本文選題：人臍帶　切入點(diǎn)：間充質(zhì)干細(xì)胞　出處：《蘇州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 第一部分:人臍帶間充質(zhì)干細(xì)胞分離純化及基本生物學(xué)特性研究 目的探討人臍帶及臍靜脈內(nèi)皮下間充質(zhì)干細(xì)胞(MSC)分離、培養(yǎng)方法,并對(duì)其生物學(xué)特性進(jìn)行初步分析。 方法無菌條件下收集足月妊娠剖宮產(chǎn)新生兒臍帶,PBS洗去血管內(nèi)的殘存瘀血,用兩種方法進(jìn)行培養(yǎng):1、組織塊培養(yǎng)剔出臍帶動(dòng)靜脈,組織剪將剩余組織剪碎至1mm3大小組織塊。直接將組織塊接種于DMEM-LG培養(yǎng)基進(jìn)行原代培養(yǎng)。2、臍靜脈內(nèi)皮/內(nèi)皮下分離培養(yǎng)將洗凈的臍靜脈灌入0.1%Ⅳ型膠原酶,37℃消化20分鐘,收集消化液離心后接種培養(yǎng)。觀察并記錄其形態(tài)學(xué)變化,細(xì)胞達(dá)到融合狀態(tài)時(shí),消化傳代進(jìn)行純化和擴(kuò)增。流式細(xì)胞儀檢測(cè)MSC的細(xì)胞表面標(biāo)志。繪制細(xì)胞生長(zhǎng)曲線,流式細(xì)胞儀測(cè)定細(xì)胞周期。 結(jié)果兩種分離方法均可獲得成纖維樣MSC。組織塊貼壁法1周后于組織塊間隙可見散在分布的紡錘型細(xì)胞,3周左右達(dá)80%融合。臍靜脈膠原酶消化法,細(xì)胞接種后第二天即可見少量形態(tài)各異的貼壁細(xì)胞,散在分布。1周左右時(shí),貼壁細(xì)胞形成集落,占優(yōu)勢(shì)的是成纖維樣MSCs及少量鋪路石樣細(xì)胞,3周左右可達(dá)80%融合。經(jīng)傳代培養(yǎng)后,可得到較為純化的成纖維樣MSC,細(xì)胞約3天即可達(dá)到70%融合,需再次傳代或凍存,細(xì)胞可傳代20代以上。分別取第4代、第10代臍帶MSC行流式細(xì)胞術(shù)表面標(biāo)記檢測(cè),結(jié)果表明細(xì)胞表型無明顯差異,高表達(dá)CD49e、CD29、CD44,低表達(dá)CD106、CD11b,不表達(dá)CD34,對(duì)第4、10代臍帶MSCs生長(zhǎng)曲線進(jìn)行分析,其平均倍增時(shí)間為33.1h, 35.2h。流式細(xì)胞儀進(jìn)行細(xì)胞周期檢測(cè)表明,臍帶MSCs有60-70%細(xì)胞處于G0-G1期。 結(jié)論組織塊貼壁法和臍靜脈膠原酶消化法均可從臍帶組織分離得到成纖維樣MSCs。該細(xì)胞具有較強(qiáng)的增殖能力,細(xì)胞表面分子檢測(cè)顯示臍帶來源的間充質(zhì)干細(xì)胞表面標(biāo)志與骨髓MSC相同。 第二部分:人臍帶間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞定向誘導(dǎo)分化的研究 目的探討bFGF聯(lián)合N2添加劑對(duì)臍帶來源MSCs向神經(jīng)細(xì)胞分化的情況,進(jìn)而為臍帶MSC向神經(jīng)移植提供理論依據(jù)。 方法取第4代MSC消化后進(jìn)行細(xì)胞爬片,細(xì)胞在玻片上生長(zhǎng)3天后,即覆蓋玻片的60%左右時(shí),進(jìn)行誘導(dǎo)分化。在誘導(dǎo)劑的培養(yǎng)液中加入30ng/ml bFGF㧏10ml/L N2添加劑。每天計(jì)數(shù)神經(jīng)元樣細(xì)胞分化率;每天取分化后的細(xì)胞爬片,用免疫組化的方法檢測(cè)神經(jīng)元樣細(xì)胞特性性標(biāo)志物Nestin、NSE、NF表達(dá),并計(jì)數(shù)陽性細(xì)胞表達(dá)率,比較不同天數(shù)bFGF對(duì)神經(jīng)細(xì)胞分化能力的差異。 結(jié)果加入誘導(dǎo)液24小時(shí)后即可見部分MSCs胞體有一定的收縮,胞體成球形或不規(guī)則,胞體折光性變強(qiáng),3天后大部分MSCs出現(xiàn)明顯形態(tài)改變,有的細(xì)胞突起進(jìn)一步伸出二級(jí)或三級(jí)分支,并可見軸突、樹突及胞體的椎樣改變,在細(xì)胞密集處突起交織成網(wǎng),4天后,絕大部分細(xì)胞形態(tài)都已發(fā)生了向神經(jīng)細(xì)胞的改變,分化率達(dá)87.7%,此后部分細(xì)胞脫落、死亡,多數(shù)細(xì)胞可存活10天以上。誘導(dǎo)分化后的第2天行免疫組化即可見Nestin表達(dá)成強(qiáng)陽性,約35.03%,于第4天達(dá)到高峰70.88%,NSE和NF第7天染色陽性率分別達(dá)到50.1%、47.51%。 結(jié)論人臍帶間充質(zhì)干細(xì)胞具有向神經(jīng)細(xì)胞分化的潛能,神經(jīng)營養(yǎng)劑N2聯(lián)合bFGF能提高臍帶間充質(zhì)干細(xì)胞分化為神經(jīng)樣細(xì)胞的分化率。
[Abstract]:Part 1: isolation and purification of human umbilical cord mesenchymal stem cells and their basic biological characteristics
Objective to investigate the isolation and culture of human umbilical cord and umbilical vein mesenchyme stem cells (MSC), and to analyze its biological characteristics.
