【作者】 项永生；

【导师】 徐如祥；

【作者基本信息】 南方医科大学 ， 神经外科， 2013， 博士

【摘要】 一.研究背景骨髓基质干细胞(Bone marrow derived mesenchymal stem cell,BMSCs)是源于骨髓的一种多潜能成体干细胞,其应用于细胞治疗的主要优势在于其可以自体移植,因而不存在胚胎干细胞移植所存在的免疫排斥、伦理学及致瘤性等方面的限制；取材相对简单,不像神经干细胞主要位于脑室下带、海马等结构中,因而细胞来源有限,获得困难。BMSCs移植治疗已经应用于脑缺血及其他一些疾病的临床研究。BMSCs移植治疗脑缺血的实验研究已经有较多文献报道。通过静脉途径、动脉途径或立体定向脑内局部注射移植BMSCs治疗大鼠大脑中动脉阻断模型(MCAO),均发现移植的细胞一方面能定向向缺血灶周围,参与损伤修复,另一方面分泌趋化因子,诱导胶质细胞、神经干细胞等向损伤部分游走,或是诱导损伤区细胞向神经干细胞分化,增殖,参与损伤修复,并促进功能的恢复。由于BMSCs具有分化成神经细胞的能力,直观上,利用移植的BMSCs发挥细胞替代作用重建神经环路是最直接的治疗方法。然而,大部分的研究发现移植的BMSCs只有相当少的一部分表达神经元或神经胶质细胞的表面标志,而即使这些少量的表达神经组织标志的细胞,目前也没有电生理的或其他方面的证据显示它们具有神经细胞的功能,更无法确定它们是否同其他的神经细胞建立了结构与功能联系。目前的研究显示,BMSCs的发挥效应机制是多方面的,如：减少缺血半影区细胞凋亡、促进内源性神经干细胞的增殖、促进缺血区新血管再生、抑制胶质瘢痕增生以及促进轴突再生等。归结起来,BMSCs治疗脑缺血的机制主要的并不是其替代了缺失的神经元,而是其在脑内缺血的微环境的刺激下,发挥了“分子工厂”的作用,分泌各种营养因子,介导了以上的治疗过程。BMSCs在骨髓中的数量稀少,因此在应用于细胞移植治疗时必须进行体外扩增以获得足够多的细胞。然而,BMSCs同大多数的成体细胞一样,会发生衰老,主要表现在两个方面：①随年龄增长,BMSCs的数量和质量均有明显下降(而老年人正是脑缺血的高发人群,是可能的需要细胞移植治疗的主体)；②BMSCs在体外传代过程中,细胞形态会逐渐发生变化(由细梭形变成宽扁平状),细胞的增殖能力、分化潜力、乃至向病灶区归巢的能力均会逐渐减弱或丧失。BMSCs发生衰老的机制尚不清楚,但目前已知的一个重要原因是BMSCs不表达端粒酶。人端粒酶是一种核糖核蛋白,其最重要的两个组份是人端粒酶催化亚单位(hTERT)和模板RNA (hTR),其中hTR持续表达,而hTERT在大部分成体细胞中并不表达。端粒酶不同于一般的DNA聚合酶,它是一种专一的逆转录酶,用自身的RNA成份的互补序列作模板,催化TTAGGG重复序列加到染色体末端,从而来维持端粒的长度。而组成染色体未端的端粒结构的维持与细胞的增殖能力密切相关。大部分的成体细胞,端粒的长度随着细胞的复制而缩短,因而也限制了细胞的增殖能力。相反,肿瘤细胞和胚胎干细胞则能通过端粒酶的活化来维持端粒的功能。由于BMSCs不表达端粒酶,因此随着细胞分裂,端粒长度逐渐缩短,最后达到10Kb左右的阈值的时候,细胞就会停止分裂,发生生长停滞。Baxter MA等研究表明,BMSCs平均端粒长度随年龄的增长而缩短(17bp/年),而在体外扩增7～10PDs (Population Doublings,群体倍增次数)平均丢失的端粒长度就约为1Kb。这种丢失的速率是惊人的,因为即便是来源于年青人的BMSCs,其生命历程中端粒缩减的长度最高也只能达到2.5～3.5Kb。因此,如果没有采取特殊的处理方法,在体外充分扩增BMSCs再回输回病人体内的过程中,实际上已经大大缩短了BMSCs的寿命。为了延长BMSCs的生命周期,或是其他一些科研目的的需要,诸多实验室进行了BMSCs永生化的探索。用人乳头状瘤病毒(HPV)E6/E7基因、猿猴病毒40(SV40)大T抗原基因或bmi-1等癌基因来修饰BMSCs,均能成功地建立永生化BMSCs细胞系。这些癌基因均能激活人端粒酶催化亚单位基因hTERT的表达,因此能够保持细胞在扩增过程中的端粒长度,然而,使用癌基因修饰所建立的BMSCs细胞系最主要的问题是染色体不稳定,有致细胞恶性变的风险,而难以用于临床移植治疗。近几年来,有多家实验室采用hTERT来转染并成功获得BMSCs永生细胞系。此法建立的细胞系在体外长期培养扩增中端粒长度没有明显缩短,保持了同原代BMSCs相似甚至更好的增殖及多向分化潜能。更为重要的是,用hTERT转染建立的BMSCs细胞系在体外长期培养后仍保持了正常的染色体组型,细胞的生长存在接触抑制,体内、体外实验均没有明显的致瘤性。除了BMSCs,其他一些成体细胞如：皮肤成纤维细胞、血管内皮细胞、皮肤角化细胞、乳腺上皮细胞、皮肤黑色素细胞、淋巴细胞、乃至由BMSCs诱导分化所得的成骨细胞,均可以通过转染hTERT基因的方法永生化。在既往的这些用hTERT基因修饰的研究中,均没有发现细胞明显的恶性变。BMSCs是否表达血管内皮生长因子受体(Vascular endothelial growth factorreceptor, VEGFR),不同的实验有不同的报道。但是成年人BMSCs是不表达VEGFR,但是表达PDGFR-A,PDGFR-B和NRP-1,VEGF可通过VEGFR共轭的PDGFR发挥其作用,而且也可以通过NRP1增强其效应。脑缺血由于脑血管的动脉粥样硬化或是脑血管的闭塞导致神经功能失调,VEGF可以促进缺血区血管内皮细胞的趋化和聚集,诱导新生血管的形成,从而增加缺血区的养分供应,减轻缺血引起的神经功能学障碍,因此,构建VEGF基因过表达的老年人BMSCs将具有重大临床应用价值。本实验将构建hTERT慢病毒载体和VEGF基因慢病毒载体,探讨以hTERT慢病毒载体和VEGF基因慢病毒载体联合修饰老年人BMSCs,建立强化表达VEGF的细胞系的可行性,并对hTERT慢病毒载体和VEGF基因慢病毒载体联合修饰的BMSCs进行表型鉴定及功能学研究。由于BMSCs含有自体的遗传信息,研究成果将有助于探索制备个体化的(patient-specific)、针对脑缺血治疗的(disease-specific)干细胞产品,也会为进一步的实验研究打下坚实的基础。二.研究目的骨髓基质细胞(BMSCs)移植是治疗脑缺血的一个有效策略,然而,BMSCs随年龄增长以及在体外扩增过程中会发生衰老,这阻碍了它的临床治疗应用。本研究拟以人端粒酶催化亚单位(hTERT)基因慢病毒表达载体和血管内皮细胞生长因子(VEGF)基因慢病毒表达载体双基因联合修饰老年人的BMSCs,探索构建强化表达VEGF的永生化BMSCs系,检测其生物学特征。由于BMSCs含有自体的遗传信息,研究成果将有助于探索制备个体化的(patient-specific)、针对脑缺血治疗的(disease-specific)干细胞产品,也会为进一步的实验研究打下坚实的基础。三.材料与方法1、hTERT基因慢病毒载体和VEGF基因慢病毒载体的构建①在体外通过PCR技术扩增hTERT与VEGF基因片段。②构建pENTR11-hTERT-IRES-EGFP和pENTR11-VEGF-IRES-EGFP入门载体。③构建pLenti6/V5-DEST-hTERT-IRES-EGFP和pLenti6/V5-DEST-VEGF-IRES-EGFP表达载体。