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VEGF综述

VEGF-A immunohistochemical and mRNA expression in tissues and its serum levels in potentially malignant oral lesions and oral squamous cell carcinomas

Seema Nayak a ,1,Madhu Mati Goel a ,?,Saumya Chandra a ,2,Vikram Bhatia a ,3,Divya Mehrotra b ,4,Sandeep Kumar c ,5,Annu Makker a ,6,S.K.Rath d ,7,S.P.Agarwal e ,8

Department of Pathology,Chhatrapati Shahuji Maharaj Medical University,Lucknow 226003,Uttar Pradesh,India

Department of Oral and Maxillofacial Surgery,Chhatrapati Shahuji Maharaj Medical University,Lucknow 226003,Uttar Pradesh,India c

Department of General Surgery,Chhatrapati Shahuji Maharaj Medical University,Lucknow 226003,Uttar Pradesh,India d

Division of Toxicology,Central Drug Research Institute,Lucknow 226001,Uttar Pradesh,India e

Department of Otolaryngology,Chhatrapati Shahuji Maharaj Medical University,Lucknow 226003,Uttar Pradesh,India

a r t i c l e i n f o Article history:

Received 26July 2011

Received in revised form 14September 2011

Accepted 10October 2011

Available online 2November 2011Keywords:VEGF-A

Oral squamous cell carcinoma Potentially malignant oral lesions Angiogenesis

Oral submucous ?brosis Immunohistochemistry CD 34

Real-time PCR ELISA

s u m m a r y

The aim of the study was to investigate whether the estimation of circulating Vascular endothelial growth factor-A (VEGF-A)levels by ELISA could be used as surrogate of VEGF-A expression in tissues of pre-malignant oral lesions (PMOLs)and oral squamous cell carcinoma (OSCC)as compared to that in healthy controls.The study samples comprised of tissue and blood samples from 60PMOLs,60OSCC,and 20healthy controls.Serum VEGF-A levels were determined by an ELISA based assay (Quantikine human VEGF;R &D System,Minneapolis USA).Tissue VEGF-A expression and microvessel density (MVD)were assessed by immunohistochemistry (IHC)using antibodies against VEGF-A and CD-34on formalin ?xed paraf?n embedded (FFPE)tissue sections.VEGF-A mRNA expression was analyzed by real-time PCR in snap frozen tissues.Serum VEGF-A levels and immunohistochemical VEGF-A expression were signi?cantly high in PMOLs and OSCC in comparison with controls.VEGF mRNA gene expression showed more than 50-fold increase in PMOLs and OSCC.VEGF-A levels in serum correlated in a linear fashion with the tissue expression in oral pre-malignant and malignant lesions,suggesting that the serum levels may serve as surrogate material for tissue expression of VEGF-A.

Introduction

Oral cancer is a major public health problem in India because of the habit of tobacco consumption in various forms like bêtel quid,khaini,pan masala and guthka.Majority of the patients already have loco-regional spread to lymph nodes at the time of initial diagnosis with dismal prognosis.Histopathologic examination is

still the gold standard for diagnostic,prognostication and follow up purposes.1,2

Vascular endothelial growth factor (VEGF)is known to be a fun-damental regulator of tumour angiogenesis and therefore is con-sidered to be an important therapeutic target.It is a potent angiogenic cytokine involved in every stage of vascular develop-ment.The multidimensional role of VEGF is based on its ability to induce various responses by endothelial cells during vascular development,including cell proliferation,migration,tumor angio-genesis,disease progression,prognosis and survival.3–9The key molecules and pathways for targeted therapy include growth fac-tor receptors,MAPK/ERK pathway and angiogenesis.For targeted therapy however a single molecule has to be identi?ed.Circulating VEGF is one such promising marker.10VEGF neutralizing antibod-ies have been reported to enhance the effect of chemotherapy and radiotherapy used for treatment of cancers.10

Among the family of VEGFs,including VEGF-A,VEGF-B,VEGF-C,VEGF-D and VEGF-F,VEGF-A is a key angiogenic factor and is most frequently used by a tumor to switch on its angiogenic phenotype.Serum VEGF-A levels have been reported to be elevated in oral

?Corresponding author.Address:Department of Pathology,Chhatrapati Shahuji

Maharaj Medical University (Erstwhile King George’s Medical University),Lucknow 226003,Uttar Pradesh,India.Tel.:+9109415010742;fax:+9105222257539.

E-mail address:madhukgmc@https://www.wendangku.net/doc/7f4554693.html, (M.M.Goel).1

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squamous cell carcinoma(OSCC)and have also been correlated with lymph node metastasis and clinical staging.11Anti-angiogenic drugs have already been added to therapeutic algorithms in a num-ber of solid tumors.There are reports of their role in recurrent and metastatic head and neck cancer and are under clinical trials.12,13 Knowledge of phenotypic expression of targeted molecules and how the level of these molecules can be assessed,is essential in or-der to decide that which patient will be bene?tted by the targeted therapy.12At present,there are no tumor characteristics or molec-ular markers that can identify patients of oral cancer who are par-ticularly likely to bene?t from anti-angiogenic treatment.The search for biomarkers includes evaluation of tissues and surrogate materials(like saliva,blood and body?uids)by molecular,geno-mic and phenotypic means.12

The present study was undertaken to investigate(1)the extent of VEGF-A expression in tissues of potentially malignant oral le-sions and oral squamous cell carcinoma and(2)to?nd out whether the estimation of serum VEGF-A levels by ELISA could be a surro-gate of VEGF-A expression in tissues of PMOLs and OSCC as com-pared to healthy controls.

Materials and methods

Patients and tissue samples

Tissue biopsies and blood samples from prospective cases of60 PMOLs,60OSCC and20healthy controls were collected from the Departments of Oral and Maxillofacial Surgery and Otolaryngology after obtaining the Institutional Ethical approval and informed written consent from the patients.Healthy oral tissues were ob-tained from patients undergoing cosmetic surgery,who otherwise did not have any infective or in?ammatory oral lesion.A small part of the tissue was snap frozen for molecular work and was stored at à80°C.The relevant clinical and demographic details were recorded.

Histopathological examination

All oral tissues were?xed in10%neutral buffered formalin and processed for histopathological examination as per standard proce-dure.5l m thick sections were cut and stained with haematoxylin and eosin(H&E).Sections were reviewed by two independent pathologists and histological diagnosis was made as per WHO criteria.

