大鼠脂肪间充质干细胞体外转染为胰岛素分泌细胞的实验研究

目的 探讨脂肪间充质干细胞(ADMSCs)体外转染成为胰岛素分泌细胞的可能性及其在不同浓度葡萄糖环境下的胰岛素分泌情况.方法 以未转染的ADMSCs作为对照组,含有PcDNA3.1的ADMSCs作为空载体组,含有PcDNA3.1-hINS的ADMSCs作为重组载体组,转染后再将重组载体组根据培养时间不同分为1、6、12、18 d组,并将重组载体18 d组根据葡萄糖浓度的不同分为高糖组和低糖组.RT-PCR扩增人胰岛素基因,并构建含有人胰岛素基因的真核表达重组载体PcDNA3.1-hINS.经离心消化法获得ADMSCs,并通过流式细胞仪检测.瞬时转染,RT-PCR测各组胰岛素DNA的转录,ELISA法检测各组胰岛素分泌量,并对重组载体18 d组行葡萄糖刺激实验,计量资料以x±s表示,多组间的比较采用方差分析,两组比较采用t检验.结果 流式细胞仪检测ADMSCs表面抗原:CD44、CD90、CD106表达阳性,CD34、CD45、CD11b表达阴性.RT-PCR法检测重组载体组有胰岛素DNA转录,对照组和空载体组均无转录.ELISA法检测重组载体1、6、12、18 d组胰岛素分泌量分别为(4.7±0.8)mIU/L、(8.8±0.5)mIU/L、(8.9±0.8) mIU/L、(8.6±0.6) mIU/L,与对照组的(1.3±0.6) mIU/L和空载体组的(1.7±0.8)mIU/L分别比较,差异均有统计学意义(t=10.09,32.64,22.20,55.53；9.23,27.56,19.43,51.25,P＜0.05)；重组载体1d组与6、12、18 d组的胰岛素分泌量分别比较,差异有统计学意义(t=12.77,12.26,13.93,P＜0.05)；而6、12、18 d组间的胰岛素分泌量比较,差异无统计学意义(F=45.67,P＞0.05).高糖组和低糖组胰岛素分泌量比较,差异有统计学意义(t=2.03,P＜0.05).葡萄糖刺激实验阴性.结论 ADMSCs成功转染为胰岛素分泌细胞,并可以稳定分泌胰岛素,虽然胰岛素分泌量不能根据葡萄糖的浓度改变而改变,但仍为干细胞治疗糖尿病提供了一种新的种子细胞.     

人碱性成纤维细胞生长因子基因转染大鼠骨髓间充质干细胞的研究

目的 观察人碱性成纤维细胞生长因子(bFGF)基因体外转染对大鼠骨髓间充质干细胞(MSCs)bFGF表达的影响.方法 密度梯度离心、贴壁法培养分离SD雄性大鼠MSCs,体外扩增,流式细胞仪检测MSCs表面抗原表达.利用慢病毒载体系统介导将具有人源性bFGF基因转染至第2代MSCs,在倒置荧光显微镜下观察转染后细胞形态和生长的变化,应用逆转录-聚合酶链反应(RT-PCR)、Western blot法鉴定bFGF在MSCs中的表达.结果 密度梯度离心、贴壁法培养分离可获得MSCs,P3代大鼠细胞利用流式细胞仪检测CD11b/c阳性细胞表达率为(13.2±0.6)%,CD34阳性细胞表达率为(1.2±0.5)%,CD44阳性细胞表达率(97.8±0.9)%,CD90阳性细胞表达率(96.8±1.4)%.MSCs转染48 h后,绿色荧光蛋白的表达明显增强.RT-PCR证实转基因MSCs表达bFGF mRNA明显增强,Western blot检测证实转基因MSCs在49 KDr出现特异性条带,而空白和空载组的MSCs则未见阳性条带.结论 采用慢病毒介导的基因转染技术可以将bFGF基因转染至MSCs中,并有外源性bFGFmRNA和蛋白的有效表达,MSCs可作为bFGF基因治疗的载体.     

