人髓核间充质干细胞的分离提纯及生物学活性鉴定的实验研究
发布时间：2019-06-21 03:05
【摘要】：目的:(1)运用贴壁法与流式细胞分选方法分离提纯人髓核间充质干细胞(nucleus pulposus mesenchymal stem cells,NPMSCs)。(2)从细胞形态、增殖特点、免疫免疫表型、三系分化等方面比较两种方法获得的髓核间充质干细胞的生物学活性。方法:收集腰椎间盘突出症患者的退变髓核组织(Pfirrmann分级均为Ⅳ级),利用酶消化法分离细胞。分别采用两种方法分离提纯NPMSCs,一组细胞采用贴壁法培养,另一组通过流式细胞分选技术利用NPMSCs表面阳性标志物CD73、CD90、CD105获得NPMSCs。将两种方法获得的NPMSCs进行体外培养扩增,分别进行形态学观察,CCK-8检测增殖能力。向成骨、成脂、成软骨诱导分化,诱导28天后分别进行茜素红染色观察其成骨能力、油红O染色观察其成脂能力、甲苯胺蓝染色观察其成软骨能力,利用Imag J软件计算染色区域所占的面积百分比。比较两组NPMSCs在形态学,免疫表型以及增殖和分化能力的差异。结果:(1)流式细胞分选仪以PE-CD73、APC-CD90、V450-CD105作为表面阳性标志,分选出CD73+、CD90+、CD105+三阳性的髓核间充质干细胞,平均获得数为(3.53±0.78)×106个,其比例约(89.67±2.52)%。贴壁法获得的髓核间充质干细胞CD73、CD90、CD105的阳性表达率,分别为(89.79±2.40)%,(94.07±2.31)%,(90.49±1.63)%。(2)经酶消化后的原代细胞在接种后5-7h贴壁,原代细胞呈长短不一的短梭形,待细胞生长达80-90%融合时,可见旋涡状生长或者以组织块为中心的漩涡形成,排列整齐。流式细胞分选方法获得的髓核间充质干细胞在接种后4-6h观察到贴壁生长,主要以旋涡状生长为主,少见散在的单个贴壁生长细胞,12-15天细胞可达到80-90%融合。(3)在培养4h-1d,流式组NPMSCs的增殖活力明显低于贴壁组NPMSCs(P0.05),第3天两组OD值无明显统计学差异(P0.05)。在第5d-13d,流式组NPMSCs增殖能力明显高于贴壁组NPMSCs(P0.05)。(4)流式组NPMSCs流式细胞仪免疫表型的检测结果显示,CD73的表达率为(98.55±0.35)%,CD90的表达率为(98.47±0.57)%,CD105的表达率为(98.20±1.24)%,表达率明显高于贴壁组NPMSCs。流式组NPMSCs造血干细胞标志物CD45、CD34及HLA-DR等的表达率均低于4%。(5)成骨诱导分化:两组NPMSCs经诱导28天后,显微镜下可见细胞内有黑色不透光区域,经茜素红染色后可见细胞表面存在大量红染的钙盐沉积,肉眼观可见流式组NPMSCs红染面积多于贴壁组NPMSCs,应用Imag J软件进行图像分析,测得流式组NPMSCs红染面积的比例明显高于贴壁组NPMSCs(P0.05)。成脂诱导分化:两组NPMSCs经成脂诱导28天后,显微镜下观察可见细胞内有大小不等的光亮圆形脂滴形成,经油红“O”染色均可见片状或点状的红染脂滴空泡,应用Imag J软件进行图像分析,测得流式组NPMSCs红染面积的比例明显高于贴壁组NPMSCs(P0.05)。成软骨诱导分化:两组NPMSCs经诱导28天后,均可见乳白色的软骨微球形成,甲苯胺蓝染色后两种细胞均可见明显蓝染的软骨细胞,Imag J软件分析发现流式组NPMSCs蓝染软骨细胞面积明显高于贴壁组NPMSCs(P0.05)。结论:本实验利用流式细胞分选技术从人退变髓核组织中获得较高纯度的NPMSCs,并能进行后续培养扩增。与贴壁法获得的NPMSCs相比,流式分选的NPMSCs具有更强的增殖与分化能力。流式细胞分选方法为研究NPMSCs的生物学特性提供了可靠的细胞分离与纯化方法。流式组获得的NPMSCs能够贴壁生长、完成三系分化诱导,提供了人髓核组织中存在间充质干细胞的依据。
[Abstract]:Objective: (1) To separate and purify human marrow-derived mesenchymal stem cells (NPMSCs) by means of the method of malapposition and flow cytometry. (2) The biological activity of the mesenchymal stem cells was obtained from the aspects of cell morphology, proliferation, immunophenotype and three-line differentiation. Methods: Retrograde nucleus pulposus of the patients with lumbar disc herniation (Pfirrmann classification of grade IV) was collected, and the cells were isolated by enzyme digestion. The NPMSCs were isolated and purified by two methods. One group of cells was cultured by an adherent method, and the other group was obtained by flow cytometry using the NPMSCs surface-positive marker CD73, CD90 and CD105. The NPMSCs obtained from the two methods were cultured in vitro to carry out morphological observation respectively, and the proliferation ability was detected by CCK-8. The osteogenic, fat-forming and chondrogenic differentiation were induced, and the bone-forming ability was observed after 28 days of induction, and the fat-forming ability was observed by the staining of the oil-red O. The cartilage-forming ability was observed by toluidine blue staining. The area percentage of the stained area was calculated by using the Imag J software. The differences of the two groups of NPMSCs in morphology, immunophenotype and proliferation and differentiation were compared. Results: (1) The expression of CD73 +, CD90 +, CD105 + 3-positive nucleus-derived mesenchymal stem cells was selected by flow cytometry in the presence of PE-CD73, APC-CD90 and V450-CD105, and the average number of obtained was (3.53-0.78) and 106, and the ratio was about 89.67 (2.52)%. The positive rate of CD73, CD90 and CD105 of the marrow-derived mesenchymal stem cells (CD73, CD90 and CD105) was (89.79-2.40)%, (94.07-2.31)%, (90.49-1.63)%, respectively. And (2) the primary cells after the enzyme digestion are adhered to the adherent cells 5-7 hours after the inoculation, the primary cells are in a short shed with different lengths, and when the growth of the cells reaches 80-90%, the primary cells can be seen in a vortex-like growth or formed in a vortex with the tissue mass as the center, and the cells are arranged in order. The bone marrow-derived mesenchymal stem cells obtained by flow cytometry were observed at 4-6 h post-inoculation, mainly in the form of vortex-like growth, rarely scattered in