Immunohistochemistry for VEGF–A

Sections were deparaf?nized in xylene followed by hydration in descending ethanol grades.Endogenous peroxidase was blocked in 3%H2O2in methanol for30min.Antigen retrieval was performed by heating specimens for15min at95°C in10mM Tris–EDTA buf-fer(pH9.0)using an EZ antigen retriever system(BioGenex,USA). Sections were then incubated with power block(BioGenex,USA) for10min to reduce nonspeci?c antibody binding.The sections were incubated overnight at4°C with primary rabbit polyclonal antibody(A-20;1:50dilution)raised against a20amino acid synthetic peptide of human VEGF-A(Santa Cruz Biotechnology Inc.,Santa Cruz,CA).Primary antibody was detected using super sensitive polymer-HRP IHC detection system(BioGenex,USA). After thorough washing with Tris buffered saline(TBS;pH7.4) sections were treated with super enhancer for20min at room tem-perature followed by incubation with poly-HRP reagent for30min at room temperature.After three washes with TBS,substrate(3,30-diaminobenzidine tetrahydrochloride was applied to the sections for5–10min in the dark.Sections were counterstained with hematoxylin,dehydrated with ethanol and xylene and mounted permanently with DPX.Negative control sections were processed by omitting primary antibody.Positive control was tissue from a previously known positive case for VEGF-A.

Interpretation of VEGF-A Immunohistochemical Expression Brown colour of VEGF staining was seen as cytoplasmic or membranous localisation.The expression was considered positive if more than25%of tumor cells of OSCC or the epithelium in PMOLs showed cytoplasmic or membranous staining.Stromal staining was not included for recording of positive expression.

Microvessel staining and counting

Microvessel staining was performed using rabbit monoclonal antibody CD-34(Santa Cruz Biotechnology,Inc.,Santa Cruz,CA). Antigen retrieval was done in0.01M citrate buffer(pH6.0)using an EZ antigen retriever system for15min at95°C.The slides were incubated overnight at4°C with primary antibody(1:50dilution). Pyogenic granuloma tissue was taken as positive control.Negative control sections were processed by omitting primary antibody. Super sensitive polymer-HRP IHC detection system(BioGenex, USA)was used for visualization of tissue antigen.Two pathologists, who were blinded to the patient history,reviewed all the slides. Microvessels were counted in?ve areas of highest vascular density at40X magni?cation.14Both single cells and cell cluster were counted.Microvessel density(MVD)was expressed as the mean number of vessels in these areas.

Determination of serum VEGF

Peripheral venous blood(5ml)was obtained from patients be-fore they underwent any surgery and from healthy volunteers.Ser-um was separated by centrifugation at3000rpm for10min and stored at-80°C until analysis.Haemolysed serum samples were not analyzed.Serum VEGF-A concentration was measured by commercially available Sandwich Enzyme-Linked Immunosorbent assay kit for human VEGF-A(Quantikine human VEGF;R&D Sys-tem,Minneapolis,USA).Assay was performed according to the manufacturer’s instruction.The detection sensitivity range of the kit was30–2000pgmlà1and showed no cross-reactivity with a series of soluble immunoactive molecules with a good reproduc-ibility.The coef?cient of variation was less than9.5%. Quantitative real-time PCR for VEGF

RNA Extraction

RNA was extracted from frozen tissue samples with Trizol reagent(Invitrogen,Carlsbad,CA).RNA puri?cation was done by DNase1treatment(Invitrogen,Ampli?cation grade).In brief,1l g of total RNA sample was treated with10X DNase I reaction buffer and DNase I(1U/10l l)and incubated for15min at room temper-ature followed by inactivation of DNase I with25mM EDTA at 65°C for10min.RNA was quanti?ed by spectrophotometer (Picodrop,UK)at260/280nm wavelength.

cDNA synthesis

250ng of the total RNA was subjected to reverse transcription using random hexamer primers with Gene AMP RNA PCR kit (Applied Biosystems,Foster city,CA)for cDNA synthesis,as per manufacturer’s instructions.Brie?y,the reaction was performed at42°C for15min,followed by denaturation at99°C for5min and cooling at4°C for5min.

234S.Nayak et al./Oral Oncology48(2012)233–239

Real-time PCR

Quantitative real-time PCR was performed using a StepOne real-time PCR system(Applied Biosystems,USA)in the presence of SYBR Green?uorescent dye according to the manufacturer’s instructions. Brie?y,the2X SYBR Green master mix containing dNTPs,ROX dye, and10l M of forward and reverse primers was dispensed into a fast optical48-well real-time PCR reaction plate(Applied Biosystems, USA).The PCR primers for VEGF-A gene were selected from a pub-lished article15and synthesized by Imperial Life Sciences,India. These were rechecked using Primer Express software3.0(Applied Biosystems,USA)and checked for homology by Blast sequence anal-ysis.Following primers sequences were used:beta-actin(internal control):forward50-GAGACCTTCAACACCCCAGCC-30;reverse50-AGACGCAGGATGGCATGGG-30(Amplicon size140bp,T m=55°C). VEGF:forward50-GCTGTCTTGGGTGCATTGG-30;reverse50-GCAG CCTGGGACCACTTG-30(Amplicon size69bp,T m=58°C).Thermal cycle conditions consisted of initial denaturation incubation at 94°C for5min followed by35cycles of94°C incubations for45s, 58°C incubations for30s and72°C incubations for30s.Fluores-cence was detected after a?nal extension at72°C for10min fol-lowed by a dissociation cycle.Quanti?cation was based on cycle number required to reach the threshold level of SYBR Green?uores-cence in the linear phase of ampli?cation.Gene expression level of VEGF-A was determined using the2àDD Ct method using beta-actin as an endogenous control.16A negative control without a template was run in parallel to assess the overall speci?city of the reaction. All reactions were run in duplicate.

Statistical analysis

Statistical analysis was performed using version15.0SPSS soft-ware for windows(SPSS,INC,Chicago,IL).Serum VEGF-A values were expressed as mean±standard deviation.For assessing pro-portional data,chi-square test was carried out.Levene’s test for equality of variance was applied to calculate difference between serum concentrations of VEGF-A in PMOL&OSCC as compared to healthy controls.Student’s t-test was used to compare the differ-ences among the various clinical variants.For all the tests,a P value<0.05was considered statistically signi?cant.Pearson’s cor-relation coef?cient was used to?nd out the correlation between serum VEGF-A ELISA and tissue VEGF-A gene expression.

