人脐带华通胶间充质干细胞向髓核细胞诱导分化的实验研究

发布时间：2018-04-27 23:05

本文选题：脐带 + 华通胶间充质干细胞　；参考：《第二军医大学》2011年硕士论文

【摘要】：研究目的:脐带华通胶间充质干细胞(Wharton’s jelly-derived mesenchymal stem cells, WJMSCs)是来源于脐带血管周围华通胶组织的间充质干细胞,具有干细胞的基本特征,即该细胞具有自我更新及向不同组织或细胞分化的能力。本研究从人脐带中分离提取华通胶间充质干细胞,观察其细胞形态,检测其增殖能力、细胞免疫表型及端粒酶活性,鉴定分离提取的细胞具有干细胞特性,为下一步通过细胞共培养的方法定向诱导分化为髓核细胞奠定基础。研究方法:取足月产健康新生儿脐带,分离华通胶组织,剪碎后用胶原酶和胰蛋白酶消化,提取细胞后加DMEM/F12培养基和胎牛血清培养,细胞融合达到80-90%时进行传代培养。观察细胞形态,测定不同代次的细胞增殖能力。取第3代细胞行端粒酶活性分析及流式细胞学检测细胞免疫表型(CD34/CD45/CD105/CD73/CD90/HLA-DR及HLA-ABC)。结果:原代培养第3-5天可见部分细胞贴壁生长,形态为梭形和多角形,1周后形成集落,2周时细胞融合达到90%。传代后细胞为梭形,形态一致,细胞呈漩涡状生长,细胞增殖能力强,5-6天即可进行传代,传至16代增殖能力未见下降。端粒酶活性测定为阳性,流式细胞分析细胞免疫表型结果表明,CD90/CD105/CD73/HLA-ABC阳性,CD34/CD45/HLA-DR阴性。结论:人脐带含有丰富的间充质干细胞,通过酶消化法可从脐带华通胶中提取间充质干细胞,该细胞呈梭形,漩涡状生长,增殖能力强。流式细胞分析检测细胞免疫表型显示细胞表达间充质干细胞免疫表型,不表达造血干细胞免疫表型,其端粒酶活性阳性。华通胶间充质干细胞由于其更强的增殖活性、更好扩增能力及更易于获取而有望成为组织工程和细胞治疗技术的理想种子细胞。研究背景和目的:椎间盘退变性疾病(degenerative disc disease, DDD)是常见病多发病,其引起的下腰痛严重影响人们的工作及生活。目前的治疗方法主要是保守治疗和外科治疗,虽能暂时缓解临床症状,但远期疗效并不理想,并可能会发生并发症。近年来,以修复椎间盘退变恢复椎间盘高度为目的的组织工程技术和细胞治疗技术逐渐被认为是具有前景的治疗方法。然而,无论是组织工程技术还是细胞治疗技术,在椎间盘退变性疾病的治疗中尚缺乏理想的种子细胞。本实验探讨人脐带华通胶间充质干细胞(Wharton’s jelly-derived mesenchymal stem cells, WJMSCs)向髓核细胞(nucleus pulposus cells, NPCs)的分化潜能,希望为椎间盘退变性疾病的治疗提供一种理想的种子细胞来源。方法:取人正常足月产婴儿脐带,分离消化脐带华通胶组织,收集培养华通胶间充质干细胞;取人正常未退变椎间盘(T12-L1),酶消化法分离培养髓核细胞。取稳定增殖的第3代华通胶间充质干细胞和髓核细胞进行共培养。CFSE标记华通胶间充质干细胞。使用带有插入层的Transwell培养板进行非接触式共培养,插入层具有0.4μm高密度孔径;下层接种华通胶间充质干细胞,上层接种髓核细胞。普通六孔板进行接触式共培养。根据共培养细胞比例不同分为3组(25:75 WJMSCs/hNPCs,50:50 WJMSCs/hNPCs,75:25 WJMSCs/hNPCs),于共培养1周后,利用MoFlo高速流式细胞分选仪分选收集接触式共培养的华通胶间充质干细胞。提取华通胶间充质干细胞总RNA进行反转录获得cDNA,利用Real-Time PCR方法检测其蛋白多糖、Ⅰ型胶原、Ⅱ型胶原、VI型胶原、SOX-9及多能聚糖等基因表达,管家基因GAPDH作为内参,单独培养的华通胶间充质干细胞作为对照,利用2-ΔΔCt方法计算基因相对表达变化。结果:细胞分选点阵图显示CFSE标记的WJMSCs与无荧光标记的髓核细胞完全分离并分选。分选后细胞进行Real-Time PCR检测华通胶间充质干细胞相对基因表达变化。经过7天共培养,结果显示,接触式共培养各组华通胶间充质干细胞的SOX-9、Ⅱ型胶原和蛋白多糖相对表达都有显著提高(P0.05),其中25:75 WJMSCs/hNPCs组上调幅度最大(SOX-9上调2429倍,Ⅱ型胶原上调9463倍,蛋白多糖上调5974倍);非接触式共培养组各组华通胶间充质干细胞的SOX-9、Ⅱ型胶原和蛋白多糖相对表达也有显著提高(P0.05),但基因上调幅度比接触式共培养组小(P0.05),其中25:75 WJMSCs/hNPCs组基因表达上调幅度最大(SOX-9上调114倍,Ⅱ型胶原上调57倍,蛋白多糖上调67倍)。共培养各组I型胶原、VI型胶原和多能聚糖的基因基因表达改变无统计学意义(P0.05)。结论:人脐带华通胶间充质干细胞具有向髓核细胞分化潜能。细胞共培养7天能够诱导华通胶间充质干细胞分化为髓核细胞,最优细胞比例为25:75 WJMSCs/NPCs。接触式共培养更有利于间充质干细胞的分化,可推测华通胶间充质干细胞移植入椎间盘髓核中,其中的髓核细胞能够诱导植入的细胞分化为髓核细胞并分泌髓核基质,以恢复椎间盘高度。因此,华通胶间充质干细胞由于其更强的增殖活性、更好扩增能力及更易于获取而有望成为治疗椎间盘退变性疾病的理想种子细胞。
