【作者】 陈苏丽；

【导师】 刘流；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 硕士

【摘要】 [研究背景及目的]研究背景：由严重创伤、广泛感染、坏死及骨肿瘤切除术所造成骨缺损的修复治疗一直是骨科临床应用中的难题。为解决体内成骨缓慢这一难题,许多学者发现用内皮细胞(human umbilical vein endothelial cells, hUVECs)与间充质干细胞(human bone marrow Mesenchymal stem cells, hBMSCs)联合培养的方法可促进或加速干细胞向成骨性方向的增殖与分化,继而加速骨质愈合。近年来,研究人员进一步发现内皮细胞可以分泌骨形态发生蛋白(Bone morphogenetic protein, BMP),而BMP是公认的可促进间充质干细胞成骨性分化从而加速骨质愈合的骨诱导因子。是否内皮细胞与间充质干细胞联合培养促进骨质愈合是由于内皮细胞可分泌产生BMP的主导作用呢?由于RNA干扰技术近年来因其独特的优点成为基因研究领域广泛应用的一种方法,我们根据siRNA高效特异抑制目的基因表达的特点,通过设计、构建针对BMP-2基因的短发夹状RNA转染hUVECs。通过RNAi静默原理观察联合培养体系中siRNA抑制BMP-2基因mRNA的转录和蛋白表达的情况。进而证实联合培养体系中hUVECs所表达的BMP-2对于hMSCs句成骨性细胞方向的增殖分化是具有主要作用的。从基因角度为组织工程和再生医学提供全新的理论知识和最佳的治疗方法,为探索研制具有高效成骨作用的组织工程人工骨提供一条全新的技术方案。目的：(1)体外分离人人骨髓间充质干细胞(hBMSCs)和人脐静脉内皮细胞(hUVECs),建立两种细胞单独及联合培养模型。(2)在广州复能基因公司的指导下,网上在线设计出针对骨形态发生蛋白(BMP-2)的siRNA序列,获得短发夹状RNA,并进一步转染hBMSCs。(3)将BMP-2蛋白特异性的shRNA转染hUVECs,检测BMP-2基因在mRNA和蛋白质水平的表达情况。(4)在利用RNAi技术抑制hUVECs上BMP-2基因表达后与hBMSCs联合培养,观察hBMSCs向成骨性方向的增殖、分化情况。[方法](1)在志愿者签署知情同意书后,严格无菌操作条件下采集志愿者骨髓液5ml,使用密度梯度离心法分离骨髓单个核细胞,并借助MSCs黏附于塑料瓶底这一特性进行纯化,相差显微镜观察形态变化。选取生长良好的第3代细胞用于进一步鉴定及联合培养,用流式细胞仪检测CD34、CD29和CD44表面抗原表达进行鉴定MSCs。(2)选用ATCC的人脐静脉血管内皮细胞系(hUVEC, Sciencell公司, catalog number:8000)作为hVECs的细胞来源,低血清培养基(500ml基础液,血清,生长因子和双抗),置于37℃,5%C02饱和湿度培养箱培养至第三代,观察各细胞形态结构。分别选用第三代人骨髓间充质干细胞和第三代人脐静脉内皮细胞以及第三代人骨髓间充质干细胞和第一部分制备的干扰后的人脐静脉内皮细胞,23块6孔板均置入插入式培养皿,建立培养液及细胞因子相互连通的联合培养装置。(3)在NCBI (http://www.ncbi.nlm.nih.gov/)的Nucleotide库中检索人骨形态发生蛋白-2(BMP-2)的mRNA序列。Dharmacon siDESIGN Center按照网页要求和提示设计shRNA,而后进行优化、纯化后筛选出其中四条有效的shRNA序列(shRNA1, shRNA2, shRNA3和shRNA4),并分别合成eGFP荧光标记的shRNA质粒载体。将随机打乱的序列作为阴性对照组。优化脂质体法将构建的干扰基因序列转染到细胞中,并采用荧光显微镜观察转染的效果,聚丙烯酰胺凝胶电泳(SDS-PAGE)、Bradford法和western blot法检测BMP-2蛋白的表达情况。(4)实验分三组,Ⅰ组：单独hBMSCs培养组；Ⅱ组：RNAi干扰hUVECs+hBMSCs联合培养组；Ⅲ组：hBMSCs+hUVECs联合培养组。分别于第4、6、8、10天每组每个时间点取6孔检测Ⅰ、Ⅱ和Ⅲ组培养体系中碱性磷酸酶(Alkaline phosphatase, ALP)及骨钙素(Osteocalin, OC)含量。通过westen-blot检测第4、6、8、10天Ⅱ、Ⅲ和Ⅰ组中BMP-2蛋白基因表达的情况,每组每个时间点取6孔。用SPSS17.0软件对各项检测值进行统计学分析。[结果](1)采用Ficoll液密度梯度离心法分离、提纯hBMSCs可达到较高的纯度。用流式细胞仪对第3代hBMSCs进行细胞表型分析鉴定,CD34低表达,CD29、 CD44高表达。(2)质粒的DNA测序结果显示,第2号质粒的碱基序列是更为符合对BMP-2具有抑制作用的shRNA片段。同时经Western Blot技术对提取的BMP-2蛋白进行检测显示以2号质粒shRNA最为有效。(3)转染纯化后的质粒到内皮细胞中,48h后荧光显微镜观察,转染效率达到60%。(4)各组碱性磷酸酶检测量随时间延长先增高后降低,各时间联合培养组ALP较高,8天时联合培养组碱性磷酸酶最高；hBMSCs组ALP基本没有变化；联合培养组和其它各组之间两两比较均有显著统计学意义(P<0.01)；各组骨钙素检测量随时间延长先增高后降低;8天时联合培养组骨钙素最高；联合培养组和其它各组之间两两比较均有显著统计学意义(P<0.01)。(5)对hUVECs中的BMP-2进行干扰后,再同hBMSCs进行联合培养与未进行干扰hUVECs与hBMSCs联合培养组相比较,并通过不同时间点的细胞生长曲线绘制、细胞液中ALP和骨钙素含量的测定结果显示,经过干扰后的细胞联合培养组中MSCs的成骨性分化增殖情况要低于未进行干扰的联合培养组。在第二部分实验中对脐静脉内皮细胞中BMP-2进行干扰后与MSCs的联合培养组仍旧显示出了MSCs的成骨性分化增殖效果要优于单独的细胞培养组。而且细胞液ALP和骨钙素含量的测定同样也是要高于单独的细胞培养组。[结论](1)成功合成并筛选出一条针对人BMP-2蛋白基因的发夹状RNA表达质粒载体,奠定了RNAi技术在实验室进一步拓展的基础。(2)通过体外转录合成的BMP-2蛋白特异性短发夹RNA成功地转染到人hUVECs中,并高效、特异性抑制了BMP-2蛋白基因的表达,达到了基因静默的目的。(3)表明了通过对脐静脉内皮细胞中BMP-2进行RNA干扰后可降低下调或减弱联合培养体系中BMSCs的成骨性增殖分化。同时干扰后也显著降低了联合培养中ALP和骨钙素的含量。(4)内皮细胞中BMP-2进行RNA干扰后在体外可抑制BMSCs向成骨细胞方向的分化。更多还原

