.The transmitters

The GPCRs are activated by a plethora of‘‘transmitters’’(Civelli et al.,2001),the first messengers that are either present in the environment or released from a cell to carry a message to a second one.These act in an endocrine,paracrine,or exocrine fashion to allow the organism to react to particular physiolog-ical challenges.Transmitters are mostly small molecules although few of them are larger polypeptides.They include biogenic amines,neuropeptides,chemokines,lipid mediators, nucleotides,amino acid and derivatives,polypeptide hor-mones,pheromones,olfactory and gustatory molecules,and some other naturally occurring chemicals such as calcium ions and protons.

Traditionally,the GPCRs are expected to exhibit specificity for the transmitters(Goldstein,1974).This specificity results from evolutionary processes that aim at diversifying the intercellular interactions.A transmitter may be the natural ligand of more than one GPCR,but those then share a higher degree of homology that groups them into a subfamily.Also a GPCR may bind more than one transmitter,but then these transmitters share structural similarities and are often part of the same synthesis pathway,as in the case of the neuropeptides synthesized from the same precursor(Douglass et al.,1984).A classic example for structurally similar ligands binding structurally related receptors are the opioid receptors and the natural opioid peptides.The concept of GPCR specificity has recently been revised and will be discussed in this review. 1.2.The physiological

importance of the G-protein-coupled receptors

Because they direct such a broad spectrum of interactions, the GPCRs participate in about every physiological response. Each cell expresses a few dozen different GPCRs,which implies that its homeostasis can be influenced by numerous transmitters.GPCR activation leads to intracellular chemical changes that may affect directly the state of the cells but can also lead to transcriptional regulation,thus making the role of the GPCRs long lasting.Consequently,every function in the organism can be affected by the activities of particular GPCRs.

For example,the cardiovascular,respiratory,muscular,and endocrine functions are directly dependent on the activities of the adrenergic,histaminergic,muscarinic,and hormone GPCRs,respectively.The organ that is possibly most dependent on GPCR activities is the brain,where practically all the GPCRs are expressed and where their activities add a slower but long lasting dimension to the synaptic transmission mechanisms that direct all brain-related responses.In addition, one will remember that most of our senses such as olfaction, taste,and vision depend directly on the activation of specific GPCRs.

A particular GPCR is often expressed in several tissues.It can be found in the periphery and in the CNS.Its roles in these tissues may ultimately be very different although the second messengers that result from its initial activation are probably the same.We only begin now to dissect the role that the activation of a particular GPCR has on the organism.In some cases,we are helped by the studies of some pathological cases (Scho¨neberg et al.,2004).Furthermore,and increasingly during the past few years,we expect that genome sequence analyses will point at GPCR structural differences(Balasu-bramanian et al.,2005)that may help explain pathological differences and consequently unravel the roles of particular GPCRs in particular tissues.But ultimately,the discovery of the predominant role(s)of a GPCR awaits the development of corresponding surrogate agonist or antagonist molecules.Only administration of such drugs to human can answer the question of the GPCR function.Fortunately,developing specific surrogate ligands for GPCRs is in the realm of medicinal chemistry.Indeed about half of the drugs presently on the market are targeting GPCRs(Drews,2000)and studies of their effects has been a driving force in our understanding of the functions of the GPCRs.Yet these are far from covering the entire supergene family,which amount to some800receptors in the human genome(Vassilatis et al.,2003).

This review will describe how the GPCRs became such an imposing supergene family and how they continue to spear-head our understanding of particular physiological responses.It will focus on the search for the ligands of the transmitter GPCRs and will proceed from specificity,to diversity,to finally complexity.

2.The orphan G-protein-coupled receptors

Most of the GPCRs started as orphans.Their discoveries stemmed from the concept that GPCRs would belong to a supergene family and thus would share sequence similarities. Homology screening techniques,low stringency hybridization (Bunzow et al.,1988,1992)soon followed by PCR-derived approaches(Libert et al.,1989),paved the way for the discoveries of new GPCRs,thus that,by the end of the 1980s,it became clear that the number of GPCRs would be large.This was confirmed,at the turn of the century,with the sequence of the genome.Today,one estimates that the number of GPCRs is about800,of which more than half are olfactory GPCRs(Vassilatis et al.,2003).

The discovery of new GPCRs found by homology screening suffers from one obvious problem,the receptors found lack their pharmacological identities,their natural ligands.They are

O.Civelli et al./Pharmacology&Therapeutics110(2006)525–532 526

all‘‘orphan’’receptors(Libert et al.,1991a,1991b).The pursuit to unravel their identities was thought to be formidable and to many an unresolvable task that would lead to unglamorous fishing expeditions.

2.1.Reverse pharmacology

In the second part of the1980s,there existed about50 transmitters,potential GPCR ligands,that had no cloned cognate receptors.Testing all of these for their binding to new GPCRs was seen as an impossible aim in an academic environment.Yet serendipity and ingenious insights,such as these that could spring from the analysis of the new GPCR tissue expression profile,proved to be successful in matching the first orphan GPCRs to known transmitters.The first deorphanized GPCRs,the5HT-1A and the D2dopamine receptors,were already reported in1988(Fargin et al.,1988; Bunzow et al.,1988).The strategies used were the same,that is,the orphan GPCR was expressed by DNA transfection in eukaryotic cells,membranes of these cells were then used as targets to determine the binding of potential transmitters.This strategy has become to be known as reverse pharmacology (Libert et al.,1991a,1991b;Mills&Duggan,1994).

During the first part of the1990s,application of the reverse pharmacology strategy led to the pharmacological character-ization of many GPCRs.These endeavors were mostly carried out in an‘‘artisanal’’fashion;the focus was on one particular receptor tested for few potential known ligands(Table1).At the same time,however,random searches for new GPCRs, using PCR-based homology screening approaches,were also in full swing,thus the overall number of orphan GPCRs was steadily increasing(Marchese et al.,1994,1998).This led us to conclude that the GPCRs outnumbered the known potential ligands and more importantly that these receptors must bind ligands that have not been thus far characterized because inactive receptors should be evolutionarily discarded(Civelli, 1998).This recognition inspired enough confidence in a few researchers to utilize orphan GPCRs as baits to isolate their natural ligands,which meant to identify novel transmitters. 2.2.The search for novel transmitters

The concept that an orphan receptor could be used as bait to identify a novel transmitter requested the application of two technologies(Civelli,1998).First,it necessitated the search for novel transmitters in tissue extracts,therefore in complex molecular mixtures.Then it necessitated the monitoring of receptor activation instead of binding.Both requirements implied the application of countless repetitive assays.Fortu-nately,such assays were existing or being developed for the

pharmaceutical industry(Wess et al.,2001).

The cloning of receptors,enzymes,and other targets of pharmaceutical interest had revolutionized drug screening (Drews,2000).From that time on,drugs could be searched for by the random screening of large libraries of synthetic compounds(Schreiber,2000).These are tested for the desired biological and in vivo activity and,if active,chemically modified to fulfill the pharmaceutical constraints of expected drugs.The screening of libraries of compounds required high-throughput assays and such assays were developed with the aim of monitoring GPCR reactivity.

The first orphan GPCR that was used for discovering a novel transmitter was ORL-1,cloned through its homology to the opioid receptors(Henderson&McKnight,1997).Its Table1

Dates in the history of GPCR deorphanization

1986The h2-adrenergic and the rhodopsin receptors share a

7-transmembrane topology.