Results

Clinical characteristics and immunohistochemical expression of VEGF-A Clinical characteristics of patient with PMOLs and OSCC and their tissue VEGF-A expression as detected by immunohistochem-istry are shown in Tables1and2,respectively.VEGF-A expression was observed in63.4%tissues of PMOLs and70%tissues of OSCC. VEGF expression was signi?cantly higher(P<0.034)in OSCC with lymph node involvement than those without lymph node.No sig-ni?cant association was found with clinical stage of the tumor and degree of differentiation in OSCC(Table2).Healthy oral tissue con-trols did not show VEGF-A expression except for some focal posi-tive staining in six cases(Fig.1A).VEGF-A staining was localized to the cytoplasm of lining epithelium in PMOLs and tumor cells in OSCC and was seldom observed in the stroma(Fig.1B–D). Among the PMOLs,VEGF-A expression appeared higher in OSMF (18/26;69.2%)as compared to LKP(20/34;58.8%).However,the difference was not statistically signi?cant.VEGF-A expression in PMOLs did not correlate signi?cantly with the status of dysplasia (Table2).MVD assessment by CD-34immunohistochemistry

Fig.1E shows the MVD in OSCC.Increased subepithelial vascu-larisation was observed in LKP(Fig.1F).Mean MVD was signi?-cantly higher in PMOLs and OSCC as compared to controls, higher in PMOLs than OSCC(Table3).Mean MVD appeared greater in PMOLs with dysplasia(23.03±8.63)than PMOLs without dys-plasia(18.15±11.33)but the difference was statistically not signif-icant(Table3).The MVD did not correlate with the VEGF-A tissue expression in any of the category.

Quantitative real-time PCR for VEGF-A expression

Quantitative real-time PCR was performed in order to validate the results of immunohistochemistry.We observed that OSCC pa-tients had signi?cantly higher level of VEGF-A gene expression,fol-lowed by OSMF and LKP,with respect to controls.The difference was statistically signi?cant(P=0.0018).VEGF-A showed53-fold higher expression in OSCC compared with the normal controls(Ta-ble4;Fig.2A).Lymph node status was also validated with Real time PCR.There was57-fold higher expression of VEGF-A in lymph node positive cases of OSCC as compared to healthy controls(Table 4;Fig.2B).

Quantikine ELISA for serum VEGF-A

Mean serum VEGF levels in OSCC,PMOLs and controls are shown in Table5.Mean VEGF levels in sera of patients with OSCC were signi?cantly higher than PMOLs(P=0.001)as well as healthy controls(P=0.009).Within the PMOL group,mean serum VEGF concentration in SMF(n=26)was503.98±398.00pg/ml,and that in LKP(n=34)was430.85±306.87pg/ml.

When the serum VEGF-A values for PMOLs versus controls were plotted for receiver operating characteristic(ROC)curve,a statisti-cally signi?cant(P<0.002)area under the curve(0.827)with a cut off value for serum VEGF-A in PMOLs as267.86pg/ml with63% sensitivity and80%speci?city was obtained(Fig.2C).Again the area under curve(0.893)of serum VEGF between OSCC versus con-trols was signi?cant(p<0.001),the cut off being445.54pg/ml with73%sensitivity and100%speci?city(Fig.2D).Considering the variable range of values in serum VEGF levels,a scatter plot for serum VEGF in OSCC and PMOLs was drawn which showed that 24cases in OSCCs were above the range of PMOLs and28cases in PMOLs were above the range of normal controls(Fig.2E). Correlation of VEGF-A ELISA and molecular expression of VEGF-A

To investigate the association between circulating VEGF levels and molecular levels of VEGF we plotted the values of serum VEGF-A by ELISA and VEGF mRNA expression by Real-time PCR from each person on X and Y axes and calculated the Pearson’s cor-relation coef?cient(Fig.2F).Correlation coef?cient and p values are mentioned on the right of the scatter plot in Fig.2F.It is evident that the VEGF tissue expression and serum expression individually were signi?cantly high in PMOLs and OSCCs but the Pearson’s correlation coef?cient was signi?cant only in leukoplakias and insigni?cant for other categories.

Discussion

The data from the experimental and clinical reports con?rms that VEGF plays a central role in regulating angiogenesis in solid tumors and is tightly associated with the angiogenic switch.17,18 This transition to a pro-angiogenic phenotype has been demon-strated to be crucial in the progression of oropharyngeal epithelial

S.Nayak et al./Oral Oncology48(2012)233–239235

dysplasia to invasive head and neck squamous cell carcinoma (HNSCC).19–21

In the present study,we studied VEGF-A tissue and serum expression in OSCCs and PMOLs as compared to healthy oral tis-sues and serum both at phenotypic and molecular level.Among the PMOLs,we included only two major clinical subgroups i.e.LKP and OSMF,as these are the most frequently biopsied lesions in our set up.Only biopsy proven cases were included in the study.Studies on production of angiogenic factors have yielded con-?icting results.22–24We observed that both VEGF-A and MVD were signi?cantly high in PMOLs irrespective of whether the PMOLs were dysplastic LKP or dysplastic OSMF.22,23We found signi?cant association of VEGF expression in OSCCs with lymph node involve-ment and no association with clinical stage and degree of differen-tiation,?ndings supported by other studies.24–26

The tumour angiogenesis is not essentially a feature of invasive tumour,but may be an early event during tumorigenesis.The ?rst evidence on angiogenesis came from assessment of MVD which was found to be signi?cantly increased in a relatively large band of pre-malignant squamous cell lesions,such as in the oral mucosa,skin,uterine cervix,vulva and anal canal.For most of them,an association was observed between MVD and VEGF expression.Further evidences came from pre-malignant lesions of glandular epithelia,in which the angiogenic switch was established by the immunohistochemical expression of VEGF in gastric metaplasia and dysplasia,atypical adenoma of the colon,atypical hyperplasia and carcinoma in situ of the breast and others.21

In a recent report,Gandolfo et al.in their study on 29cases of leukoplakia and 45cases of OSCC showed increased immunohisto-chemical expression of VEGF-A in non tumoural epithelial borders of OSCC (i.e.areas adjacent to tumour)and leukoplakia with dys-plasia than in leukoplakia without dysplasia.27They concluded that demonstration of expression of epithelial VEGF-A and sub-basal vascularization could be an additional aid for evaluation of the severity of potentially malignant oral lesions in routine biop-sies.The VEGF-A expression in potentially malignant oral mucosal lesions showed that angiogenesis is induced by epithelia in early stages of cancerization.27Their ?ndings support our observation on increased subepithelial vascularisation and signi?cantly increased VEGF-A expression in oral leukoplakias (Fig.1F).How-ever,their study did not include cases of OSMF.

