【作者】 石东文；

【导师】 吴军；

【作者基本信息】 第三军医大学 ， 外科学， 2006， 博士

【摘要】 研究背景和目的严重创伤、感染、肿瘤等原因所致的骨缺损是临床常见的疑难问题,骨组织工程被认为是修复骨缺损的理想方法,在将来的临床中具有广阔的应用前景。种子细胞作为骨组织工程的最重要环节之一,近年来进行了大量的研究,一般认为,理想的骨组织工程种子细胞需要满足三个基本条件:①可靠的细胞来源,取材方便,易于体外扩增;②具有特定的分化表型或定向分化潜能;③能够适应受区环境,组织相容性好,不引发移植排斥反应。目前可用于骨组织工程的种子细胞主要包括:成骨细胞,胚胎干细胞和骨髓间充质干细胞。其中成骨细胞是成体终末细胞,增殖潜能有限,来源困难。胚胎干细胞则受到伦理学的限制,成骨分化的调控机制也较为复杂。骨髓间充质干细胞（MSCs）具有多向分化潜能和自我更新能力,易于取材和体外培养扩增,可进行自体移植等特有的生物学特性,因而被组织工程领域视为最佳的种子细胞。并且在自体移植修复骨缺损动物模型和异基因移植修复免疫缺陷动物骨缺损模型实验中取得了良好的效果。但深入的研究表明,hMSCs作为种子细胞的临床应用依然面临着许多需要解决的问题。例如MSCs的多向分化机制及其调控,MSCs与移植受体之间的相互作用,表型鉴定和自体移植细胞数量不足等问题。其中自体移植来源不足是限制临床应用的最为突出的问题。这是由于大段骨缺损的修复需要大量种子细胞,而hMSCs在骨髓中含量很少,只占骨髓中单个核细胞的0.01—0.001%,并随着年龄的增长逐渐减少,体外培养扩增平均只有38PD（population doublings）,并且难以在短时间内大量扩增。解决这一问题的主要途径应包括两个方面:一是增强hMSCs的增殖活力,二是应用异体或异种MSCs。端粒是染色体末端串连、重复的TTAGGG序列,在正常细胞分裂过程中会逐渐变短。当端粒在一个或多个染色体中缩短到某一极限值时,细胞衰老和生长停止就发生了。端粒的长度是由端粒酶维持的。端粒酶逆转录酶（hTERT）是端粒酶的一个接触反应亚单位,大量实验表明,外源性hTERT基因导入细胞可提高端粒酶活性,延缓细胞的衰老Simonsen JL等将hTERT转入hMSCs筛选到的hMSCs-T细胞克隆在体外培养200PD以上,细胞未发现明显的衰老现象、保持了多向分化潜能并没有致瘤倾向。这说明用hTERT基因转染修饰hMSCs可以达到延长hMSCs生命周期、保持多向分化潜能的目的。尽管近年来研究表明hMSCs免疫源性较低,但hMSCs并非完全的免疫赦免细胞,异基因移植必然要面临免疫排斥问题。免疫抑制剂的应用已经使组织器官移植取得了巨大的进步,但因全身免疫功能降低,导致肿瘤和严重感染的问题难以克服。新型免疫调节分子CTLA-4Ig通过阻断抗原特异性T细胞活化过程起重要作用的B7-CD28共刺激通路,可诱导特异性免疫耐受。近年研究表明,CTLA-4Ig的全身应用和局部基因导入在器官移植中均取得了良好的效果。本课题组前期研究曾经用逆转录病毒载体介导CTLA-4Ig基因转染hMSCs,被修饰细胞及其分化后代能够持续分泌CTLA-4Ig蛋白,保持了体外培养分化成骨能力,而且在异种动物体内能够成骨。逆转录病毒载体介导的基因转染能够将外源基因整合到细胞基因组中并传向子代,使目的蛋白得以持续表达。对于细胞基因改造,筛选获得新细胞株和某些疾病的基因治疗是一种理想的转染方式,但CTLA-4Ig在体内的长期持续表达也可能带来对全身免疫功能的不利影响。而腺病毒是一种安全,高效的转染方式,目的基因只在被转染细胞本身短暂表达。而组织工程种子细胞只须在细胞移植入体内早期的一定时间内表达CTLA-4Ig,诱导特异性免疫耐受即可达到降低免疫排斥反应的目的。因此,本课题拟应用逆转录病毒将hTERT基因导入hMSCs,以筛选获得hTERT—hMSCs细胞克隆或细胞群;然后再通过腺病毒介导CTLA4Ig基因转染hTERT—hMSCs细胞。以期获得保持成骨分化潜能,体外培养具有旺盛的增殖活力,能够在异基因移植受体移植局部表达分泌CTLA4Ig蛋白,诱导特异性免疫耐受的组织工程种子细胞。实验方法1.采用Percoll密度梯度离心法从健康成人骨髓中分离人骨髓间充质干细胞;DMEM/F12+10%FBS常规培养,细胞生长至80%-100%融合时以1:3传代;FACS检测第3代细胞的CD105、CD34表达。2.hTERT重组逆转录病毒载体的构建和鉴定用质粒提取试剂盒从DH5α大肠杆菌中分别提取pGRN145质粒和pLXSN质粒,限制性内切酶EcoRⅠ酶切鉴定。用凝胶回收试剂盒从pGRN145质粒和pLXSN质粒酶切产物中回收纯化hTERT和线形化pLXSN DNA片段。将pLXSN线性片段去磷酸,用Quick Ligation^TMKit连接hTERT和pLXSN DNA片段,连接产物转化DH5α大肠杆菌,接种于氨苄青霉素抗性的LB培养基,37℃培养过夜。提取质粒,BamHⅠ酶切鉴定,并送样测序鉴定。3.hTERT重组逆转录病毒包装和病毒滴度的测定,用质粒提取试剂盒提取新构建的pLXSN-hTERT重组质粒,测定浓度,脂质体介导转染PT67细胞,应用G418抗性,克隆环和有限稀释法筛选阳性克隆。用3T3细胞按常规方法测定病毒滴度。选择滴度最高的PT67细胞进行下一步实验。4.hTERT重组逆转录病毒感染hMSCs细胞,将病毒滴度最高的hTERT—PT67克隆细胞用丝裂霉素处理,与第2代hMSCs共培养以感染hMSCs,应用G418筛选出抗性细胞群hTERT—hMSCs。5.免疫细胞化学方法检测hTERT—hMSCs细胞中hTERT蛋白表达,以hMSCs为对照。6.端粒酶活性检测试剂盒（TRAPEZE Telomerase Detection Kit）检测hTERT—hMSCs端粒酶活性,以hMSCs为对照。7.CTLA4Ig重组腺病毒（Adv-CTLA4Ig-EGFP）直接感染hMSCs和hTERT—hMSCs。倒置荧光显微镜观察绿色荧光蛋白（EGFP）的表达,流式细胞仪（FACS）检测转染效率。8.免疫细胞化学检测CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs细胞中CTLA4Ig蛋白表达。Western blotting检测CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs细胞培养液中CTLA4Ig蛋白。9.