黄芩苷诱导大鼠骨髓间充质干细胞体外分化为胰岛素分泌细胞

目的 体外诱导大鼠骨髓间质干细胞（BMSCs）分化为胰岛样细胞。方法 采用分步法体外诱导后，间接免疫荧光法鉴定诱导前后细胞nestin、胰岛素蛋白表达；RT-PCR法检测诱导前后胰岛转录因子mRNA表达；ELISA检测诱导后细胞葡萄糖刺激的胰岛素分泌。结果 免疫荧光染色显示诱导5h，nestin阳性细胞为（44．6±7．3）％。诱导24h，nestin阳性细胞增至（61．8±8．4）％。此后，nestin阳性细胞数目开始下降，诱导第14天后，nestin表达基本消失；同时诱导后的细胞可以表达胰岛素蛋白。另一方面，RT-PCR结果显示诱导后细胞可以表达胰岛素-1、葡萄糖转运子-2及其转录因子mRNA。ELISA结果显示不同浓度的葡萄糖刺激的胰岛素分泌量不同，5mmol／L和25mmol／L葡萄糖刺激的胰岛素分泌量分别为（25．53±6．49）和（53．26±7．56）mU／L，而诱导前MSCs不具备上述特点。结论 大鼠BMSCs体外可以诱导成为胰岛素分泌细胞。     

体外转染GFP-Bcl-2基因对低氧条件下间充质干细胞细胞周期的影响

目的探讨低氧条件下诱导骨髓来源的间充质干细胞（MSCs）向目标细胞分化为相关疾病提供了潜在的临床治疗途径。方法将表达GFP-Bcl-2的慢病毒载体转染大鼠骨髓MSCs使其过量表达（GFP-Bcl-2-MSCs）。在低氧环境下诱导其分化并检测细胞凋亡和增殖。采用流式细胞术检测MSCs和GFP-Bcl-2-MSCs在低氧条件下MSCs细胞细胞周期分布。采用Western blot检测不同组细胞cyclinD1、cyclinE及PCNA表达变化。结果在体外低氧诱导条件下,Bcl-2基因对MSCs有明显的抗凋亡功能。与空载体对照组相比：实验组（GFP-Bcl-2-MSCs）细胞凋亡率下降27%,细胞增殖率升高了58%。但细胞周期分布及cyclinD1、cyclinE、及PCNA的表达无明显差异。结论研究证实了抗凋亡基因修饰的MSCs（GFP-Bcl-2-MSCs）在低氧环境下可抑制细胞凋亡,但细胞周期的相关机制需要进一步研究。     

绿色荧光蛋白基因逆转录病毒载体转染人骨髓间充质干细胞及表达的研究

目的　绿色荧光蛋白 (EGFP)基因的逆转录病毒载体转染人骨髓间充质干细胞(MSCs)及其表达情况。方法　pEGFP与逆转录病毒载体经过酶切、连接等构建重组的EGFP 逆转录病毒载体 ,转染PA3 17细胞 ,用G418筛选出抗性克隆 ,获病毒上清 (滴度达到 8.5× 10 5cfu/ml)并感染人MSCs。流式细胞仪测定转染效率后加入成骨细胞分化培养夜 (地塞米松 (10 -7mol/L)、β 甘油磷酸钠 (10mmol/L)、维生素C(5 0mg/L ) ) ,2周后观察转染细胞的分化情况。 结果　 48h后细胞转染效率为 7% ,G418筛选 2周后约 97%细胞出现抗性克隆 ,表达维持 4周。基因转染细胞能够分化为成骨细胞。结论　重组EGFP逆转录病毒载体能转染MSCs ,且不影响细胞的功能。     