a single adherent growth cell, and the 12-15 day cells could be fused to 80-90%. (3) The proliferation of NPMSCs in the flow group was significantly lower than that of the group NPMSCs (P0.05). There was no significant difference between the two groups (P0.05). The proliferation ability of NPMSCs in flow group was significantly higher than that of NPMSCs (P0.05). (4) The expression rate of CD73 was (98.55-0.35)%, the expression rate of CD90 (98.47-0.57)% and the expression rate of CD105 (98.20-1.24)%, and the expression rate was higher than that of NPMSCs in the group. The expression rate of CD45, CD34 and HLA-DR in the flow group of NPMSCs was lower than 4%. (5) osteogenic induction and differentiation: after 28 days of induction of the two groups of NPMSCs, there was a dark opaque region in the cells under the microscope, and a large amount of red-stained calcium salt was found on the surface of the cells after the red staining, and the naked eye view showed that the red-stained area of the NPMSCs in the flow group was more than that of the adherent group NPMSCs, Image analysis was carried out using the Imag J software, and the proportion of NPMSCs in the flow group was significantly higher than that of the group NPMSCs (P0.05). To induce differentiation: two groups of NPMSCs were induced by liposuction for 28 days, and the visible cells of the two groups were observed under the microscope to form bright circular lipid droplets of different sizes, and the red-dyed fat drop vacuoles in the form of flake or dot were observed through the oil-red "O" dyeing, and the image analysis was carried out using the Imag J software. The proportion of NPMSCs in the flow group was significantly higher than that of the group NPMSCs (P0.05). Cartilage-induced differentiation: After 28 days of induction, the two groups of NPMSCs were found to be milk-white cartilage microballoon, and both cells were visible blue-stained chondrocytes after toluidine blue staining, and the Imag J software analysis found that the area of NPMSCs blue-stained chondrocytes in the flow group was significantly higher than that of the adherent group NPMSCs (P0.05). Conclusion: In this experiment, the high-purity NPMSCs were obtained by flow cytometry, and the subsequent culture and amplification could be carried out. Compared with the NPMSCs obtained by the malapposition method, the flow-sorted NPMSCs has stronger proliferation and differentiation ability. The flow cytometric method provides a reliable method for cell separation and purification for the study of the biological characteristics of NPMSCs. The NPMSCs obtained by the flow-type group can grow on the wall and complete the three-line differentiation induction, and provide the basis for the presence of the mesenchymal stem cells in the human nucleus pulposus tissues.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.5
本文编号：2503738
[Abstract]:Objective: (1) To separate and purify human marrow-derived mesenchymal stem cells (NPMSCs) by means of the method of malapposition and flow cytometry. (2) The biological activity of the mesenchymal stem cells was obtained from the aspects of cell morphology, proliferation, immunophenotype and three-line differentiation. Methods: Retrograde nucleus pulposus of the patients with lumbar disc herniation (Pfirrmann classification of grade IV) was collected, and the cells were isolated by enzyme digestion. The NPMSCs were isolated and purified by two methods. One group of cells was cultured by an adherent method, and the other group was obtained by flow cytometry using the NPMSCs surface-positive marker CD73, CD90 and CD105. The NPMSCs obtained from the two methods were cultured in vitro to carry out morphological observation respectively, and the proliferation ability was detected by CCK-8. The osteogenic, fat-forming and chondrogenic differentiation were induced, and the bone-forming ability was observed after 28 days of induction, and the fat-forming ability was observed by the staining of the oil-red O. The cartilage-forming ability was observed by toluidine blue staining. The area percentage of the stained