Fas配体诱导婴幼儿血管瘤内皮细胞凋亡的实验研究
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摘要
血管瘤是婴幼儿最常见的良性肿瘤,文献统计约有4%～12%白种新生儿出现血管瘤,男女发病比例为1:3～5,早产低体重儿发病率更高,可高达23%,血管瘤好发头面颈部(60%),其次为四肢(25%)和躯干(15%)。目前血管瘤发病机制仍不清楚,研究显示血管瘤内皮细胞表达增殖细胞核抗原(Proliferating Cell NuclearAntigen,PCNA)、葡萄糖转运蛋白—1(Glucose Transporter-1,GLUT-1)以及淋巴内皮生长因子(Lymphatic Endothelial Hyaluronan Receptor-1,LYVE-1)等,并表达内皮祖细胞标志CD133,提示血管瘤内皮细胞可能处于细胞发育的幼稚阶段,是不成熟的内皮细胞,推测这是血管瘤快速增殖的原因。研究还证实血管瘤的自行消退是内皮细胞凋亡所致,目前已发现多种凋亡调控蛋白和因子在血管瘤组织中表达,如Bcl-2家族的Bax,Bad,Bcl-xl,Bak基因等。本课题前期研究发现Fas蛋白在血管瘤组织中大量表达,并且定位于内皮细胞;血管瘤组织中除存在Fas蛋白大量表达,还有表达Fas配体(Fas Ligand,FasL)的活化T细胞数量增多,由此推断血管瘤内皮细胞凋亡可能是表达FasL的活化T细胞诱导并通过Fas/FasL途径发生,本课题旨在探讨表达FasL的活化T细胞能否诱导血管瘤内皮细胞通过Fas/FasL途径发生凋亡。
第一部分婴幼儿血管瘤内皮细胞培养及生物学特性
研究目的:探讨体外分离培养和纯化婴幼儿血管瘤内皮细胞的方法,对体外培养的细胞进行鉴定并检测其纯度,观察培养细胞的生物学特性。
研究方法:收集第二军医大学附属长海医院和上海交通大学附属新华医院整形外科手术切除的14例新鲜婴幼儿血管瘤组织,无菌条件下立即放置于含有D-Hank's液的培养管中,放置于4℃冰盒中保存,运至细胞培养室,采用改良组织块法进行细胞培养,细胞培养成功后,在相差显微镜下刮除非内皮细胞;内皮细胞铺满皿底,运用免疫组化Envision法对内皮细胞进行鉴定;流式细胞仪检测内皮细胞纯度;微格法对培养细胞进行计数并绘制细胞生长曲线。
研究结果:14例标本中有8例成功培养出血管瘤内皮细胞,培养的内皮细胞有两种形态:一种是形态较规则的圆形,核居中,细胞境界清晰;一种是形态较规则的短梭形,核小,形态扁长,体积较小,生长速度较快,细胞融合后呈现内皮细胞特有“铺路石”特征。细胞免疫组化鉴定第Ⅷ因子相关抗原标记、CD31、CD34均为阳性,证实为内皮细胞,培养的细胞具有“管腔形成”现象,流式细胞仪检测传代培养的内皮细胞CD34(+)数为75.27%±3.73%。微格法细胞计数绘制的细胞生长曲线显示,细胞数量在接种后7天左右进入快速增殖期,第11～12天左右细胞数量达到最高值,此后细胞数量进入稳定期,持续3天左右逐渐进入细胞数量衰减期,细胞传代最多传至第3代。
研究结论:采用改良组织块法可以分离培养婴幼儿血管瘤内皮细胞,培养的细胞经鉴定为内皮细胞,传代后细胞纯度较高,培养的细胞有“管腔形成”现象。
第二部分婴幼儿血管瘤内皮细胞表达Fas、FasL的检测
研究目的:运用流式细胞仪和荧光定量RT-PCR法检测培养的血管瘤内皮细胞Fas、FasL的表达。
研究方法:将传代的血管瘤内皮细胞和复苏后传代的脐静脉内皮细胞株接种至培养皿,常规培养1周后,用0.25%胰蛋白酶消化细胞,离心(1000 r/min,5min)收集细胞,流式细胞仪检测血管瘤和脐静脉内皮细胞表达Fas(+)、FasL(+)细胞数,并和Jurkat细胞进行对照;荧光定量PCR法检测血管瘤和脐静脉内皮细胞Fas、FasL mRNA表达量,并和人T淋巴细胞白血病细胞(Jurkat细胞)Fas mRNA表达量进行比较。Fas引物:上游5'-TTGCTAGATTATCGTCCAAAAGTGT-3',下游5'-GCACTTGGTGTTGCTGGTGAGT-3',产物大小205bp。FasL引物:上游5'-TTCAGCTCTTCCACCTACAGAAGGA-3',下游5'-TCACTCCAGAAAGCAGGACAATTC-3';产物大小219bp。内参:上游5'-ACCACAGTCCATGCCATCAC-3',下游5'-TCCACCACCCTGTTGCTGTA-3',产物大小450bp。反应条件设置为:95℃3min,{95℃20s,61℃20s,72℃30s}×50个循环,72℃5min,55℃—95℃(溶解曲线)。
研究结果:流式细胞仪检测培养的血管瘤内皮细胞中Fas(+)细胞数为90.97%±2.36%,FasL(+)细胞数4.17%±1.75%,脐静脉内皮细胞Fas(+)细胞数为25.07%±7.60%,FasL(+)细胞数为4.45%±0.50%,荧光定量PCR结果显示血管瘤内皮细胞FasmRNA的2~(-ΔΔCT)值为1.260±0.721,FasLmRNA表达量为0.038±0.022,脐静脉内皮细胞FasmRNA为0.354±0.170,FasLmRNA表达量为0.022±0.011。血管瘤FasmRNA表达量与Jurkat细胞表达量1.448±0.059的比值为0.87,差异无显著性(p＞0.05)和脐静脉内皮细胞表达量的比值为3.56,差异有显著性(p＜0.01)。
研究结论:婴幼儿血管瘤内皮细胞高度表达Fas,而FasL的表达量很低或几乎不表达。
第三部分可溶性FasL诱导婴幼儿血管瘤内皮细胞凋亡的实验
研究目的:探讨不同剂量sFasL诱导婴幼儿血管瘤内皮细胞凋亡的情况;观察100ng/ml sFasL诱导人血管瘤内皮细胞凋亡及中和抗体NOK-2阻断sFasL诱导凋亡的作用,并和人脐静脉细胞和人Jurkat细胞进行对比;Western Blot法检测sFasL诱导血管瘤内皮细胞发生凋亡后,活化Caspase3,8,9的表达。
研究方法:(1)体外培养的人血管瘤内皮细胞培养基中分别加入5ng/ml、20ng/ml、100ng/ml三种不同剂量的sFasL,与细胞分别培养6h、18h和36h后,流式细胞仪检测血管瘤内皮细胞凋亡细胞数,常规培养的血管瘤内皮细胞凋亡细胞数作阴性对照;(2)体外培养的人血管瘤内皮细胞和人脐静脉细胞系培养基中分别加100ng/ml剂量的sFasL、100ng/ml sFasL+10ng/μl中和抗体NOK-2;人Jurkat细胞株培养基中加入5ng/ml sFasL,5ng/ml sFasL+10ng/μl中和抗体NOK-2,三种细胞加入不同的作用因素常规培养18h后,用流式细胞仪检测三种细胞凋亡细胞数,三种细胞常规培养的凋亡细胞数作阴性对照,观察sFasL诱导血管瘤内皮细胞凋亡的情况以及中和抗体NOK-2对sFasL诱导凋亡的阻断作用,并和sFasL诱导人脐静脉内皮细胞、Jurkat细胞凋亡情况进行对比。(3) sFasL诱导血管瘤内皮细胞凋亡后,Western Blot法检测活化Caspase3,8,9的表达,常规培养的血管瘤内皮细胞活化Caspase3,8,9表达量作阴性对照。
研究结果:结果显示:(1) 5ng/ml的sFasL作用于血管瘤内皮细胞6h、18h、36h后,凋亡细胞数分别为:9.92%±0.84%,10.79%±2.01%,10.33%±0.98%;20ng/ml的sFasL作用于细胞6h、18h、36h后,内皮细胞凋亡细胞数分别为:11.03%±1.62%,11.36%±1.13%,11.46%±1.67%;100 ng/ml的sFasL作用于细胞6h、18h、36h后,内皮细胞凋亡细胞数分别为:11.68%±1.63%,14.86%±0.40%,16.40%±1.08%。(2) sFasL作用三种不同细胞18h后,脐静脉内皮细胞凋亡细胞数为14.53%±1.49%,血管瘤内皮细胞的凋亡细胞数为18.12%±0.24%;而sFasL作用于人Jurkat细胞株18h后,其凋亡细胞数达61.59%±11.37%;三种细胞间以人Jurkat细胞凋亡细胞数最高,血管瘤内皮细胞其次,脐静脉内皮细胞凋亡数最少(p均＜0.01)。中和抗体NOK-2阻断sFasL诱导细胞凋亡后三种不同细胞凋亡情况如下:血管瘤内皮细胞未阻断与阻断后的凋亡细胞数分别为18.12%±0.24%、12.85%±0.93%,两者之间差异显著(p＜0.01);脐静脉内皮细胞未阻断与阻断后的凋亡细胞数分别为14.53%±1.49%、11.17%±1.19%,两者之间差异显著(p＜0.01)。人Jurkat细胞未阻断与阻断后的凋亡细胞数分别为61.59%±11.37%、16.59%±4.66%,两者之间差异显著(p＜0.01)。(3) sFasL诱导血管瘤内皮细胞凋亡后,Caspase 8的两个活化条带43kDa和18kDa的灰度比值(灰度值/β-actin灰度)分别为0.50、0.08;阴性对照组仅出现43kDa的活化条带,其灰度比值为0.09;凋亡诱导组Caspase3有19kDa和17kDa两条活化条带出现,它们灰度比值分别为0.26、0.16;对照组无活化片断出现。凋亡诱导组Caspase 9产生37kDa、35kDa和17kDa三条活化条带,它们灰度比值分别为0.43、0.04、0.07,阴性对照组也出现同样的活化条带,它们灰度比值分别为0.44、0.04、0.07,凋亡诱导组和阴性对照组活化条带灰度比值之间差异不明显。