骨形态发生蛋白-2和转化生长因子-β1双基因共表达对骨髓基质细胞向软骨细胞分化基因表达的影响

目的 观察骨形态发生蛋白-2(BMP-2)和转化生长因子-β1 (TGF-β1)双基因真核共表达载体对兔骨髓基质细胞(MSCs)向软骨细胞分化mRNA表达的影响。方法 pIRES-BMP-2-TGF-β1、pIREES-BMP-2 and pIRES-TGF-β1质粒通过质脂体介导转染MSCs,采用荧光定量逆转录-聚合酶链反应(RT-PCR)方法检测MSCs内Ⅱ型胶原和蛋白多糖mRNA的表达。结果 转染后2、4d双基因组MSCs内的Ⅱ型胶原和蛋白多糖mRNA表达量60.4±10.1、606.0±88.5和42.6±6.0、411.2±73.6均明显高于转染后单一BMP-2(28.7±4.0、236.7±48.5和26.9±4.3、208.2±36.7)及TGF-β1(30.9±4.54、205.5±38.7和28.4±3.7、184.9±30.9)组(P＜0.05)。结论 pIRES-BMP-2-TGF-β1双基因真核共表达载体诱导MSCs向软骨细胞分化的作用强于单一基因BMP-2及TGF-β1。     

多途径将骨髓间充质干细胞制备成类β细胞的体外实验研究

目的 通过诱导法和转基因法将小鼠骨髓间充质干细胞(BMSCs)制备成类β细胞.方法 在体外分离、培养小鼠BMSCs,运用细胞因子诱导剂诱导和共培养诱导,将BMSCs 诱导分化为类β细胞,通过逆转录病毒载体将外源性胰岛素基因转入小鼠BMSCs,使其表达外源性胰岛素基因;获得分泌胰岛素的类β细胞;通过腺病毒载体将PDX-1...     

腺病毒介导入BMP-2对骨髓间质干细胞成骨能力的影响

目的　探讨腺病毒介导的人骨形态发生蛋白 (BMP 2 )转染对体外培养骨髓间质干细胞(MSCs)成骨能力的影响。　方法　取日本大耳白兔 2 0只自双侧股骨大转子取材培养MSCs ,左侧来源细胞为实验组 ,右侧为对照组。以复制缺陷重组腺病毒介导人BMP 2基因转染MSCs后 ,用ALP检测两组细胞的ALP活性 ;骨钙素RT PCR检测两组细胞Ⅰ型胶原的表达 ;BGP放免分析检测两组细胞的骨钙素含量。　结果　转基因组和对照组ALP分泌量 (U/L)分别为 7 0 1± 0 5 9、5 2 3± 0 5 5 ;RT PCRⅠ型胶原的表达为 1 3 5± 0 12、3 65± 0 3 7;骨钙素 (ng/ml)为 2 4 5 0± 0 93和 15 45± 1 81。两组间差异均有显著性 (P <0 0 5 )。　结论　用腺病毒介导人BMP 2蛋白作用后的MSCs具有成骨细胞的生物学特性。腺病毒介导人BMP 2转基因可以提高MSCs的体外成骨能力。     

小鼠CXCR4腺病毒的构建及其转染对MSCs向受损肝脏归巢的影响

目的:观察小鼠CXCR4腺病毒(Ad-mCXCR4)转染对骨髓间充质干细胞(MSCs)受损肝脏归巢的影响。方法:分离小鼠MSCs并体外扩增、鉴定;从小鼠肝脏组织中获得CXCR4目的基因,用同源重组法构建腺病毒载体Ad-mCXCR4并鉴定;用Ad-mCXCR4转染MSCs,以转染空载体Ad-vector和未转染的MSCs为对照,然后用Western blot法检测各组细胞CXCR4蛋白的表达;小鼠注射CCl4诱导肝损伤模型后随机分为3组,分别通过尾静脉注射转染Ad-mCXCR4,Ad-vector和未转染的MSCs,48 h后用激光共聚焦观察各组MSCs归巢到受损肝组织的情况。结果:成功构建小鼠CXCR4腺病毒载体Ad-mCXCR4;MSCs转染Ad-mCXCR4后CXCR4蛋白呈高表达,而转染Ad-vector和未转染的MSCs无CXCR4蛋白表达;注射未转染MSCs组小鼠肝脏未见绿色荧光蛋白阳性细胞,注射Ad-mCXCR4-MSCs组小鼠较注射转染Ad-vector-MSCs组小鼠肝脏绿色荧光蛋白阳性细胞明显增加[(21.25±1.56)vs.(5.42±0.81)](P<0.01)。结论:基因修饰可提高MSCs的CXCR4表达,高表达CXCR4的MSCs向受损肝脏归巢增加。     