用倒置光学显微镜观察hMSCs、CTLA4Ig-hMSCs、hTERT-hMSCs和CTLA4Ig-hTERT-hMSCs的形态及其生长情况。10.FACS测定hMSCs、CTLA4Ig-hMSCs、hTERT-hMSCs和CTLA4Ig-hTERT-hMSCs的细胞周期。11.用成骨诱导培养基（含地塞米松、维生素C和β-GP的DMEM/F12）进行。CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs的成骨诱导培养,培养3周后进行成骨特异性标志检测。方法包括:用钙的四环素荧光标记法和茜素红染色检测钙盐沉积,碱性磷酸酶染色和骨钙素免疫细胞化学检测方法检测成骨特异性标记蛋白。12.组织工程骨构建:采用单向接种法将成骨诱导1周的CTLA4Ig-hMSCs和hMSCs接种到脱钙骨基质（DBM）上,继续诱导培养1周后应用,用前计算单位重量DBM所吸附的细胞数量。13.CTLA4Ig-hMSCs和hMSCs细胞成骨动物实验动物分组:新西兰大白兔20只,雌雄不限,体重2.5—3.5千克,随机分为4组,A组:DBM+CTLA4Ig—hMSCs,B组:DBM+hMSCs,C组:DBM,D组:空白。每组5只,采用兔桡骨缺损动物模型进行骨缺损修复实验。术后2天、2周、4周、8周、12周进行X线摄片观察骨缺损部位成骨修复情况。实验结果1.分离培养的原代hMSCs呈纺锤形或三角形,类似纤维细胞,旋涡样排列;第3代细胞FACS检测结果显示:分离培养的细胞90.8%表达CD105表达,96.2%CD34为阴性。证明该细胞为高纯度的hMSCs。2.pGRNl45质粒用EcoRⅠ单酶切获得一条11.5Kb的载体片断和一条3.5Kb的hTERT目的片断;pLXSN质粒EcoRⅠ单酶切获得一条5.9Kb的线性片断,将回收纯化的hTERT和线形化pLXSN DNA片段经过连接,转化和酶切鉴定,获得pLXSN—hTERT重组质粒。测序鉴定结果证明pLXSN—hTERT重组质粒有3402个碱基与Genebank中hTERT序列cDNA的3402个碱基匹配率为100%。3.pLXSN—hTERT重组质粒转染的PT67细胞经过G418抗性筛选获得产病毒hTERT—PT67包装细胞,测定细胞培养上清（病毒液）病毒滴度为1.2×10⁴。4.pLXSN-hTERT—PT67包装细胞与hMSCs共培养5天,G418抗性筛选30天。存活细胞命名为hTERT-hMSCs。细胞培养至第46代,细胞形态无明显变化,生长良好。5.hTERT-hMSCs和hMSCs细胞hTERT免疫细胞化学染色结果显示,两种细胞hTERT蛋白阳性表达均在细胞核,hTERT-hMSCs细胞核全部深染,计数10个高倍视野阳性率99%,hMSCs阳性率65%。6.端粒酶活性检测结果显示,hMSCs细胞有50bp端粒酶活性产物条带,但未形成6碱基梯度递增条带,hTERT-hMSCs细胞TRAP产物扩增梯度递增条带:50bp、56bp、62bp、68bp、74bp等明显可见。说明未经转染的hMSCs有低水平的端粒酶活性。经hTERT基因转染的hMSCs,即hTERT-hMSCs细胞端粒酶活性明显增强。7.CTLA4Ig重组腺病毒（AdvCTLA4Ig-EGFP）感染hMSCs后48小时,感染hTERT—hMSCs后24小时可见少量细胞表达绿色荧光,随后逐渐增多,两种细胞的绿色荧光蛋白表达高峰均在第5天,第28天绿色荧光开始减少。流式细胞仪检测结果,hMSCs感染率为81.14%,hTERT—hMSCs感染率为83.75%。8.免疫细胞化学结果显示,大多数CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs在胞浆中可见CTLA4-Ig蛋白阳性表达,核周围最明显。第28天阳性细胞数目减少。Western blotting显示CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs细胞培养液中有CTLA4Ig蛋白存在。9.细胞形态观察可见hMSCs、CTLA4Ig-hMSCs、hTERT-hMSCs和CTLA4Ig-hTERT-hMSCs均以长梭形细胞为主,有少量细胞呈三角形。随着细胞的多次传代,hMSCs逐渐变宽变短,生长变慢。而hTERT-hMSCs保持长梭形,突起增多,三角形和多角形的细胞稍增多。生长速度无明显改变。CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs与转染CTLA4Ig基因前相比形态无明显改变,但生长速度减慢。10.细胞周期检测结果1—12代hMSCs G0/G1逐渐增多G2/M和S期细胞逐渐减少,统计分析相差显著。说明细胞增殖能力逐渐减弱;1—18代hTERT—hMSCs G0/G1细胞虽然也显示逐渐增多G2/M和S期细胞逐渐减少,但统计结果显示无明显差异,说明hTERT—hMSCs增殖能力没有随着传代次数的增加而明显减弱。用双因素等重复方差分析分别对hMSCs和hMSCs-TERT两组细胞的G0/G1、G2/M和S期进行对比,结果三组P值均小于0.01,说明hMSCs-TERT细胞增殖能力比hMSCs细胞增殖能力强。hMSCs和CTLA4Ig-hMSCs,hTERT-hMSCs和CTLA4Ig-hTERT-hMSCs细胞周期对比结果显示,CTLA4基因转染可使细胞生长速度减慢。11.CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs成骨诱导培养过程中,前2周细胞形态和生长速度无明显改变;2周后细胞逐渐变宽变短,多角形细胞增多,细胞有聚集成团的趋势,第3—4周时大多数细胞为多角形且聚集成团。四环素荧光标记见到团块状细胞中有钙盐沉积;茜素红染色钙结节呈橙红色;碱性磷酸酶（AKP）染色呈强阳性;免疫细胞化学检测骨钙素表达阳性。12.本实验构建的组织工程骨,每克DBM上吸附、生长的细胞约5×10⁵个。13.CTLA4Ig-hMSCs异种动物移植DBM+CTLA4Ig-hMSCs组在术后2周、4周骨缺损处呈现低密度云雾影,8周时呈高密度影,与骨组织相似,12周可见完全骨化,髓腔再通。DBM+MSCs组前4周和DBM+CTLA4Ig-hMSCs组相似,但8周时缺损处密度明显低于DBM+MSCs-C组,12周骨缺损仍未完全愈合。DBM组早期有云雾状影,随后逐渐消失。