BMP-2因素对血管内皮细胞共培养干细胞成骨分化以及相关基因表达的影响研究

发布时间：2018-01-17 20:20

  本文关键词：BMP-2因素对血管内皮细胞共培养干细胞成骨分化以及相关基因表达的影响研究　出处：《昆明医科大学》2012年博士论文　论文类型：学位论文

  更多相关文章： 血管内皮细胞 骨髓间充质干细胞 Bmi-1 Runx2 联合培养BMP-2 RNA干扰

【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(P0.01)。 (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(P0.01)。 (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(P0.01)。 (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(P0.05)；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(P0.01)。 (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(P0.01)。 [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。
[Abstract]:[research background and purpose]
Bone defect caused by various types of congenital or acquired causes is one of the common clinical diseases, and repair such bone defects has been a problem difficult to solve. Using tissue engineering technology in vitro osteogenesis after transplantation is the bone defect except for surgery a new repair method. Bone marrow mesenchymal stem cells (bone marrow Mesenchymal stem cells, BMSCs) as a body is easy to obtain stem cells, which can differentiate into various types of cells.BMSCs can differentiate into bone cells, cartilage cells under appropriate induction conditions, fat cells and other related cells. At present about the single type of cultured bone marrow mesenchymal stem cells and its osteogenic differentiation have made great progress, but there are still slow cell proliferation, osteogenic bone defects of low efficiency, long cycle and so on.
Studies have found that vascular endothelial cells (vein endothelial cells, VECs) in BMSCs co cultured with the bone morphogenetic protein (Bone morphogenetic, protein, BMP), promote osteoblast differentiation and stimulate osteoblasts and osteogenic cells before release of vascular endothelial growth Naga Innoko (vascular endothelial growth factor, VEGF, and VEGF) in promoting the proliferation of endothelial cells and plays an important role in angiogenesis and the formation process. But the research from the gene level of vascular endothelial cells on bone marrow mesenchymal stem cells into osteoblasts has not been reported.
This study adopts BMP-2 RNAi technology principle of hUVECs gene silence, respectively using hUVECs and hBMSCs normal hUVECs and BMP-2 gene silence in the construction of joint training system, detected by fluorescence quantitative PCR and quantitative culture influence system were hUVECs to hBMSCs Bmi-1 and Runx2 gene; verification of hUVECs in hBMSCs proliferation and osteogenic effect; to investigate the effects of Bmi-1 and Runx2 gene BMP-2 factor hUVECs of hBMSCs, to determine whether BMP-2 is a major factor in the expression of Bmi-1 and Runx2 gene regulation of hUVECs hBMSCs. This study was hUVECs hBMSCs co cultured seed cells in vitro in bone tissue engineering provides a theoretical basis for the cultivation and differentiation, and expect the research results to explore new methods in order to solve the seed cells of tissue engineering scaffolds in proliferation, adhesion and osteogenic differentiation of the problem.
[method]
(1) extraction of volunteer bone marrow, bone marrow mononuclear cells were isolated by density gradient centrifugation, and with the help of hBMSCs (?) the characteristics of wall growth were purified in phase contrast microscopy stem cell morphological changes. HBMSCs cells were cultured to the third generation, flow cytometric detection of CD34, CD29, expression of CD44 surface antigen identification of hBMSCs;
(2) hUVECs cells were cultured in vitro, and hUVECs cells were identified by immunohistochemical staining. After third generations, the expression of BMP-2 protein on 4,6,8,10 day was detected by Western Blotting protein assay.
(3) four groups of shRNA interference sequence of BMP-2 gene, the shRNA sequence design good insertion plasmid using liposome transfection method to construct the interference gene sequence was transfected into vascular endothelial cells, the expression of.Western Blotting protein effect detection of BMP-2 vascular endothelial cells were observed by fluorescence microscope. The transfection and identification of hUVECs cell BMP-2 RNA silencing effect.
(4) hUVECs cells of neonatal ECM+10% fetal bovine serum amplification to the third generation and the third generation of hBMSCs cells at the ratio of 1:1 to establish DMEM+10% fetal bovine serum for co culture medium group. The hUVECs cells and third RNAi treated hBMSCs cells according to the ratio of 1:1 to DMEM+10% fetal bovine serum for co culture interference group. Set up a separate hBMSCs culture group, hUVECs alone group was negative control group respectively. On day 4,6,8,10 is to observe the morphological changes by microscope, count the number of plates were counted hBMSCs; statistical analysis on the test value with SPSS17.0 software.
(5) were in the 4,6,8,10 days each time point for 6 hole detection alone hBMSCs group, co culture group and co culture group (Alkaline alkaline phosphatase phosphatase interference, ALP) and Osteocalcin (Osteocalin, OC). The content of the statistical analysis on the test value with SPSS17.0 software.
(6) on the day of 4,6,8,10, 6 holes were collected from each group at each time point. Western Blotting was used to detect the expression of BMP-2 protein in the hUVECs group, the co culture group and the co culture interference group respectively.
(7) using fluorescence quantitative PCR (fluorescence quantitative PCR, FQ-PCR) detection day 4,6,8,10 hBMSCs alone group, co culture of expression of Bmi-1 and Runx2 gene hBMSCs interference group in the group and joint training, each group of each time point for 6 holes. Statistical analysis of the measured value by SPSS17.0 software.