[Abstract]:Objective: Wharton s jelly-derived mesenchymal stem cells, WJMSCs) is a mesenchymal stem cell derived from the tissue of Huatong gum around the umbilical cord. It has the basic characteristics of stem cells, that is, the cells have the ability to renew themselves and differentiate into different tissues or cells. This study is from the human umbilical cord. The cell morphology, the proliferation ability, the cell immunophenotype and the telomerase activity of the cells were detected by the isolation and extraction of the mesenchymal stem cells, and the identification of the isolated cells had the characteristics of stem cells. It laid the foundation for the next step to differentiate into nucleus pulposus cells by the method of cell co culture.
Methods: the healthy newborn umbilical cord was harvested for full month, and Huatong gum was isolated and digested with collagenase and trypsin after shredding. After the extraction of cells, the cells were cultured with DMEM/F12 medium and fetal bovine serum, and the cell fusion reached 80-90%. The cell morphology was observed and the proliferation ability of the cells in the same generation was measured. Third generation cells were used for telomere. Enzyme activity analysis and flow cytometry were used to detect cellular immunophenotype (CD34/CD45/CD105/CD73/CD90/HLA-DR and HLA-ABC).
Results: on the 3-5 day of primary culture, some cells were found to grow on the wall, form a spindle shape and polygon. After 1 weeks, the cells formed a colony. After 2 weeks, the cell fusion reached the shape of the 90%. and the cells were in the same shape. The cells were whirlpool, and the cell proliferation ability was strong. The proliferation ability of the 16 generation was not decreased. The telomerase activity was not decreased. The positive immunophenotype of flow cytometry showed that CD90/CD105/CD73/HLA-ABC was positive and CD34/CD45/HLA-DR was negative.
Conclusion: human umbilical cord is rich in mesenchymal stem cells, and mesenchymal stem cells can be extracted from Huatong gum by enzyme digestion. The cells are spindle shaped, whirlpool and proliferating. Flow cytometry analysis and detection of cell immunophenotype show that cells express mesenchymal stem cell immunophenotype and do not express hematopoietic stem cell immunophenotype. Telomerase activity is positive. Huatong glue mesenchymal stem cells are expected to become ideal seed cells for tissue engineering and cell therapy because of their stronger proliferative activity, better amplification and easier access.
