發(fā)布時(shí)間：2018-04-27 23:05

  本文選題：臍帶 + 華通膠間充質(zhì)干細(xì)胞�。� 參考：《第二軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：研究目的:臍帶華通膠間充質(zhì)干細(xì)胞(Wharton’s jelly-derived mesenchymal stem cells, WJMSCs)是來源于臍帶血管周圍華通膠組織的間充質(zhì)干細(xì)胞,具有干細(xì)胞的基本特征,即該細(xì)胞具有自我更新及向不同組織或細(xì)胞分化的能力。本研究從人臍帶中分離提取華通膠間充質(zhì)干細(xì)胞,觀察其細(xì)胞形態(tài),檢測其增殖能力、細(xì)胞免疫表型及端粒酶活性,鑒定分離提取的細(xì)胞具有干細(xì)胞特性,為下一步通過細(xì)胞共培養(yǎng)的方法定向誘導(dǎo)分化為髓核細(xì)胞奠定基礎(chǔ)。 研究方法:取足月產(chǎn)健康新生兒臍帶,分離華通膠組織,剪碎后用膠原酶和胰蛋白酶消化,提取細(xì)胞后加DMEM/F12培養(yǎng)基和胎牛血清培養(yǎng),細(xì)胞融合達(dá)到80-90%時(shí)進(jìn)行傳代培養(yǎng)。觀察細(xì)胞形態(tài),測定不同代次的細(xì)胞增殖能力。取第3代細(xì)胞行端粒酶活性分析及流式細(xì)胞學(xué)檢測細(xì)胞免疫表型(CD34/CD45/CD105/CD73/CD90/HLA-DR及HLA-ABC)。 結(jié)果:原代培養(yǎng)第3-5天可見部分細(xì)胞貼壁生長,形態(tài)為梭形和多角形,1周后形成集落,2周時(shí)細(xì)胞融合達(dá)到90%。傳代后細(xì)胞為梭形,形態(tài)一致,細(xì)胞呈漩渦狀生長,細(xì)胞增殖能力強(qiáng),5-6天即可進(jìn)行傳代,傳至16代增殖能力未見下降。端粒酶活性測定為陽性,流式細(xì)胞分析細(xì)胞免疫表型結(jié)果表明,CD90/CD105/CD73/HLA-ABC陽性,CD34/CD45/HLA-DR陰性。 結(jié)論:人臍帶含有豐富的間充質(zhì)干細(xì)胞,通過酶消化法可從臍帶華通膠中提取間充質(zhì)干細(xì)胞,該細(xì)胞呈梭形,漩渦狀生長,增殖能力強(qiáng)。流式細(xì)胞分析檢測細(xì)胞免疫表型顯示細(xì)胞表達(dá)間充質(zhì)干細(xì)胞免疫表型,不表達(dá)造血干細(xì)胞免疫表型,其端粒酶活性陽性。華通膠間充質(zhì)干細(xì)胞由于其更強(qiáng)的增殖活性、更好擴(kuò)增能力及更易于獲取而有望成為組織工程和細(xì)胞治療技術(shù)的理想種子細(xì)胞。 研究背景和目的:椎間盤退變性疾病(degenerative disc disease, DDD)是常見病多發(fā)病,其引起的下腰痛嚴(yán)重影響人們的工作及生活。目前的治療方法主要是保守治療和外科治療,雖能暫時(shí)緩解臨床癥狀,但遠(yuǎn)期療效并不理想,并可能會(huì)發(fā)生并發(fā)癥。近年來,以修復(fù)椎間盤退變恢復(fù)椎間盤高度為目的的組織工程技術(shù)和細(xì)胞治療技術(shù)逐漸被認(rèn)為是具有前景的治療方法。然而,無論是組織工程技術(shù)還是細(xì)胞治療技術(shù),在椎間盤退變性疾病的治療中尚缺乏理想的種子細(xì)胞。本實(shí)驗(yàn)探討人臍帶華通膠間充質(zhì)干細(xì)胞(Wharton’s jelly-derived mesenchymal stem cells, WJMSCs)向髓核細(xì)胞(nucleus pulposus cells, NPCs)的分化潛能,希望為椎間盤退變性疾病的治療提供一種理想的種子細(xì)胞來源。 