【作者】 杨瑞年；

【导师】 刘流；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 博士

【摘要】 [研究背景及目的]由各类先天或后天原因造成的骨缺损是临床上的常见疾病之一,而此类骨缺损的修复一直是较难解决的问题。运用组织工程技术体外成骨后移植是这类骨缺损除手术外新的修复方法。骨髓间充质干细胞(bone marrow Mesenchymal stem cells, BMSCs)作为体内一种较易获得的干细胞,其具有分化为多种不同类型细胞的潜能。BMSCs在适当诱导条件下能够分化为骨细胞、软骨细胞、脂肪细胞等各类相关细胞。目前关于体外培养单一种类的骨髓间充质干细胞并诱导其成骨分化的研究取得了很多进展,但仍然存在细胞增殖慢、成骨效率低、成骨周期长等缺点。目前有研究发现血管内皮细胞(vein endothelial cells, VECs)在与BMSCs共培养可以通过骨形态发生蛋白(Bone morphogenetic protein, BMP),促进成骨分化的同时刺激成骨细胞及成骨前细胞释放血管内皮生长因子(vascular endothelial growth factor, VEGF),而VEGF在促进内皮细胞的增殖的同时,也在血管发生和形成过程中发挥着重要作用。但目前从基因水平研究血管内皮细胞对骨髓间充质干细胞成骨分化作用仍未见报道。本研究采用RNAi技术原理静默hUVECs的BMP-2基因,分别采用正常hUVECs和BMP-2基因静默的hUVECs与hBMSCs构建联合培养体系,利用荧光定量PCR技术定量检测培养体系中各组hUVECs对hBMSCs的Bmi-1和Runx2基因的影响；验证hUVECs对hBMSCs内增殖和成骨诱导促进作用；探讨hUVECs的BMP-2因子对hBMSCs的Bmi-1和Runx2基因的影响,明确BMP-2是否是hUVECs调控hBMSCs的Bmi-1和Runx2基因表达的主要因素。本研究为hUVECs共培养hBMSCs在骨组织工程中的种子细胞体外培养分化提供理论依据,并期望研究成果能够探索出新的方法从而解决组织工程种子细胞在支架材料上增殖、黏附和成骨分化的难题。[方法](1)抽取志愿者骨髓液,使用密度梯度离心法分离骨髓单个核细胞,并借助hBMSCs(?)壁生长的特性进行纯化,在相差显微镜观察干细胞形态变化。将hBMSCs传代扩增培养至第三代,流式细胞仪检测CD34、CD29、CD44表面抗原表达,鉴定hBMSCs;(2)取hUVECs细胞进行体外培养,并运用免疫组化染色方法鉴定hUVECs细胞,培养至第三代后用Western Blotting蛋白检测方法分别检测第4、6、8、10天BMP-2蛋白的表达情况。(3)设计四组BMP-2基因的shRNA干扰序列,将所设计好的shRNA序列插入质粒载体,运用脂质体转染法将构建的干扰基因序列转染到血管内皮细胞内,并采用荧光显微镜观察质粒转染的效果。Western Blotting检测血管内皮细胞BMP-2蛋白的表达,鉴定对hUVECs细胞BMP-2的RNA静默的效果。(4)将hUVECs细胞用ECM+10%新生胎牛血清扩增至第三代后与第三代hBMSCs细胞按1：1比例建立以DMEM+10%胎牛血清为培养基的联合培养组。将经RNAi处理的hUVECs细胞和第三代hBMSCs细胞按1：1比例建立以DMEM+10%胎牛血清为培养基的联合培养干扰组。设置单独hBMSCs培养组、单独hUVECs培养组为阴性对照组。分别于第4、6、8、10天相差显微镜观察形态变化,用计数板计数各组hBMSCs数量；用SPSS17.0软件对各项检测值进行统计学分析。(5)分别于第4、6、8、10天每组每个时间点取6孔检测单独hBMSCs组、联合培养组和联合培养干扰组中碱性磷酸酶(Alkaline phosphatase, ALP)及骨钙素(Osteocalin, OC)含量。用SPSS17.0软件对各项检测值进行统计学分析。(6)分别于第4、6、8、10天每组每个时间点取6孔应用Western Blotting方法检测单独hUVECs组、联合培养组和联合培养干扰组中hUVECs的BMP-2蛋白表达情况。(7)采用荧光定量PCR法(fluorescence quantitative PCR, FQ-PCR)检测第4、6、8、10天单独hBMSCs组、联合培养组和联合培养干扰组中hBMSCs的Bmi-1和Runx2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)采用密度梯度离心法结合贴壁培养法分离、提纯hBMSCs,所获得细胞的纯度较高,达到实验要求。用流式细胞仪对提取纯化的第3代hBMSCs进行细胞表型分析鉴定,CD34低表达,CD29、CD44高表达,符合hBMSCs的表型特征。(2)对所培养hUVECs行免疫组化染色鉴定,符合该细胞表型,Western Blotting检测结果显示hUVECs能正常表达BMP-2蛋白。(3)2号质粒转染后的细胞BMP-2蛋白表达量明显降低,本研究设计合成的shRNA干扰质粒对hUVECs细胞BMP-2蛋白表达的静默是有效的。质粒3ug、脂质体10ul,转染6小时是最佳转染条件,转染效率约为60%。(4)培养体系中hBMSCs的形态变化结果显示各时间点联合培养组hBMSCs呈现出一定的成骨分化表现,联合培养干扰组hBMSCs成骨分化的形态表现弱于联合培养组,单独hBMSCs组未见成骨分化表现。(5)各组别细胞数目随时间延长先增高后降低,各时间点联合培养组细胞数目最高,联合培养干扰组次之,单独hBMSCs最低。各组之间两两比较,差异均有显著统计学意义(P<0.01)。(6)Western Blotting检测结果显示联合培养组hUVECs对BMP-2的表达较单独hUVECs组增高。联合培养干扰组对BMP-2的表达显著降低,达到实验对RNA干扰的要求。(7)联合培养组和联合培养干扰组内碱性磷酸酶检测量随时间延长先增高后降低,单独hBMSCs组的碱性磷酸酶含量在各时间点变化较小。各时间点联合培养组ALP最高,联合培养干扰组次之,单独hBMSCs最低。各组之间两两比较,差异均有显著统计学意义(P<0.01)。(8)联合培养组和联合培养干扰组内骨钙素检测量随时间延长先增高后降低；单独hBMSCs组的骨钙素含量在各时间点变化较小；各时间点联合培养组骨钙素检测量最高,联合培养干扰组次之,单独hBMSCs组最低。各组之间两两比较,差异有显著统计学意义(P<0.01)。(9)干细胞Bmi-1基因表达量在联合培养组和联合培养干扰组内按时间增长逐渐上升；单独hBMSCs组的Bmi-1基因表达量在各时间点变化较小；各时间点联合培养组和联合培养干扰组Bmi-1基因表达较高,单独hBMSCs组最低；相同时间点联合培养组和联合培养干扰组之间比较,差异无统计学意义(P>0.05)；单独hBMSCs组与联合培养组和联合培养干扰组之间比较,差异有显著统计学意义(P<0.01)。(10)联合培养组和联合培养干扰组内Runx2基因表达量随时间延长逐渐增高；单独hBMSCs组的Runx2基因表达量在各时间点变化较小；各时间点联合培养组Runx2基因表达量最高,联合培养干扰组次之,单独hBMSCs组最低；各组之间两两比较,差异均有显著统计学意义(P<0.01)。[结论](1)采用密度梯度离心法分离以及贴壁法纯化的hBMSCs,经流式细胞仪表型鉴定为骨髓来源的干细胞,可在后期联合培养中应用。(2)脐静脉血管内皮细胞能正常表达BMP-2蛋白。实验中构建的质粒序列正确有效,能够达到实验对BMP-2的RNA干扰要求。(3)联合培养体系中骨髓间充质干细胞和脐静脉血管内皮细胞有相互促进增殖的作用。两种细胞联合培养相容性好,未见抑制现象。(4)联合培养体系中骨髓间充质干细胞分泌碱性磷酸酶和骨钙素增加,向成骨细胞方向分化速度显著加快。(5)血管内皮细胞能提高联合培养体系中骨髓间充质干细胞的Bmi-1表达,这与骨髓间充质干细胞的增殖密切相关。(6)联合培养体系对骨髓间充质干细胞Bmi-1的表达有促进作用,但此信号通路上游的调控并不是通过BMP-2而完成。(7)骨髓间充质干细胞在联合血管内皮细胞培养时成骨分化速度加快,这与其Runx2基因表达增加重要相关。(8)骨髓间充质干细胞Runx2基因的表达与血管内皮细胞分泌的BMP-2密切相关,这证明在联合培养体系中存在BMP-2/Runx2通路。但BMP-2不是联合培养体系中Runx2通路上游唯一的影响因子。更多还原