[results]
(1) by density gradient centrifugation and adherent cell culture separation, purification of hBMSCs, the cells with high purity, meet the requirement of experiment. The purification of the extract by flow cytometry third generation hBMSCs were analyzed to identify the cell phenotype, the low expression of CD34, CD29, high expression of CD44, hBMSCs. The phenotypic characteristics of character
(2) the immuno histochemical staining of the cultured hUVECs was identified, which conformed to the phenotype of the cell. The results of Western Blotting detection showed that hUVECs could express the BMP-2 protein normally.
(3) the expression of BMP-2 protein decreased significantly after transfection of plasmid 2. The shRNA interference plasmid designed in this study is effective for the silence of BMP-2 protein expression in hUVECs cells. Plasmid 3UG, liposome 10ul and transfection for 6 hours are optimal transfection conditions, and the transfection efficiency is about 60%..
(4) the morphological changes of hBMSCs in the culture system showed that hBMSCs showed a certain osteogenic differentiation at different time points. In the co culture interference group, the osteogenic differentiation of hBMSCs was weaker than that of the co culture group, and there was no osteogenic differentiation in the hBMSCs group alone.
(5) the number of cells in each group increased first and then decreased with the time prolonging, each time point of maximum number of cells co culture group, co culture interference group, alone hBMSCs minimum. 22 comparison between groups, the difference was statistically significant (P0.01).
(6) Western Blotting detection showed that the expression of BMP-2 in hUVECs group was higher than that in single hUVECs group. The expression of BMP-2 in the co culture interference group was significantly reduced, which reached the requirement of RNA interference.
(7) co culture group and co culture group in alkaline phosphatase detection interference amount increased firstly and then decreased with time prolonging, alkaline phosphatase hBMSCs alone group at each time point were relatively small. Each time point of joint training group had the highest ALP, co culture interference group, alone hBMSCs minimum. 22 comparison between the groups was statistically significant. There was significant difference (P0.01).
(8) co culture group and co culture group osteocalcin detection interference first increased and then decreased with time prolonging; osteocalcin content of hBMSCs only groups at each time point changes little; each time point of the co culture group was the highest detection of osteocalcin co culture, interference group and single hBMSCs group is the lowest. 22 comparison between the groups. The difference was statistically significant (P0.01).
(9) stem cell Bmi-1 gene expression increased gradually in the co culture group and co culture group interference according to the time of growth; Bmi-1 gene expression of hBMSCs alone group at each time point changes little; each time point of joint training and joint training group the expression of Bmi-1 gene interference group was higher, hBMSCs alone was the lowest; at the same time comparison between co culture group and the co cultured interference group, the difference was not statistically significant (P0.05); hBMSCs alone group and co culture group and co culture between interference group comparison, the difference was statistically significant (P0.01).
(10) joint training group and the co cultured interference group Runx2 expression increased with time gradually; the Runx2 gene alone group hBMSCs expression at each time point changes little; each time point of the co culture group the expression of Runx2 gene was the highest, joint training of interference group, hBMSCs alone group was the lowest; comparison between groups 22, the difference was statistically significant (P0.01).
[Conclusion]
(1) hBMSCs purified by density gradient centrifugation and adherent method was identified as bone marrow derived stem cells by flow cytometry.
(2) the umbilical vein endothelial cells can express the BMP-2 protein normally. The plasmid sequence constructed in the experiment is correct and effective, and can meet the RNA interference requirements of the experimental BMP-2.
(3) in the combined culture system, bone marrow mesenchymal stem cells and umbilical vein endothelial cells promote each other's proliferation. The two kinds of cells have good compatibility and no inhibition phenomenon.
(4) bone marrow mesenchymal stem cells (MSCs) secreted alkaline phosphatase and osteocalcin in the combined culture system, and the velocity of osteoblast differentiation was accelerated significantly.
(5) vascular endothelial cells can improve the Bmi-1 expression of bone marrow mesenchymal stem cells in the joint culture system, which is closely related to the proliferation of bone marrow mesenchymal stem cells.
(6) the combined culture system can promote the expression of Bmi-1 in bone marrow mesenchymal stem cells, but the upstream regulation of this signal pathway is not accomplished through BMP-2.
(7) bone marrow mesenchymal stem cells (MSCs) have accelerated osteogenic differentiation at the combined culture of vascular endothelial cells, which are closely related to the increase in the expression of Runx2 gene.
(8) the expression of Runx2 gene in bone marrow mesenchymal stem cells is closely related to the BMP-2 secreted by vascular endothelial cells. This proves that there is BMP-2/Runx2 pathway in the co culture system, but BMP-2 is not the only upstream factor in the Runx2 pathway of co culture system.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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