DBM组和空白组在观察全程骨缺损逐渐缩小,但在术后12周仍留有明显的骨缺损。结论1.成功构建了hTERT重组逆转录病毒载体pLXSN—hTERT。2.筛选获得了能够产生hTERT重组逆转录病毒的包装细胞系hTERT—PT67。3.逆转录病毒载体介导的hTERT基因转导hMSCs细胞（hTERT—hMSCs）,与hMSCs细胞相比,端粒酶活性明显增强,增殖能力提高,生命周期延长。4.CTLA4Ig基因转导的hMSCs（CTLA4Ig-hMSCs）和hTERT与CTLA4Ig双基因转导的hMSCs（CTLA4Ig-hTERT-hMSCs）均可表达和分泌CTLA4-Ig蛋白。表达持续时间在4周以上。5.腺病毒介导的CTLA4Ig基因转染hMSCs和hTERT-hMSCs可使细胞群体倍增时间延长,生长速度变慢。6.CTLA4Ig-hMSCs和CTLA4Ig-hTERT-hMSCs均能够在体外诱导分化为成骨细胞。说明腺病毒介导的CTLA4Ig基因转染使hMSCs保持了成骨分化潜能。7.用CTLA4Ig-hMSCs作为种子细胞构建的组织工程骨在异种移植动物体内成骨效果优于hMSCs。CTLA4Ig-hTERT-hMSCs在异种移植动物体内成骨效果有待于进一步研究。更多还原

【Abstract】 Background:Massive bone defect,caused by severe trauma,infection,tumor and such diseases like these,is a very common but difficult clinical work,and now engineering bone tissue is considered to be a promising method to treat this problem,which will be of great value in future clinic research.As one of the most crucial part of engineering bone tissue,seed cells have been widely studied in recent years.Generally,proper seed cells for engineering bone tissue should meet following demands:First,having reliable and convenient cell source and facilitating expansion in vitro;Second,bearing specific differentiation phenotype or oriented differentiation potential;Third,having favorable histocompatibility with recipient and not inducing transplantation rejection.Up to now,osteoblasts,embryonic stem cells and bone marrow-derived mesenchymal stem cells（MSCs） are three available candidates for engineering bone tissue,in which osteoblasts and embryonic stem cells are not very satisfying.Osteoblasts,as adult terminal cells have limited proliferation potential and scarce sources.Due to some ethnical reasons and complicated regulatory mechanism of osteogenesis,embryonic stem cells are excluded as well.Compared with the other two cells, MSCs,which own multi-oitented differentiative potential and self-renewal capability,have convenient sources and can expand in vitro cell culture,is regarded as the optimal seed cell of engineering bone tissue.What’s more,in both normal animal models of autotransplantation and immunodeficient animal models of allotransplantation,MSCs were proved to be effective to repair bone defects.However,further researches indicate that,to be applied in clinic as the seed cell, hMSCs still have a long way to go.For example,there are problems such as how to control their multi-oriented differentation,the interaction between hMSCs and recipients, phenotype identification and the insufficient cell number of autotransplantation,among which,the insufficient cell number is the main problem which limits their application in clinic.This is because that massive bone defects require massive seed cells,while hMSCs only account for 0.01—0.001%of mononuclear cells in bone marrow,and