【Abstract】 [Objective and background] Bone defect which were caused by various types of congenital or acquired reasons is a common disease in plastic surgery. The rehabilitation of maxillary defects has been a difficult problem in clinical. The current and new treatment of this disease is constructing tissue engineered bone in vitro.Bone marrow Mesenchymal stem cells have the potential of multiline differentiation and are the early development of the mesoderm cells. They can not only differentiate into mesoderm from the same Mesenchymal cells, but also break mesoderm boundaries, differentiate into mesodermal tissue, such as fat cells, bone cells, cartilage cells, cardiac cells, nerve cells, muscle cells, tendon cells and astrocytes, etc. Currently, we have make great progress in the study that induce pure BMSCs to osteoblastic differentiation.However, this way exist some problems, such as a long cycle into bone, low efficiency to formation bone, cells easy to aging and other shortcomings.Researchers found VECs have the ability of secreting bone morphogenetic protein, stimulating osteoblasts and their precursor cells to secrete vascular endothelial growth factor when it promotes osteoblast differentiation, and the vascular endothelial growth factor play a very important role in the process of angiogenesis and the formation of vascular. It can promote endothelial cells proliferation and angiogenesis. These studies have shown that endothelial cells can support bone marrow Mesenchymal stem cells to change into bone. However, there is still lack of research from the level of genes in vascular endothelial cells of bone marrow mesenchymal stem cells into osteoblasts differentiation with specific gene signaling mechanism.Our research silented hUVECs’BMP-2genes expression with RNAi technology.We used the normal hUVECs、hUVECs with BMP-2gene silented and hBMSCs constructing co-cultue system，Then we analysis influence of each part in training system hUVECs hBMSCs Bmi-1and Runx2genes expression with fluorescence quantitative PCR technology;we confirmed the influence of the hUVECs to hBMSCs in proliferation and bone induction mechanism promotion; discusseed the hUVECs’ BMP-2factors on hBMSCs Bmi-1and Runx2genes expression;clear BMP-2is hUVECs regulation hBMSCs Bmi-1and the main factors Runx2gene expression; provide the data and the theoretical basis for For hVECs and hBMSCs co-culture system ueing in bone tissue engineering seed cells research. This study can provide the data and theoretical basis of aplling hBMSCs and hUVECs to the joint co-culture system in bone tissue engineering seed cells research.