④慢病毒载体pLenti6/V5-DEST-hTERT-IRES-EGFP和pLenti6/V5-DEST-VEGF-IRES-EGFP的包装、表达及分析。2、探讨以hTERT基因慢病毒载体和VEGF基因慢病毒载体联合修饰老年人BMSCs,建立强化表达VEGF的细胞系的可行性①老年人BMSCs购自生物公司,或从临床志愿者(老年人)抽取骨髓,密度梯度离心法结合贴壁法获取BMSCs。②分组：BMSCs未修饰组、空载慢病毒修饰组、VEGF基因修饰组、hTERT基因修饰组、hTERT/VEGF双基因修饰组(各基因修饰组以杀稻瘟筛选稳定表达克隆)。③荧光显微镜和透射电镜观察不同代别细胞形态的变化；流式细胞技术法检测不同代别细胞表面标记物的变化；RT-PCR法检测细胞hTERT和VEGF mRNA水平；免疫细胞荧光化学法和Western blot法检测细胞蛋白表达水平。3、以hTERT/VEGF双基因修饰的BMSCs表型及功能学研究①分组：BMSCs未修饰组、空载慢病毒修饰组、VEGF基因修饰组、hTERT基因修饰组、hTERT/VEGF双基因修饰组②流式细胞仪检测各组细胞表型特征；透射电镜观察细胞超微结构；共聚焦显微镜观察经免疫荧光细胞染色标记的重组细胞；MTT实验和平板克隆实验检测细胞生长和增殖能力；单细胞划痕实验和Transwell小室法检测细胞迁移侵袭能力；RHG条带染色体组型分析评估染色体稳定性；裸鼠接种肿瘤形成实验检验重组细胞的致瘤性；TRAP-ELISA法检测细胞端粒酶活性和细胞分泌VEGF水平。4、所有原始数据用x±s表示,用SPSS13.0软件进行One Way ANOVA统计学分析,组间比较采用LSD法。P<0.05为差异有统计学意义。四.结果1、成功构建hTERT基因慢病毒载体和VEGF基因慢病毒载体。①以pEGFP-hTERT、pEGFP-VEGF(165)质粒(本实验室现有)为模板成功扩增hTERT基因和VEGF基因；②先用EcoR I、Not I酶切pENTR11入门载体和pIRES载体,从pIRES载体切下的含IRES序列的片段用T4DNA连接酶连入pENTR11构建pENTR11-IRES,重组质粒转化DH5a感受态细胞,筛选后,PCR、酶切及测序鉴定,成功获得pENTR11-IRES。③再用Nco I、EcoR I酶切pENTR11-IRES质粒和hTERT基因或VEGF基因PCR产物,将hTERT片段或是VEGF片段用T4DNA连接酶连入载体获得pENTR11-hTERT-IRES或是pENTR11-VEGF-IRES质粒,经酶切、测序鉴定；最后用Xbo I、Not I酶切pENTR11-hTERT-IRES或是pENTR11-VEGF-IRES质粒和带绿色荧光标记的EGFP基因PCR产物,T4DNA连接酶连接构建pENTR11-hTERT-IRES-EGFP或是pENTR11-VEGF-IRES-EGFP质粒,经酶切、测序鉴定；成功获得pENTR11-hTERT-IRES-EGFP和pENTR11-VEGF-IRES-EGFP入门载体。④将制备的pENTR11-hTERT-IRES-EGFP和pENTR11-VEGF-IRES-EGFP入门克隆、表达载体pLenti6/V5-DEST与GatewayTM LR ClonaseTM enzyme II mix混合反应,构建携带双基因的慢病毒表达载体pLenti6/V5-DEST-hTERT-IRES-EGFP和pLenti6/V5-DEST-VEGF-IRES-EGFP,将连接产物转化进Stb13TM E.coli细胞中,筛选阳性克隆鉴定。成功获得pLenti6/V5-DEST-hTERT-IRES-EGFP和pLenti6/V5-DEST-VEGF-IRES-EGFP目的基因的表达载体。⑤用Lipofectamine2000将pLenti6/V5-DEST-hTERT-IRES-EGFP或(和)pLenti6/V5-DEST-VEGF-IRES-EGFP目的基因质粒直接转染入COS-7细胞系进行瞬时表达,获得重组蛋白,验证构建的慢病毒载体表达情况,结果满意。2、成功获得强化表达VEGF永生化BMSCs细胞系,并对其进行鉴定。①利用ViraPowerTM Lentiviral Expression System将纯化的重组质粒和包装混合物共转染293FT细胞系,从293FT细胞系收集病毒上清液,进行滴度测定；在合适的MOI下用病毒上清转染骨髓基质细胞；用杀稻瘟筛选产生稳定表达pLenti6/V5-DEST-hTERT-IRES-EGFP或(和)pLenti6/V5-DEST-VEGF-IRES-EGFP的细胞株；经RT-PCR和Western-blot检测确定mRNA和蛋白正确表达和具有较高的活性。②光学显微镜下观察,原代细胞刚接种时悬浮于DMEM液体培养基中,呈圆形,细胞核位于胞浆中央。一般于接种后2-4h原代细胞开始贴壁,48h后大部分原代细胞贴壁。更换培养液去除未贴壁细胞后可见贴壁细胞形态多样,呈梭形或多角形,细胞核较大、扁圆形,胞浆可向不同方向伸出伪足。贴壁原代细胞以分散的集落克隆方式迅速增殖,1W左右每个集落约含有数百个细胞,细胞形态基本为成纤维细胞样的长梭形。传代细胞一般于接种后1-2h开始贴壁,24h内完全贴壁,刚传代时细胞呈宽大扁平的多边形,6-7d细胞完全汇合,细胞变细长,形态呈成纤维细胞样。③透射电镜观察不同代别BMSCs细胞超微结构,没有发现明显的结构改变。④流式细胞技术法检测不同代别的BMSCs细胞表面标记物CD29、CD34. CD44、CD45、CD90和CD105的表达水平,原代细胞CD44、CD90呈阳性表达,CD29呈弱阳性表达,而CD34、CD45、CD133基本不表达；传代细胞(P3,P9) CD44、CD90呈阳性表达,CD29呈弱阳性表达,而CD34、CD45、CD133基本不表达,和原代细胞基本一致。⑤RT-PCR、Western-blot和免疫荧光细胞化学法检测确定不同组别mRNA和蛋白正确表达和具有较高的活性。3.成功构建hTERT/VEGF双基因修饰的BMSCs细胞系并对其表型及功能学研究①流式细胞技术法检测不同组别的BMSCs细胞表面标记物CD29、CD34、 CD44、CD45、CD90和CD105的表达水平,各组别均出现CD44、CD90呈阳性表达,CD29呈弱阳性表达,而CD34、CD45、CD133基本不表达。②透射电镜观察不同组别的BMSCs细胞超微结构,结果表明,表达VEGF基因的BMSCs细胞出现WP小体,质膜小泡和分泌vWF等,其他组别均不表达。③激光共聚焦显微镜观察不同组别的BMSCs细胞蛋白的表达,结果表明,各组别BMSCs细胞均能表达CD31和UEA-1,而只有表达VEGF的BMSCs细胞才能表达vWF蛋白。④RHG条带染色体分析法分析不同组别的BMSCs细胞核型的变化,结果表明,各组别BMSCs细胞核型一致,没有出现遗传性变异。⑤MTT实验和平板克隆实验检测不同组别的BMSCs细胞生长和增殖能力,结果表明,表达hTERT基因BMSCs细胞生长和增殖能力明显增强。⑥单细胞划痕实验和Transwell小室法检测不同组别的BMSCs细胞迁移侵袭能力,结果表明,表达hTERT基因或是VEGF基因的BMSCs细胞迁移能力增强,而各组BMSCs细胞均没有出现侵袭能力。⑦裸鼠接种肿瘤形成实验检验不同组别的BMSCs细胞的致瘤性,结果表明,各组别BMSCs细胞均不具有致瘤性。