what’s worse, they will decreases with age,expansion in vitro culture is only 38PD（population doublings）on average and they are hard to expand in a short time.The feasible solution comprises of two aspects:one is to enhance the proliferation activity of MSCs,the other is to apply allogeneic or xenogeneic MSCs.As we know,telomere is a clustered and repeated TTAGGG sequence in chromosome ends,which will gradually shorten during cell division.When telomere in one or more chromosomes is shorten to its extreme,cell senescence and growth stop will occur.The length of telomere is sustained by telomerase and human telomerase reverse transcriptase （hTERT） is contact reaction of subunit of telomerase.Some researchers reported that ectopic expression of hTERT in MSCs could extend their life-span and maintain their osteogenic potential.Simonsen JLetc.transferred hTERT into hMSCs and obtained hMSCs-T cell clone which showed no obvious senescent phenomena,maintained multipotential differentiation,had no tendency to cause tumor after more than 200PD in vitro culture,which indicated hMSCs with hTERT transgene could prolong their life span and maintain multi-differential potential.Although recent studys indicate that MSCs are low hypoimmunogenic,they are not completely immune privileged cells,So allogenic MSCs transplantation still faces immune rejection problem.Tissue and organ transplantations have made great progress with the application of immunosuppressive drugs,but immunosuppressive drugs would decrease the immune function and lead to tumor and severe infection.As we know,there is no method that can induce donor specific,complete and permanent immune tolerance.Among numerous studies of immune tolerance,CTLA4 is one of the most important focus points. CTLA4 can bond B7 molecule on APC competitively with CD28 and block the B7-CD28 costimulatory pathway,which inhibits T cell full-activation and induces T cell anergy, apoptosis or clone deletion,resulting in immune tolerance.CTLA4Ig,a fusion protein of CTLA4 extracellular domain and partial segment of IgG- Fc,has the same function with CTLA4.Our previous study has modified MSCs with CTLA4Ig gene and screend out transfected cells.The modified MSCs can secrete CTLA4Ig protein to induce immune tolerance,which has been demonstrated in rat model.Thingking about security and graft requisition,adenovirus vector is safe and has efficient infectivity,we design to transfer CTLA4Ig gene by adenovirus vector in this study.Objective1.To construct the retroviral hTERT expression vector.2.To prepare the retroviral hTERT expression vector transferred hMSCs （hTERT-hMSCs）.3.To prepare CTLA4Ig gene transferred hTERT-hMSCs（CTLA4Ig-hTERT-hMSCs）.4.To study the biological characters