We observed an increased expression of VEGF-A in OSMF cases also at both phenotypic and molecular levels in tissue biopsies and sera as well.This seems to be an important ?nding for predicting progression of OSMF to OSCC because we see a good number of OSCC with previous history of OSMF.The biopsies from OSMF also quite often show epithelial dysplasia.We could not ?nd any pub-lished report in the literature on the circulating and tissue VEGF-A expression in cases of oral sub mucous ?brosis.

VEGF-A expression showed some focal VEGF positivity in the epithelium of normal or healthy mucosa in 6/20(30%)cases.These ?ndings are supported by those of Johnstone and Logon 22who observed an epithelial positivity of VEGF-A in 4/10(40%)healthy oral mucosal biopsies,suggesting the signi?cant upregulation from normal oral mucosa through dysplasia to OSCC.Further the pres-ence of signi?cant Pearson’s coef?cient between tissue VEGF and serum VEGF in leukoplakias may be indicative of serum VEGF being derived from pre-malignant oral mucosal lesions.These ?nd-ings of positive VEGF expression in normal mucosa and pre-malig-nant lesions may possibly be re?ective of an angiogenic switch from non angiogenic phenotype to an angiogenic phenotype.18

Mean MVD was higher in PMOLs than OSCC,although the dif-ference was statistically insigni?cant.The association of MVD with VEGF-A was also statistically insigni?cant.Our results suggest that tissue VEGF and MVD are independent discriminants in PMOLs and carcinomas,?ndings supported by Li et al.28Some workers on the

Table 1

Clinical characteristics of patients of PMOLs and OSCC.Clinical

characteristics PMOL (n =60)number (%)OSCC (n =60)number (%)Gender Male 50(83.3)52(86.6)Female

10(16.7)8(13.4)Topological site Buccal mucosa 38(63.4)12(20)Tongue 14(23.4)20(33.4)Palate

4(6.6)6(10)Gingivo-buccal sulcus 4(6.6)4(6.6)Tonsil 06(10)Lip

08(13.4)Floor of mouth 02(3.3)Retromolar region 02(3.3)Differentiation WD –36(60)MD –22(36.6)PD

–2(3.3)Tumor stage Stage I–II –34(56.6)Stage III–IV –26(43.3)Node involvement Present –24(36.6)Absent –

36(63.3)Metastasis Present –16(26.6)Absent –

44(73.3)PMOLs 60;(LKP 34,OSMF 26)

–Dysplasia Present a

48(80)–Absent

12(20)

–

WD =well differentiated,MD =moderately differentiated,PD =poorly differentiated.a

24cases of leukoplakia (LKP)with dysplasia and 24cases of Oral Sub Mucous Fibrosis (OSMF)with dysplasia.b

LKP =10,OSMF =2.

Table 2

VEGF-A immunohistochemical expression in controls,PMOLs and OSCC.Groups VEGF negative VEGF positive v 2

P value Controls 146PMOLs 2238 3.360.67OSMF 818 3.480.62LKP 1420 2.090.148OSCC

42 5.000.025Differentiation in OSCC WD 24120.56

0.755

MD 166PD 20Tumor stage Stage I–II 2014 2.330.127

Stage III–IV 224Node involvement Present 222 4.470.034

Absent 2016Metastasis Present 142 1.590.207

Absent 2816PMOLs 20141880.340.558Dysplasia Present 30180.03

0.850

Absent

WD =well differentiated,MD =moderately

differentiated,PD =poorly

differentiated.

236

S.Nayak et al./Oral Oncology 48(2012)233–239

contrary have shown signi?cant association of MVD with VEGF expression.24–26MVD in all these studies was done using antibody against CD 31whereas we used antibody to CD 34in the present study.Kademani et al.in their study,showed statistically signi?-cant association of CD 34expression with risk of cervical lymph node metastasis.29

Circulating VEGF estimation has been reported to be a reliable,convenient and noninvasive surrogate marker of angiogenic activ-ity and a promising novel approach for prognostication in cancer patients which otherwise is not afforded by conventional clinico-pathologic prognostic indicators.30Mean serum levels of VEGF-A in the present study were proportionately high in PMOLs and carcinomas with high cut off values as shown in the results.Again at the transcriptional level,VEGF-A gene expression was very high in LKP (47-fold),OSMF (33-fold)and carcinomas (53-fold)as com-pared to normal controls.Only a few studies till date have corre-lated the combined serum VEGF with tissue VEGF and MVD.Some have only done tissue expression and others have done only serum expression.Shang et al.showed circulating VEGF-A levels to be signi?cantly raised in oral carcinomas (31cases)as compared to healthy controls (10cases).11In this report however,they did not look for tissue https://www.wendangku.net/doc/7f4554693.html,ter the same group,showed correla-tion of the serum VEGF expression with tissue VEGF-A expression and microvessel density emphasing on the role of VEGF upregula-tion in the tumour tissue and the sera as well.31In another study on circulating VEGF in sera of oral and oropharyngeal

cancer

Immunohistochemical staining of VEGF-A and CD-34in OSCC and PMOLs.(A)VEGF-A epithelial positivity in a positivity in OSMF with dysplasia in epithelial and subepithelial zone X 100.(D)VEGF-A epithelial OSCC X 200.(F)CD-34positivity in blood vessels of LKP X 200.

Table 3

Immunohistochemical assessment of MVD versus tissue expression of VEGF-A in PMOLs and OSCC.