研究结论:(1) sFasL在体外可诱导婴幼儿血管瘤内皮细胞发生凋亡,凋亡诱导作用和sFasL剂量相关,与作用时间关系不明显。(2) sFasL能够诱导不同Fas(+)靶细胞发生凋亡,但不同细胞的凋亡细胞数不同:人Jurkat细胞的凋亡细胞数最高,血管瘤内皮细胞其次,脐静脉内皮细胞凋亡数最少;sFasL中和抗体NOK-2可阻断sFasL诱导不同细胞的凋亡。(3) sFasL诱导婴幼儿血管瘤内皮细胞凋亡后,Caspase 8和Caspase 3有活化条带出现,而Caspase 9活化条带与阴性对照组差异不明显,表明sFasL能够诱导血管瘤内皮细胞凋亡,并且是通过Fas/FasL途径发生凋亡。
第四部分人淋巴瘤T细胞诱导婴幼儿血管瘤内皮细胞凋亡的实验
研究目的:检测IL-12刺激后人淋巴瘤T细胞株(H9细胞)FasL的表达量,观察表达FasL的H9细胞在体外诱导婴幼儿血管瘤内皮细胞发生凋亡的能力。
研究方法:(1)将25ng/ml的IL-12加入H9细胞培养液中,使其刺激活化,用流式细胞仪检测表达FasL细胞的数量并使用荧光定量RT-PCR检测H9细胞FasLmRNA的表达量,未刺激活化组的FasL细胞数量和FasLmRNA表达量作阴性对照。(2)实验分两组:A组预先用25ng/ml的IL-12加入H9细胞培养液中培养12h,然后将活化H9细胞与人血管瘤内皮细胞共培养18h。B组预先用25ng/ml的IL-12加入H9细胞培养液中培养12h,然后加入10ng/μl sFasL中和抗体NOK-2孵育1h,再和人血管瘤内皮细胞共培养18h,流式细胞仪检测血管瘤内皮细胞凋亡细胞数,未活化H9细胞与血管瘤内皮细胞共培养的凋亡细胞数作阴性对照。
研究结果:(1) IL-12刺激活化H9细胞后,其FasL(+)细胞数为46.65%±3.26%,与未刺激H9细胞表达FasL的4.51%±0.85%相比,有显著差异(p＜0.01)。荧光定量PCR法测IL-12刺激后的细胞FasLmRNA表达量(2~(-ΔΔCT)值)为:0.6±0.32,与未刺激H9细胞FasLmRNA表达量(2~(-ΔΔCT)值)0.097±0.01相比,差异明显(p＜0.01)。(2)活化H9细胞诱导人血管瘤内皮细胞凋亡细胞数为26.02%±1.70%,未活化H9细胞诱导人血管瘤内皮细胞凋亡细胞数为13.69%±1.68%,NOK-2阻断活化后H9细胞诱导人血管瘤内皮细胞凋亡细胞数为12.54%±1.67%,活化H9细胞诱导的HEC凋亡率显著高于未活化组和NOK-2阻断组(p＜0.01)。
研究结论:(1) IL-12能够活化H9细胞并表达FasL;(2) IL-12刺激后的H9细胞能够诱导血管瘤内皮细胞凋亡,并且这种凋亡诱导作用可被sFasL中和抗体NOK-2阻断。
Hemangioma is the most common tumor of infancy and childhood,occuring in 4%to 12%of white infants.These lesions are three to five times more common in girls than in boys,there is an increased frequency of hemangiomas in premature infants,with a reported incidence of 23%in neonates who weigh less than 1200g.Hemangioma occur most often in the craniofacial region(60%),followed by the trunk(25%) and extremities(15%),little is understood about the pathogenesis of infantile hemangiomas to date.Several studies have revealed that expression of proliferating cell nuclear antigen(PCNA),erythrocyte-type glucose transporter protein- 1(GLUT- 1) and lymphatic endothelial hyaluronan receptor- 1(LYVE- 1) as well as CD 133,an important marker for endothelial progenitor cells(EPCs) in endothelial cell of infantile hemangioma,which indicated the endothelial cells in infantile hemangioma are arrested in an early developmental stage of vascular differentiation.The immature,incompletely differentiated immunophenotype of proliferating infantile hemangioma may contribute to their rapid growth.Many studies suggested that programmed to death have a important role in hemangioma,a lot of regulatory proteins of apoptosis have been found to express in infantile hemangioma,such as bcl-2,bax、bad、bak、C-fos.In our previous study,we found the number of Fas(+) cells can be observed to increased from the middle proliferating stage to the late proliferating stage of infantile hamangioma,similar variance have been found in the number cells of T cell of infantile hernangioma,which expressed FasL antigen.We make the hypothesis that the apoptosis of endothelial cell in IH may be caused mainly by T cells expressing FasL protein,aiming at the Fas antigen of IH cells and mediated by Fas-FasL pathway.
Part one Culture and identification of endothelial cells from IH and observation of biological character in vitro
Objective:To explore the method of cultivation and purification the endothelial cells from infantile hemangiomas in vitro,immunohistochemical stain was performed to identificate the cell cultured form IH,the purity of endothelial cell was detected by flow cytometry analysis,the biological character of the cultured cell has been observed in vitro.