Birth of the GPCR family concept and recognition that

homology screening approaches could lead to new

GPCRs.

1987Identification of G-21,the first orphan GPCR using low

stringency hybridization.

1988The first deorphanizations:G-21is recognized as the

5-HT1A receptor,RGB-2as the dopamine D2receptor.

Introduction of the reverse pharmacology approach.

1989Development of the PCR-based homology screening

approach.

1990–1995Orphan GPCRs are identified by homology screening.

The‘‘artisanal’’era of GPCR deorphanization.

The following GPCRs are characterized:Adenosine A1,

A2a,A2b,A3;adrenergic a1A,a1d,a2b,a2c,b1,b3;

anaphylatoxin C3a,C5a;angiotensin AT1b;bombesin

BB1,BB3;cannabinoid CB1,CB2;chemokine CCR1,

CCR2,CCR3,CCr4,CCR5,CXCR2,CXCR3,CXCR4;

cholecystokinin CCKa;dopamine D1,D2,D3,D4,D5;

follicle-stimulating hormone;formyl-peptide FPR2;

galanin type2;gonadotropin-releasing hormone;

histamine H2;lysophosphatidic acid;melanocortin MC1,

MC2,MC3,MC4,MC5;melatonin ML1a,ML1B;

muscarinic acetylcholine M3,M4,M5;neurokinin NK1,

NK3;neuropeptide YY1,YY1-like,YY4;neurotensin

NTR2;opioid and A;prostanoid EP1,EP2,EP3,EP4,DP,

FP,IP;protease-activated2,3;purinoceptor P2Y1,P2Y3,

P2Y4,P2Y6,P2Y8;serotonin5-HT1a,5-HT1b,5-HT1d,

5-HT1e,5-HT1f,5-HT2a,5-HT2b,5-HT4,5-HT5a,

5-HT5b,5-HT6,5-HT7;somatostatin SST1,SST2,SST3,

SST4,SST5;thyrotropin-stimulating hormone;

vasopressin V1b,V2.

1995Discovery of the first novel natural ligand of an orphan

GPCR:nociceptin/orphanin FQ.Demonstration that

orphan GPCRs can be used to identify new transmitters. 1996–2005The‘‘industrial’’era of GPCR deorphanization.

The following GPCRs are identified:leukotriene B4,C4,

D4;latrotoxin;sphingosine1-phosphate;lysophosphatidic

acid;melanin-concentrating hormone;urotensin II;motilin;

neuromedin U;UDP-glucose;sphingosylphosphorylcholine;

histamine3;prostaglandin D2;neuropeptide FF,AF;

RFamide-related protein1,3;lysophosphatidylcholine;

adenosine diphosphate;psychosine;trace amines;

5-oxo-ETE;bile acids;bovine adrenal medulla peptide22;

relaxin;relaxin-3;bradykinin;pyroglutamylated

arginine-phenylalanine-amide peptide;cortistatin;medium

and long fatty acids;nicotinic acid;proton;h-alanine;

a-ketoglutarate.

1998–2004Discovery of8novel neuropeptide/receptor system:

Orexins/hypocretins,prolactin-releasing peptide,apelin,

ghrelin,metastatin,neuropeptides B/W,prokineticins1/2,

neuropeptide S.

The dates presented in this table refer to these mentioned in text.The GPCR lists were derived from Marchese et al.(1998)and Saito and Civelli (2005).

O.Civelli et al./Pharmacology&Therapeutics110(2006)525–532527

activation was monitored by quantifying intracellular decreases in cAMP levels,which could be measured in newly developed scintillation proximity assays.Because phylogenic analyses classified ORL-1as a peptidergic GPCR and because ORL-1is expressed in the CNS,peptidergic brain tissue extracts were prepared,purified,and fractionated.Fractions were tested for their abilities to inhibit adenylyl cyclase activity in cells that were stably transfected with ORL-1.A17-residue long peptide was ultimately isolated,named orphanin FQ or nociceptin (OFQ/N)(Meunier et al.,1995;Reinscheid et al.,1995).Its structural similarities to the opioid peptides immediately attracted considerable attention,yet it has been proven not to bind the opioid receptors(Reinscheid et al.,1998).

The second successful attempt at discovering novel trans-mitter through orphan GPCRs screened over50different orphan GPCRs by measuring their abilities to induce intracel-lular calcium release when subjected to peptidic extracts.One receptor did respond and led to the characterization of two peptides,the orexins(Oxs)(Sakurai et al.,1998),also identified through an RNA subtraction approach as hypocretins (Hcrts)(de Lecea et al.,1998).This was immediately followed by the discovery of two novel peptides,prolactin-releasing peptide,and apelin as the natural ligands of the orphan GPCRs GPR10and APJ,respectively(Hinuma et al.,1998;Tatemoto et al.,1998).

These successes proved the validity of high-throughput screening of orphan GPCRs.It is therefore not surprising that the pharmaceutical industry became its major proponent (Hinuma et al.,1999).Consequently,orphan GPCRs began to be screened randomly against large libraries of ligands, setting the stage for the‘‘industrial’’period of deorphanization (Wise et al.,2004).These libraries contained all the ligands that had not been matched to any receptor molecules but also many molecules that are known to exist in cells.This led in a few years to the deorphanization of some40GPCRs(Saito& Civelli,2005).

2.3.Physiological roles of the

orphan G-protein-coupled receptors

The impact that the deorphanization of GPCRs had and continues to have on our understanding of the organism’s function is fundamental.Deorphanizing a GPCR opens the opportunity to combine anatomical studies on the site of synthesis with that on the sites of action of the system and thus to gain a full understanding of the localization of the system. Most often,the sites of the orphan GPCR expression serve as primary indication of the role of the system.The matching of orphan GPCRs to previously known transmitters has permitted such precise anatomical analyses.Genetic ablations of the orphan GPCRs can also help to understand these new receptor systems(Morita et al.,2004;Piao et al.,2004).But the most striking results came from the discoveries of novel peptides. Our understanding of several physiological responses has greatly gained from these discoveries.

Sleep is1physiological response that has been impacted by the orphan GPCR research(Civelli,2005,in press).While GABA,noradrenalin,and histamine were known to regulate sleep,the discoveries of Hcrts/Oxs have proven that neuropep-tides have a prominent role.Inactivation of one of the Hcrts/ Oxs receptor induces narcolepsy(Chemelli et al.,1999;Lin et al.,1999).More recently,3other orphan GPCR systems,the PrRP(Lin et al.,2002),neuropeptide S(Xu et al.,2004a, 2004b),and urotensin II(Huitron-Resendiz et al.,2005) systems have also been shown to regulate some aspects of sleep.Finally,the prokineticin system has been shown to be a major regulator of the circadian rhythm(Cheng et al.,2002).

Another physiological response that has been shown to depend on the activity of orphan GPCRs is feeding(Xu et al., 2004a,2004b).While this response was understood as relying on the release of several peptides,in particular leptin(Zhang et al.,1994),melanocyte-stimulating hormone(Marks&Cone, 2001),and neuropeptide Y(Gehlert,1999),it has been shown that the deorphanized GPCR systems of ghrelin(Kojima et al., 1999;Tschop et al.,2000),melanin-concentrating hormone (Shimada et al.,1998;Marsh et al.,2002),and the Oxs/Hcrts (Willie et al.,2001)play important roles in the central regulation of food intake.