Background and objective: degenerative disc disease (DDD) is a common disease, and the cause of lower back pain seriously affects people's work and life. Current treatment methods are mainly conservative and surgical treatment, although it can temporarily relieve the clinical symptoms, but the long-term effect is not ideal, and may occur concurrent. In recent years, tissue engineering and cell therapy for repair of disc degeneration and disc height have gradually been considered as a promising treatment. However, no ideal seed cells are still lacking in the treatment of degenerative diseases of intervertebral disc by tissue engineering and cell therapy. The differentiation potential of Wharton 's jelly-derived mesenchymal stem cells, WJMSCs from human umbilical cord to nucleus pulposus cells (nucleus pulposus cells, NPCs) is expected to provide an ideal seed cell source for the treatment of intervertebral disc degeneration disease.
Methods: the human umbilical cord of normal full-term birth was taken and the Huatong gum tissue was isolated and digested and the Huatong mesenchymal stem cells were collected and cultured. The normal and non degenerative intervertebral discs (T12-L1) were collected and cultured, and the nucleus pulmedulla cells were isolated and cultured by enzyme digestion. The third generations of mesenchymal stem cells and nucleus medullary cells, which were stable and proliferated, were used for the co culture of.CFSE labelled Huatong glue. The Transwell culture plate with the insertion layer was used for non contact co culture. The insertion layer had a high density of 0.4 mu m; the lower layer was inoculated with the mesenchymal stem cells of Huatong gum, the upper layer inoculated the nucleus pulposus cells and the common six hole plates were coculture. The co culture cells were divided into 3 groups (25:75 WJMSCs/hNPCs, 50:50 WJMSC). S/hNPCs, 75:25 WJMSCs/hNPCs), after 1 weeks of co culture, a MoFlo high speed flow cell sorting instrument was used to collect the contact co cultured Huatong mesenchymal stem cells. The total RNA of the Huatong mesenchymal stem cells was extracted to reverse transcriptional cDNA, and the Real-Time PCR method was used to detect the proteoglycan, type I collagen, type II collagen, VI collagen, SO. The gene expression of X-9 and poly glycan, the housekeeping gene GAPDH was used as the internal parameter, the individual cultured Huatong mesenchymal stem cells were used as the control, and the gene relative expression changes were calculated by the 2- Delta Delta Ct method.
Results: the cell separation dot matrix showed that the CFSE labeled WJMSCs was completely separated and selected from the non fluorescent labeled nucleus pulposus cells. After the separation, the cells were selected for Real-Time PCR to detect the relative gene expression changes of the Huatong mesenchymal stem cells. After 7 days co culture, the results showed that the SOX-9 and type II of the contact cultured cells were cultured in each group. The relative expression of collagen and proteoglycan was significantly increased (P0.05), in which the 25:75 WJMSCs/hNPCs group was up to the maximum up range (SOX-9 up 2429 times, the type II collagen up up 9463 times, and the protein polysaccharide up up 5974 times), and the relative expression of SOX-9, type II collagen and proteoglycan in the non-contact co culture group was also significantly increased. High (P0.05), but the range of gene up-regulation was smaller than that of the contact co culture group (P0.05), in which the expression of 25:75 WJMSCs/hNPCs gene expression was up to the maximum (114 times higher than SOX-9, 57 times up regulation of type II collagen, and 67 times the up regulation of protein polysaccharide). All groups of I collagen were cultured, and the gene expression of VI collagen and polysaccharide was not statistically significant (P0.05).
Conclusion: human umbilical cord Huatong mesenchymal stem cells have the potential to differentiate into nucleus pulmedulla cells. The 7 days of co culture can induce Huatong mesenchymal stem cells to differentiate into nucleus pulposus cells. The optimal cell ratio is 25:75 WJMSCs/NPCs. contact co culture, which is more beneficial to the differentiation of mesenchymal stem cells. In the nucleus pulposus of the intervertebral disc, the nucleus pulposus cells can induce the implanted cells to differentiate into nucleus pulposus cells and secrete the nucleus pulposus matrix to restore the height of the intervertebral disc. Therefore, Huatong MSCs are expected to be ideal seeds for the treatment of degenerative diseases of the intervertebral disc because of their stronger proliferation activity. Cell.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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