Methods collected in sterile condition of full-term pregnancy cesarean section newborn umbilical cord blood PBS, residual wash vessel, by two kinds of methods of culture: 1, tissue culture from umbilical cord vein tissue, cut the remaining tissue cut to size 1mm3 tissue. Direct tissue inoculated on DMEM-LG medium for primary culture.2, human umbilical vein endothelial / endothelial isolation will wash the umbilical vein into 0.1% collagenase digestion, 37 degrees 20 minutes, medium collected after inoculation. The centrifugal digestive fluid to observe and record the change of morphology, the cells reached confluence, digestion and passage were purified and amplified. Cell surface markers by flow cytometry the detection of MSC. Cell growth curve and cell cycle were determined by flow cytometry.
The results can be obtained two kinds of separation methods of fibroblast like MSC. tissue adherent to the tissue after 1 weeks of spindle cells scattered in space distribution, about 3 weeks to 80%. Fusion of umbilical vein collagenase digestion. Cells were inoculated second days after a small amount of different forms of adherent cells scattered in.1 week, adherent cell colony formation, the dominant is fibroblast like MSCs and a small amount of cobblestone like cells, about 3 weeks up to 80% fusion. After subculture, can obtain more purified fibroblast like MSC cells, about 3 days can reach 70% confluence, again passage or cryopreservation. Cells can be passaged for more than 20 generations respectively. The fourth generation, cytometry to detect surface markers of umbilical cord MSC tenth generation line flow, results showed no significant difference of cell phenotype, expression of CD49e, CD29, CD44, low expression of CD106 and CD11b, the expression of CD34 on the 4,10 MSCs growth curve of umbilical cord The average doubling time was 33.1h, and the cell cycle detection of 35.2h. flow cytometry showed that there were 60-70% cells in the umbilical cord MSCs in G0-G1 stage.
Conclusion tissue block adherence and umbilical vein collagenase digestion can get fibroblast like MSCs. from umbilical cord tissue. The cell has strong proliferative ability. Cell surface molecular detection shows that umbilical cord derived mesenchymal stem cells surface markers are the same as bone marrow MSC.
The second part: A Study on the directed differentiation of human umbilical cord mesenchymal stem cells into neural cells
Objective to investigate the differentiation of umbilical cord derived MSCs into neural cells by bFGF combined with N2 additive, and to provide a theoretical basis for the transplantation of umbilical cord MSC to the nerve.
Methods fourth MSC after digestion of cell smear, cell growth after 3 days in the slides, which covered the 60% coverslips, differentiation of 30ng/ml bFGF in the culture medium. Adding inducer? 10ml/L N2 additive. Differentiation of neuron like cells count rate per day; every day take differentiated cell climbing slices immunohistochemical method was used to detect characteristics of neuron like cell markers Nestin, NSE, NF expression, and the expression rate of positive cells count, the difference of different days of bFGF on the differentiation of neural cells.
The addition of inducing medium after 24 hours of the visible part of MSCs cell bodies have a certain degree of contraction, cell body into spherical or irregular, cell body index becomes strong, 3 days after the most MSCs appear obvious morphological changes, some cell protrusions further extend two or three branches, and visible axons, dendrites and somata of vertebra like change, interwoven into a network in the cell dense protrusions 4 days later, most cells have been changed into neural cells, the differentiation rate reached 87.7%, then part of cell loss, death, most cells can survive for 10 days. Second days after induction of differentiation of immune group showed that Nestin expressed strong positive, about 35.03% to fourth days to reach the peak of 70.88%, NSE and NF for seventh days the positive staining rate reached 50.1%, 47.51%.
Conclusion human umbilical cord mesenchymal stem cells have the potential to differentiate into neurons. Neurotrophic agent N2 combined with bFGF can improve the differentiation rate of umbilical cord mesenchymal stem cells into neuron like cells.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329.2

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相關(guān)期刊論文 前3條

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本文編號(hào)：1599646