of CTLA4Ig-hTERT-hMSCs both in vitro and in vivo.Materials and methods1.MSCs were isolated from bone marrow of human iliac crest by a modificative procedure of Pittenger and density gradient centrifugation of Percoll and were cultured by routine methods.The symbols CD105/CD34 at passage 3 were detected by FACS.2.Construction recombination plasmid pLXSN-hTERT by using molecular biological technique.3.Package of hTERT-retroviral vector and determination of titer:pLXSN-hTERT was transfected into PT67 cells mediated by lipoplast2000.The positive clones were screened by G418,clone line and limiting dilution assay.TCID50 was used to determine the virus titer.4.hTERT-retroviral vector infection human MSCs:PT67 cells involved hTERT-retroviral vector and passage2 hMSCs were cocultured.The hTERT—hMSCs were screened with G418.5.The expression of hTERT protein in hTERT—hMSCs was immunocytologically demonstrated.6.The telomerase activity was detected with TRAPEZE Telomerase Detection Kit.7.Both hMSCs and hTERT—hMSCs were infected by Adv-CTLA4Ig-EGFP.The expression of EGFP was observed under the inverse fluorescent microscope and the infection rate was detected by FACS.8.The expression of CTLA4IG protein in both CTLA4Ig- hMSCs and hTERT—hMSCs was detected by Immunocytochemistry,and CTLA4IG protein in cell culture fluid was detected by Western Blot.9.It was observed with Inverted microscope that morphous and growth state of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs,and CTLA4Ig-hTERT-hMSCs.10.The cells cycles of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs,and CTLA4Ig-hTERT-hMSCs had been studied by FACS method.11.Osteogenesis of CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs were detected by calcium salts dying,alkaline phosphatase dying and osteocalcin immunocytochemistry.12.Engineering bone tissue was prepared by DBH seeded by CTLA4Ig -hMSCs.13.Osteogenesis of engineering bone tissue in radius defect of rabbits was observed by X-ray on designed time points（i.e.,2-days,2,4,8,12weeks post-surgery）.Results:1.Like fibrocyte,cultured primary hMSCs showed an atractoid or triangular shape and arrayed in whirpool.FACS detection indicated that 90.8%of isolated cells expressed CD105 and 96.2%were CD34 negetive.2.Plasmid pGRN145 was cut into a 11.5kb vecter fragment and a 3.5kb hTERT target fragment,Plasmid pLXSN was cut into a 5.9kb linear fragment by EcoR I resPectively. Then we gained recombinant plasmid pLXSN—hTERT by ligating hTERT and pLXSN. DNA sequencing illustrated that 3402 bases of recombinant plasmid pLXSN—hTERT matched with 3402 bases of hTERT sequence of gene bank.The matching rate was 100%.3.We used recombinant plasmid pLXSN—hTERT to transfect PT67 cells and obtained hTERT—PT67 