Other physiological responses that have been found to be regulated by orphan GPCR systems include anxiety as it relates to stress,which is impacted by the activity of OFQ/N(Jenck et al.,1997;Koster et al.,1999)or NPS(Xu et al.,2004a,2004b).

It should be emphasized that,due their novelty,our understanding of the function of the novel neuropeptide systems is still at its infancy.Novel systems such as these are initially tested on the basis of receptor localization.Most of these orphan GPCRs are expressed in several parts of the CNS, their activation should affect the behavioral responses related to these CNS centers.Consequently,the effects that administra-tion of the novel neuropeptide has on behavior depend on the assays used.A novel neuropeptide may modulate a behavioral response in a totally new fashion and that may lead to the discovery of a different function for that neuropeptide.The Hcrt/Ox effect on sleep is an example of such discovery (Chemelli et al.,1999;Lin et al.,1999).It is therefore likely that the deorphanized GPCRs may point at new behavioral or physiological responses that will enlarge our understanding of the function of the organism.

3.The surprises of the reverse pharmacology approach

GPCRs have been deorphanized at a rate of7–8per year from1999until2004(Civelli,2005).This was mostly the result of large-scale random screenings of practically all molecules known to exist in cells.The primary pharmacolog-ical constraint of these screening endeavors was that active compounds exhibit affinities to the orphan GPCRs that are defined by the investigators.But there is no definite rule for predicting the affinity constant of a natural ligand at a particular receptor.Biogenic amines,for example,activate their cognate receptors with potencies that are mostly in the micromolar range while most peptides do so in the nanomolar range. Moreover,the level of receptor expression in a transfected cell can affect ligand potency.Consequently,one has to remember

O.Civelli et al./Pharmacology&Therapeutics110(2006)525–532 528

that the reverse pharmacology approach is based on the investigator’s set standards and therefore subject to artefacts (i.e.,‘‘made by the art’’).

These screening endeavors took place mainly in pharma-ceutical companies.From a pharmaceutical standpoint,finding a compound that can activate a GPCR is the key to opening the door for the drug discovery process(Robas et al.,2003a, 2003b).It is not a prerequisite that this compound is the genuine transmitter.In doing so,many orphan GPCRs were matched to undoubtedly genuine transmitters,but some were matched to only surrogate ligands.For example,the orphan GPCR PUMA-G/HM74was matched to nicotinic acid(Soga et al.,2003;Tunaru et al.,2003),a success with therapeutic implications,but one that leaves open the door for the search of the natural ligand.What grew from the intense search for natural and surrogate ligands by reverse pharmacology is that the number of potential genuine transmitters wound down.At that point,the technology began to be pushed to its limits and transmitters began to be found that are unexpected.This is further compounded by some recent discoveries showing that some GPCRs are activated by several transmitters that are chemically unrelated(Civelli,2005,in press).

3.1.The unexpected transmitters

An‘‘unexpected’’transmitter could best be defined as a naturally occurring molecule that was not expected to exert its action through a specific receptor.For example,UDP-glucose was found to activate the orphan GPCR KIAA0001with affinities in the100-nM range(Chambers et al.,2000).UDP-glucose was known to be a glucosyl donor in the biosynthesis of carbohydrates.Whether it acts as a transmitter has still to be shown.Other surprising examples are succinate and a-ketoglutarate,which are known as citric acid cycle intermedi-ates but were shown to activate the orphan GPCRs,GPR91 and99,respectively,with affinities in the25-to70-A M range (He et al.,2004).Succinic acid was identified not by random screening of defined ligands but through the purification of kidney extracts.Succinate was known to have a role on the reabsorption of phosphate and glucose in the proximal tubule and to stimulate gluconeogenesis.In GPR91-deficient mice, succinate was unable to induce hypertension,thus demonstrat-ing that its activity relies on GPR91activation(He et al.,2004). So there is no doubt that succinate is a genuine activator of GPR91,although the question remains whether it serves as a transmitter.Citric acid intermediates are not expected to be secreted in a regulated manner.They may be released upon mechanical stress or cell death,which would infer that some GPCRs are used as monitors of metabolic breakdown or global injury.

3.2.The promiscuous G-protein-coupled receptors

Another issue regarding pharmacological selectivity has arisen from some of the results of the application of reverse pharmacology.Subfamilies of GPCRs usually bind one or several closely related ligands.Three opioid receptors all bind opioid peptides and have evolved their structure to insure that they do not bind OFQ/N(Reinscheid et al.,1998;Meng et al., 1998).Catecholamine receptors are structurally related as are their ligands.Yet the adrenergic and dopaminergic systems are viewed as separate,although it has been shown that adrenaline and noradrenalin can efficiently activate the dopamine D4 receptor(Lanau et al.,1997).But this does not hold for a recently discovered GPCR subfamily,the Mas-related GPCRs (Mrgs or sensory neuron-specific receptors;SNSRs).This is a family of orphan GPCRs that is predominantly expressed in dorsal root ganglions and thus might have a role in nociception.Variable numbers of Mrgs exist in human,rat, and mouse making any attempt at orthologous classification difficult(Zylka et al.,2003).Being part of a subfamily,one could have expected that the Mrgs would bind similar transmitters.Instead,the Mrgs have been paired to a variety of structurally diverse transmitters:RFamide peptides for some mouse Mrgs(Dong et al.,2001);BAM22(Lembo et al.,2002) and cortistatin(Robas et al.,2003a,2003b)for2human Mrgs; adenine(Bender et al.,2002)for a rat Mrg;and h-alanine (Shinohara et al.,2004)for an Mrg found in human,rat,and mouse.The matched transmitters are specific to particular Mrgs and activate them efficiently.For example,the RFamides or BAM22peptides have affinities in the low nanomolar range (Lembo et al.,2002;Han et al.,2002),which is the range that is expected for peptides binding to GPCRs.So can it be that there are GPCR subfamilies that have a broad spectrum of transmitters and if so what does it imply for their function? From a physicochemical standpoint,if a molecule contains a motif that permits its interaction with a receptor,this interaction will take place.The issue is whether the receptor and that surrogate ligand will be in a position to interact in vivo.But this cannot be answered by reverse pharmacology.

3.3.The non-selective G-protein-coupled receptors

Finally,when considering the outcomes of reverse pharma-cology that go against one of the tenets of pharmacology,one has also to consider the case of a receptor that is not selective in its ligand recognition.Such a receptor is the orphan GPCR GPRC6A.One has come to accept that glutamate,glycine,or GABA receptors do not bind other amino acids.GPRC6A,on the other hand,can be activated by a series of basic l-a-amino acids with only a preference for basic amino acids(Well-endorph et al.,2005).For example,it is activated by l-Arg, with an affinity of about50A M.The concentration of l-Arg in the plasma is in the100-A M range.So,if Arg acts in an endocrine fashion,the receptor would be constantly and strongly activated.In addition,it would be surprising that any metabolic change would result in a high enough increase in the concentration of l-Arg to account for a significant difference in signaling.This suggests that l-Arg and the other GPRC6A ligands act in a paracrine fashion.Furthermore, because GPRC6A belongs to the receptor family that include the calcium sensing receptor(Brown,1999),it may sense free amino acid concentrations.Such a notion would help explain the promiscuity of GPRC6A in ligand recognition.