Method:Small fragments of fresh operative specimens were obtained from 14 infantile hemangiomas that came form Shanghai Changhai hospital,Second Military Medical University and Shanghai Xinhua hospital,Shanghai Jiaotong University.Pieces of fresh operative hemangiomas tissue were submerged in D-hank's medium and stored at 4℃,then transport it from operative room to cell culture room.The endothelial cells of IH were cultured by the explants of specimens.The explants was removed from the dishes after the endothelial cell grew out.The cell colonies,which did not have the gross morphology of endothelial cells(under inverted phase contrast microscopy),were detached by scraping and removed from the dish by aspiration.The colonies that exhibited the characteristic cobblestone morphology of endothelial cells were allowed to proliferate in the dish for 1 week,selected as before.After 3-4 weeks culture,the primary culture cell grew the major of dish.Immunohistochemical staining of anti-CD31、anti-CD34 and anti-factorⅧassociated protein was performed respectively to identificate the EC origin of isolated cells.The purity of endothelial cell was detected with FITC-CD34 by flow cytometry analysis.The number of cultured cells was counted with micro grid technique and growth curve of cell was drawled.
Results:In all 14 explants,endothelial cells of IH from 8 specimens grew in tissue culture.The morphology of cultured endothelial cells cold be divided into 2 types:the round cell with well defined,centrally located nuclei and distinct cell borders,the shape cell,which exhibited cobblestone morphology after confluence.Cultured EC were homogenously positive for factorⅧassociated protein、CD31、CD34 immunohistochemical staining,indicated the EC origin.Formation tube was found in endothelial cell of IH.The number of CD34(+) cell was 75.27%±3.73%in endothelial cell by flow cytometry analysis.Grow curve based on microgrid technique indicated EC began to proliferated rapidly after 7 days of plating,the number of EC was maximum at 11 to 12 days of plating,follow this,the number of EC began to plateau in grow for 3 days,then decreased slowly.The primary cell can be subculture for no more than 3 eras
Conclusion:The EC of IH can be isolated and cultured successfully by modified explant method,verified the endothelial origin by immunohistochemical staining,the purity of EC is high in the subculture cell,and Formation tube was found in endothelial cell of IH.
Part two Expression of Fas and Fas ligand on endothelial cell of IH
Objective:The flow cytometry analysis and fluorescencere quantitative reverse-transcriptase polymerase chain reaction(RT-PCR) assay were used to detect the expression of Fas and Fas ligand on endothelial cell of IH.
Method:the EC of IH and umbilical vein were subculfivated and plated on plastic dish,after 1 week of culture,the cells were digested with 0.25%trypsin solution and centrifuged(1000r/min,5 min),After harvesting,the cells were detected the Fas and FasL antigen of expression on EC of IH and umbilical vein by FACS,fluorescencere quantitative PCR was performed on cell to detect Fas and FasLmRNA expression levels. The results were compared with that of Jurkat cells.Fas primers were 5'- TTGCTAGATTATCGTCCAAAAGTGT-3'and 5'- GCACTTGGTGTTGCTGGTGAGT-3',amplification product was 205 bp;FasL primers were 5'-TTCAGCTCTTCCACCTACAGAAGGA-3' and 5'-TCACTCCAGAAAGCAGGACAATTC-3',amplification product was 219 bp,β-actin primers were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3',amplification product was 450 bp,amplification was performed under the following conditions:95℃3 min,{95℃20s61℃20s72℃30s}×50cycles,72℃5rain,55℃—95℃(melt curve).