⑧TRAP-ELISA法检测不同组别的BMSCs细胞端粒酶活性和细胞分泌VEGF水平,结果表明,表达hTERT基因的BMSCs端粒酶活性增强,hBMSC-T及hBMSC-V&T组的相对端粒酶活性(RTA)分别为57.9±3.1和55.3±1.3,而表达VEGF基因的hBMSCs分泌VEGF增加,hBMSC-V及hBMSC-V&T组的细胞上清液中VEGF分泌量分别为3935±154pg/ml和3749.8±164pg/ml。五.结论本实验将构建hTERT慢病毒载体和VEGF基因慢病毒载体,探讨以hTERT慢病毒载体和VEGF基因慢病毒载体联合修饰老年人BMSCs,建立强化表达VEGF的细胞系的可行性,并对hTERT慢病毒载体和VEGF基因慢病毒载体联合修饰的BMSCs进行表型鉴定及功能学研究。通过上述研究我们得到了以下结论：1、hTERT基因慢病毒载体和VEGF基因慢病毒载体联合修饰老年人BMSCs,建立强化表达VEGF的BMSCs细胞系是可行的,具有重要的临床价值。2、hTERT基因慢病毒载体和VEGF基因慢病毒载体联合修饰老年人BMSCs,能够表达BMSCs细胞表面具有的特征,在体外能够无限制的扩增,具有分泌VEGF蛋白的功能。3、hTERT基因慢病毒载体和VEGF基因慢病毒载体联合修饰老年人BMSCs细胞,具有较快的生长增殖能力,较强的迁移能力,不具有侵袭性和致瘤性,细胞染色体核型稳定。更多还原

【Abstract】 Background:Bone marrow derived mesenchymal stem cell (BMSCs) is a kind of multipotent adult stem cellsderived from bone marrow, the main advantage of its application in cell therapy is autologous transplantation, not transplantation immune rejection, not ethics and induced tumor limitations,compared withembryonic stem celltransplantation; andBMSCs is relatively simple, unlimitedandeasy to obtain,compared with neural stem cells, which were mainly located in the subventricular zone, hippocampus and other structures. Clinical study of BMSCs transplantation has been used in cerebral ischemia and some other diseases.There are many studies in the treatment with brain ischemia throughBMSCs transplantation. The BMSCs transplanted treatment with rat cerebral MCAO by vein, artery and stereotactic local injection had found that transplanted cells could directe to repair the focal ischemic area, andon the other handindirect to repair the focal ischemic area inducing glial cells and neural stem cells walking to the injured area or differentiation and proliferation of the local neural stem cells, through secreting chemokines. It is an important way to reconstruct neural circuit with BMSCs transplantation becauseBMSCs have the ability to differentiate into neural cells. However, most studies found that BMSCs transplantation only hadfew expression of neural or glial cell surface markers, and even a few expression of nerve tissue marker cells, there is no electrophysiological or other evidence shows they have nerve cell function, but can not determine whether they are with other nerve cells established contact.The present studies showed that, BMSCs plays the effect mechanism in many aspects, such as reducing apoptosis of ischemic penumbra cells, promoting proliferation of endogenous neural stem cells, promoting angiogenesis in ischemic region, inhibiting scar of glial proliferation and promoting axon regeneration. In sum, BMSCs are not replaced the missing neurons in the treatment with cerebral ischemia, but played a " molecular factory " role to mediate the above treatment process by secreting various neurotrophic factorsin brain microenvironment.BMSCs are scarce in bone marrow, which should be cell amplification in vitro in order to obtain enough cells in cell transplantation therapy. However, BMSCs will slow death the same as most somatic cell, mainly in two aspects:①With the increase of age, the quantity and quality of BMSCs decreased obviously (the elder is higher risk population inbrain ischemia, is subject to