O.Civelli et al./Pharmacology&Therapeutics110(2006)525–532529

4.The impact of the orphan

G-protein-coupled receptor research

The search for the ligands of orphan GPCRs has impacted the basic and therapeutic fields.Although matching known trans-mitters to their respective GPCRs had the most success,it is the discovery of novel transmitters from tissue extracts that has had perhaps the most impact.By the mid-1990s,¨90transmitters were known;since then,a dozen new transmitters have been found and one expects that the remaining120orphan GPCRs will lead to the discovery of at least50more transmitters(Civelli et al.,2001).The deorphanization of GPCRs has revolutionized the discovery of novel transmitters and in turn these have revolutionized many fields of biomedical research in which they have been implicated.For example,the novel neuropeptides found as ligands of orphan GPCRs have changed our under-standing of the mechanisms that regulate sleep or food intake.

Orphan GPCR research has also dramatically impacted drug discovery.The GPCRs are de facto preferential drug targets. Although,as mentioned above,most of the GPCR deorphaniza-tions that took place until1995were achieved in academia, many of those deorphanized GPCRs were already targets of drug

development programs.They had been sought using traditional pharmaceutical techniques,often membrane binding assays.The deorphanization successes allowed the pharmaceutical industry to develop defined assays based on cloned receptors.The discovery of OFQ/N showed that orphan GPCRs could open the door to untapped drug targets.Furthermore,this line of research required high-throughput techniques that were in use in the pharmaceutical industry.It is therefore not surprising that the pharmaceutical industry was enthusiastic to espouse the reverse pharmacology approach(Wise et al.,2004).From1995,the majority of the GPCR deorphanizations have been carried out in pharmaceutical companies,and in this respect,Takeda Chemical Industries should be recognized as the most successful entity at discovering novel transmitters.

So there is no doubt that orphan GPCRs are used as potential drug targets.That there is no marketed drug directed at any of the ones that have been deorphanized since1995is of no surprise knowing the length of time required to bring a drug on the market.But are there drugs directed at any of these new deorphanized GPCRs in the pipeline?This is an answer that would,of course,require knowledge of proprietary drug discovery programs.Yet,one can foresee that several orphan GPCRs are being used as targets in drug discovery programs (Table2).The orphan GPCRs that bind novel neuropeptides represent pharmaceutical targets that may approach therapeutic needs from a totally novel standpoint.They have the potential to not only treat known indications but also to,possibly,define new ones.If pursued,they can lead to drugs with unmatched competitive advantages.They can also lead surprises that could prove of great benefits as,for example,OFQ/N that has been shown to be a potent antitussic agent(McLeod et al.,2001). Deorphanized GPCRs represent also assay targets.The targets listed in Table1B,for example,were recognized of therapeutic interest but could not be developed for drug screening.It is the deorphanization research that made this aim possible.If one considers that the entire number of biochemical targets that have led to marketed drugs are fewer than500,one has to recognize that the orphan GPCRs are an outstanding source of novel pharmaceutical targets.Whether drugs will ultimately reach patients can only be hoped.

Acknowledgments

This work was supported by NIH grants MH60231, DK63001,DK70619,and the Stanley Medical Research Institute.

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Table2

Foreseen impact of orphan GPCR research on drug discovery since1995 A.Novel transmitter systems with therapeutic implications

OFQ/N Anxiety

Hcrt/Ox Narcolepsy/obesity

Ghrelin Obesity

Prokineticin2Sleep

NPS Arousal/anxiety

B.Assay systems of therapeutic interest

Receptor Ligand Indication

SCL-1/GPR24MCH Obesity/CNS

GPR14Urotensin II High blood pressure P2Y12ADP Thrombosis CysLT1LTD4Asthma

CysLT2LTC4/LTD4Asthma

NPGPR NPFF and NPAF Pain

TA1Trace amines Depression

GPR40M and L chain fatty acids Diabetes

HM74Nicotinic acid Dyslipidemia

The therapeutic indications are derived from the authors’knowledge of the biology of the different systems.Deorphanized GPCR systems for which the authors do not foresee therapeutic indications of significant market size are not mentioned.

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脐带干细胞综述

脐带间充质干细胞的研究进展 间充质干细胞（mesenchymal stem cells,MSC S ）是来源于发育早期中胚层 的一类多能干细胞[1-5]，MSC S 由于它的自我更新和多项分化潜能，而具有巨大的 治疗价值 ,日益受到关注。MSC S 有以下特点：(1)多向分化潜能，在适当的诱导条件下可分化为肌细胞[2]、成骨细胞[3、4]、脂肪细胞、神经细胞[9]、肝细胞[6]、心肌细胞[10]和表皮细胞[11, 12]；(2)通过分泌可溶性因子和转分化促进创面愈合；(3) 免疫调控功能，骨髓源（bone marrow ）MSC S 表达MHC-I类分子，不表达MHC-II 类分子，不表达CD80、CD86、CD40等协同刺激分子，体外抑制混合淋巴细胞反应，体内诱导免疫耐受[11, 15]，在预防和治疗移植物抗宿主病、诱导器官移植免疫耐受等领域有较好的应用前景；(4)连续传代培养和冷冻保存后仍具有多向分化潜能，可作为理想的种子细胞用于组织工程和细胞替代治疗。1974年Friedenstein [16] 首先证明了骨髓中存在MSC S ，以后的研究证明MSC S 不仅存在于骨髓中，也存在 于其他一些组织与器官的间质中：如外周血[17]，脐血[5]，松质骨[1, 18]，脂肪组织[1]，滑膜[18]和脐带。在所有这些来源中，脐血(umbilical cord blood)和脐带(umbilical cord)是MSC S 最理想的来源，因为它们可以通过非侵入性手段容易获 得，并且病毒污染的风险低，还可冷冻保存后行自体移植。然而，脐血MSC的培养成功率不高[19, 23-24]，Shetty 的研究认为只有6%，而脐带MSC的培养成功率可 达100%[25]。另外从脐血中分离MSC S ，就浪费了其中的造血干/祖细胞(hematopoietic stem cells/hematopoietic progenitor cells,HSCs/HPCs) [26, 27]，因此，脐带MSC S (umbilical cord mesenchymal stem cells, UC-MSC S )就成 为重要来源。 一．概述 人脐带约40 g, 它的长度约60–65 cm, 足月脐带的平均直径约1.5 cm[28, 29]。脐带被覆着鳞状上皮，叫脐带上皮，是单层或复层结构，这层上皮由羊膜延续过来[30, 31]。脐带的内部是两根动脉和一根静脉，血管之间是粘液样的结缔组织，叫做沃顿胶质，充当血管外膜的功能。脐带中无毛细血管和淋巴系统。沃顿胶质的网状系统是糖蛋白微纤维和胶原纤维。沃顿胶质中最多的葡萄糖胺聚糖是透明质酸，它是包绕在成纤维样细胞和胶原纤维周围的并维持脐带形状的水合凝胶，使脐带免受挤压。沃顿胶质的基质细胞是成纤维样细胞[32]，这种中间丝蛋白表达于间充质来源的细胞如成纤维细胞的，而不表达于平滑肌细胞。共表达波形蛋白和索蛋白提示这些细胞本质上肌纤维母细胞。 脐带基质细胞也是一种具有多能干细胞特点的细胞，具有多项分化潜能，其 形态和生物学特点与骨髓源性MSC S 相似[5, 20, 21, 38, 46]，但脐带MSC S 更原始，是介 于成体干细胞和胚胎干细胞之间的一种干细胞，表达Oct-4, Sox-2和Nanog等多