Results:FCM detection showed that Fas expression rate of IH was 90.97%±2.36% and FasL expression rate was 4.17%±1.75%,Fas expression rate of HUVEC was 25.07%±7.60%and FasL expression rate was 4.45%±40.50%;results of RT-PCR showed that the level of Fas mRNA of IHEC was 1.260±0.721 and FasLmRNA was 0.038±0.022, the level of Fas mRNA of HUVEC was 0.354±0.170 and FasLmRNA was 0.022±0.011. The level of Fas mRNA of IHEC compared with that of Jurkat cells 1.448±0.059,there were no significant difference between IHEC and Jurkat cells.
Conclusion:The expression of Fas antigen on IHEC is high,but that of FasL antigen on IHEC is low.
Part three IHEC apoptosis induced by soluble Fas ligand
Objective:To investigate the effect of IHEC apoptosis induced by different doses of sFasL,To examined the susceptivity of apoptosis on IHEC,HUVEC line and Jurkat cell line induced by sFasL,as well as the effect of FasL neutralizing antibody NOK-2 block the apoptosis induced by sFasL.Western Blot analysis was carried out to determine the protein of active Caspase 3,8,9 after IHEC apoptosis induced by sFasL.
Method:5 ng/ml、20 ng/ml、100 ng/ml doses of sFasL were added to EC medium respectively,all of the above cells were cultured for 6h,18h and 36h,then IHECs were collected carefully and stained with PI and Annexin V-FITC conjugate,apoptosis of IHECs was measured by FACScan.Apoptosis of normal grown IHECs without sFasL were negative control group;IHECs and HUVECs were incubated with sFasL at 100ng/ml,in another group IHECs and HUVECs were incubated with sFasL at 100ng/ml and neutralizing FasL monoclonal antibody NOK-2(10ng/μl),Jurkat cells were incubated with sFasL at 5ng/ml,in another group Jurkat cells were incubated with sFasL at 5ng/ml and neutralizing NOK-2(10ng/μl),all of the above cells were cultured for 18h,then,apoptosis of IHECs,HUVECs and Jurkat cells were measured by FACScan.Apoptosis of normal grown IHECs,HUVECs and Jurkat cells were negative control group respectively.Western Blot analysis was performed to detect the protein of active Caspase 3,8,9 after IHEC apoptosis had been induced by sFasL for 18h.The protein of active Caspase 3,8,9 of norm grown IHECs was negative control group.
Results:Results shows:(1)The total apoptotic rate of IHECs were 9.92%±0.84%; 10.79%±2.01%;10.33%±0.98%after incubation with 5 ng/ml sFasL for 6h、18h、36h; The total apoptotic rate of IHECs were 11.03%±1.62%;11.36%±1.13%;11.46%±1.67% after incubation with 20 ng/ml sFasL for 6h、18h、36h;the total apoptotic rate of IHECs were 11.68%±1.63%;14.86%±0.40%;16.40%±1.08%after incubation with 100 ng/ml sFasL for 6h、18h、36h。(2) The apoptotic rate of IHECs were 18.12%±0.24 %;12.85±0.93%after incubation with 100 ng/ml sFasL and 100ng/ml sFasL+NOK-2 (10ng/μl) for 18h,the difference were significant(p＜0.01);The apoptotic rate of HUVECs were 14.53%±1.49%,11.17%±1.19%after incubation with 100 ng/ml sFasL and 100ng/ml sFasL+ 10ng/μl NOK-2 for 18h,the difference were significant(p＜0.01); The apoptoticrate of Jurkat cells were 61.59%±11.37%;16.59%±4.66%after incubation with 5 ng/ml sFasL and 5ng/ml sFasL+ 10ng/μl NOK-2 for 18h,the difference were significant(p＜0.01)。(3) the apoptotic rate of IHECs induced by sFasL that was 18.12%±0.24%was higher than that of HUVECs 15.87%±0.48%(p＜0.01),and was lower than that of Jurkat cells 61.59%±11.37%(p＜0.01 ).(4) After IHEC had been induced by 100ng/ml sFasL for 18h,Western Blot showed that the fragment of Caspase 8 were 43kDa and 18kDa,which relative gray scale to that ofβ-actin were 0.5,0.08,compare with the control group0.09,0,the difference were significant(p＜0.01);The fragment of Caspase 3 were 19kDa and 17kDa,which relative gray scale to that ofβ-actin were 0.26, 0.16,the control group did not express those fragments;The fragment of Caspase9 were 37kDa,35kDa and 17kDa,which relative gray scale to that ofβ-actin were 0.43,0.04 and 0.07,compare with the control group0.44,0.04,0.07,the difference were no significant.
Conclusion:IHEC apoptosis can be induced by sFasL,the viability of apoptosis is in dose-dependant manner,no correlate with time.The susceptibility to sFasL among Jurkat cells,IHECs and HUVEC is vary,Jurkat cells was highest,lower was IHECs and lowest was HUVECs.The apoptotic effect of sFasL on cells can be blocked by NOK-2,IHECs apoptosis had been induced by sFasL,Western Blot detect the expression of active Caspase 8,3,but not Caspase 9,indicated that the apoptosis of IHEC is by Fas/FasL pathway.
Part four Apoptosis of infantile hemangioma endothelial cells induced by H9 cells line
Objective:To detect the expression of FasL on H9 cells line after treated with IL-12 and exminated the apoptosis of IHECs co-cultured with H9 cells after treated with IL-12.
Method:(1) H9 cells line were treated with 25ng/μl IL-12 for 12h,then H9 cells were collected and to detect the FasL expression by FCAScan,as well as the FasLmRNA expression by RT-PCR,H9 cells untreated with IL-12 were negative control group.(2) IHECs were subjected to co-culture with H9 cells for 18h which prior to treat with IL-12 (25ng/μl) for 12h;IHECs were co-cultured with H9 cells which prior to treat with IL-12(25ng/μl) for 12h then were neutralizing NOK-2 for 1h.The apoptosis of IHECs was detected by FACScan,the negative control group was IHECs co-cultured with H9 cells untreated with IL- 12.
