【摘要】：目的:(1)運用貼壁法與流式細(xì)胞分選方法分離提純?nèi)怂韬碎g充質(zhì)干細(xì)胞(nucleus pulposus mesenchymal stem cells,NPMSCs)。(2)從細(xì)胞形態(tài)、增殖特點、免疫免疫表型、三系分化等方面比較兩種方法獲得的髓核間充質(zhì)干細(xì)胞的生物學(xué)活性。方法:收集腰椎間盤突出癥患者的退變髓核組織(Pfirrmann分級均為Ⅳ級),利用酶消化法分離細(xì)胞。分別采用兩種方法分離提純NPMSCs,一組細(xì)胞采用貼壁法培養(yǎng),另一組通過流式細(xì)胞分選技術(shù)利用NPMSCs表面陽性標(biāo)志物CD73、CD90、CD105獲得NPMSCs。將兩種方法獲得的NPMSCs進(jìn)行體外培養(yǎng)擴增,分別進(jìn)行形態(tài)學(xué)觀察,CCK-8檢測增殖能力。向成骨、成脂、成軟骨誘導(dǎo)分化,誘導(dǎo)28天后分別進(jìn)行茜素紅染色觀察其成骨能力、油紅O染色觀察其成脂能力、甲苯胺藍(lán)染色觀察其成軟骨能力,利用Imag J軟件計算染色區(qū)域所占的面積百分比。比較兩組NPMSCs在形態(tài)學(xué),免疫表型以及增殖和分化能力的差異。結(jié)果:(1)流式細(xì)胞分選儀以PE-CD73、APC-CD90、V450-CD105作為表面陽性標(biāo)志,分選出CD73+、CD90+、CD105+三陽性的髓核間充質(zhì)干細(xì)胞,平均獲得數(shù)為(3.53±0.78)×106個,其比例約(89.67±2.52)%。貼壁法獲得的髓核間充質(zhì)干細(xì)胞CD73、CD90、CD105的陽性表達(dá)率,分別為(89.79±2.40)%,(94.07±2.31)%,(90.49±1.63)%。(2)經(jīng)酶消化后的原代細(xì)胞在接種后5-7h貼壁,原代細(xì)胞呈長短不一的短梭形,待細(xì)胞生長達(dá)80-90%融合時,可見旋渦狀生長或者以組織塊為中心的漩渦形成,排列整齊。流式細(xì)胞分選方法獲得的髓核間充質(zhì)干細(xì)胞在接種后4-6h觀察到貼壁生長,主要以旋渦狀生長為主,少見散在的單個貼壁生長細(xì)胞,12-15天細(xì)胞可達(dá)到80-90%融合。(3)在培養(yǎng)4h-1d,流式組NPMSCs的增殖活力明顯低于貼壁組NPMSCs(P0.05),第3天兩組OD值無明顯統(tǒng)計學(xué)差異(P0.05)。在第5d-13d,流式組NPMSCs增殖能力明顯高于貼壁組NPMSCs(P0.05)。(4)流式組NPMSCs流式細(xì)胞儀免疫表型的檢測結(jié)果顯示,CD73的表達(dá)率為(98.55±0.35)%,CD90的表達(dá)率為(98.47±0.57)%,CD105的表達(dá)率為(98.20±1.24)%,表達(dá)率明顯高于貼壁組NPMSCs。流式組NPMSCs造血干細(xì)胞標(biāo)志物CD45、CD34及HLA-DR等的表達(dá)率均低于4%。(5)成骨誘導(dǎo)分化:兩組NPMSCs經(jīng)誘導(dǎo)28天后,顯微鏡下可見細(xì)胞內(nèi)有黑色不透光區(qū)域,經(jīng)茜素紅染色后可見細(xì)胞表面存在大量紅染的鈣鹽沉積,肉眼觀可見流式組NPMSCs紅染面積多于貼壁組NPMSCs,應(yīng)用Imag J軟件進(jìn)行圖像分析,測得流式組NPMSCs紅染面積的比例明顯高于貼壁組NPMSCs(P0.05)。成脂誘導(dǎo)分化:兩組NPMSCs經(jīng)成脂誘導(dǎo)28天后,顯微鏡下觀察可見細(xì)胞內(nèi)有大小不等的光亮圓形脂滴形成,經(jīng)油紅“O”染色均可見片狀或點狀的紅染脂滴空泡,應(yīng)用Imag J軟件進(jìn)行圖像分析,測得流式組NPMSCs紅染面積的比例明顯高于貼壁組NPMSCs(P0.05)。成軟骨誘導(dǎo)分化:兩組NPMSCs經(jīng)誘導(dǎo)28天后,均可見乳白色的軟骨微球形成,甲苯胺藍(lán)染色后兩種細(xì)胞均可見明顯藍(lán)染的軟骨細(xì)胞,Imag J軟件分析發(fā)現(xiàn)流式組NPMSCs藍(lán)染軟骨細(xì)胞面積明顯高于貼壁組NPMSCs(P0.05)。結(jié)論:本實驗利用流式細(xì)胞分選技術(shù)從人退變髓核組織中獲得較高純度的NPMSCs,并能進(jìn)行后續(xù)培養(yǎng)擴增。與貼壁法獲得的NPMSCs相比,流式分選的NPMSCs具有更強的增殖與分化能力。流式細(xì)胞分選方法為研究NPMSCs的生物學(xué)特性提供了可靠的細(xì)胞分離與純化方法。流式組獲得的NPMSCs能夠貼壁生長、完成三系分化誘導(dǎo),提供了人髓核組織中存在間充質(zhì)干細(xì)胞的依據(jù)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R681.5

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