cell transplantation therapy);②In cell amplification processin vitro, BMSCs cell morphology will graduallychange (from fine spindle to wide flat), and gradually lose the ability to cell proliferation, differentiation potential, and homing to focus area.The senescence mechanism of BMSCs is complex and not yet clarified in research. It is well known that BMSCs did not express telomerase. Human telomerase is a ribonucleoprotein, which two most important componentsare the human telomerase catalytic subunit (hTERT) and RNA (hTR) template, and hTR sustained expression, while hTERT is not expressed in most adult cells. Telomerase is different from the general DNA polymerase, which is a specialized reverse transcriptase, using the complementary sequence RNA component itself as a template, catalytic TTAGGG sequences to chromosome ends, so as to maintain telomere length. The telomere structure and composition of chromosome end is closely related to cell in the maintenance and proliferation. Most adult somatic cells’telomere length decreases with cell replication, and limites cell proliferation. In contrast, tumor cells and embryonic stem cells sustain telomere functionthrough the activation of telomerase.BMSCs are not express telomerase, telomere length is gradually shortened with cell division. If it reaches about10Kb, cells will stop dividing and growth arrest. Study on the Baxter MA showed that BMSCs’mean telomere length decreased with the growth of the age (17bp/year), and in vitro amplification of7～10PDs (Population Doublings, population doubling times) the average loss of telomere length is about1Kb. The loss rate is amazing, even in young BMSCs, their life experience telomere reduce the length of the highest can only reach2.5-3.5Kb. Therefore, if you do not take the special processing method, in vitro fully amplified BMSCs back to the patient in the process, actually has greatly shortened the life of BMSCs.In order to extend life cycle of BMSCs, or some other scientific purposes, many labshave in biochemical research with BMSCsimmortalization. There were able to successfully establish BMSCs’immortalizationcell lines by modified BMSCs with the human papilloma virus (HPV) E6/E7gene, simian virus40(SV40) large T antigen gene or Bmi-1gene. These cancer gene could activate expression of human telomerase catalytic subunit gene hTERT, andsustain cells’telomere length in the amplification process, however, the major problems of BMSCs cell lines using a cancer gene modified was chromosome instability, often caused cells malignant, and not for transplantation.In recent years, there are many laboratories using hTERT transfection and successfully obtained BMSCs immortalization cell lines. The established cell lines were not significantly reduced telomere length in vitro amplification, and maintain the same primary BMSCs similar or even better proliferation and multi-directional differentiation potential. More importantly, BMSCs cell line was established by using hTERT transfected in vitro maintained normal karyotype, cell growth inhibition in vitro and in vivo, contact, experiments were not obvious tumorigenicity.In