【Abstract】 [Study background and Objective]Study background:The repair therapy of bone defect result from trauma, infectation,necrosis and bone neoplasms is always a tough problem of orthopedics. In order to resolve this issue, many scientists find that human bone marrow Mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (hUVECs) co-culture maybe stimulate the proliferation and differentiation of hBMSCs and accelerate the bone healing. In recent years, researchers found vascular endothelial cells(VECs) have the ability of secreting bone morphogenetic protein, wich is the best bone induced factor all of the world. The effect of secreting BMP of hUVECs whether is a key role in hBMSCs and hUVECs co-culture? According to the specific inhibition of BMP-2gene by siRNA, we designed siRNA targeting BMP-2and gain small hairpin loop RNA, and transfected in hUVECs. To demonstrate the role of BMP-2in hUVECs by observate the expression of BMP-2gene in h UVECs after inhibition. So, we can provide a new knowledge and best therapy for tissue engineering and regeneration Medicine.[Objective]:(1) To construct hBMSCs and/or hUVECs co-culture system isolated from the bone marrow of hunman in vitro.(2) According to the guidance of FuNeng company in GuangZhou, we designed siRNA targeting BMP-2and gain small hairpin loop RNA, and transfected in hUVECs.(3) To detect the expression of BMP-2mRNA and protein after transfection by special shRNA on hUVECs. (4) To observe the inhibition effects of shRNA targeting BMP-2on the proliferation, adhesion and migration of cultivation of the hUVECs and hBMSCs co-culture.[Methods](1) We extracted5millilitre of a volunteer’s bone marrow fluid and isolated the bone marrow mononuclear cells by the way of density gradient centrifugation. And we purified the MSCs by its characteristic of adhesion to the plastic bottom. In order to identify the MSCs, We cultured MSCs to passage to the third generation and then we detected CD34, CD29, and CD44’s surface antigen expression by flow cytometry.(2) We were selected hUVECs of ATCC(hUVEC, Sciencell company, catalog number:8000) as the source of hVECs. To observe the morphosis of all kinds of cells in complete medium(500millilitre of basic fluid, blood serum, growth factor and double antigen) at37centigrade after the third generation of passage in5percent saturated humidity incubator. The third generation of passage of hBMSCs, hUVECs and interferential hUVECs were to establish the system of cultivation of the co-culture in the6wells of23plates.(3) BMP-2mRNA sequence of hUVECs was retrieved in nucleotide library of NCBI. BMP-2specific shRNA(shRNAl, shRNA2, shRNA3and shRNA4) was designed and chosed on Dharmacon siDESIGN Center. And further, four kinds of shRNA were synthesized the plasmid vector of marked eGFP. In addition, the gene sequence of random was synthesized to negative control. The gene sequence of interference was transfected in hUVECs by pure method. To observe the transfection effects by fluorescence microscope, to detect the expression of BMP-2by SDS-PAGE, Bradford and Western Blot.