package cells after G418 resistant screening.The virus tilter of cell culture supernatant was 1.2×10⁴.4.After 5 days coculture of pLXSN—hTERT—PT67 package cells and hMSCs and 30 days G418 resistant screening,the survival cells were named hTERT-hMSCs.The cells didn’t show obvious cell shape change when they were cultured to 46^th passage.5.Immunocytochemical stain of hTERT showed that both hTERT-hMSCs and hMSCs expressed hTERT protein in cell nucleus.The whole nucleus of hTERT-hMSCs was deeply stained,and the positive staining of hTERT-hMSCs was 99%while hMSCs was 65%under 10times highpower field.6.Telomerase activity detection indicated that compared with untransfected hMSCs, hMSCs with hTERT transgene had stronger telomerase activity.7.48hours after AdvCTLA4Ig-EGFP infecting hMSCs and 24 hours after AdvCTLA4Ig-EGFP infecting hTERT—hMSCs,we observed that both a few hMSCs and hTERT—hMSCs expressed green fluorescence,and the cell number gradually increased. The expression peak of green fluorescent protein in both cells was at the fifth day,and the green fluorescence began to decrease at the 28th day.Flow cytometry indicated that the infection rate of hMSCs was 81.14%,while hTERT-hMSCs was 83.75%.8.Immunocytochemistry detection demonstrated that most of CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs positively expressed CTLA4Ig in cytoplasm,especially around nucleus,hMSCs that expressed hTERT were small,long spindleshape while hMSCs that negatively expressed hTERT were mainly large,flat shape.Western blotting indicated that CTLA4Ig protein existed in culture solution of both CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs.9.We observed that most of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs were long spindle—shaped and a few were triangular,hMSCs gradually became wide and short along with passage,and the growth velocity slowed down, while hTERT-hMSCs remained long spindle—shaped and the growth velocity changed little.The shape of CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs showed little change before and after CTLA4Ig transfection,however,the growth velocity slowed down.10.Detection of cell cycle1-12 passage hMSCs in G0/G1 phase increased while in G2/M and S phase decreased gradually,and statistical analysis showed significant difference,which indicated that proliferative capability of hMSCs weakened gradually.1-18 passage hTERT—hMSCs showed similar change tendency but statistical analysis showed insignificant difference, which suggested that proliferative capability of hTERT-hMSCs changed little accompanying passage.Statistical analysis demonstrated that hTERT—hMSCs had higher proliferative capability than hMSCs did.Cell cycle comparison between hMSCs and