addition to BMSCs, other somatic cells skin fibroblasts, vascular endothelial cells, skin cells, mammary epithelial cells, skin melanoma cells, lymphocytes, and by BMSCs induced differentiation of the osteoblast, can beimmortalizationby transfecting hTERT gene. In the previous studies, there were not found significant malignant cellsin usingthe hTERT gene modified.Whether BMSCs express vascular endothelial growth factor receptor (VEGFR), different experiments have different reports.BMSCs are not expressed in adult VEGFR, but expressed PDGFR-A, PDGFR-B and NRP-1. VEGF play important role through the VEGFR conjugate of PDGFRand enhance the effect by NRP1. Cerebral ischemia due to cerebral vascular atherosclerosis or cerebral vascular occlusion cause nerve dysfunction, VEGF can promote ischemic vascular endothelial cell chemotaxis and aggregation, induced the formation of new blood vessels, thus increasing the nutrient supply to the ischemic area, reduce ischemia caused by functional disorder of God, therefore, will be of great value in clinical application construction of VEGF gene overexpression of BMSCs in the elderly.This experiment will construct the hTERT lentiviral vector and VEGF gene lentiviral vector, to study the effect of hTERT lentiviral vector and VEGF gene lentiviral vector combined modification in elderly patients with BMSCs, establish the feasibility of enhancing cell lines expressing VEGF, and the hTERT lentiviral vector and VEGF gene slow virus vector modified BMSCs studies phenotype identification and function. Because the genetic information of BMSCs containing autologous, research results will be helpful to explore the preparation of individual (patient-specific), for the treatment of cerebral ischemia (disease-specific) stem cell products, but also a solid foundation for further experimental study.Objectives:BMSCs transplantation is an effective strategy for the treatment of cerebral ischemia, however, BMSCs will slow death with aging and amplification process in vitro, which hinders the treatment application. This study intends to human telomerase catalytic subunit hTERTgene lentiviral expression vector and the expression of VEGF gene lentiviral expression vector of BMSCs gene modified the elderly, explore the construction of enhanced expression of VEGF permanent biochemical BMSCs, detection of its biological characteristics. Because the genetic information of BMSCs containing autologous, research results will be helpful to explore the preparation of individual (patient-specific), for the treatment of cerebral ischemia (disease-specific) stem cell products, but also a solid foundation for further experimental study.Methods:1.Construction of lentivirus vector containing hTERT gene and VEGF gene.①The amplification of hTERT and VEGF gene fragments by PCR in vitro.②Construction of pENTR11-hTERT-IRES-EGFP and pENTR11-VEGF-IRES-EGFP entry vector.③Construction of pLenti6/V5-DEST-hTERT-IRES-EGFP和pLenti6/V5-DEST-VEGF-IRES-EGFPexpression vector.