重组pcDNA3.1-hBMP-2转染人骨髓基质干细胞和对其增殖及血管内皮生长因子表达的影响

目的构建人骨形成蛋白-2(hBMP-2)真核表达载体pcDNA3.1-hBMP-2,转染人骨髓基质干细胞(MSCs),探讨基因转染对其增殖和血管内皮生长因子(VEGF)表达的影响. 方法利用重组DNA和基因克隆技术构建重组载体pcDNA3.1-hBMP-2;细胞培养和基因转染技术体外转染人MSCs;免疫细胞化学、原位杂交和蛋白印迹法检测细胞BMP-2的表达;通过流式细胞仪和VEGF探针原位杂交分析其对细胞增殖和VEGF表达的影响. 结果转染后细胞在mRNA水平和蛋白质水平均表达BMP-2;转染后S期细胞比例增多,提示细胞DNA的合成增加;BMP-2基因转染上调细胞VEGF的表达. 结论在脂质体介导下,pcDNA3.1-hBMP-2转染MSCs获得成功.基因转染后能促进细胞增殖并将通过使VEGF的表达增加促进血管再生,为进一步骨缺损的基因治疗及构建组织工程骨奠定了实验基础.     

SDF-1/CXCR4轴在骨髓间充质干细胞移植促进胰岛再生中的作用

目的 观察同种异体骨髓间充质干细胞(MSCs)移植入受体后,基质细胞衍生因子(SDF)-1/CXCR4轴在促进残存胰岛及其周围新生血管增殖中的作用.方法 对大鼠MSCs进行体外培养、鉴定.链脲佐菌素(STZ)诱导的糖尿病大鼠随机分为A组(MSCs移植组)、B组(MSCs移植+SDF-I/CXCR4轴阻断剂AMD组)和C组(糖尿病对照组),另设D组(正常大鼠对照组).移植MSCs后第30天取出各组大鼠胰腺和血清,胰腺组织采用苏木素-伊红(HE)染色和免疫组织化学法观察CD31、增殖细胞核抗原(PCNA)、胰腺干细胞标志物(PDX)-1在胰腺组织的表达水平.血糖仪检测血糖水平、放免法检测胰岛素水平、酶联免疫吸附试验(ELISA)检测SDF-1水平.结果 (1)A组残存胰岛周围可见新生血管,CD31、PCNA、PDX-1染色阳性率分别为(71.2±5.3)%、(76.5±4.5)%、(69.8±6.7)%;B组残存胰岛周围基本未见新生血管,CD31、PCNA、PDX-1染色阳性率分别为(7.4±2.1)%、(5.5±3.7)%、(8.8±2.9)%,两组比较差异有统计学意义(P＜0.05).(2)移植后第25天,A组血糖浓度基本正常,低于B组和C组,而胰岛素水平明显高于B组和C组(P＜0.05).(3)A组与B组血清SDF-1水平差异无统计学意义(P＞0.05),但都明显高于C组(P＜0.05).结论 MSCs促进胰岛再生和新生血管形成,AMD3100能抑制MSCs的作用,进而提示SDF-1/CXCR4轴在胰岛再生和血管形成中具有重要作用.