[Method]1We extracted 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. The hUVECs which were ordered were cultured in vitro, were identified by immunohistochemical staining method. Cells were cultured to the third generation,then the expression of BMP-2protein was verifid in4th、6th、8th、10th day by western blot method.3. We design four BMP-2gene interfering sequences and decorate it into plasmid Using the method of optimized liposome,we transfect cells with preconstructed interference gene sequence.Then we observed the effect of transfection by fluorescence microscope, detected the expression of BMP-2protein by western blot method, and identied RNA silent effect of hUVECs cells’BMP-2.4. The co-culture of the third generation hMSCs and HUVECs was established in the rate of1:1, and DMEM with10percent FBS were used as the medium of the co-culture system. The co-culture of the third generation hMSCs and HUVECs with RNAi treatment was established in the rate of1:1, and DMEM with10percent FBS were used as the medium of the co-culture RNAi system. 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 make a statistical analysis about the test value with software SPSS17.0.5. Alkaline phosphatase and osteocalin were detected in hBMSCs group, co-culture group and co-culture rnai group at4th、6th、8th、10th day. And we make a statistical analysis about the test value with software SPSS17.0.6.The expression of Bmp-2were detected in hUVECs group, co-culture group and co-culture rnai group at4th、6th、8th、10th day by western blot method.4The expression of Bmil and Runx2gene were detected in hBMSCs group, co-culture group and co-culture rnai group at4th、6th、8th、10th day by fluorescence quantitative PCR method. And we make a statistical analysis about the test value with software SPSS17.0.[Result] l.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 hBMSCs,which are cultured with FBS, are elongated spindle and small. The Primary generation cells grow into groups at4to5days. The third generation of bone marrow Mesenchymal stem cells forms a single, and into the spindle, vortex-like distribution, there is no cell overlap. The hBMSCs grow in logarithmic at4-6days, and into platform at8-10days. HUVECs grow as monolayer, polygonal shape, cobblestone-like arrangement. The hBMSCs have clear boundary and rich cell slurry,nuclei were round or oval. Occasionally.they show dual-core and confluent at5th. From first generation to fourth generation, the hBMSCs grow faster,2-3days can be passage.3. The target cells is affirmed as hUVECs by Immunohistochemical staining.Protein test shows they can normally express BMP-2protein.4.The cells BMP-2protein expression is decreased obviously after no.2plasmid transfected. The design of the shRNA interference plasmid is effective to silence hUVECs cells BMP-2protein expression. The best tranfection conditions is3ug Plasmid, l0ul liposomes and tranfect6hours, the efficiency is about60%5.The form change results of hBMSCs in Culture system are shown that the hBMSCs in co-culture group has a certain osteoblast differentiation performance at Each time point, The co-culture rnai group showed weaker osteoblast differentiation performance,there was not a obviously osteoblast differentiation performance in hBMSCs group.6.The number of hBMSCs in each group gradually increased with time, and the co-culture group’s cell number was the highest at all time; the co-culture rnai group was lower than co-culture group;the hBMSCs group was lowest.The comparisons between all groups were statistically significant(P<0.01).7. Western-blot test shows that Bmp-2expression of hUVECs in the co-culture group was higher than its’in the hUVECs group.8. The amount of alkaline phosphatase in co-culture group and co-culture RNAi group gradually increased with time, The amount of alkaline phosphatase in hBMSCs group had a small changes at each time point。 