MVD (Mean ±SD)

P value

MVD (Mean ±SD)P value

VEGF Positive

VEGF Negative Healthy controls 10.42±6.27(n =20)15.00±4.25(n =6)8.45±6.31(n =14)0.145PMOLs

22.06±9.23(n =60)0.00122.64±10.25(n =38)21.04±7.50(n =22)0.655Dysplasia positive 23.03±8.63(n =48)0.25323.94±8.99(n =30)21.53±8.29(n =18)0.521Dysplasia negative 18.15±11.33(n =12)17.80±14.61(n =8)18.85±.77(n =4)0.928OSCC

18.96±10.90(n =60)

0.030

19.38±9.8(n =42)

16.95±13.58(n =18)

0.585

patients the results did not correlate with the tumour stage.They observed that vide variety of VEGF serum levels made this marker dif?cult to handle for initial diagnostics and monitoring of therapy.6,32Tong et al.showed that head and neck squamous cell

ROC Curve in PMOL Vs Control

t y

1.00

.75

ROC Curve in OSCC vs Control

1.00

.75

A E B

Statistically significant level was represented by *. p value * 0.01-0.05 (significant), ** 0.001-0.01 (very significant),<0.001 (extremely significant)

V E G F A c o n c e n t r a t i o n (p g /m l )

real-time PCR for VEGF-A gene expression in tissues LKP and OSMF with respect to controls.(B)Bar involvement with respect to controls.(C)ROC curves for VEGF-A of OSCC,PMOLs and controls.(F)Scatter plot for their correlation coef?cients.

Table 4

Quantitative real-time PCR results of VEGF-A gene expression as fold of internal control gene (b -actin)expression in tissue samples of PMOL,OSCC and normal controls.Group

2àDD Ct (fold change)SE P value Healthy tissues (n =20)10.700424–PMOLs (n =50)OSMF (n =26)33.51756 6.7435010.0003LKP (n =24)

47.6413814.340210.0074OSCC (n =30)

53.13897.817710.0018OSCC with lymph node positivity (n =14)

57 6.310.0102OSCC without lymph nodes (n =16)

9.87

0.0007

Table 5

Serum VEGF-A levels in PMOLs,OSCC,normal controls as measured by Sandwich ELISA.Groups

Mean serum VEGF-A (pg/ml)Levene’s test for equality of variance between two variables

P value Controls 187.91±106.75OSCC/controls (n =60/20)380.009PMOLs 462.54±344.76PMOL/controls (n =60/20)380.019OSCC 1264.08±1216.70

OSCC/PMOLs (n =60/60)

0.001

238S.Nayak et al./Oral Oncology 48(2012)233–239

carcinoma cell proliferation was signi?cantly decreased by VEGF siRNA silencing;implying the presence of an autocrine–paracrine VEGF growth loop and biphasic role of VEGF;both pro-angiogenic and pro-tumorigenic functions in head and neck tumorigenesis.33 To conclude,the data in the present study clearly shows that VEGF-A expression was consistently upregulated in PMOLs and OSCC in comparison to healthy controls,both in tissues and sera of the patients at molecular and phenotypic levels.Therefore VEGF serum estimation might be a good surrogate of tissue VEGF status of both PMOLs and OSCCs.However before the serum VEGF-A can be really be translated into a practical biomarker,this would re-quire a well controlled large scale,multicentric study in different geographical locations along with clinico-epidemiological,aetio-logical factors and follow up studies for serum VEGF expression in post treatment cases.

Source of funding

Authors gratefully acknowledge the?nancial support from In-dian Council of Medical Research(ICMR),New Delhi and Council of Science&Technology,UP,India.

Con?ict of interest statement

None.

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生物标志物

泥炭沉积的类脂化合物（正构烷烃、脂肪醇、脂肪酸、甾酮、三萜类化合物和类异戊二烯、直链酯类等）、纤维素中C，H，O 同位素，以及泥炭腐殖化度和孢粉、生物化石等都是恢复古环境的良好指标。虽然泥炭的这些气候代用指标能够反演古环境的相对干湿、冷暖，但并不能定量地给出温度值的大小。 1、GDGTs（甘油二烷基甘油四醚脂）研究较多的GDGTs化合物主要包括类异戊二烯类（GDGT-0~GDGT-4）和支链类（I~III）两大类，类异戊二烯GDGTs被认为是古菌细胞质膜中所特有，是古菌存在的生物标志化合物。与该指标的相关内容：（1）CBT：环化指数（the Cyclisation ratio of Branched Tetraethers）（2）MBT：甲基化指数（the Methylation index of Branched Tetraethers （3）研究发现支链GDGTs 结构中甲基个数（MBT指数）主要受当地年平均大气温度（MAAT）影响，其次受环境pH影响；支链GDGTs结构中环戊烷个数（CBT指数）主要受环境pH控制。（4）环化指数（CBT）/甲基化指数（MBT）是近年来根据支链四醚膜类脂（GDGTs）提出的定量化重建土壤pH和陆地年平均大气温度（MAAT）的生物标志物指标。（5）Weijers等人提出的MBT/CBT 指标在近海、湖泊沉积中都得到了较好应用，并依此将MBT/CBT 指标应用到泥炭沉积中，讨论了指标在泥炭沉积中的适用性和应用潜力。文章发表在2007年的《Geochimica et Cosmochimica Acta》上。（6）许云平等利用GDGTs来重建全新世渤海湾有机碳的来源及沉积能量（2010年国家自然科学基金项目）。由GDGTS衍生出的指标BIT比值可用作湖相、河口、滨浅海环境沉积物中判识有机质来源的重要指标。（7）高效液相色谱-质谱仪（HPLC-MS）进行GDGTs分析（当前存在的主要问题）。 2、脱-A-三萜烯系列化合物（属脂肪族）脱-A-三萜类是地质体中重要的生物标志化合物，已在石油和各种沉积物中多有报道，认为是高等植物三萜类经光化学和/或微生物氧化使得A环丢失的降解产物。该系列化合物在沉积物中的出现一方面说明被子植物的输入，另一方面显示A环的丢失是高等植物五环三萜类较为普遍的转换途径。与该指标的相关内容（1）可反映气候的干湿、温度高低以及沼泽水位的高低；（2）研究发现，该指标在泥炭中的积累与沼泽发育期生物群落结构组成差异密不可分；（3）脱-A-三萜烯变化序列与植被群落结构演替具有相关性（可以与孢粉、植物大化石的结果相互验证）（4）GC-MS分析采用惠普6890气相色谱与HP5973质谱联用仪