Abstract：

Objective To investigate the role of stromal cell derived factor-1 (SDF-1)/CXCR4axis in recipients' remnant islets regeneration and neovascularization after the transplantation of allogeneic bone marrow mesenchymal stem cells (MSCs). Methods MSCs were isolated from SD rats, cultured in vitro and identified by testing the phenotypes with flow cytometry ( FCM ). The diabetic rats induced by streptozotozin were randomly divided into group A ( MSCs transplant group), group B ( MSCs transplant +AMD group) and group C ( DM control group). Group D serve as the normal control. The pancreata were removed and blood serum was retrieved from each group simultaneously at the 13th day after MSCs transplant. The expression of CD31, proliferating cell nuclear antigen (PCNA) and PDX-1 in each group of pancreas tissue was detected by using immunohistochemistry, and the morphological changes in the isletswere observed by Hematoxylin and Eosin (HE) staining. Serum glucose and insulin levels were determined by blood glucose monitor, radioimmunoscintigraphy, and SDF-1 in serum was by enzyme linked immunosorbent assay (ELISA). Results Neovascularization was observed in the remnant islets of the recipient pancreatic tissue and CD31 -positive cells (71.2 ± 5.3 ) %, PCNA-positive cells ( 76. 5 ± 4. 5 ) %, PDX-1-positive cells (69. 8 ±6. 7)% were highly expressed in group A. As compared with group A, seldom-positive cells[CD31 (7.4±2. 1)%, PCNA (5.5 ±3.7)% and PDX-1 (8.8 ±2.9)%]and rarely neovascularization were observed in group B (P ＜0. 05 ). Serum glucose level in group A was lower than that in group B and group C, but serum insulin level in group A was significantly higher than that in group B and group C (P ＜ 0. 05 ). There was no significant difference between group A and group B in serum SDF-1level ( P ＞ 0. 05 ), but that was higher in groups A and B than in group C ( P ＜ 0. 05 ). Conclusion Obviously, MSCs promote recipient neovascularization surrounding the islets, which enhances the proliferation and regeneration of remnant islets. AMD 3100 has the function of intervening SDF-1/CXCR4 axis,which inhibits the effect of MSCs on promoting islets regeneration. It is suggested that SDF-1/CXCR4 axis may play an important role in vascularization and islets regeneration.     

大鼠胰腺次全切除后导管细胞同源盒基因-1表达规律研究

目的 研究大鼠胰腺大部分切除术后,残余胰腺导管上皮细胞转录因子胰-十二指肠同源盒基因(PDX)-1表达规律,探讨PDX-1表达的意义。方法手术切除大鼠胰腺约90％组织,不同时间点处死动物后取材,解剖显微镜下游离胰腺导管,逆转录-聚合酶链反应(RT-PCR)与Western blot检测导管上皮中PDX-1 mRNA和蛋白表达。结果实验组手术后第2天与第3天导管上皮细胞PDX-1蛋白表达显著升高,高于对照组两倍以上,与对照组比较差异有显著性(P<0.05),第5天到第7天逐渐恢复到对照组水平;实验组在各时间点PDX-1 mRNA表达水平与对照组无明显差异(P>0.05)。结论残余胰腺再生过程中导管上皮细胞中出现PDX-1高表达,表明胰腺干细胞参与残余胰腺再生,且PDX-1表达受转录后调控。     

腺病毒介导的NT-3基因在骨髓间质干细胞中的表达

目的:研究腺病毒介导的NT-3基因在培养的大鼠骨髓间质干细胞(mesenchymal stem cells,MSCs)中的表达.方法:在293细胞中培养扩增NT-3重组腺病毒(adenovirus vector for NT-3,Ad-NT-3),测定病毒滴度,然后用Ad-NT-3感染传代培养的MSCs,RT-PCR技术检测NT-3基因的表达.结果:Ad-NT-3扩增后获得了较高滴度的病毒,MSCs经Ad-NT-3感染后有NT-3 mRNA的转录.结论:腺病毒介导的NT-3基因可转入培养的MSCs并高效表达,为NT-3基因治疗的研究奠定了基础.     

