本文選題：臍血 + 間充質(zhì)干細(xì)胞�。� 參考：《蚌埠醫(yī)學(xué)院》2012年碩士論文

【摘要】：背景：周圍神經(jīng)損傷缺損的臨床治療是外科領(lǐng)域尚未解決的世界難題。自體神經(jīng)移植是治療周圍神經(jīng)缺損的標(biāo)準(zhǔn)方法，但會(huì)造成供區(qū)神經(jīng)損傷。通過(guò)組織工程化人工神經(jīng)修復(fù)周圍神經(jīng)缺損是目前臨床治療研究的熱點(diǎn)。人工神經(jīng)的種子細(xì)胞為許旺細(xì)胞(Schwann cells,SCs)，其來(lái)源問(wèn)題尚未解決。人臍血間充質(zhì)干細(xì)胞(Human Umbilical Cord Blood Mesenchymal Stem Cells,HUCBMSCs)作為人骨髓間充質(zhì)干細(xì)胞（Mesenchymal stem cells,MSCs）的一種替代來(lái)源，在體外誘導(dǎo)培養(yǎng)條件下可以向類SCs分化，但還需進(jìn)一步進(jìn)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(dǎo)HUCBMSCs定向分化為類SCs的方法，并優(yōu)化誘導(dǎo)方案；進(jìn)一步將誘導(dǎo)成功的類SCs復(fù)合去細(xì)胞神經(jīng)基膜管體外共培養(yǎng)，示蹤。 方法：取人臍血標(biāo)本，6%羥乙基淀粉(Hetastarch, HES)沉降紅細(xì)胞，再使用人淋巴細(xì)胞分離液Ficoll（密度為1.077g/mL）分離臍血單個(gè)核細(xì)胞，在Mesencult完全培養(yǎng)基中培養(yǎng)，流式細(xì)胞儀對(duì)培養(yǎng)細(xì)胞進(jìn)行表型測(cè)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(xì)胞進(jìn)行定向誘導(dǎo)分化，免疫細(xì)胞化學(xué)法、RT-PCR、Western Blotting等方法對(duì)誘導(dǎo)后的細(xì)胞進(jìn)行神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物S100b、GFAP、P75鑒定；采用反復(fù)凍融振蕩洗滌法制備去細(xì)胞坐骨神經(jīng)基膜管，將誘導(dǎo)后類SCs使用微量注射器注入去細(xì)胞基膜管中置入六孔板體外培養(yǎng)0、1、2W，然后取出標(biāo)本行HE染色檢測(cè)。 結(jié)果：分離培養(yǎng)出的細(xì)胞低表達(dá)或不表達(dá)CD34，高表達(dá)CD44、CD73，免疫細(xì)胞化學(xué)檢測(cè)發(fā)現(xiàn)，誘導(dǎo)4d后幾乎所有細(xì)胞都表達(dá)神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物S100b(98±1.63%)、GFAP (95.33±2.05%)和P75(90.67±1.7%)，其中較多細(xì)胞表現(xiàn)出SCs經(jīng)典的雙極、梭形形態(tài)，同時(shí)RT-PCR、Western Blotting在基因和蛋白水平證明誘導(dǎo)后細(xì)胞表達(dá)神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物S100b、GFAP和P75，而未分化細(xì)胞不表達(dá)；HE染色檢測(cè)結(jié)果顯示去細(xì)胞基膜管中細(xì)胞可以存活并有遷移趨勢(shì)。 結(jié)論： 1.人臍血中可以成功分離出MSCs，HUCBMSCs在體外具有較強(qiáng)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(xì)胞。 3.類SCs可以在去細(xì)胞基膜管中存活、遷移，，有望成為新的種子細(xì)胞來(lái)源且為下一步人工神經(jīng)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(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.28

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