有限稀釋法分離鼠髓核間充質(zhì)干細(xì)胞及對(duì)細(xì)胞活性的影響
發(fā)布時(shí)間：2019-04-25 20:46
【摘要】：背景椎間盤(pán)退行性疾病(degenerative disc disease,DDD)是腰痛的主要原因,給患者帶來(lái)巨大痛苦,也給家庭和社會(huì)造成巨大的經(jīng)濟(jì)負(fù)擔(dān),而目前仍缺乏有效的治療手段。隨著細(xì)胞治療的發(fā)展,干細(xì)胞移植成為治療椎間盤(pán)退變的研究熱門(mén)。然而,椎間盤(pán)惡劣的微環(huán)境使外源性干細(xì)胞難以存活,成了研究的瓶頸。近年來(lái),隨著髓核來(lái)源干細(xì)胞——髓核間充質(zhì)干細(xì)胞(nucleus pulposus mesenchymal stem cells,NPMSCs)的發(fā)現(xiàn)及其更好的椎間盤(pán)微環(huán)境適應(yīng)力的證實(shí),椎間盤(pán)退變的細(xì)胞治療開(kāi)啟了新的一頁(yè)。然而,目前存在多種干細(xì)胞篩選方式,使得NPMSCs的分離純化尚未得到共識(shí)。其中,控制細(xì)胞種植密度實(shí)現(xiàn)干細(xì)胞分離的有限稀釋法,有助維持細(xì)胞活性及干性潛能,因其操作便捷,費(fèi)用較低得到推廣應(yīng)用。既往研究報(bào)道已經(jīng)利用該方法成功分離獲得牙周膜干細(xì)胞、單克隆臍血間充質(zhì)干細(xì)胞、齒齦間充質(zhì)干細(xì)胞和髕腱干細(xì)胞。但有限稀釋法是否適用于NPMSCs的體外分離,尚未見(jiàn)文獻(xiàn)報(bào)道。目的本研究通過(guò)高、中、低細(xì)胞接種密度(高密度接種處理為對(duì)照組,低、中密度處理即有限稀釋法為實(shí)驗(yàn)組)分離培養(yǎng)NPMSCs,從細(xì)胞形態(tài)、增殖、集落形成、遷移、細(xì)胞周期、多系誘導(dǎo)分化、免疫表型和干性基因表達(dá),評(píng)估其對(duì)NPMSCs的生物學(xué)活性影響,進(jìn)一步優(yōu)化分離和純化NPMSCs,為干細(xì)胞治療DDD及組織工程種子細(xì)胞的探索打下堅(jiān)實(shí)基礎(chǔ)。方法取三月齡SD雄性大鼠髓核組織,體外培養(yǎng),P1代以細(xì)胞種植密度分為三組:低密度組(5個(gè)細(xì)胞/cm2)、中密度組(100個(gè)細(xì)胞/cm2)、高密度組(10000個(gè)細(xì)胞/cm2)。各組繼續(xù)培養(yǎng)皆以10000個(gè)細(xì)胞/cm2進(jìn)行傳代,觀察細(xì)胞形態(tài)并檢測(cè)細(xì)胞周期分布、集落形成、遷移、增殖和多系誘導(dǎo)分化能力,干性基因(Oct4、Sox2 和 Nanog)及干細(xì)胞免疫表型(CD29、CD34、CD44、CD45、CD90)表達(dá)情況。結(jié)果細(xì)胞外觀上,各組NPMSCs均貼壁生長(zhǎng),局部旋渦樣排列。低密度組細(xì)胞為集落樣增殖,均一長(zhǎng)梭形外觀;高、中密度組細(xì)胞中出現(xiàn)多角形、類圓形細(xì)胞,細(xì)胞形態(tài)異質(zhì)性增大,出現(xiàn)寬大扁平且折光性差的細(xì)胞。相比另外兩組,低密度組細(xì)胞具有更強(qiáng)的集落形成能力、遷移能力,并且有更多的細(xì)胞進(jìn)入S期,更少的細(xì)胞滯留在G0/G1期(P0.05)。三組細(xì)胞均能夠進(jìn)行成骨、成脂、成軟骨誘導(dǎo)分化,但低密度組細(xì)胞三系分化能力較其他兩組增強(qiáng)(P0.05)。低密度組細(xì)胞的干性相關(guān)基因Nanog、Oct4和Sox2表達(dá)量均高于其余兩組(P0.05)。低、中密度組免疫表型CD29、CD44和CD90的陽(yáng)性率高于高密度組(P0.05),而CD34、CD45在三組間無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論本研究首次應(yīng)用有限稀釋法(5個(gè)細(xì)胞/cm2)從髓核中分離得到活性良好且干性潛能得以維持的NPMSCs,更好地應(yīng)用于干細(xì)胞移植及組織工程相關(guān)研究,為椎間盤(pán)退變的研究奠定基礎(chǔ)。
[Abstract]:Background Intervertebral disc degenerative disease (degenerative disc disease,DDD) is the main cause of low back pain, which brings great pain to patients, and also causes great economic burden to family and society. However, there is still no effective treatment. With the development of cell therapy, stem cell transplantation has become a hot topic in the treatment of intervertebral disc degeneration. However, the poor microenvironment of intervertebral disc makes it difficult for exogenous stem cells to survive, which has become the bottleneck of research. In recent years, with the discovery of nucleus pulposus-derived mesenchymal stem cells (nucleus pulposus mesenchymal stem cells,NPMSCs) and its better microenvironmental adaptation to intervertebral disc, the cell therapy of intervertebral disc degeneration has opened a new page. However, there are a variety of stem cell screening methods, so the isolation and purification of NPMSCs has not been agreed. The limited dilution method, which can control the density of cells to separate stem cells, is helpful to maintain the cell activity and dry potential. Because of its convenient operation and low cost, it has been popularized and applied. It has been reported that periodontal ligament stem cells, umbilical cord blood mesenchymal stem cells, gingival mesenchymal stem cells and patellar tendon stem cells have been successfully isolated by this method. However, whether the finite dilution method is suitable for in vitro separation of NPMSCs has not been reported in the literature. Objective to isolate and culture NPMSCs, from cell morphology, proliferation, colony formation, migration and cell cycle by high, middle and low cell density (high density inoculation as control group, low and medium density treatment i.e. limited dilution method as experimental group), and through cell morphology, proliferation, colony formation, migration, cell cycle, cell morphology, proliferation, colony formation, migration, cell cycle. Multi-line differentiation, immunophenotype and dry gene expression were used to evaluate the biological activity of NPMSCs, and further optimization of isolation and purification of NPMSCs, laid a solid foundation for the treatment of DDD and tissue engineering seed cells by stem cells. Methods Nucleus pulposus tissue of 3-month-old male SD rats was cultured in vitro. P1 generation was divided into three groups: low density group (5 cells / cm2), medium density group (100 cells / cm2) and high density group (10000 cells / cm2). Cell morphology and cell cycle distribution, colony formation, migration, proliferation and multilineage inducing differentiation ability, stem gene (Oct4,Sox2 and Nanog) and stem cell immunophenotype (CD29,) were observed in each group by 10000 cells / cm2. CD34,CD44,CD45,CD90). Results on the surface of the cells, NPMSCs was adherent to the wall and arranged in a local vortex-like manner. The cells in the low density group showed colony-like proliferation with a long spindle-shaped appearance, and in the high and middle density groups, polygonal and circular cells appeared, and the heterogeneity of the cell morphology increased, and the cells with wide flattened and poor refractive property appeared in the high-density and middle-density groups. Compared with the other two groups, the cells in the low density group had stronger colony forming ability and migration ability, and more cells entered the S phase and fewer cells remained in the G0 / G1 phase (P0.05). All the cells in the three groups were able to induce differentiation by osteogenesis, lipogenesis and chondrogenesis, but the differentiation ability of the three cell lines in the low density group was higher than that in the other two groups (P0.05). The expression levels of Nanog,Oct4 and Sox2 in the low density group were higher than those in the other two groups (P0.05). The positive rates of immunophenotype CD29,CD44 and CD90 in the low and middle density groups were higher than those in the high density group (P0.05), but there was no significant difference in CD34,CD45 among the three groups (P0.05). Conclusion in this study, the limited dilution method (5 cells / cm2) was first used to isolate NPMSCs, from nucleus pulposus and maintain its dry potential, which could be used in stem cell transplantation and tissue engineering. It lays a foundation for the study of intervertebral disc degeneration.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.5
[Abstract]:Background Intervertebral disc degenerative disease (degenerative disc disease,DDD) is the main cause of low back pain, which brings great pain to patients, and also causes great economic burden to family and society. However, there is still no effective treatment. With the development of cell therapy, stem cell transplantation has become a hot topic in the treatment of intervertebral disc degeneration. However, the poor microenvironment of intervertebral disc makes it difficult for exogenous stem cells to survive, which has become the bottleneck of research. In recent years, with the discovery of nucleus pulposus-derived mesenchymal stem cells (nucleus pulposus mesenchymal stem cells,NPMSCs) and its better microenvironmental adaptation to intervertebral disc, the cell therapy of intervertebral disc degeneration has opened a new page. However, there are a variety of stem cell screening