Increased PDX-1 expression is associated with outcome in patients with pancreatic cancer

BACKGROUND: During pancreatic development, pancreatic duodenal homeobox gene-1 (PDX-1) is expressed in pancreatic duct cells that have the potential to differentiate into islets. Therefore, PDX-1 is thought to be a marker of de-differentiated cells with the capacity to redifferentiate into several pancreatic cell types. We analyzed PDX-1 expression in human pancreatic cancer specimens, pancreatic cancer cell lines, and the effects of forced expression of PDX-1 in pancreatic cancer cells. METHODS: Thirty-five pancreatic adenocarcinomas were immunohistochemically stained with a polyclonal rabbit antibody against mouse PDX-1. Correlations with tumor characteristics were made with chi-squared analysis. The influence of clinicopathologic factors on survival was assessed. The expression of PDX-1 in pancreatic cancer cells was examined. Replication-deficient recombinant adenoviruses were constructed by the cosmid-adenoviral DNA terminal protein complex method. PANC-1 cells were infected with Ad-pdx-1 or Ad-LacZ. PANC-1 cells that were infected with adenovirus were used in a cell growth assay and a migration assay and for morphologic analysis. RESULTS: Interestingly, 43% of pancreatic cancers were positive for PDX-1 expression, and 57% of pancreatic cancers were negative (normal pancreatic exocrine tissue shows little or no staining for PDX-1). Lymph node metastasis (P =.02) and histologic grade (P =.04) were correlated significantly with PDX-1 expression. Patients with positive PDX-1 had a significantly worse prognosis than those patients with negative PDX-1 (P =.02). Importantly, PDX-1 was an independent variable that effected overall survival (P =.03). Pancreatic cancer cell lines showed no PDX-1 expression. There were no significant differences in cell proliferation or morphologic condition between Ad-pdx-1- and Ad-lacZ-infected PANC-1 cells. However, Ad-pdx-1-infected PANC-1 cells did show a significantly higher migration rate than Ad-lacZ-infected PANC-1 cells. CONCLUSIONS: Re-expression of PDX-1 may represent a return to a more de-differentiated state by more aggressive pancreatic cancers and may also represent an important new tumor marker for these aggressive cancers.     

Enhanced cytotoxicity of RIPTK gene therapy of pancreatic cancer via PDX-1 co-delivery

BACKGROUND: Using in vivo mouse models, we have demonstrated that the insulin promoter-driven suicidal gene therapy (RIPTK) could be used in the treatment of mouse insulinoma and human pancreatic cancer cells. However, limitations of this therapy include tumor cells lack of sufficient PDX-1 protein and low levels of transgene expression mediated by liposome delivery system. The purpose of this study was to determine 1) whether transient transfection of PDX-1 into selected pancreatic cancer cells would lead to increased RIPTK cytotoxicity, and 2) whether an adenoviral delivery system would increase the overall RIPTK gene expression in vitro. MATERIAL AND METHODS: RIPlacZ and RSVlacZ plasmid DNA as well as AdCMVlacZ and AdRIPlacZ were used in transfection assays in human pancreatic cancer cell lines PANC-1 and MIA PaCa2 (n = 8). An expression plasmid DNA containing the mouse PDX-1 cDNA was also used. LacZ reporter assays were performed. RIPTK genes constructed either in plasmid or in adenoviral vectors were used in cytotoxic assays. RT-PCR assays were used to determine PDX-1 expression levels. RESULTS: PDX-1 protein was detected in the human pancreatic ductal carcinoma cell line PANC-1, a little in MIA PaCa2 cells. Liposome mediated (L) RSVlacZ and RIPlacZ transfection in PANC-1 cells resulted in 10.1% and 9.3% transgene expression, respectively. Co-delivery of PDX-1 had no significant effect on RSVlacZ expression (9.3%, P = NS) but significantly increased RIPlacZ gene expression (14.9% P < 0.05). Adenoviral mediated (Ad) RIPlacZ transgene was highly expressed in PANC-1 cells (66.1%) and the reporter activity was further enhanced when PDX-1 was co-delivered (70.2%, P < 0.05). Liposomal transfection of MIA PaCa2 cells using RSVlacZ and RIPlacZ reporter genes resulted in 9.3% and 1.0% gene expression, respectively. Co-transfection of PDX-1 in these cells resulted in a significant activation of RIPlacZ gene expression (14.5%, P < 0.05) with no effects on RSVlacZ treated cells (9.8%). AdCMVlacZ and AdRIPlacZ significantly increased reporter activities in MIA PaCa2 cells (63.0% and 9.8%, respectively). Transfection of PDX-1 also significantly enhanced the AdRIPlacZ activities (46.0%, P < 0.05), with no significant effect in AdCMVlacZ treated cells (68.2%). The cytotoxic effect of liposome-RIPTK/ganciclovir (GCV) in PANC-1 cells was 18.6% and increased to 22.8% when PDX-1 was co-transfected into the cells (P = NS). MIA PaCa2 cells treated with RIPTK alone resulted in 4.9% cell death and increased to 18.2% when exogenous PDX-1 was co-delivered (P < 0.05). The AdRIPTK gene delivery with GCV treatment caused significant cytotoxic effect in PANC-1 (29.3%) and MIA PaCa2 (12.4%) compared with untreated cells. The cytotoxic effects were further increased to 43.4% and 29.4% in PANC-1 and MIA PaCa2 cells, respectively, when PDX-1 was co-transfected (P < 0.05 for both). CONCLUSIONS: These data demonstrated that adenoviral mediated gene delivery resulted in a significant increase of transgene expression compared with liposomal delivery systems. RIPTK mediated cytotoxicity was also significantly enhanced via co-delivery of exogenous PDX-1 in these cells. Thus, these results also indicated that PDX-1 plays critical roles in insulin promoter activation and demonstrated that PDX-1 production is essential for insulin promoter-directed gene therapy.     