脐带血造血干细胞库管理办法(试行)

脐带血造血干细胞库管理办法（试行） 第一章总则 第一条为合理利用我国脐带血造血干细胞资源，促进脐带血造血干细胞移植高新技术的发展，确保脐带血 造血干细胞应用的安全性和有效性，特制定本管理办法。 第二条脐带血造血干细胞库是指以人体造血干细胞移植为目的，具有采集、处理、保存和提供造血干细胞 的能力，并具有相当研究实力的特殊血站。 任何单位和个人不得以营利为目的进行脐带血采供活动。 第三条本办法所指脐带血为与孕妇和新生儿血容量和血循环无关的，由新生儿脐带扎断后的远端所采集的 胎盘血。 第四条对脐带血造血干细胞库实行全国统一规划，统一布局，统一标准，统一规范和统一管理制度。 第二章设置审批 第五条国务院卫生行政部门根据我国人口分布、卫生资源、临床造血干细胞移植需要等实际情况，制订我 国脐带血造血干细胞库设置的总体布局和发展规划。 第六条脐带血造血干细胞库的设置必须经国务院卫生行政部门批准。 第七条国务院卫生行政部门成立由有关方面专家组成的脐带血造血干细胞库专家委员会（以下简称专家委

员会），负责对脐带血造血干细胞库设置的申请、验收和考评提出论证意见。专家委员会负责制订脐带血 造血干细胞库建设、操作、运行等技术标准。 第八条脐带血造血干细胞库设置的申请者除符合国家规划和布局要求，具备设置一般血站基本条件之外， 还需具备下列条件： （一）具有基本的血液学研究基础和造血干细胞研究能力； （二）具有符合储存不低于1 万份脐带血的高清洁度的空间和冷冻设备的设计规划； （三）具有血细胞生物学、HLA 配型、相关病原体检测、遗传学和冷冻生物学、专供脐带血处理等符合GMP、 GLP 标准的实验室、资料保存室； （四）具有流式细胞仪、程控冷冻仪、PCR 仪和细胞冷冻及相关检测及计算机网络管理等仪器设备； （五）具有独立开展实验血液学、免疫学、造血细胞培养、检测、HLA 配型、病原体检测、冷冻生物学、 管理、质量控制和监测、仪器操作、资料保管和共享等方面的技术、管理和服务人员； （六）具有安全可靠的脐带血来源保证； （七）具备多渠道筹集建设资金运转经费的能力。 第九条设置脐带血造血干细胞库应向所在地省级卫生行政部门提交设置可行性研究报告，内容包括：

卫生部办公厅关于印发《脐带血造血干细胞治疗技术管理规范(试行)

卫生部办公厅关于印发《脐带血造血干细胞治疗技术管理规 范(试行)》的通知 【法规类别】采供血机构和血液管理 【发文字号】卫办医政发[2009]189号 【失效依据】国家卫生计生委办公厅关于印发造血干细胞移植技术管理规范(2017年版)等15个“限制临床应用”医疗技术管理规范和质量控制指标的通知 【发布部门】卫生部(已撤销) 【发布日期】2009.11.13 【实施日期】2009.11.13 【时效性】失效 【效力级别】部门规范性文件 卫生部办公厅关于印发《脐带血造血干细胞治疗技术管理规范（试行）》的通知 （卫办医政发〔2009〕189号） 各省、自治区、直辖市卫生厅局，新疆生产建设兵团卫生局： 为贯彻落实《医疗技术临床应用管理办法》，做好脐带血造血干细胞治疗技术审核和临床应用管理，保障医疗质量和医疗安全，我部组织制定了《脐带血造血干细胞治疗技术管理规范（试行）》。现印发给你们，请遵照执行。 二〇〇九年十一月十三日

脐带血造血干细胞 治疗技术管理规范（试行） 为规范脐带血造血干细胞治疗技术的临床应用，保证医疗质量和医疗安全，制定本规范。本规范为技术审核机构对医疗机构申请临床应用脐带血造血干细胞治疗技术进行技术审核的依据，是医疗机构及其医师开展脐带血造血干细胞治疗技术的最低要求。 本治疗技术管理规范适用于脐带血造血干细胞移植技术。 一、医疗机构基本要求 （一）开展脐带血造血干细胞治疗技术的医疗机构应当与其功能、任务相适应，有合法脐带血造血干细胞来源。 （二）三级综合医院、血液病医院或儿童医院，具有卫生行政部门核准登记的血液内科或儿科专业诊疗科目。 1.三级综合医院血液内科开展成人脐带血造血干细胞治疗技术的，还应当具备以下条件： （1）近3年内独立开展脐带血造血干细胞和（或）同种异基因造血干细胞移植15例以上。 （2）有4张床位以上的百级层流病房，配备病人呼叫系统、心电监护仪、电动吸引器、供氧设施。 （3）开展儿童脐带血造血干细胞治疗技术的，还应至少有1名具有副主任医师以上专业技术职务任职资格的儿科医师。 2.三级综合医院儿科开展儿童脐带血造血干细胞治疗技术的，还应当具备以下条件：

卫生部关于印发《脐带血造血干细胞库设置管理规范(试行)》的通知

卫生部关于印发《脐带血造血干细胞库设置管理规范(试行)》的通知 发文机关：卫生部(已撤销) 发布日期： 2001.01.09 生效日期： 2001.02.01 时效性：现行有效 文号：卫医发(2001)10号 各省、自治区、直辖市卫生厅局： 为贯彻实施《脐带血造血干细胞库管理办法（试行）》，保证脐带血临床使用的安全、有效，我部制定了《脐带血造血干细胞库设计管理规范（试行）》。现印发给你们，请遵照执行。 附件：《脐带血造血干细胞库设置管理规范（试行）》 二○○一年一月九日 附件： 脐带血造血干细胞库设置管理规范（试行） 脐带血造血干细胞库的设置管理必须符合本规范的规定。 一、机构设置 （一）脐带血造血干细胞库（以下简称脐带血库）实行主任负责制。 （二）部门设置 脐带血库设置业务科室至少应涵盖以下功能：脐带血采运、处理、细胞培养、组织配型、微生物、深低温冻存及融化、脐带血档案资料及独立的质量管理部分。 二、人员要求