生物标志物

生物标志物科技名词定义中文名称：生物标志物英文名称：biomarker 定义：用于监测和评价能够导致生物有机体的生物化学和生理学改变的化学污染物。所属学科：海洋科技（一级学科）；海洋科学（二级学科）；环境海洋学（三级学科）本内容由全国科学技术名词审定委员会审定公布生物标志物：在亚个体和个体水平上既可以测定污染物暴露水平，也可以测定污染物效应的生理和生化指标。对于疾病研究，生物标志物一般是指可供客观测定和评价的一个普通生理或病理或治疗过程中的某种特征性的生化指标，通过对它的测定可以获知机体当前所处的生物学过程中的进程。检查一种疾病特异性的生物标志物，对于疾病的鉴定、早期诊断及预防、治疗过程中的监控可能起到帮助作用。寻找和发现有价值的生物标志物已经成为目前研究的一个重要热点。自1994年蛋白质组概念提出，定量蛋白质组学已经成为蛋白质组学研究的热点和中心。定量蛋白质组学便是检测正常与疾病状态下组织全部表达蛋白质在量上的差别。定量蛋白质组学中的蛋白质定量技术也成为发现生物标志物的重要途径。生物标志物是生物体受到严重损害之前，在不同生物学水平（分子、细胞、个体等）上因受环境污染物影响而异常化的信号指标。它可以对严重毒性伤害提供早期警报。这种信号指标可以是细胞分子结构和功能的变化、可以是某一生化代谢过程的变化或生成异常的代谢产物或其含量，可以是某一生理活动或某一生理活性物质的异常表现，可以是个体表现出的异常现象，可以是种群或群落的异常变化，可以是生态系统的异常变化。生物标志物分类从功能上一般分为: 接触（暴露）生物标志物（biomarker of exposure）; 效应生物标志物

生物标志物_biologicalmarker_

倍,经χ2检验,差异均有显著性;二项分布拟合与Edward检验均显示,扬中胃癌的发病存在明显的家庭聚集性,符合多基因遗传方式;先证者家庭成员发生胃癌的危险性显著高于均衡可比的对照家庭成员,核心家系成员间患病率的差异,可能与胃癌遗传易感性和家庭内环境因素暴露的差异有关[5,6]。分析胃癌家族史在家庭聚集性中的作用,结果显示(资料未列出):先证者家系有胃癌家族史的比例为28134%(761/2685),对照家系胃癌家族史的比例为2170%(69/2557),两者差异有极显著性,χ2 =64612,P=01001;同样,胃癌病例有家族史的比例为41175%(291/697),也显著高于非胃癌对照家族史的比例11186%(539/4545),表明遗传易感性因素在胃癌发生中有重要地位。同时,也应该看到,以肿瘤发病率为观察研究的终点指标,对遗传易感性作用相对较弱的散发性肿瘤而言,敏感性较低,出现一些难于解释的阴性结果,需要借助分子遗传学、分子生物学技术,准确判断肿瘤早期生物学表型与遗传易感性(基因型)之间的关系。根据国内外现有流行病学资料:胃癌是在多种环境和遗传因素长时间、多步骤、交互作用下的结果[2,7],无论是外源性致癌物,或是机体产生的内源性致癌物,都要通过宿主遗传易感性因素(研究比较成熟的是各种代谢酶基因多态性)的作用,才能最终导致癌变,因此,有必要采用分子流行病学方法,进一步阐明在致癌物代谢的各条通路中,易感基因及其多态性所起的作用[8212],我们已经利用在扬中胃癌高发区获得的环境暴露与基因多态性资料,对此进行了探讨。有关结果将另文报道。参考文献 1李茂森,耿昌友,朱阳春,等.扬中市1991～1995年恶性肿瘤发病及死亡情况调查研究1肿瘤,1997,17:47724781 2C orrea P1Human gastric carcinogenesis:a multistep and multifactorial process2first American cancer s ociety award lecture on cancer epidemiology and prevention1Cancer Res,1992,52:6735267401 3Perera FP1Environment and cancer:who are susceptible?Science, 1997,278:1068210731 4S tadtlander CT,W aterbor JW1M olecular epidemiology,pathogenesis and prevention of gastric cancer1Carcinogenesis,1999,20:2195222081 5Nagase H,Ogino K,Y oshida I,et al1Family history2related risk of gastric cancer in Japan:a hospital2based case2control study1Jpn J Cancer Res,1996,87:1025210281 6La Vacchia C,Negri E,Franceschi S,et al1Family history and the risk of stomach and colorectal cancer1Cancer,1992,70:502551 7T oy oshima H,Hayashi S,Hashim oto S,et al1Familial aggregation and covariation of diseases in a Japanese rural community:com paris on of stomach cancer with other diseases1Ann E pidemiol,1997,7:44624511 8K ato S,Onda M,M atsukura N,et al1G enetic polym orphisms of the cancer related gene and Helicobacter pylori in fection in Japanese gastric cancer patients1An age and gender matched case2control study1Cancer, 1996,77:1654216611 9K ato S,Onda M,M atsukura N,et al1Helicobacter pylori in fection and genetic polym orphisms for cancer2related genes in gastric carcinogenesis1 Biomed Pharmacother,1997,51:14521491 10Ng EK,Sung JJ,Ling TK,et al1Helicobacter pylori and the null genotype of glutathione2S2trans ferase2mu in patients with gastric adenocarcinoma1Cancer,1998,82:26822731 11National Institute of Environmental Health Science.Research on environment2related disease1Environmental G enome Project119981 Available from:http://w w w1niehs1nih1g ov/envgenom1 12沈靖.人类基因组计划与肿瘤预防研究面临的机遇.肿瘤,2000, 20:682721 (收稿日期:2000202220) (本文编辑:邵隽一) ?名词小词典? 生物标志物(biological marker) 能够反映致病因素或毒物从暴露到效应过程各个环节性质的特异性生物分子,如DNA、蛋白质、酶、脂质、糖类等。生物标志物的确定和检测是流行病学研究中的关键问题,因为这种确定和检测可被用来进行病因探讨、危险因素的评价、致病因子致病机理的研究、人群易感性评估、疾病流行规律的掌握、疾病防治措施的研究和评估等。生物标志物大致上可分为两大类,一类是根据表型和基因型的特点分为表型生物标志物和基因型生物标志物,前者包括蛋白质、多肽、脂质、糖类和其他在血清和体液中可检测到的特异性分子,后者主要包括基因类型及突变型、DNA加合物、DNA多态性等;另一类是根据致病因子作用机体的过程,可划分为暴露生物标志物、作用生物标志物、效应生物标志物等。随着分子生物学理论和技术的深入发展,研究生物标志物的技术手段日趋先进、完善。现可用先进的核酸研究技术、蛋白质研究技术、酶学研究技术、免疫学研究技术等检测和研究生物标志物。 (方福德100005北京市中国医学科学院基础医学研究所) (收稿日期:2000209219) (本文编辑:邵隽一) ? 6 3 ?中华预防医学杂志2001年1月第35卷第1期　Chin J Prev M ed,January2001,V ol35,N o. 1

