【摘要】： 目的:近年來研究表明骨髓間充質(zhì)干細(xì)胞(Bone marrow stromal cells ,BMSCs)可以體外誘導(dǎo)分化為神經(jīng)細(xì)胞,因此BMSCs有望成為神經(jīng)細(xì)胞移植的種子細(xì)胞來治療神經(jīng)系統(tǒng)疾病,但BMSCs定向誘導(dǎo)分化率低,且分化后的細(xì)胞存活時(shí)間短而不能有效增殖以滿足移植的需要。如何能夠找到穩(wěn)定高效的誘導(dǎo)方法及提高誘導(dǎo)后神經(jīng)細(xì)胞的存活率是需要解決的一個(gè)關(guān)鍵問題。單唾液酸四己糖神經(jīng)節(jié)苷脂(monosialotetrahexosyl ganglioside ,GM1)在神經(jīng)細(xì)胞的發(fā)育、分化、修復(fù)和信號(hào)轉(zhuǎn)導(dǎo)中有相當(dāng)重要的作用。研究表明, GM1有誘導(dǎo)BMSCs分化為神經(jīng)元樣細(xì)胞的作用,本文就不同濃度GM1對(duì)BMSCs的誘導(dǎo)分化作用作一探討。 方法:以全骨髓培養(yǎng)法分離成人BMSCs,進(jìn)行原代和傳代培養(yǎng)。用流式細(xì)胞儀檢測(cè)BMSCs表面CD44, CD45, CD90,CD105的表達(dá)以鑒定純度。以含有10μg/ml、60μg/ml、90μg/ml GM1的DMEM無血清培養(yǎng)基誘導(dǎo)第五代的BMSCs,陰性對(duì)照組不加任何誘導(dǎo)劑。至誘導(dǎo)后7d觀察細(xì)胞形態(tài)變化并計(jì)數(shù),誘導(dǎo)后6h以免疫細(xì)胞化學(xué)法測(cè)定各組細(xì)胞神經(jīng)細(xì)胞特異性表面標(biāo)志神經(jīng)元特異性烯醇化酶(NSE)、神經(jīng)元特異性微管相關(guān)蛋白(MAP-2),以鑒定是否誘導(dǎo)為神經(jīng)元樣細(xì)胞并計(jì)算分化百分率。各組間分化百分率進(jìn)行比較和統(tǒng)計(jì)學(xué)處理。并用MTT法測(cè)定誘導(dǎo)后0.5h、6h、24h、3d細(xì)胞生長(zhǎng)狀況。 結(jié)果:細(xì)胞接種2天后給予PBS沖洗,全量換液可見貼壁細(xì)胞呈集落狀生長(zhǎng)。8～10天后細(xì)胞可基本鋪滿瓶底,為均一梭狀細(xì)胞。以1:3比例傳代,傳代后1天左右細(xì)胞可以恢復(fù)活力并貼壁生長(zhǎng),5～6天后可鋪滿瓶底。傳代至4～5代時(shí)細(xì)胞形態(tài)比較均一,混雜細(xì)胞較少。流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面標(biāo)志CD44(+),CD90(+), CD105(+),CD45(-),基本證實(shí)BMSCs的純度。BMSCs經(jīng)預(yù)誘導(dǎo)后,細(xì)胞體積增大,形態(tài)變長(zhǎng)。加入誘導(dǎo)液30～40 min后,細(xì)胞開始發(fā)生形態(tài)變化,胞體向內(nèi)收縮,呈球形或圓形,并向周圍長(zhǎng)出突起,有立體感,折光性增強(qiáng);誘導(dǎo)4 h后,細(xì)胞形態(tài)發(fā)生明顯改變,胞體進(jìn)一步收縮形成突起,胞體立體感增強(qiáng),周圍出現(xiàn)光暈,可見到很多雙極和多極細(xì)胞;6 h后大多數(shù)細(xì)胞形成錐形、三角形等不規(guī)則形狀,突起增多變長(zhǎng),細(xì)胞間突起相互連接,交錯(cuò)成網(wǎng),呈現(xiàn)典型的神經(jīng)細(xì)胞樣形態(tài)。到誘導(dǎo)后3天,神經(jīng)元樣細(xì)胞增多不明顯。5天后有少量細(xì)胞懸浮死亡,突起延長(zhǎng)多交織成網(wǎng)狀;7天時(shí)細(xì)胞形態(tài)與5天時(shí)基本相同,但懸浮死亡細(xì)胞較多。陰性對(duì)照組細(xì)胞仍呈均一長(zhǎng)梭狀細(xì)胞,但細(xì)胞生長(zhǎng)速度明顯減慢。細(xì)胞誘導(dǎo)6 h免疫細(xì)胞化學(xué)檢測(cè)不同濃度GM1組和陰性對(duì)照組細(xì)胞均有MAP-2,NSE表達(dá),但未檢測(cè)到GFAP的表達(dá)。10μg/ml、60μg/ml、90μg/ml GM1組NSE、MAP-2細(xì)胞陽(yáng)性率均較陰性對(duì)照組明顯高(P0.05)。組間比較:60μg/ml、90μg/ml組NSE、MAP-2細(xì)胞陽(yáng)性率均較10μg/ml組高(P0.05),60μg/ml、90μg/ml組細(xì)胞陽(yáng)性率差異不顯著(P0.05)。MTT結(jié)果顯示經(jīng)GM1誘導(dǎo)后的神經(jīng)元樣細(xì)胞比陰性對(duì)照組生長(zhǎng)狀態(tài)好。 結(jié)論 1、不同濃度GM1均具有誘導(dǎo)BMSCs向神經(jīng)細(xì)胞分化的作用,誘導(dǎo)6 h后BMSCs向神經(jīng)細(xì)胞分化的分化率最高。 2、中、高濃度GM1誘導(dǎo)BMSCs分化為神經(jīng)元樣細(xì)胞的百分率較高,誘導(dǎo)后的神經(jīng)元樣細(xì)胞生長(zhǎng)狀態(tài)較好。
[Abstract]:Objective: in recent years, studies have shown that Bone marrow stromal cells (BMSCs) can be induced to differentiate into nerve cells in vitro, so BMSCs is expected to be the seed cell of the nerve cell transplantation to treat nervous system diseases, but the differentiation rate of BMSCs is low, and the survival time of the differentiated cells is short and can not be effectively increased. Monosialotetrahexosyl Ganglioside (GM1), a single sialic acid four hexose Ganglioside (GM1), is of considerable importance in the development, differentiation, repair and signal transduction of nerve cells. The research shows that GM1 has the function of inducing BMSCs to differentiate into neuron like cells. In this paper, the induction and differentiation of BMSCs with different concentrations of GM1 are discussed.