（一）脐带血库主任应具有医学高级职称。脐带血库可设副主任，应具有临床医学或生物学中、高级职称。 （二）各部门负责人员要求 1．负责脐带血采运的人员应具有医学中专以上学历，2年以上医护工作经验，经专业培训并考核合格者。 2．负责细胞培养、组织配型、微生物、深低温冻存及融化、质量保证的人员应具有医学或相关学科本科以上学历，4年以上专业工作经历，并具有丰富的相关专业技术经验和较高的业务指导水平。 3．负责档案资料的人员应具相关专业中专以上学历，具有计算机基础知识和一定的医学知识，熟悉脐带血库的生产全过程。 4．负责其它业务工作的人员应具有相关专业大学以上学历，熟悉相关业务，具有2年以上相关专业工作经验。 （三）各部门工作人员任职条件 1．脐带血采集人员为经过严格专业培训的护士或助产士职称以上卫生专业技术人员并经考核合格者。 2．脐带血处理技术人员为医学、生物学专业大专以上学历，经培训并考核合格者。 3．脐带血冻存技术人员为大专以上学历、经培训并考核合格者。 4．脐带血库实验室技术人员为相关专业大专以上学历，经培训并考核合格者。 三、建筑和设施 （一）脐带血库建筑选址应保证周围无污染源。 （二）脐带血库建筑设施应符合国家有关规定，总体结构与装修要符合抗震、消防、安全、合理、坚固的要求。 （三）脐带血库要布局合理，建筑面积应达到至少能够储存一万份脐带血的空间；并具有脐带血处理洁净室、深低温冻存室、组织配型室、细菌检测室、病毒检测室、造血干/祖细胞检测室、流式细胞仪室、档案资料室、收/发血室、消毒室等专业房。 （四）业务工作区域应与行政区域分开。

脐带血间充质干细胞的分离培养和鉴定

脐带血间充质干细胞的分离培养和鉴定 【摘要】目的分离培养脐带血间充质干细胞并检测其生物学特性。方法在无菌条件下用密度梯度离心的方法获得脐血单个核细胞，接种含10%胎牛血清的DMEM培养基中。单个核细胞行贴壁培养后，进行细胞形态学观察，绘制细胞生长曲线，分析细胞周期，检测细胞表面抗原。结果采用Percoll(1.073 g/mL)分离的脐血间充质干细胞大小较为均匀，梭形或星形的成纤维细胞样细胞。细胞生长曲线测定表明接后第5天细胞进入指数增生期，至第9天后数量减少;流式细胞检测表明50%～70%细胞为CD29和CD45阳性。结论体外分离培养脐血间充质干细胞生长稳定，可作为组织工程的种子细胞。 【关键词】脐血;间充质干细胞;细胞周期;免疫细胞化学 Abstract: Objective Isolation and cultivation of mesenchymal stem cells (MSCs) in human umbilical cord in vitro, and determine their biological properties. Methods The mononuclear cells were isolated by density gradient centrifugation from human umbilical cord blood in sterile condition, and cultured in DMEM medium containing 10% fetal bovine serum. After the adherent mononuclear cells were obtained, the shape of cells were observed by microscope, then the cell growth curve, the cell cycle and the cell surface antigens were obtained by immunocytochemistry and flow cytometry methods. Results MSCs obtained by Percoll (1.073 g/mL) were similar in size, spindle-shaped or star-shaped fibroblasts-liked cells. Cell growth curve analysis indicated that MSCs were in the exponential stage after 5d and in the stationary stages after 9d. Flow cytometry analysis showed that the CD29 and CD44 positive cells were about 50%～70%. Conclusions The human umbilical cord derived mesenchymal stem cells were grown stably in vitro and can be used as the seed-cells in tissue engineering. Key words:human umbilical cord blood; mesenchymal stem cells; cell cycle; immunocytochemistry 间充质干细胞(mesenchymal stem cells，MSCs)在一定条件下具有多向分化的潜能，是组织工程研究中重要的种子细胞来源。寻找来源丰富并不受伦理学制约的间充质干细胞成为近年来的研究热点[1]。脐血(umbilical cord blood, UCB)在胚胎娩出后，与胎盘一起存在的医疗废物。与骨髓相比，UCB来源更丰富，取材方便，具有肿瘤和微生物污染机会少等优点。有人认为脐血中也存在间充质干细胞(Umbilical cord blood-derived mesenchymal stem cells，UCB-MSCs)。如果从脐血中培养出MSCs，与胚胎干细胞相比，应用和研究则不受伦理的制约，蕴藏着巨大的临床应用价值[2,3]。本研究将探讨人UCB-MSCs体外培养的方法、细胞的生长曲线、增殖周期和细胞表面标志等方面，分析UCB-MSCs 作为间充质干细胞来源的可行性。

脐带血干细胞检测

脐带血干细胞检测 对每份脐血干细胞进行下列检测： ①母体血样做梅毒、HIV和CMV等病原体检测，这一检测使脐血干细胞适合于其它家庭成员应用。如任何一种病原体测试阳性，需重复测定。 ②每份脐血干细胞样本同时检测确定没有微生物污染。 ③细胞活性检测、有核细胞数、CD34+细胞数、集落形成试验等。CD34是分子量115KD 的糖蛋白分子，使用特定单克隆抗体（抗-CD34）确定，脐血祖细胞的大部分，包括体外培养产生造血集落的细胞都包含在表达CD34抗原的细胞群中。 ④HLA组织配型、ABO血型。 一、采血方式及其优点 再生缘生物科技公司采用最严谨的封闭式血袋收集法，避免在收集脐带血液时可能遭受微生物污染的发生，且以最少之操作步骤，收集最大量之脐带血液方式，在产房内即可完成。 二、脐带血处理与保存 脐带血收集于血袋，经专人运送至再生缘生物科技公司之无菌细胞分离实验室后，由专业的技术人员于完全无菌的环境下，依标准操作程序将血液进行分离，收集具有细胞核的细胞，其中含有丰富的血液干细胞，经加入冷冻保护剂和适当品管检测后，并进行以最适合

血液干细胞的冷冻降温程序方式，进行细胞冷冻程序，达到避免细胞受到冷冻过程之伤害。完成后，冷冻细胞立刻保存于摄氏零下196度的液态氮槽中。所有操作程序记录和细胞保存相关数据，均由计算机条形码系统追踪确认，完全符合国际脐带血库之标准操作程序和品管要求。 母亲血液之检测 为确保所操作和保存的脐带血液细胞，符合国际血液操作规范，并提供客户最大的保障，对于产妇血液必须同时进行一些病毒传染病的检测，以确保没有下列病毒，如艾滋病毒(HIV)、C型肝炎病毒(HCV)、人类T细胞淋巴病毒(HTLV)和梅毒(syphilis)，同时对于B型肝炎病毒(HBV)和巨细胞病毒(CMV)加以侦测和纪录，作为将来可能应用脐带血细胞时之必要参考数据并符合卫生医疗之要求。 脐带血细胞之品管 对于所保存之脐带血细胞均进行多项操作流程监控和品管检测，如微生物污染检测、血液细胞浓度、细胞存活率、细胞活性测定等，每一步骤均有详细之纪录，在操作方法和使用仪器方面均定期进行验证和校验，以符合国际医疗标准。 三、实验室、贮存处所介绍 再生缘生物科技公司拥有符合美国联邦标准(FED-STD-209E)和中华民国优良药品制造标准（一区、二区、三区）的生物安全实验室和无菌操作设备，在专业的技术人员依标准操作程序下进行血液分离和保存步骤，保障客户珍贵样品和权益。 分离后之细胞将依浓度分装入4-6个冷冻管，计算机降温冷冻完成后，即由食品工业发展研究所国家细胞库专业液态氮库房人员，将冷冻细胞分别存放于二个不同的脐带血细胞专属液态氮槽中保存，在安全机制上更有保障。液态氮库房拥有五吨的液态氮供应系统，每一液氮槽均有自动充填装置和异常警报系统，和每日值勤人员监控，确保冷冻细胞处于最佳的冷冻状态。 四、安全管制措施 脐带血液经快递送达无菌细胞分离实验室后，每一步骤均有专业技术人员操作和监督，并将所有分析数值详细填于具有条形码管制之分析表格和计算机数据表中，利用条形码和读码系统确认样品之专一性，避免人为失误，且便于追溯和数据品管。 在冷冻细胞保存上