methods, so the isolation and purification of NPMSCs has not been agreed. The limited dilution method, which can control the density of cells to separate stem cells, is helpful to maintain the cell activity and dry potential. Because of its convenient operation and low cost, it has been popularized and applied. It has been reported that periodontal ligament stem cells, umbilical cord blood mesenchymal stem cells, gingival mesenchymal stem cells and patellar tendon stem cells have been successfully isolated by this method. However, whether the finite dilution method is suitable for in vitro separation of NPMSCs has not been reported in the literature. Objective to isolate and culture NPMSCs, from cell morphology, proliferation, colony formation, migration and cell cycle by high, middle and low cell density (high density inoculation as control group, low and medium density treatment i.e. limited dilution method as experimental group), and through cell morphology, proliferation, colony formation, migration, cell cycle, cell morphology, proliferation, colony formation, migration, cell cycle. Multi-line differentiation, immunophenotype and dry gene expression were used to evaluate the biological activity of NPMSCs, and further optimization of isolation and purification of NPMSCs, laid a solid foundation for the treatment of DDD and tissue engineering seed cells by stem cells. Methods Nucleus pulposus tissue of 3-month-old male SD rats was cultured in vitro. P1 generation was divided into three groups: low density group (5 cells / cm2), medium density group (100 cells / cm2) and high density group (10000 cells / cm2). Cell morphology and cell cycle distribution, colony formation, migration, proliferation and multilineage inducing differentiation ability, stem gene (Oct4,Sox2 and Nanog) and stem cell immunophenotype (CD29,) were observed in each group by 10000 cells / cm2. CD34,CD44,CD45,CD90). Results on the surface of the cells, NPMSCs was adherent to the wall and arranged in a local vortex-like manner. The cells in the low density group showed colony-like proliferation with a long spindle-shaped appearance, and in the high and middle density groups, polygonal and circular cells appeared, and the heterogeneity of the cell morphology increased, and the cells with wide flattened and poor refractive property appeared in the high-density and middle-density groups. Compared with the other two groups, the cells in the low density group had stronger colony forming ability and migration ability, and more cells entered the S phase and fewer cells remained in the G0 / G1 phase (P0.05). All the cells in the three groups were able to induce differentiation by osteogenesis, lipogenesis and chondrogenesis, but the differentiation ability of the three cell lines in the low density group was higher than that in the other two groups (P0.05). The expression levels of Nanog,Oct4 and Sox2 in the low density group were higher than those in the other two groups (P0.05). The positive rates of immunophenotype CD29,CD44 and CD90 in the low and middle density groups were higher than those in the high density group (P0.05), but there was no significant difference in CD34,CD45 among the three groups (P0.05). Conclusion in this study, the limited dilution method (5 cells / cm2) was first used to isolate NPMSCs, from nucleus pulposus and maintain its dry potential, which could be used in stem cell transplantation and tissue engineering. It lays a foundation for the study of intervertebral disc degeneration.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 張燕;陶暉;顧韜;李歡迎;李海峰;吳劍宏;何R，
本文編號(hào)：2465446
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