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  本文選題：臍血 + 間充質干細胞　； 參考：《蚌埠醫(yī)學院》2012年碩士論文

【摘要】：背景：周圍神經損傷缺損的臨床治療是外科領域尚未解決的世界難題。自體神經移植是治療周圍神經缺損的標準方法，但會造成供區(qū)神經損傷。通過組織工程化人工神經修復周圍神經缺損是目前臨床治療研究的熱點。人工神經的種子細胞為許旺細胞(Schwann cells,SCs)，其來源問題尚未解決。人臍血間充質干細胞(Human Umbilical Cord Blood Mesenchymal Stem Cells,HUCBMSCs)作為人骨髓間充質干細胞（Mesenchymal stem cells,MSCs）的一種替代來源，在體外誘導培養(yǎng)條件下可以向類SCs分化，但還需進一步進行研究。 目的：本研究旨在建立HUCBMSCs的分離、培養(yǎng)、純化及鑒定體系，探討體外誘導HUCBMSCs定向分化為類SCs的方法，并優(yōu)化誘導方案；進一步將誘導成功的類SCs復合去細胞神經基膜管體外共培養(yǎng)，示蹤。 方法：取人臍血標本，6%羥乙基淀粉(Hetastarch, HES)沉降紅細胞，再使用人淋巴細胞分離液Ficoll（密度為1.077g/mL）分離臍血單個核細胞，在Mesencult完全培養(yǎng)基中培養(yǎng)，流式細胞儀對培養(yǎng)細胞進行表型測定，向成骨、成脂方向分化鑒定其多向分化的潛能；取第3代細胞進行定向誘導分化，免疫細胞化學法、RT-PCR、Western Blotting等方法對誘導后的細胞進行神經膠質細胞標志物S100b、GFAP、P75鑒定；采用反復凍融振蕩洗滌法制備去細胞坐骨神經基膜管，將誘導后類SCs使用微量注射器注入去細胞基膜管中置入六孔板體外培養(yǎng)0、1、2W，然后取出標本行HE染色檢測。 結果：分離培養(yǎng)出的細胞低表達或不表達CD34，高表達CD44、CD73，免疫細胞化學檢測發(fā)現(xiàn)，誘導4d后幾乎所有細胞都表達神經膠質細胞標志物S100b(98±1.63%)、GFAP (95.33±2.05%)和P75(90.67±1.7%)，其中較多細胞表現(xiàn)出SCs經典的雙極、梭形形態(tài)，同時RT-PCR、Western Blotting在基因和蛋白水平證明誘導后細胞表達神經膠質細胞標志物S100b、GFAP和P75，而未分化細胞不表達；HE染色檢測結果顯示去細胞基膜管中細胞可以存活并有遷移趨勢。 結論： 1.人臍血中可以成功分離出MSCs，HUCBMSCs在體外具有較強的增殖、自我更新能力，還具有多向分化潛能。 2. HUCBMSCs在體外可以定向分化為類許旺細胞。 3.類SCs可以在去細胞基膜管中存活、遷移，，有望成為新的種子細胞來源且為下一步人工神經移植提供依據(jù)。
[Abstract]:Background: the clinical treatment of peripheral nerve injury defect is an unsolved world problem in the field of surgery.Autologous nerve transplantation is the standard method for the treatment of peripheral nerve defect, but it can cause nerve injury in donor area.The repair of peripheral nerve defect by tissue engineering artificial nerve is the focus of clinical treatment.The seed cells of artificial nerve were Schwann cells and SCsN.Human Umbilical Cord Blood Mesenchymal Stem cells (HUCBMSCs), as an alternative source of mesenchymal stem cells (MSCs), can differentiate into SCs like stem cells in vitro, but further research is needed.Objective: to establish the system of isolation, culture, purification and identification of HUCBMSCs, to explore the method of inducing HUCBMSCs to differentiate into SCs in vitro, and to optimize the induction scheme.Furthermore, the successfully induced SCs-like neural basement tube was co-cultured in vitro.Methods: umbilical cord blood mononuclear cells were isolated from human umbilical cord blood samples from 6% Hetastarch-Hetsea (HES-) erythrocytes. The mononuclear cells from human umbilical cord blood were isolated by Ficolll (density 1.077g / mL) and cultured in Mesencult culture medium. The phenotypes of cultured cells were determined by flow cytometry (FCM).The differentiation potential of the cells in the direction of osteogenesis and adipogenesis was evaluated, and the third generation cells were selected for directional differentiation, and the glial marker S100bGFAPP75 was identified by immunocytochemistry and RT-PCRX Western Blotting.The acellular sciatic nerve basal membrane tube was prepared by repeated freeze-thaw oscillatory washing method. The induced SCs was injected into the acellular basal membrane tube with a micro syringe and cultured in vitro with a six-hole plate. The specimens were examined by HE staining.Results: after 4 days of induction, almost all of the cells expressed low or no CD34, and high expression of CD44-tir CD73. Almost all the cells expressed glial cell marker S100b(98 鹵1.63 + GFAP95.33 鹵2.05 and P75 + 90.67 鹵1.7, among which more cells showed the classic bipolar of SCs.At the same time, the expression of glial cell markers S100bGFAP and P75 was confirmed by RT-PCR Western Blotting at the level of gene and protein. The results of HE staining showed that the cells in the acellular basement membrane tube could survive and migrate.Conclusion:1.MSCs can be successfully isolated from human umbilical cord blood and have strong proliferation, self-renewal ability and multidirectional differentiation potential in vitro.2.HUCBMSCs can differentiate into Schwan-like cells in vitro.3.SCs like can survive and migrate in acellular basement membrane tube, which may become a new seed cell source and provide the basis for the next artificial nerve transplantation.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329.28

【參考文獻】

相關期刊論文 前10條

1 寧仁德;周建生;;改良凍融制備成年兔周圍神經去細胞材料的研究[J];蚌埠醫(yī)學院學報;2009年04期

2 王婧,劉開彥;人骨髓間充質干細胞體外分化為雪旺樣細胞的方法[J];北京大學學報(醫(yī)學版);2003年02期

3 丁志清;范偉杰;黃長征;;組織工程神經橋接物的研究進展[J];中外醫(yī)療;2009年22期

4 丁海,胡汝麒,周建生;大鼠坐骨神經去細胞基膜管的組織形態(tài)學研究[J];解剖與臨床;2005年02期

5 肖玉周;周維永;周建生;;體外誘導兔骨髓間充質干細胞分化為類許旺細胞的初步研究[J];解剖與臨床;2008年01期

6 范秀波;劉天慶;郝永杰;劉洋;馬學虎;崔占峰;;臍帶血來源間充質干細胞體外分離培養(yǎng)條件的優(yōu)化[J];生物化學與生物物理進展;2008年08期

7 劉岱;趙玉明;晏曉青;崔雅寧;王曉軍;徐軍;;人臍帶血間充質干細胞分離培養(yǎng)體系的優(yōu)化篩選[J];中國組織工程研究與臨床康復;2009年10期

8 王劍,杜勁松,張信英,王德生;成人骨髓基質干細胞體外誘導為許旺細胞的實驗研究[J];中風與神經疾病雜志;2003年06期

9 顧玉東;周圍神經缺損的基本概念與治療原則[J];中華手外科雜志;2002年03期

10 王建云,劉小林,朱家愷,鄧宇斌,向劍平,李佛保;改良法體外誘導骨髓基質干細胞分化為類許旺細胞[J];中華顯微外科雜志;2004年03期

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