④Packaging, expression and analysis of lentiviral vectors pLenti6/V5-DEST-hTERT-IRES-EGFP and pLenti6/V5-DEST-VEGF-IRES-EGFP.2. To study the effect of hTERT gene lentiviral vector and VEGF gene lentiviral vector combined modification in elderly patients with BMSCs, establish the feasibility of enhancing cell lines expressing VEGF.①The old BMSCs was purchased from biotech companies, or from clinical volunteers (aged) bone marrow adherent method to obtain BMSCs, combined with density gradient centrifugation.②Grouping:unmodified BMSCs group, empty lentiviral modification group, VEGF gene modification group, hTERT gene modification group, hTERT/VEGF double gene modification group (each gene modification group stable expression clones were selected by blasticidin).③Fluorescence microscope and transmission electron microscope observecells’ changes in different generation cell morphology; flow cytometry method detectescells’ changes indifferent generation cell surface markers; RT-PCR method detectes cell hTERT and VEGF level of mRNA; fluorescence immune cell chemical method and Western blot method detecteexpression ofcell protein.3. Study on modification of BMSCs phenotype and function in hTERT/VEGF double gene①Grouping:unmodified BMSCs group, empty lentiviral modification group, VEGF gene modification group, hTERT gene modification group, hTERT/VEGF double gene modification group.②Flow cytometry method detectescells’changes indifferent group cell surface markers; transmission electron microscope observescells’ changes in different group cell ultramorphology; confocal microscopy observescells’ changes in different groupcell markers by immunofluorescence; MTT assay and colony assay observe cell growth and proliferation; single cell scratch assay and transwell chamber assay observecell migration and invasion; RHG band karyotypeanalysis observes evaluation of chromosome stability; nude mice inoculated tumor formation experiment of recombinant cell tumorigenicity; the level of VEGF secreting cells telomerase activity and cell was detected by TRAP-ELISA.4.Data are expressed as mean SD. Statistical comparisons were performed using one-way ANOVA (LSD). Statistical significance was set at P<0.05. All statistical analysis was operated with SPSS13.0software.Results:1.Successful construction of hTERT gene lentiviral vector and VEGF gene lentiviral vector.①Successful amplified hTERT gene and VEGF gene by using pEGFP-hTERT, pEGFP-VEGF165plasmid (our laboratory existing) as a template.②Using EcoR I and Not I enzyme cut pENTR11entry vector and pIRES vector, and IRES sequence fragment from pIRES vector cut with T4DNA ligase connected into pENTR11to construct pENTR11-IRES, the recombinant plasmid was transformed into DH5acompetent cell, screening, PCR, enzyme digestion and sequencing, the success of the pENTR11-IRES.③Using Nco I, EcoR I pENTR11-IRES plasmid and hTERT gene or VEGF gene PCR product, hTERT fragment or VEGF fragments with T4DNA ligase cloned into vector pENTR11-hTERT-IRES or pENTR11-VEGF-IRES plasmid, by enzyme digestion, sequencing; finally, Xbo I, Not I restriction enzyme pENTR11-hTERT-IRES or pENTR11-VEGF-IRES plasmid and belt EGFP PCR gene product green fluorescent marker, T4DNA ligase to construct pENTR11-hTERT-IRES-EGFP or pENTR11-VEGF-IRES-EGFP plasmid by restriction enzyme digestion, sequencing, pENTR11-hTERT-IRES-EGFP and pENTR11-VEGF-IRES-EGFP; successful entry vector.