方法:取人正常足月產(chǎn)嬰兒臍帶,分離消化臍帶華通膠組織,收集培養(yǎng)華通膠間充質(zhì)干細(xì)胞;取人正常未退變椎間盤(T12-L1),酶消化法分離培養(yǎng)髓核細(xì)胞。取穩(wěn)定增殖的第3代華通膠間充質(zhì)干細(xì)胞和髓核細(xì)胞進(jìn)行共培養(yǎng)。CFSE標(biāo)記華通膠間充質(zhì)干細(xì)胞。使用帶有插入層的Transwell培養(yǎng)板進(jìn)行非接觸式共培養(yǎng),插入層具有0.4μm高密度孔徑;下層接種華通膠間充質(zhì)干細(xì)胞,上層接種髓核細(xì)胞。普通六孔板進(jìn)行接觸式共培養(yǎng)。根據(jù)共培養(yǎng)細(xì)胞比例不同分為3組(25:75 WJMSCs/hNPCs,50:50 WJMSCs/hNPCs,75:25 WJMSCs/hNPCs),于共培養(yǎng)1周后,利用MoFlo高速流式細(xì)胞分選儀分選收集接觸式共培養(yǎng)的華通膠間充質(zhì)干細(xì)胞。提取華通膠間充質(zhì)干細(xì)胞總RNA進(jìn)行反轉(zhuǎn)錄獲得cDNA,利用Real-Time PCR方法檢測其蛋白多糖、Ⅰ型膠原、Ⅱ型膠原、VI型膠原、SOX-9及多能聚糖等基因表達(dá),管家基因GAPDH作為內(nèi)參,單獨(dú)培養(yǎng)的華通膠間充質(zhì)干細(xì)胞作為對(duì)照,利用2-ΔΔCt方法計(jì)算基因相對(duì)表達(dá)變化。 結(jié)果:細(xì)胞分選點(diǎn)陣圖顯示CFSE標(biāo)記的WJMSCs與無熒光標(biāo)記的髓核細(xì)胞完全分離并分選。分選后細(xì)胞進(jìn)行Real-Time PCR檢測華通膠間充質(zhì)干細(xì)胞相對(duì)基因表達(dá)變化。經(jīng)過7天共培養(yǎng),結(jié)果顯示,接觸式共培養(yǎng)各組華通膠間充質(zhì)干細(xì)胞的SOX-9、Ⅱ型膠原和蛋白多糖相對(duì)表達(dá)都有顯著提高(P0.05),其中25:75 WJMSCs/hNPCs組上調(diào)幅度最大(SOX-9上調(diào)2429倍,Ⅱ型膠原上調(diào)9463倍,蛋白多糖上調(diào)5974倍);非接觸式共培養(yǎng)組各組華通膠間充質(zhì)干細(xì)胞的SOX-9、Ⅱ型膠原和蛋白多糖相對(duì)表達(dá)也有顯著提高(P0.05),但基因上調(diào)幅度比接觸式共培養(yǎng)組小(P0.05),其中25:75 WJMSCs/hNPCs組基因表達(dá)上調(diào)幅度最大(SOX-9上調(diào)114倍,Ⅱ型膠原上調(diào)57倍,蛋白多糖上調(diào)67倍)。共培養(yǎng)各組I型膠原、VI型膠原和多能聚糖的基因基因表達(dá)改變無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論:人臍帶華通膠間充質(zhì)干細(xì)胞具有向髓核細(xì)胞分化潛能。細(xì)胞共培養(yǎng)7天能夠誘導(dǎo)華通膠間充質(zhì)干細(xì)胞分化為髓核細(xì)胞,最優(yōu)細(xì)胞比例為25:75 WJMSCs/NPCs。接觸式共培養(yǎng)更有利于間充質(zhì)干細(xì)胞的分化,可推測華通膠間充質(zhì)干細(xì)胞移植入椎間盤髓核中,其中的髓核細(xì)胞能夠誘導(dǎo)植入的細(xì)胞分化為髓核細(xì)胞并分泌髓核基質(zhì),以恢復(fù)椎間盤高度。因此,華通膠間充質(zhì)干細(xì)胞由于其更強(qiáng)的增殖活性、更好擴(kuò)增能力及更易于獲取而有望成為治療椎間盤退變性疾病的理想種子細(xì)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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