生物标志物监测环境污染研究新进展

广东化工 2010年第4期· 150 · https://www.wendangku.net/doc/7f4554693.html, 第37卷总第204期生物标志物监测环境污染研究新进展姜元臻 (中山市环境监测站，广东中山 528400) [摘要]生物标志物在环境污染监测方面的应用日益重要，文章侧重于对生物标志物在此方面的应用进行全面阐述，包括：生物标志物的定义及分类，生物标志物的特征及优势，生物标志物在检测环境污染的应用，最后还提出了生物标志物在环境监测方向的展望。 [关键词]生物标志物；环境污染；生物监测 [中图分类号]O65 [文献标识码]A [文章编号]1007-1865(2010)04-0150-03 New Advances of Study on Monitoring Environmental Pollution by Biomarkers Jiang Yuanzhen (Zhongshan Environmental Monitoring Station, Zhongshan 528400, China) Abstract: Biomarkers is becoming more and more important in the application of environmental monitoring. The article focased on a comprehensive exposition of biomarker application in this regard, which included definition and classification of biomarker, characteristics and advantages of biomarker, biomarker’s application in the detection of environmental pollution, finally made an outlook of biomarker in the direction of environmental monitoring. Keywords: biomarker；environmental pollution；biomonitoring 1 生物标志物概述 1.1 生物标志物的定义目前，中国的环境监测工作还主要是针对环境中化学成分的存在量进行检测。物理化学监测虽然能清楚地知道环境中各化学成分的具体含量及其变化，但却不能直接反应环境对生物所造成的毒害作用。另外，由于环境中的许多污染物含量很低，相互混合，体系复杂，仅用化学因子监测的手段往往不能够全面的反映环境的污染状况。在环保观念日益增强的今天，社会对环境评价的全面性和准确性的要求也日益增高，这就要求建立一个综合的、多手段的、多参数的环境监测体系以实现快速、高效、准确地对环境状况作出全面的评价。而生物监测正好补充了理化监测的不足。生物标志物是生物体受到严重损害之前，在分子、细胞、个体或种群水平上因受环境污染物影响而产生异常变化的信号指标。一种标志物应能敏感有效地反映出生物体发生严重损伤之前的生物变化，并能准确评估生物体所处的污染状态及其潜在危害，为环境污染提供早期警报。随着分子生物学理论和技术的迅速发展，生物标志物(biomaker)的研究作为一个崭新的领域逐渐引起了国内外共同关注[1]。1987年美国国家科学院首先将生物标志物定义为由生物体或样品可测出由外来化合物导致的细胞学或生物化学组份或过程、以及结构或功能的变化[2]。Benson和DiGiulo[3]认为生物标志物是在生物个体所测得的生物化学、生理学或病理学反应，而这些生物学反应能给出环境污染物的暴露，或由暴露所引起的亚致死效应资料。生物指示物(Bioindicators)自上世纪70年代污染生态学中出现并一直沿用至今。最初只是将耐污的生物物种称为指示生物(Indicator species或Bioindicator)，随着污染生态学的野外研究和实验室毒性试验研究，逐渐将生物指示物的应用范围扩大至污染生态学的不同生物学组织层次，小至分子水平，大至生态系统结构与功能，包括发生在分子、生物化学、生理、病理组织、生物个体、种群、群落和生态系统等不同生物学组织水平上的生物学效应，从生物学的角度为环境质量的监测和评价提供依据。简单地讲，生物标志物就是可衡量环境污染物的暴露及效应的生物反应。一个理想的生物标志物应具备化学特异性，能够微量鉴定、试验费用低廉、检验快速，与环境样品中污染物有量的相关性等。寻找理想的生物标志物一直是环境监侧、环境毒理学及环境医学领域研究的重要内容。 1.2 生物标志物的分类和各种类型的生物标志物从功能上看，生物标志物一般可分为三类[4]，即暴露生物标志物(Biomarkers of exposure)，反应或毒性效应生物标志物(Biomarkers of responser or toxic effect)，易感性生物标志(Biomarkers of susceptibility)。 1.2.1 暴露生物标志物暴露生物标志物指示机体经化学品的暴露，即污染物引起的物体的反应，如指示对重金属暴露的金属硫蛋白(MTs)，但此类标志物不能指示污染物的毒性效应，有助于研究生物对化学分析方法很难检测到的的环境中的不稳定化合物的暴露。暴露生物标志物一般依靠测定体液和组织中特定化学物质或者其代谢物，或者与生物分子相互作用形成的产物。 1.2.2 反应或毒性效应生物标志物效应标志物是指在一定的环境暴露作用下，生物体产生相应的可测定的生理生化变化或其它病理方面的改变，即指示污染物对生物体健康状况的损害效应，如指示DNA损伤的DNA 加合物(DNA-adducts)，它可能是生物机体中某一内源性成分或测定机体功能容量，产生疾病或障碍的改变等。确定化学物质的生物学效应的生物标志物很多，从最简单的标志物如监测体重变化至复杂的标志物如采用免疫化学技术测定特定同功酶[5]。酶活性抑制持久，因此，可作为重要的效应生物标志物。如血细胞数和血细胞损伤的检测可提供各种资料，出现姊妹染色单体交换指示染色体潜在损伤，可由环氧乙烯暴露引起；缺乏特有淋巴细胞指示免疫抑制，可由二恶英(TCDD)等化学物质引起。HSP70家族是序列最保守并且对污染物的应激反应最为显著的一类应激蛋白。沈骅等[6]以鲫鱼为实验动物，Cu，EDAT-Cu，Zn，Pb，Cd，染料橙(HC Orange 1)及两种金属同时进行长期低浓度暴露，在不同浓度下，应激蛋白HSP70被不同程度地诱导，并有明显的剂量效应关系。研究发现，在低于国家渔业水质标准的浓度下，HSP70仍然有显著的诱导表达，说明水体中污染物在低于现行渔业水质标准的浓度下，长期暴露仍然会对鱼类产生一定的损伤。HSP70比传统的生长、繁殖等生物指标更为敏感。 1.2.3 易感性生物标志物易感性标志物是指当生物体暴露于某种特定的外源化合物时，由于其先天遗传性或后天获得性缺陷而反映出其反应能 [收稿日期] 2009-07-31 [作者简介]姜元臻(1982-)，男，山东人，硕士，主要从事环境监测方面的工作。