Methods: full bone marrow culture was used to isolate adult BMSCs and carry out primary and subculture. Flow cytometry was used to detect the expression of CD44, CD45, CD90 and CD105 on the surface of BMSCs to identify the purity. Fifth generation BMSCs was induced by the DMEM serum-free medium containing 10 mu g/ml, 60 mu g/ml and 90 u g/ml GM1. Cell morphological changes were observed and counted. After induction, 6h was induced by immunocytochemical method to determine neuron specific enolase (NSE) and neuron specific microtubule related protein (MAP-2), to identify whether the neuron like cells were induced as neuron like cells and to calculate the percentage of differentiation. The percentage of differentiation among groups was compared. The growth status of 0.5h, 6h, 24h and 3D after induction was determined by MTT.
Results: after 2 days of inoculation, the cells were given PBS irrigation, and the full volume of the cells could be seen that the adherent cells were colonialized for.8 to 10 days. The cells could be basically covered with the bottom of the bottle. The cells were homogeneous fusiform cells. The cells were passaged in the 1:3 ratio. The cells could be rejuvenated and adhered to the wall for about 1 days after the passage. After 5~6 days, the cells could be filled with the bottom of the bottle. The cell morphology was compared to the 4~5 generation. The cell surface markers CD44 (+), CD90 (+), CD105 (+) and CD45 (-) were detected by flow cytometry, which basically confirmed that the cell volume increased and the morphology became longer after preinduction of the purity of BMSCs. After adding the inducer 30~40 min, the cells began to produce morphologic changes, the cell bodies contracted inward, and they were spherical or round, and protruded around them. After 4 h, the cell morphology changes obviously, the cell body is further contracted to form the protuberance, the body stereoscopic sense is enhanced, the halo around the cell appears, and a lot of bipolar and multipolar cells can be seen. After 6 h, most of the cells form a cone, triangle and other irregular shapes, the protuberances grow and grow, the intercellular protuberances are connected to each other. In the 3 day after induction, a small number of cell suspension died and the protuberance was more interwoven into the net, and the cell morphology was basically the same at the time of 7 days, but the number of dead cells was more in suspension, but the cell growth was still a long spindle cell, but the cell growth was growing at 3 days after induction. The cell induced 6 h immunocytochemistry test showed that the cells of different concentration GM1 and negative control group had MAP-2, NSE expression, but the expression of GFAP was not.10 g/ml, 60 mu g/ml, 90 u g/ml GM1 NSE, and the positive rate of MAP-2 cells was higher than that of the negative control group. The rate of sex was higher than that of the 10 g/ml group (P0.05), 60 g/ml, and the positive rate of cell positive rate in 90 mu g/ml group was not significant (P0.05).MTT results showed that the growth state of neuron like cells induced by GM1 was better than that of the negative control group.
conclusion
1, different concentrations of GM1 all have the effect of inducing BMSCs to differentiate into neurons. After 6 h induction, BMSCs has the highest differentiation rate to neural cells.
2, medium, high concentration of GM1 induced BMSCs to differentiate into neuron like cells, and the neuron like cells grew better after induction.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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