Results:(1) FACS showed the FasL expression rate on H9 cells treated with IL-12 was 46.65%±3.26%,was higher than that of untreated group 4.51%±0.85%,the difference was significant(p＜0.01 ).RT-PCR results showed the FasLmRAN expression rate on H9 cells treated with IL-12 was 0.6±0.32,was higher than that of untreated group 0.097±0.01, the difference was significant(p＜0.01).(2)The apoptosis of IHECs co-cultured with H9 cells treated with IL-12 was 26.02%±41.70%,which was higher than that of IHECs co-cultured with H9 cells untreated with IL-12 13.69%±1.68%(p＜0.01 ),it also was higher than that oflHECs co-cultured with H9 cells neutralized with NOK-2(p＜0.01 ).
Conclusion:H9 cells line stimulated with IL-12 can express FasL.The apoptosis of IHECs can induced by H9 cells treated with IL-12 and the apoptotic effect of H9 cells expressed FasL can be blocked by sFasL neutralizing antibody NOK-2.
第一部分婴幼儿血管瘤内皮细胞培养及生物学特性
研究目的:探讨体外分离培养和纯化婴幼儿血管瘤内皮细胞的方法,对体外培养的细胞进行鉴定并检测其纯度,观察培养细胞的生物学特性。
研究方法:收集第二军医大学附属长海医院和上海交通大学附属新华医院整形外科手术切除的14例新鲜婴幼儿血管瘤组织,无菌条件下立即放置于含有D-Hank's液的培养管中,放置于4℃冰盒中保存,运至细胞培养室,采用改良组织块法进行细胞培养,细胞培养成功后,在相差显微镜下刮除非内皮细胞;内皮细胞铺满皿底,运用免疫组化Envision法对内皮细胞进行鉴定;流式细胞仪检测内皮细胞纯度;微格法对培养细胞进行计数并绘制细胞生长曲线。
研究结果:14例标本中有8例成功培养出血管瘤内皮细胞,培养的内皮细胞有两种形态:一种是形态较规则的圆形,核居中,细胞境界清晰;一种是形态较规则的短梭形,核小,形态扁长,体积较小,生长速度较快,细胞融合后呈现内皮细胞特有“铺路石”特征。细胞免疫组化鉴定第Ⅷ因子相关抗原标记、CD31、CD34均为阳性,证实为内皮细胞,培养的细胞具有“管腔形成”现象,流式细胞仪检测传代培养的内皮细胞CD34(+)数为75.27%±3.73%。微格法细胞计数绘制的细胞生长曲线显示,细胞数量在接种后7天左右进入快速增殖期,第11～12天左右细胞数量达到最高值,此后细胞数量进入稳定期,持续3天左右逐渐进入细胞数量衰减期,细胞传代最多传至第3代。
研究结论:采用改良组织块法可以分离培养婴幼儿血管瘤内皮细胞,培养的细胞经鉴定为内皮细胞,传代后细胞纯度较高,培养的细胞有“管腔形成”现象。
第二部分婴幼儿血管瘤内皮细胞表达Fas、FasL的检测
研究目的:运用流式细胞仪和荧光定量RT-PCR法检测培养的血管瘤内皮细胞Fas、FasL的表达。
研究方法:将传代的血管瘤内皮细胞和复苏后传代的脐静脉内皮细胞株接种至培养皿,常规培养1周后,用0.25%胰蛋白酶消化细胞,离心(1000 r/min,5min)收集细胞,流式细胞仪检测血管瘤和脐静脉内皮细胞表达Fas(+)、FasL(+)细胞数,并和Jurkat细胞进行对照;荧光定量PCR法检测血管瘤和脐静脉内皮细胞Fas、FasL mRNA表达量,并和人T淋巴细胞白血病细胞(Jurkat细胞)Fas mRNA表达量进行比较。Fas引物:上游5'-TTGCTAGATTATCGTCCAAAAGTGT-3',下游5'-GCACTTGGTGTTGCTGGTGAGT-3',产物大小205bp。FasL引物:上游5'-TTCAGCTCTTCCACCTACAGAAGGA-3',下游5'-TCACTCCAGAAAGCAGGACAATTC-3';产物大小219bp。内参:上游5'-ACCACAGTCCATGCCATCAC-3',下游5'-TCCACCACCCTGTTGCTGTA-3',产物大小450bp。反应条件设置为:95℃3min,{95℃20s,61℃20s,72℃30s}×50个循环,72℃5min,55℃—95℃(溶解曲线)。
研究结果:流式细胞仪检测培养的血管瘤内皮细胞中Fas(+)细胞数为90.97%±2.36%,FasL(+)细胞数4.17%±1.75%,脐静脉内皮细胞Fas(+)细胞数为25.07%±7.60%,FasL(+)细胞数为4.45%±0.50%,荧光定量PCR结果显示血管瘤内皮细胞FasmRNA的2~(-ΔΔCT)值为1.260±0.721,FasLmRNA表达量为0.038±0.022,脐静脉内皮细胞FasmRNA为0.354±0.170,FasLmRNA表达量为0.022±0.011。血管瘤FasmRNA表达量与Jurkat细胞表达量1.448±0.059的比值为0.87,差异无显著性(p＞0.05)和脐静脉内皮细胞表达量的比值为3.56,差异有显著性(p＜0.01)。
研究结论:婴幼儿血管瘤内皮细胞高度表达Fas,而FasL的表达量很低或几乎不表达。
第三部分可溶性FasL诱导婴幼儿血管瘤内皮细胞凋亡的实验
研究目的:探讨不同剂量sFasL诱导婴幼儿血管瘤内皮细胞凋亡的情况;观察100ng/ml sFasL诱导人血管瘤内皮细胞凋亡及中和抗体NOK-2阻断sFasL诱导凋亡的作用,并和人脐静脉细胞和人Jurkat细胞进行对比;Western Blot法检测sFasL诱导血管瘤内皮细胞发生凋亡后,活化Caspase3,8,9的表达。
研究方法:(1)体外培养的人血管瘤内皮细胞培养基中分别加入5ng/ml、20ng/ml、100ng/ml三种不同剂量的sFasL,与细胞分别培养6h、18h和36h后,流式细胞仪检测血管瘤内皮细胞凋亡细胞数,常规培养的血管瘤内皮细胞凋亡细胞数作阴性对照;(2)体外培养的人血管瘤内皮细胞和人脐静脉细胞系培养基中分别加100ng/ml剂量的sFasL、100ng/ml sFasL+10ng/μl中和抗体NOK-2;人Jurkat细胞株培养基中加入5ng/ml sFasL,5ng/ml sFasL+10ng/μl中和抗体NOK-2,三种细胞加入不同的作用因素常规培养18h后,用流式细胞仪检测三种细胞凋亡细胞数,三种细胞常规培养的凋亡细胞数作阴性对照,观察sFasL诱导血管瘤内皮细胞凋亡的情况以及中和抗体NOK-2对sFasL诱导凋亡的阻断作用,并和sFasL诱导人脐静脉内皮细胞、Jurkat细胞凋亡情况进行对比。(3) sFasL诱导血管瘤内皮细胞凋亡后,Western Blot法检测活化Caspase3,8,9的表达,常规培养的血管瘤内皮细胞活化Caspase3,8,9表达量作阴性对照。