胎盘干细胞与脐带血干细胞的区别

胎盘干细胞与脐带血干细胞的区别 干细胞是一类具有自我复制和多向分化能力的原始的未分化的细胞，它可以向多种类型细胞分化，并具有相应的功能，可以用来修复和替代受到损伤，病变的组织和器官。目前主要的成体干细胞来源有胎盘来源、脐带来源、脐带血来源和骨髓来源的干细胞，本文主要介绍一下胎盘来源的干细胞和脐带血来源的干细胞的区别： 1、分离部位不同：胎盘干细胞是从胎盘组织中分离提取的干细胞，脐带血干细胞是从脐带里面血液中分离提取的干细胞。 2、种类不同：胎盘中的干细胞主要指的是间充质干细胞，而脐带血干细胞主要指的是造血干细胞。 3、分化能力不同：1）胎盘干细胞分化能力强，在特定的诱导条件下可以分化成血管干细胞、神经干细胞、肝干细胞等多种类型的干细胞，从而修复受损和病变的组织和器官2）脐带血干细胞可以在体内向红细胞、血小板等各种血液细胞分化。 4、数量不同：1）胎盘体积大，从中提取的干细胞数量丰富，并可以在体外培养扩增，扩增培养的子细胞数量达十亿个，可以供成人多次使用2）脐带血干细胞的数量要根据抽取的脐带血多少而定，不可以在体外培养扩增，一份脐带血干细胞可以供40kg以下患者一次使用。 5、治疗使用：1）胎盘干细胞目前在治疗脑瘫、糖尿病、肝硬化、心血管疾病等诸多疾病都显示了良好的效果2）脐带血干细胞可以治疗白血病，再生障碍性贫血等血液系统疾病。不过对于儿童白血病多以先天性为主，所以对于这种情况，自己的脐带血干细胞是不能使用的，需要配型使用捐献的脐血干细胞。 6、配型方面：二者自体使用都不需要配型，如果异体使用，胎盘干细胞由于免疫源性低的特点，所以配型成功率非常高，有血缘关系的亲属都可以使用；脐带血干细胞与父母配型有1/2的几率，与兄弟姐妹有1/4的几率。

脐带血造血干细胞库技术规范

脐带血造血干细胞库技术规范(试行) I 脐带血造血干细胞库(简称脐带血库)的质量控制 1 规章制度和操作规程 脐带血库必须制定脐带血采集、制备、检测、库存、选择和发放的规章制度、操作规程。 1.1 脐带血供者筛选标准和咨询。 1.2 脐带血采集和运送。 1.3 脐带血制备、冷冻、库存。 1.4 标签。 1.5 传染性疾病、人类组织相容性抗原（HLA）分型、造血干细胞和其他检测。 1.6 库存脐带血和脐带血检测标本的确认。 1.7 脐带血的发放。 1.8 脐带血库与移植机构之间的运输。 1.9 数据管理、申请查询、供者与受者配型、脐带血的选择。 1.10 移植随访资料的收集和分析。 1.11 人员培训和继续教育。 1.12 材料、试剂和设备。 1.13 不合格产品、操作错误和事故的报告。 1.14 卫生清洁。 1.15 保密制度。 2 规章制度和操作规程的执行 2.1有脐带血库主任的签字及开始实施的日期。 2.2各项规章制度和操作规程修改，须由脐带血库主任或文件起草人进行审查、签字并标明日期。 2.3 规章制度和操作规程应置于方便工作人员随时取用的位置。 2.4 存档的各种规程和标准记录应长期保留。 2.5 如认为本技术规范不适应当前发展，允许各脐带血库根据情况适当调整，但应报脐带血造血干细胞库专家委员会备案，备案期为30天。专家委员会如不同意备案的规章制度和操作规程，应在备案期间内通知备案单位停止执行。 3 质量控制 3.1 应有专门的质量控制规程，以便对脐带血库工作人员在常规操作中所使用的规程、试剂、设备和材料进行质量控制。 3.2 脐带血库内部的质量控制 3.2.1应由脐带血库主任或指定专人进行质量控制。 3.2.2脐带血库内部的质量控制包括质量评估，改进和修正的措施，错误和事故的处理。 3.2.2.1 脐带血库必须有不合格产品的记录和报告。 3.2.2.2 脐带血库主任应定期召开质量评价会议，对错误和事故进行评价；对重大事故应及时处理。 3.2.2.3 脐带血库主任必须签发对规程的修正。

脐带血干细胞的基础与应用研究

生命科学 Chinese Bulletin of Life Sciences 第18卷 第4期2006年8月 Vol. 18, No. 4Aug., 2006 脐带血干细胞的基础与应用研究 顾东生, 刘 斌, 韩忠朝* （中国医学科学院中国协和医科大学血液学研究所实验血液学国家重点实验室，天津 300020） 摘　要：作为造血干/祖细胞(hematopoietic stem cells/hematopoietic progenitor cells, HSCs/HPCs)的另一 来源，脐带血已经应用于临床治疗多种恶性和非恶性疾病。脐带血中HSCs/HPCs 的质与量是决定其临床应用效果的最重要因素。同时，脐带血中还存在多种非造血的干细胞和前体细胞，如间充质干细胞(mesenchymal stem cells, MSCs)、内皮前体细胞(endothelial progenitor cells, EPCs)和非限制性体干细胞(unrestricted somatic stem cells, USSCs)等，这些细胞可能会在未来的细胞治疗和再生医学中发挥重要作用。本综述还讨论了脐带血的临床应用及HSCs/HPCs 的体外扩增、增加HSCs 归巢和再植能力等提高其临床应用能力的相关研究。关键词：脐带血；造血干细胞；移植 中图分类号：R322.2; R323.3; Q813 文献标识码：A The research and application of cord blood stem cells GU Dong-Sheng, LIU Bin, HAN Zhong-Chao* (State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China) Abstract: Umbilical cord blood (UCB), as an alternative source of hematopoietic/progenitor stem cells (HSCs/HPCs), has been used clinically for a large number of malignant and non-malignant disorders. The quality and quantity of HSC and HPC may be the most important factors on which the capacity of UCB to perform clinical function depends. Other non-HSCs/HPCs, such as mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and unrestricted somatic stem cells (USSCs), also present in cord blood, which may play a future role in cell therapy and regenerative medicine. This review also covers the efforts to expand HSCs and HPCs ex vivo and recent studies on attempts to enhance the homing and engrafting capability of HSCs as means to enhance the clinical utility of UCB. Key words: umbilical cord blood; hematopoietic stem cells; transplantation 收稿日期：2006-04-25 基金项目：“863”计划(2003AA205060)；“973”项目子项(2001C B5101) 作者简介：顾东生(1981—)，男，硕士研究生；刘 斌(1973—)，男，硕士，助理研究员；韩忠朝(1953—)，男，博士，教授，博士生导师，*通讯作者。 文章编号 ：1004-0374(2006)04-0323-05 1988年，Broxmeyer 首先以实验证明脐带血(umbilical cord blood, UCB)中富含造血干细胞(hematopoietic stem cells, HSCs)。法国Gluckman 等[1]在巴黎圣路易斯医院为一位患有先天性再生不良性贫血的儿童实施了世界上首例脐带血移植术，并取得成功。从此，人们对于一直被当成废弃物丢掉 的胎盘和脐带血有了全新的评价和认识，至今，各国学者对脐带血的基础研究和临床应用进行了大量工作并取得很大成绩。本文对脐带血中存在的多种干/祖细胞的生物学特性及临床应用研究进行综述。1　脐带血干细胞 在现阶段，脐带血之所以能够应用于临床治疗