人骨形成蛋白7基因重组2型腺相关病毒载体的构建及其在骨髓间充质干细胞中的表达

目的构建人骨形成蛋白7基因（hBMP7）重组腺相关病毒载体,并观察其在兔骨髓间充质干细胞中的表达。方法将骨形成蛋白7基因片段克隆入穿梭质粒pUC18获得重组质粒pUC18-hBMP7。KpnⅠ和鼠Ⅱ双酶切质粒pUC18-hBMP7／与pSNAV,用T4DNA连接酶连接分别回收的两片段后转化大肠杆菌DH5α感受态细胞,获得重组质粒PSNAV—hBMP7／,转染BHK-21细胞,筛选培养,用能表达Rap和Cap的重组Ⅰ型单纯疱疹病毒HSVl-rc／△UL2感染此细胞,裂解细胞收获病毒液。采用氯仿处理-PEG／NaCl沉淀-氯仿抽提法分离浓缩、纯化与测定病毒滴度。用rAAV2-hBMP7／和rAAV2-EGFP在体外分别转染兔骨髓间充质干细胞。流式细胞仪、RT-PCR和Western—blot方法检测兔骨髓间充质干细胞中hBMP-7基因的转录和表达。结果成功构建具有感染活性的重组腺相关病毒载体rAAV2-hBMP7／,病毒载体对兔骨髓间充质干细胞早期转染效率可达99．8％,hBMP7／在兔骨髓间充质干细胞中可得到转录和表达。结论成功构建了人骨形成蛋白7基因重组腺相关病毒载体,rAAV2-hBMP7／载体在体外可转染兔骨髓间充质干细胞,并获得较高的转染效率。     

骨髓间充质干细胞分化为血管内皮细胞的实验研究

目的：探讨骨髓间充质干细胞（MSCs)分化为肉芽组织中血管内皮细胞的可能性。及其参与创面修复的可能机制。方法：抽取小型香猪的骨髓，经密度梯度离心分离、纯化MSCs，体外培养扩增后，应用溴脱氧尿嘧啶（BrdU)标记技术标记细胞。于已抽取骨髓的小型香猪背部制作全层皮肤缺损创面，即刻以纤维蛋白胶为载体，将已标记的MSC回植到供体动物创面上。术后2、4、6、8、12周切取创面组织，行BrdU和因子Ⅷ（FⅧ）免疫组织化学染色，进行对比观察。结果：BrdU阳性的MSCs多聚集在创面肉芽组织中的小血管周围，且有个别血管内皮细胞也呈现BrdU 阳性。部分BrdU阳性的MSCs胞浆中亦有FⅧ表达。结论：创面愈合过程中，MSCs与肉芽组织中小血管的形成密切相关。在创面微环境下，MSCs可分化为血管内皮细胞，并参与创面修复。