研究结果:结果显示:(1) 5ng/ml的sFasL作用于血管瘤内皮细胞6h、18h、36h后,凋亡细胞数分别为:9.92%±0.84%,10.79%±2.01%,10.33%±0.98%;20ng/ml的sFasL作用于细胞6h、18h、36h后,内皮细胞凋亡细胞数分别为:11.03%±1.62%,11.36%±1.13%,11.46%±1.67%;100 ng/ml的sFasL作用于细胞6h、18h、36h后,内皮细胞凋亡细胞数分别为:11.68%±1.63%,14.86%±0.40%,16.40%±1.08%。(2) sFasL作用三种不同细胞18h后,脐静脉内皮细胞凋亡细胞数为14.53%±1.49%,血管瘤内皮细胞的凋亡细胞数为18.12%±0.24%;而sFasL作用于人Jurkat细胞株18h后,其凋亡细胞数达61.59%±11.37%;三种细胞间以人Jurkat细胞凋亡细胞数最高,血管瘤内皮细胞其次,脐静脉内皮细胞凋亡数最少(p均＜0.01)。中和抗体NOK-2阻断sFasL诱导细胞凋亡后三种不同细胞凋亡情况如下:血管瘤内皮细胞未阻断与阻断后的凋亡细胞数分别为18.12%±0.24%、12.85%±0.93%,两者之间差异显著(p＜0.01);脐静脉内皮细胞未阻断与阻断后的凋亡细胞数分别为14.53%±1.49%、11.17%±1.19%,两者之间差异显著(p＜0.01)。人Jurkat细胞未阻断与阻断后的凋亡细胞数分别为61.59%±11.37%、16.59%±4.66%,两者之间差异显著(p＜0.01)。(3) sFasL诱导血管瘤内皮细胞凋亡后,Caspase 8的两个活化条带43kDa和18kDa的灰度比值(灰度值/β-actin灰度)分别为0.50、0.08;阴性对照组仅出现43kDa的活化条带,其灰度比值为0.09;凋亡诱导组Caspase3有19kDa和17kDa两条活化条带出现,它们灰度比值分别为0.26、0.16;对照组无活化片断出现。凋亡诱导组Caspase 9产生37kDa、35kDa和17kDa三条活化条带,它们灰度比值分别为0.43、0.04、0.07,阴性对照组也出现同样的活化条带,它们灰度比值分别为0.44、0.04、0.07,凋亡诱导组和阴性对照组活化条带灰度比值之间差异不明显。
研究结论:(1) sFasL在体外可诱导婴幼儿血管瘤内皮细胞发生凋亡,凋亡诱导作用和sFasL剂量相关,与作用时间关系不明显。(2) sFasL能够诱导不同Fas(+)靶细胞发生凋亡,但不同细胞的凋亡细胞数不同:人Jurkat细胞的凋亡细胞数最高,血管瘤内皮细胞其次,脐静脉内皮细胞凋亡数最少;sFasL中和抗体NOK-2可阻断sFasL诱导不同细胞的凋亡。(3) sFasL诱导婴幼儿血管瘤内皮细胞凋亡后,Caspase 8和Caspase 3有活化条带出现,而Caspase 9活化条带与阴性对照组差异不明显,表明sFasL能够诱导血管瘤内皮细胞凋亡,并且是通过Fas/FasL途径发生凋亡。
第四部分人淋巴瘤T细胞诱导婴幼儿血管瘤内皮细胞凋亡的实验
研究目的:检测IL-12刺激后人淋巴瘤T细胞株(H9细胞)FasL的表达量,观察表达FasL的H9细胞在体外诱导婴幼儿血管瘤内皮细胞发生凋亡的能力。
研究方法:(1)将25ng/ml的IL-12加入H9细胞培养液中,使其刺激活化,用流式细胞仪检测表达FasL细胞的数量并使用荧光定量RT-PCR检测H9细胞FasLmRNA的表达量,未刺激活化组的FasL细胞数量和FasLmRNA表达量作阴性对照。(2)实验分两组:A组预先用25ng/ml的IL-12加入H9细胞培养液中培养12h,然后将活化H9细胞与人血管瘤内皮细胞共培养18h。B组预先用25ng/ml的IL-12加入H9细胞培养液中培养12h,然后加入10ng/μl sFasL中和抗体NOK-2孵育1h,再和人血管瘤内皮细胞共培养18h,流式细胞仪检测血管瘤内皮细胞凋亡细胞数,未活化H9细胞与血管瘤内皮细胞共培养的凋亡细胞数作阴性对照。
研究结果:(1) IL-12刺激活化H9细胞后,其FasL(+)细胞数为46.65%±3.26%,与未刺激H9细胞表达FasL的4.51%±0.85%相比,有显著差异(p＜0.01)。荧光定量PCR法测IL-12刺激后的细胞FasLmRNA表达量(2~(-ΔΔCT)值)为:0.6±0.32,与未刺激H9细胞FasLmRNA表达量(2~(-ΔΔCT)值)0.097±0.01相比,差异明显(p＜0.01)。(2)活化H9细胞诱导人血管瘤内皮细胞凋亡细胞数为26.02%±1.70%,未活化H9细胞诱导人血管瘤内皮细胞凋亡细胞数为13.69%±1.68%,NOK-2阻断活化后H9细胞诱导人血管瘤内皮细胞凋亡细胞数为12.54%±1.67%,活化H9细胞诱导的HEC凋亡率显著高于未活化组和NOK-2阻断组(p＜0.01)。
研究结论:(1) IL-12能够活化H9细胞并表达FasL;(2) IL-12刺激后的H9细胞能够诱导血管瘤内皮细胞凋亡,并且这种凋亡诱导作用可被sFasL中和抗体NOK-2阻断。
Hemangioma is the most common tumor of infancy and childhood,occuring in 4%to 12%of white infants.These lesions are three to five times more common in girls than in boys,there is an increased frequency of hemangiomas in premature infants,with a reported incidence of 23%in neonates who weigh less than 1200g.Hemangioma occur most often in the craniofacial region(60%),followed by the trunk(25%) and extremities(15%),little is understood about the pathogenesis of infantile hemangiomas to date.Several studies have revealed that expression of proliferating cell nuclear antigen(PCNA),erythrocyte-type glucose transporter protein- 1(GLUT- 1) and lymphatic endothelial hyaluronan receptor- 1(LYVE- 1) as well as CD 133,an important marker for endothelial progenitor cells(EPCs) in endothelial cell of infantile hemangioma,which indicated the endothelial cells in infantile hemangioma are arrested in an early developmental stage of vascular differentiation.The immature,incompletely differentiated immunophenotype of proliferating infantile hemangioma may contribute to their rapid growth.Many studies suggested that programmed to death have a important role in hemangioma,a lot of regulatory proteins of apoptosis have been found to express in infantile hemangioma,such as bcl-2,bax、bad、bak、C-fos.In our previous study,we found the number of Fas(+) cells can be observed to increased from the middle proliferating stage to the late proliferating stage of infantile hamangioma,similar variance have been found in the number cells of T cell of infantile hernangioma,which expressed FasL antigen.We make the hypothesis that the apoptosis of endothelial cell in IH may be caused mainly by T cells expressing FasL protein,aiming at the Fas antigen of IH cells and mediated by Fas-FasL pathway.