The co-culture group’s ALP was the highest at each time point。; the co-culture rnai group was lower than co-culture group;the hBMSCs group was lowest.The comparisons between all groups were statistically significant(P<0.01).9.The amount of osteocalcin in co-culture group and co-culture RNAi group gradually increased with time, The amount of alkaline phosphatase in hBMSCs group had a small changes at each time point。 The co-culture group’s osteocalcin was the highest at each time point。; the co-culture rnai group was lower than co-culture group;the hBMSCs group was lowest.The comparisons between all groups were statistically significant(P<0.01).10.The expression of Bmi-1gene in co-culture group and co-culture RNAi group gradually increased with time, The expression of Bmi-1gene in hBMSCs group had a small changes at each time point。 The expression of Bmi-1gene in co-culture group was the highest at each time point。; the co-culture rnai group was lower than co-culture group; the hBMSCs group was lowest.The comparisons between the hBMSCs group and other groups were statistically significant(P<0.01), but The comparison between the co-culture group and co-culture RNAi group was not statistically significant(P>0.05).11.The expression of Runx-2gene in co-culture group and co-culture RNAi group gradually increased with time, The expression of Runx-2gene in hBMSCs group had a small changes at each time point。The expression of Runx-2gene in co-culture group was the highest at each time point。; the co-culture rnai group was lower than co-culture group; the hBMSCs group was lowest.The comparisons between the all groups and other groups were statistically significant(P<0.01).[Conclusion]1.We can get higher purity hBMSCs cultured with FBS by the way of Ficoll density gradient centrifugation, which is used to isolate and purify hBMSCs.2.hUVECs can normally express BMP-2protein. Four plasmids constructed are all correct and No.2have the best effect.The result is up to standard of RNA interference requirements.3.1t shows good compatibility about the co-culture of hBMSCs and hUVECs. hUVECs can promote hBMSCs multiplication.4. Alkaline phosphatase and osteocalcin which were secreted by hBMSCs increased in the co-culture system and hBMSCs’s Osteogenic Differentiation was speed up.5.hUVECs can enhance Bmi-1expression of hBMSCs in co-culture system, this is closely related to hBMSCs multiplication.6. Bmi-1gene expression of hBMSCs increased in the co-culture system, but this Signaling pathways is not regulated by Bmp2.7.hUVECs can enhance Bmi-1expression of hBMSCs in co-culture system, this is closely related to hBMSCs’s Osteogenic Differentiation.8.hUVECs can enhance Runx2expression of hBMSCs in co-culture system by Bmp2,this improve that Bmp2/Runx2Signaling pathways exist in co-culture system.but Bmp2is not only one factor in Runx2pathways.更多还原