胎盘干细胞和脐带血干细胞是否需要都保存

胎盘干细胞和脐带血干细胞是否需要都保存？ 近来,保存胎盘干细胞热潮在京城悄然兴起，引起了人们对于保存胎盘干细胞和脐带血干细胞的一番讨论，即究竟该保存哪个更有价值呢？是否需要都保存呢？针对这一问题，记者特意查找了大量专业性新闻报导及相关信息，并就该问题咨询了北京博雅干细胞库技术人员，以求寻获一个明确的答案。 记者了解到，在保存胎盘干细胞热潮兴起之前，保存脐带血干细胞或捐献 脐带血干细胞的观念早已深入人心，而且已经占据了一定的市场。并且大多数人都已知晓利用脐带血干细胞能够治愈血液系统疾病等等诸多好处。但是对于胎盘干细胞在生物医学领域的重大用途却知之甚少。 现如今，胎盘干细胞同脐带血干细胞一样被人们所熟知，可是面临的问题 却使得人们不知该如何选择，那就是胎盘干细胞和脐带血干细胞是否应该都保存呢? 针对此问题,记者经过一番周折终于可以比较专业的为大家解答这个问题了。其实，保存胎盘主要是保存从胎盘中提取的间充质干细胞，而保存脐带血是保存脐带血中的造血干细胞。那么，胎盘间充质干细胞和脐带血造血干细胞有何区别呢？首先，胎盘中含有的间充质干细胞非常丰富，这种间充质干细胞属于多能干细胞，具有强大的增殖能力和多向分化潜能，能培养发育成人体的各个组织器官与神经系统。而脐带血中的造血干细胞含量较少，这种造血干细胞属于单能干细胞，不能进行体外扩增，不能分化成为其他的干细胞。其次，胎盘间充质干细胞治疗疾病范围比较广泛，如对于治疗心、脑血管疾病、神经系统疾病、肝脏疾病、骨组织病、角膜损伤、烧伤烫伤、肌病等多种疾病都有不错的疗效。造血干细胞主要针对治疗血液系统疾病和免疫系统疾病具有较好的疗效。最后，记者想告诉大家的是，胎盘间充质干细胞和脐带血造血干细胞两者同样都具有很高的医学价值，都能治疗多种疾病，虽然造血干细胞治疗疾病范围有限且不能进行体外扩增，导致干细胞数量仅可供30公斤以下儿童一次使用，但是胎盘间充质干细胞可以和造血干细胞共移植治疗血液系统疾病，并可供成人使用。另外，两者在采集的时候都不会给产妇和新生儿带来任何不适的感觉和产生任何不良的影响，并且在未来生物医学研究发展过程中，都将起到不可或缺的作用，并将推动再生医学的发展。那么，人们更应该保存哪个呢？记者建议，如果经济条件许可的话，建议人们将两者一起保存起来，以备将来不时之需。

脐血干细胞移植实施方案

附件3 脐带血造血干细胞移植实施方案

(一）准备 患者准备 1、身体准备全面体检和实验室检查； 2、心理准备移植病人大多数对治疗方法及过程缺乏了解，又因长期接受化疗，造成很大的痛苦，病人对移植既抱有希望，又有焦虑和恐惧的心理。因此，在移植前护理人员应主动与病人及家属进行交谈，尽可能做好心理。 物品准备： 病人入舱前，舱内所有物品包括药品、被服、纸张、卫生材料、医疗器械都要经过灭菌处理后，由传递窗送入无菌舱内。 病人在舱内的生活用品，经灭菌处理后入舱。 环境准备： 无菌层流舱: ?患者舱：100级 ?护士站、治疗室等：1000级 ?手消毒间、备无菌餐间：10，000级 ?更衣间、药浴间：100，000级 ------舱内压力递减 患者入住前环境准备 1、彻底卫生清洁： 2、熏蒸24小时：每立方米用高锰酸钾5mg+40%甲醛10ml，熏蒸24小时，通风24小时。 3、入住前的全面消毒液擦拭。 4、空气培养：达标。目前选用平皿沉降法检测； 5、入室物品一律消毒灭菌：可以高压灭菌或适合环氧乙烷消毒的物品，一律灭菌后进舱，须浸泡消毒的物品要确保浸泡消毒的效果可靠。 患者入住后无菌全环境的保持 （一）入住后患者要求： 1、每日以KL-98消毒液洗头、洗脸、擦身、洗脚，早晚各一次（20分钟）。 2、每日以KL-98消毒液于晨起、睡前、便后坐浴一次（20分钟）。

3、睡前、饭前、饭后（进食任何饮食后）认真漱口。 4、3%双氧水擦洗鼻前庭、外耳道每日三次，然后用碘伏消毒液擦拭，再涂以红霉素软膏等。 5、抗菌及抗病毒的眼药水交替点眼，每日三次。 6、经常以含KL-98消毒液棉球擦手（代替洗手）。 （二）入住后环境要求： 1、净化舱内地面、所有物品表面每日消毒液擦拭一次，发现有污染随时擦拭消毒。 2、室内墙壁隔天消毒液擦拭一次。 3、被服高压消毒更换每日一次。 4、空气喷雾消毒每日一次。 5、坐便桶、污水桶每日更换消毒一次。 （三）无菌饮食要求： 1、食物新鲜，彻底洗净、煮熟、微波炉消毒7分钟。 2、水时须做成水果羹后微波炉消毒，或须经消毒后用无菌刀削皮后方可食用。 3、饼干、馒头放微波炉隔水蒸7分钟。 4、饮水均须用开水经舱内电热水瓶二次沸腾后方可饮用。 5、餐具严格消毒。 工作人员入室要求： 严格控制入室人员。医护人员入室前先淋浴，更换清洁衣裤，戴清洁帽子。在缓冲间用肥皂洗手，清水冲净后，再用手快速消毒剂擦手，然后更换无菌拖鞋进入更衣间。戴一次性无菌手套，按无菌操作要求穿无菌分体式隔离衣，戴无菌口罩，进入消毒间再次消毒手，更换无菌拖鞋方可进入护士站。如果进入病人所在的百级层流病房，还需戴无菌手套，穿无菌隔离衣，更换无菌拖鞋方可进入。(二）预处理 定义：是指在输注造血干细胞前对病人进行的大剂量化疗或放疗。 目的：尽可能杀灭病人体内的异常细胞或肿瘤细胞，最大限度减少复发；破坏病人免疫系统，为造血干细胞的植入提供条件，防止移植物被排斥；为造血干细胞的植入、生长提供必要的空间。