Part one Culture and identification of endothelial cells from IH and observation of biological character in vitro
Objective:To explore the method of cultivation and purification the endothelial cells from infantile hemangiomas in vitro,immunohistochemical stain was performed to identificate the cell cultured form IH,the purity of endothelial cell was detected by flow cytometry analysis,the biological character of the cultured cell has been observed in vitro.
Method:Small fragments of fresh operative specimens were obtained from 14 infantile hemangiomas that came form Shanghai Changhai hospital,Second Military Medical University and Shanghai Xinhua hospital,Shanghai Jiaotong University.Pieces of fresh operative hemangiomas tissue were submerged in D-hank's medium and stored at 4℃,then transport it from operative room to cell culture room.The endothelial cells of IH were cultured by the explants of specimens.The explants was removed from the dishes after the endothelial cell grew out.The cell colonies,which did not have the gross morphology of endothelial cells(under inverted phase contrast microscopy),were detached by scraping and removed from the dish by aspiration.The colonies that exhibited the characteristic cobblestone morphology of endothelial cells were allowed to proliferate in the dish for 1 week,selected as before.After 3-4 weeks culture,the primary culture cell grew the major of dish.Immunohistochemical staining of anti-CD31、anti-CD34 and anti-factorⅧassociated protein was performed respectively to identificate the EC origin of isolated cells.The purity of endothelial cell was detected with FITC-CD34 by flow cytometry analysis.The number of cultured cells was counted with micro grid technique and growth curve of cell was drawled.
Results:In all 14 explants,endothelial cells of IH from 8 specimens grew in tissue culture.The morphology of cultured endothelial cells cold be divided into 2 types:the round cell with well defined,centrally located nuclei and distinct cell borders,the shape cell,which exhibited cobblestone morphology after confluence.Cultured EC were homogenously positive for factorⅧassociated protein、CD31、CD34 immunohistochemical staining,indicated the EC origin.Formation tube was found in endothelial cell of IH.The number of CD34(+) cell was 75.27%±3.73%in endothelial cell by flow cytometry analysis.Grow curve based on microgrid technique indicated EC began to proliferated rapidly after 7 days of plating,the number of EC was maximum at 11 to 12 days of plating,follow this,the number of EC began to plateau in grow for 3 days,then decreased slowly.The primary cell can be subculture for no more than 3 eras
Conclusion:The EC of IH can be isolated and cultured successfully by modified explant method,verified the endothelial origin by immunohistochemical staining,the purity of EC is high in the subculture cell,and Formation tube was found in endothelial cell of IH.
Part two Expression of Fas and Fas ligand on endothelial cell of IH
Objective:The flow cytometry analysis and fluorescencere quantitative reverse-transcriptase polymerase chain reaction(RT-PCR) assay were used to detect the expression of Fas and Fas ligand on endothelial cell of IH.
Method:the EC of IH and umbilical vein were subculfivated and plated on plastic dish,after 1 week of culture,the cells were digested with 0.25%trypsin solution and centrifuged(1000r/min,5 min),After harvesting,the cells were detected the Fas and FasL antigen of expression on EC of IH and umbilical vein by FACS,fluorescencere quantitative PCR was performed on cell to detect Fas and FasLmRNA expression levels. The results were compared with that of Jurkat cells.Fas primers were 5'- TTGCTAGATTATCGTCCAAAAGTGT-3'and 5'- GCACTTGGTGTTGCTGGTGAGT-3',amplification product was 205 bp;FasL primers were 5'-TTCAGCTCTTCCACCTACAGAAGGA-3' and 5'-TCACTCCAGAAAGCAGGACAATTC-3',amplification product was 219 bp,β-actin primers were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3',amplification product was 450 bp,amplification was performed under the following conditions:95℃3 min,{95℃20s61℃20s72℃30s}×50cycles,72℃5rain,55℃—95℃(melt curve).
Results:FCM detection showed that Fas expression rate of IH was 90.97%±2.36% and FasL expression rate was 4.17%±1.75%,Fas expression rate of HUVEC was 25.07%±7.60%and FasL expression rate was 4.45%±40.50%;results of RT-PCR showed that the level of Fas mRNA of IHEC was 1.260±0.721 and FasLmRNA was 0.038±0.022, the level of Fas mRNA of HUVEC was 0.354±0.170 and FasLmRNA was 0.022±0.011. The level of Fas mRNA of IHEC compared with that of Jurkat cells 1.448±0.059,there were no significant difference between IHEC and Jurkat cells.
Conclusion:The expression of Fas antigen on IHEC is high,but that of FasL antigen on IHEC is low.