④The preparation of pENTR11-hTERT-IRES-EGFP and pENTR11-VEGF-IRES-EGFP entry clone, expression vector pLenti6/V5-DEST and Gateway TM LR ClonaseTM enzyme Ⅱ mix mixed reaction, construction with poison slow disease table with double gene as carrier pLenti6/V5-DEST-hTERT-IRES-EGFP and pLenti6/V5-DEST-VEGF-IRES-EGFP, will connect the product into Stb13TM E.coli cells, screening positive clone identification. Successful expressing pLenti6/V5-DEST-hTERT-IRES-EGFP and pLenti6/V5-DEST-VEGF-IRES-EGFP vector.⑤Lipofectamine2000pLenti6/V5-DEST-hTERT-IRES-EGFP or (and) the transient expression of pLenti6/V5-DEST-VEGF-IRES-EGFP gene plasmid was transfected into COS-7cell line, the expression of recombinant protein, lentivirus vector verification of construction, with satisfactory results.2. Successfully enhanced and identified expression of VEGF immortalization BMSCs cell lines①Using ViraPowerTM Lentiviral Expression System recombinant plasmid and packaging purified mixtures were transfected into293FT cell line, collect the virus supernatant from293FT cell line, for titer determination; in the appropriate MOI with the virus supernatant transfection of bone marrow stromal cells; screening to produce stable expression of pLenti6/V5-DEST-hTERT-IRES-EGFP or by blasticidin (and) pLenti6/V5-DEST-VEGF-IRES-EGFP cell line; the RT-PCR and Western-blot testing to determine the mRNA and protein expression and has higher activity.②Observation of optical microscope, primary cell Gang inoculation suspended in a DMEM liquid culture medium, the nucleus was round, located in cytoplasm of central. In general,2-4h after inoculation of primary cells became adherent,48h after most primary cell wall. The medium was changed after removal of nonadherent cells adherent cells can be seen in many forms, fusiform or polygonal, nucleus large, oblate, cytoplasmic can extend lamellipodia in different directions. Adherent primary cells to disperse the colony clonal rapid proliferation,1W around each colony contains about hundreds of cells, cell morphology was long fusiform fibroblast-like. Passage cells begin to adherent to1-2h after inoculation,24h completely adherent, passaged cells showed a broad flat polygon,6-7d cells completely confluent, cells become elongated, morphology were fibroblast-like.③There were not obvious structure changed in the ultrastructure of different generation BMSCs cells by transmission electron microscope.④The expression level of flow cytometry assay for the detection of different generations of BMSCs cell surface markers CD29, CD34, CD44, CD45, CD90and CD105, CD44, primary cultured cells showed positive expression of CD90, CD29was weakly positive expression of CD34, CD45, CD133, and almost no expression; cell line (P3, P9) CD44the positive expression of CD29, CD90, weakly positive expression, while CD34, CD45, CD133almost no expression, and primary cells are basically the same.