生物标志物

生物标志物（biomarker）思路总结：第一部分：Biomarkers Introduction 1.什么是生物标志物？生物标志物是生物体内对于单个或多个环境压力（污染物）和非化学的。生物标志物反应，因此能够提供不良反应的早期预警。生物标志物反应通常在亚致命毒性范围内观察到，作为对生长、繁殖和生存影响的前兆。它们包括正常生理功能的细微变化，如生殖行为、疾病复原力和捕食能力，这些能力会对生物体的长期生存和生殖输出产生深远影响;最终，这些会影响生态系统健康 (生物体受到严重损害之前，在不同生物学水平（分子、细胞、个体等）上因受环境污染物影响而异常化的信号指标。它可以对严重毒性伤害提供早期警报。这种信号指标可以是细胞分子结构和功能的变化、的变化或生成异常的代谢产物或其含量，可以是某一生理可以是个体表现出的异常现象，可以是种群或群落的异常变化，可以是

系统的异常变化。 ) 2. 生物标志物的选择原则: 1) 生物标志物必须能够表明暴露于污染物。 2) 生物标记物的反应必须能够与对个体和生态系统的影响联系起来 3) 必须具有足够的灵敏度，即所选标志物的水平与外接触水平要有剂量-反应关系，在无害效应接触水平下仍能维持这种关系。 3. 生物标志物的具体应用：种群或群体-- 但在这一水平上的影响通常是由于许多个体适应度的变化而产生的。对于一个个体的有机体来说，接触污染物是有害的，因为它们改变了个体的正常生理，而这些变化通常可以在分子或亚细胞水平上测量。在分子或亚细胞水平上的损伤是由酶过程、蛋白质表达和功能的变化、突变或Pollutant Exposure Molecular Sub-cellular (organelle) Cellular Tissue Systematic (organ) Organism Population Community Ecosystem ‘Early’ biomarker signals ( rapid ) ‘Later’ effects ( slow )

生物标志物概述

生物标志物概述随着近年来进入环境的化学物质种类和数量的增加，作为食物链顶端以及生物圈重要组成部分的人类的健康受到越来越多的威胁。对这些化学物质在生物圈中产生的多种生物效应、环境影响和生态风险进行必要的评估已成为环境科学一个重要的研究内容和必要趋势。尤其是近年来，环境污染问题被更多的提及和关注，对于环境污染的监测也逐步受到关注。用化学方法分析等传统方法监测环境污染，虽然可以快速检测其反应产物及对生物的致死性影响，但也很难评估其潜在的毒性。传统的检测方法所得的各类数据，虽然对污染物的致死浓度评估具有重要作用，但在探索能够反映低浓度早期影响等亚致死效应参数上却没有太大的意义。因此，就需要我们发展能够反映亚致死效应参数的生物标志物，来更加准确地进行对污染物的评估预测。随着分子生物学理论和技术的发展，作为能够反映污染物危害效应的早期生物信号，生物标志物的研究作为一个全新的领域引起了国内外预防医学界的共同关注。生物标志物在早期预测以及预报污染物效应，反映被污染物侵入或正处于污染环境中的生物体从健康到疾病这一连续谱上的确切位置等这些方面有着很大的优势。因此，只要随时掌握标志物的情况，就可以较为准确地预防甚至避免减轻环境污染的危害。因此生物标志物作为目前毒理学领域研究的热点,被称为环境医学延伸发展到分子水平的重要里程碑。同时，生物标志物在分子流行病学、分子毒理学、劳动卫生学、环境医学等诸多领域均具有极其重要的价值[1]。迄今，国内外已有大量有关环境科学和医学科学中的生物标志物的研究报道和文件综述[2]。在我国，生物标志物虽然已经引起重视，也已经有相应的研究机构，但发展并不迅速可观，也没有足够的关于生物标志物的综合概述类资料。

生物监测与综合生物标志物响应

4生物监测与综合生物标志物响应目前常见的水体监测方法按监测指标分类主要有理化监测（利用水体相关的理化指标进行的监测）、生物监测（利用水体中的水生生物的组织、个体、种群及群落相关的指标进行的监测）两大类（表）。单独使用一种监测方法或只测定某一类指标会有许多的不足（Derrien et al 2017），只有将上述理化监测与生物监测方法结合起来使用，才能更具体地反映水体污染的实际情况，并采取针对性的措施来解决水体污染问题（Li et al 2011）。生物标志物是指通过测量体液、组织或整个生物体, 能够表征对一种或多种化学污染物的暴露和其效应的生化、细胞、生理、行为或能量上的变化（余建新和吴采樱2002）。分子生物标志物能直接反映外源污染物与细胞靶分子特别是生物大分子等的相互作用及其影响，因其具有检测周期短、高效、实用、灵敏度高等特点而被广泛应用于污染物的监测及早期预警中（周驰和李纯厚2007；Livingstone 1993），而多种生物标志物的联合使用则可以反映更大范围内环境污染的程度（孟范平2013）。本实验所用的评估方法为综合生物标志物响应法（Integrated Biomarker Response，IBR），其最早由法国生态毒理学家Benoit Beliaeff 和Thierry Burgeot 于2002年首创（Beliaeff et al 2002），在2013年经Wilfried Sanchez和Thierry Burgeot等进一步完善后推出本实验所采用的第二版综合生物标志物响应法（Sanchez et al 2013），其具体计算过程如下：（1）标准化：Y i =log（X i / X 0 ） X i ：生物标志物指标在各站位上测定结果的平均值； X 0 ：对照组生物标志物指标的测定结果； Y i ：标准化值；（2）均一化：Z i =（Y i -μ）/ σ μ：生物标志物标准化值Y i 在所有站位上的总平均值； σ：生物标志物标准化值Y i 在所有站位上的总标准差； Z i ：均一化值；（3）赋值：A=Z i -Z 0 ； Z 0 ：对照站位该标志物的均一化值；