(4) The experiment were divided into three groups, I group:Cultivation of hBMSCs; II group:Cultivation of the co-culture after RNAi; IIIgroup:Cultivation of hBMSCs and hUVECs co-culture;. The separate cultured hBMSCs and hUVECs as a negative control group.We observed the morphological changes by phase contrast microscope in4th、6th、8th、10th day, and counted the number of each hBMSCs group by count plate; we detected alkaline phosphatase and osteocalin in three culture groups(I, III and IV group) at4th、6th、8th、10th day. We detected the expression of BMP-2gene in hUVECs groups(II, III and IV group) at4th、6th、8th、10th day. At last, we make a statistical analysis about the test value with software SPSS17.0.[Results](1) We make an analysis and identification on third-generation hMSCs’s cell phenotype By flow cytometry, the expression of CD34is negative and the expression of CD29、CD44are positive; We can get higher purity hBMSCs by the way of Ficoll density gradient centrifugation, which is used to isolate and purify hBMSCs.(2) The results of DNA plasmid sequence were shown that the second plasmid sequence is the best inhibition effects for BMP-2. To measure the expression of BMP-2by Western Blot, the level is similar to the second plasmid.(3) After transfection by pure plasmid on hUVECs at48hours, the transfection efficiency is60precent by fluorescence microscope.(4) The amount of alkaline phosphatase in each group gradually increased with time, and the co-culture groups ALP is the highest at all time; The ALP of Mesenchymal stem cells group and umbilical vein endothelial cells group did not change; The comparisons between co-culture group and other groups were statistically significant(P<0.01). The osteocalcin detected in each group was gradually increased with time. The highest osteocalcin of co-culture group is detected at8th day. The comparisons between co-culture group and other groups were statistically significant(P<0.01).(5) IVgroup comparison of IIIgroup revealed the ALP and osteocalin of hBMSCs is lower by cell growth curve. And the proliferation, adhesion and migration of cultivation of hBMSCs co-culture at IVgroup is better than I group. Moreover, the level of ALP and osteocalin is also higher than I group.[Conclusions](1) One BMP-2specific shRNA plasmid vector was successful synthesized in vitro through MessageMuterTM shRNAi Production Kit. It can supply stable RNAi technique for following experiments.(2) Small hairpin RNA targeting BMP-2of hUVECs efficiently and specifically inhibits the expression of BMP-2.(3) To determine the osteoblast differentiation status of the hBMSCs is down-regulation after RNAi in the co-culture(IVgroup). Meanwhile, IVgroup also reveal low ALP and osteocalin level.(4) The results of study is also suggested that shRNA targeting BMP-2of hUVECs maybe inhibit the osteoblast differentiation status of the hBMSCs in cultivation of the co-culture.更多还原