area was calculated by using the Imag J software. The differences of the two groups of NPMSCs in morphology, immunophenotype and proliferation and differentiation were compared. Results: (1) The expression of CD73 +, CD90 +, CD105 + 3-positive nucleus-derived mesenchymal stem cells was selected by flow cytometry in the presence of PE-CD73, APC-CD90 and V450-CD105, and the average number of obtained was (3.53-0.78) and 106, and the ratio was about 89.67 (2.52)%. The positive rate of CD73, CD90 and CD105 of the marrow-derived mesenchymal stem cells (CD73, CD90 and CD105) was (89.79-2.40)%, (94.07-2.31)%, (90.49-1.63)%, respectively. And (2) the primary cells after the enzyme digestion are adhered to the adherent cells 5-7 hours after the inoculation, the primary cells are in a short shed with different lengths, and when the growth of the cells reaches 80-90%, the primary cells can be seen in a vortex-like growth or formed in a vortex with the tissue mass as the center, and the cells are arranged in order. The bone marrow-derived mesenchymal stem cells obtained by flow cytometry were observed at 4-6 h post-inoculation, mainly in the form of vortex-like growth, rarely scattered in a single adherent growth cell, and the 12-15 day cells could be fused to 80-90%. (3) The proliferation of NPMSCs in the flow group was significantly lower than that of the group NPMSCs (P0.05). There was no significant difference between the two groups (P0.05). The proliferation ability of NPMSCs in flow group was significantly higher than that of NPMSCs (P0.05). (4) The expression rate of CD73 was (98.55-0.35)%, the expression rate of CD90 (98.47-0.57)% and the expression rate of CD105 (98.20-1.24)%, and the expression rate was higher than that of NPMSCs in the group. The expression rate of CD45, CD34 and HLA-DR in the flow group of NPMSCs was lower than 4%. (5) osteogenic induction and differentiation: after 28 days of induction of the two groups of NPMSCs, there was a dark opaque region in the cells under the microscope, and a large amount of red-stained calcium salt was found on the surface of the cells after the red staining, and the naked eye view showed that the red-stained area of the NPMSCs in the flow group was more than that of the adherent group NPMSCs, Image analysis was carried out using the Imag J software, and the proportion of NPMSCs in the flow group was significantly higher than that of the group NPMSCs (P0.05). To induce differentiation: two groups of NPMSCs were induced by liposuction for 28 days, and the visible cells of the two groups were observed under the microscope to form bright circular lipid droplets of different sizes, and the red-dyed fat drop vacuoles in the form of flake or dot were observed through the oil-red "O" dyeing, and the image analysis was carried out using the Imag J software. The proportion of NPMSCs in the flow group was significantly higher than that of the group NPMSCs (P0.05). Cartilage-induced differentiation: After 28 days of induction, the two groups of NPMSCs were found to be milk-white cartilage microballoon, and both cells were visible blue-stained chondrocytes after toluidine blue staining, and the Imag J software analysis found that the area of NPMSCs blue-stained chondrocytes in the flow group was significantly higher than that of the adherent group NPMSCs (P0.05). Conclusion: In this experiment, the high-purity NPMSCs were obtained by flow cytometry, and the subsequent culture and amplification could be carried out. Compared with the NPMSCs obtained by the malapposition method, the flow-sorted NPMSCs has stronger proliferation and differentiation ability. The flow cytometric method provides a reliable method for cell separation and purification for the study of the biological characteristics of NPMSCs. The NPMSCs obtained by the flow-type group can grow on the wall and complete the three-line differentiation induction, and provide the basis for the presence of the mesenchymal stem cells in the human nucleus pulposus tissues.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.5
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