Part three IHEC apoptosis induced by soluble Fas ligand
Objective:To investigate the effect of IHEC apoptosis induced by different doses of sFasL,To examined the susceptivity of apoptosis on IHEC,HUVEC line and Jurkat cell line induced by sFasL,as well as the effect of FasL neutralizing antibody NOK-2 block the apoptosis induced by sFasL.Western Blot analysis was carried out to determine the protein of active Caspase 3,8,9 after IHEC apoptosis induced by sFasL.
Method:5 ng/ml、20 ng/ml、100 ng/ml doses of sFasL were added to EC medium respectively,all of the above cells were cultured for 6h,18h and 36h,then IHECs were collected carefully and stained with PI and Annexin V-FITC conjugate,apoptosis of IHECs was measured by FACScan.Apoptosis of normal grown IHECs without sFasL were negative control group;IHECs and HUVECs were incubated with sFasL at 100ng/ml,in another group IHECs and HUVECs were incubated with sFasL at 100ng/ml and neutralizing FasL monoclonal antibody NOK-2(10ng/μl),Jurkat cells were incubated with sFasL at 5ng/ml,in another group Jurkat cells were incubated with sFasL at 5ng/ml and neutralizing NOK-2(10ng/μl),all of the above cells were cultured for 18h,then,apoptosis of IHECs,HUVECs and Jurkat cells were measured by FACScan.Apoptosis of normal grown IHECs,HUVECs and Jurkat cells were negative control group respectively.Western Blot analysis was performed to detect the protein of active Caspase 3,8,9 after IHEC apoptosis had been induced by sFasL for 18h.The protein of active Caspase 3,8,9 of norm grown IHECs was negative control group.
Results:Results shows:(1)The total apoptotic rate of IHECs were 9.92%±0.84%; 10.79%±2.01%;10.33%±0.98%after incubation with 5 ng/ml sFasL for 6h、18h、36h; The total apoptotic rate of IHECs were 11.03%±1.62%;11.36%±1.13%;11.46%±1.67% after incubation with 20 ng/ml sFasL for 6h、18h、36h;the total apoptotic rate of IHECs were 11.68%±1.63%;14.86%±0.40%;16.40%±1.08%after incubation with 100 ng/ml sFasL for 6h、18h、36h。(2) The apoptotic rate of IHECs were 18.12%±0.24 %;12.85±0.93%after incubation with 100 ng/ml sFasL and 100ng/ml sFasL+NOK-2 (10ng/μl) for 18h,the difference were significant(p＜0.01);The apoptotic rate of HUVECs were 14.53%±1.49%,11.17%±1.19%after incubation with 100 ng/ml sFasL and 100ng/ml sFasL+ 10ng/μl NOK-2 for 18h,the difference were significant(p＜0.01); The apoptoticrate of Jurkat cells were 61.59%±11.37%;16.59%±4.66%after incubation with 5 ng/ml sFasL and 5ng/ml sFasL+ 10ng/μl NOK-2 for 18h,the difference were significant(p＜0.01)。(3) the apoptotic rate of IHECs induced by sFasL that was 18.12%±0.24%was higher than that of HUVECs 15.87%±0.48%(p＜0.01),and was lower than that of Jurkat cells 61.59%±11.37%(p＜0.01 ).(4) After IHEC had been induced by 100ng/ml sFasL for 18h,Western Blot showed that the fragment of Caspase 8 were 43kDa and 18kDa,which relative gray scale to that ofβ-actin were 0.5,0.08,compare with the control group0.09,0,the difference were significant(p＜0.01);The fragment of Caspase 3 were 19kDa and 17kDa,which relative gray scale to that ofβ-actin were 0.26, 0.16,the control group did not express those fragments;The fragment of Caspase9 were 37kDa,35kDa and 17kDa,which relative gray scale to that ofβ-actin were 0.43,0.04 and 0.07,compare with the control group0.44,0.04,0.07,the difference were no significant.
Conclusion:IHEC apoptosis can be induced by sFasL,the viability of apoptosis is in dose-dependant manner,no correlate with time.The susceptibility to sFasL among Jurkat cells,IHECs and HUVEC is vary,Jurkat cells was highest,lower was IHECs and lowest was HUVECs.The apoptotic effect of sFasL on cells can be blocked by NOK-2,IHECs apoptosis had been induced by sFasL,Western Blot detect the expression of active Caspase 8,3,but not Caspase 9,indicated that the apoptosis of IHEC is by Fas/FasL pathway.
Part four Apoptosis of infantile hemangioma endothelial cells induced by H9 cells line
Objective:To detect the expression of FasL on H9 cells line after treated with IL-12 and exminated the apoptosis of IHECs co-cultured with H9 cells after treated with IL-12.
Method:(1) H9 cells line were treated with 25ng/μl IL-12 for 12h,then H9 cells were collected and to detect the FasL expression by FCAScan,as well as the FasLmRNA expression by RT-PCR,H9 cells untreated with IL-12 were negative control group.(2) IHECs were subjected to co-culture with H9 cells for 18h which prior to treat with IL-12 (25ng/μl) for 12h;IHECs were co-cultured with H9 cells which prior to treat with IL-12(25ng/μl) for 12h then were neutralizing NOK-2 for 1h.The apoptosis of IHECs was detected by FACScan,the negative control group was IHECs co-cultured with H9 cells untreated with IL- 12.
Results:(1) FACS showed the FasL expression rate on H9 cells treated with IL-12 was 46.65%±3.26%,was higher than that of untreated group 4.51%±0.85%,the difference was significant(p＜0.01 ).RT-PCR results showed the FasLmRAN expression rate on H9 cells treated with IL-12 was 0.6±0.32,was higher than that of untreated group 0.097±0.01, the difference was significant(p＜0.01).(2)The apoptosis of IHECs co-cultured with H9 cells treated with IL-12 was 26.02%±41.70%,which was higher than that of IHECs co-cultured with H9 cells untreated with IL-12 13.69%±1.68%(p＜0.01 ),it also was higher than that oflHECs co-cultured with H9 cells neutralized with NOK-2(p＜0.01 ).
Conclusion:H9 cells line stimulated with IL-12 can express FasL.The apoptosis of IHECs can induced by H9 cells treated with IL-12 and the apoptotic effect of H9 cells expressed FasL can be blocked by sFasL neutralizing antibody NOK-2.
引文
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