CTLA4Ig-hMSCs,and comparison hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs indicated that CTLA4 gene transfection could slow down cell proliferationAll of hMSCs,CTLA4Ig-hMSCs,hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs were diploid cells.11.During the course of osteogenesis in vito,the shape and growth speed of CTLA4Ig-hMSCs and CTLA4Ig -hTERT-hMSCs changed little in the first 2 week;then the cells became wide and short,cells with polygonal shape increased,and the cells had the tendency to cluster together.During 3 to 4 weeks,most of cells were polygonal and clustered.Calcareous infarct could be detected by tetracycline dying in cell clump;AKP dying was strong positive;the expression of osteocalcin could be detected by immunocytochemistry.12.In our experiment,there were about 5×10⁵ cells attached to per gram DBM.13.CTLA4Ig-hMSCs xenotransplantationLow density vague shadow in bone defect could be seen 2 weeks and 4 weeks post-surgery in group treated with DBM+CTLA4Ig-hMSCs under X-ray;at 8 weeks,the shadow was high-density,similar with bone tissue;at 12 weeks,the bone defect was completely ossified,and medullary cavity was recanalized.Compared with group treated with DBM+CTLA4Ig-hMSCs,in group treated with DBM+MSCs,similar shadow appeared in the first 4 weeks,but at 8 weeks,the shadow density was apparently lower than the former;the bone defect didn’t heal completely at 12 weeks.Group treated with DMB showed vague shadow early,but the shadow disappeared gradually.During the observation, bone defects diminished in both group treated with DBM and blank group,but obvious bone defects still remained 12 weeks post-surgery.Conclusions:1.We successfully constructed recombinant retroviral expression vector-pLXSN—hTERT.2.We obtained package cell lineage hTERT—PT67 which has the ability to produce hTERT recombinant retrovirus.3.Compared with hMSCs,hTERT—hMSCs show stronger telomerase activity,higher proliferative capability and longer life-span..4.Expression and secretion of CTLA4Ig protein could be detected in both CTLA4Ig-hMSCs and CTLA4Ig-hTERT-hMSCs,which last more than 4 weeks.5.The times of population doublings could be prolonged and growth speed slowed after hMSCs and hTERT—hMSCs had been modified by adenovirus mediated-CTLA4 gene transfection6.Both hTERT-hMSCs and CTLA4Ig-hTERT-hMSCs could be induced to differentiate into osteoblast in vitro,which indicates that adenovirus mediated-CTLA4 gene transfection don’t alter hMSCs’ osteogenesis potential.7.CTLA4Ig-hMSCs have better ostogenesis effects than hMSCs as seed cells for engineering bone tissue on xenotransplantation animals.The ostogenesis effects of CTLA4Ig -hTERT-hMSCs on xenotransplantation animals need further studying.更多还原