⑤RT-PCR, Western-blot and immunofluorescence assay to determine the different groups of mRNA and protein expression and has higher activity.3. Successful construction of hTERT/VEGF modified BMSCs cell line and studies on the phenotype and function①The expression level of flow cytometry assay for the detection of different groups of BMSCs cell surface markers CD29, CD34, CD44, CD45, CD90and CD105, each group was CD44, the positive expression of CD90, CD29was weakly positive expression of CD34, CD45, CD133, and almost no expression.②Transmission electron microscope was used to observe the ultrastructure of BMSCs cells, and different groups showed that, WP bodies were detected VEGF gene expression in BMSCs cells, plasma membrane vesicles and secretion of vWF, the other groups were not expressed.③the laser confocal microscopy of BMSCs cell protein expression of different groups, each group results show that, BMSCs cells could express CD31and UEA-1, but only the expression of VEGF BMSCs cells to express vWF protein.④RHG banding chromosome analysis BMSCs analysis of karyotype of different groups of changes, results show that, the group BMSCs cell karyotype, no genetic variation.⑤The MTT experiment and plate cloning test in different groups of the BMSCs cell growth and proliferation, results showed that, hTERT gene expression in BMSCs cell growth and proliferation was significantly increased.⑥BMSCs cells of different groups of detecting the single cell scratch assay and Transwell chamber assay invasion and migration, results show that, expression of hTERT gene or VEGF gene of BMSCs cell migration ability enhancement, and BMSCs cell groups were no invasion.⑦Tumorigenicity, different groups of BMSCs cells to nude mice tumor formation experiment test results show that, the group BMSCs cells are not tumorigenic.⑧The level of VEGF, the secretion of TRAP-ELISA method for the detection of different groups of BMSCs cells telomerase activity and cell showed that, hTERT gene expression or enhance telomerase activity of BMSCs cells with VEGF gene, VEGF secretion increased.Conclusions:The experiment will construct the hTERT lentiviral vector and VEGF gene lentiviral vector, in order to study the effect of hTERT lentiviral vector and VEGF gene lentiviral vector combined modified elderly patients with BMSCs, establish the feasibility of enhancing cell lines expressing VEGF, and the hTERT lentiviral vector and VEGF lentiviral vector modified BMSCs studies phenotype identification and function. Therefore, we found that the following conclusions: 1. Establishedenhanced VEGF expression BMSCs cell line with hTERT and VEGF gene lentivirus vector combined modified elderly patients with BMSCs, which has important clinical value.2.Enhanced VEGF expression BMSCs cell line with hTERT and VEGF gene lentivirus vector combined modified elderly patients with BMSCs expresses the characteristics of the surface of BMSCs cells, can be amplified in vitro without limit, and can secrete VEGF protein function.3. Enhanced VEGF expression BMSCs cell line with hTERT and VEGF gene lentivirus vector combined modified elderly patients with BMSCs has fast cell proliferation, strong ability of migration, notinvasiveness and tumorigenicity, and chromosome karyotype stability.更多还原