【摘要】：目的：研究人臍帶間充質(zhì)干細(xì)胞(MSCs)的生物學(xué)特性及丹參等藥物誘導(dǎo)MSCs向神經(jīng)細(xì)胞分化的可能性，為神經(jīng)細(xì)胞移植、臍帶組織庫的建立及中藥丹參作為神經(jīng)誘導(dǎo)劑提供理論依據(jù)。方法：從人臍帶華爾通膠(Wharton's jelly)分離培養(yǎng)MSCs，培養(yǎng)、檢測人臍帶來源的MSCs的細(xì)胞表面標(biāo)記，評價其生物學(xué)特性；丹參和β—巰基已醇誘導(dǎo)人臍帶來源的MSCs向神經(jīng)細(xì)胞分化；用免疫細(xì)胞化學(xué)方法檢測神經(jīng)細(xì)胞相關(guān)蛋白標(biāo)記的表達(dá)；用光鏡、掃描電子顯微鏡監(jiān)測丹參誘導(dǎo)后細(xì)胞形態(tài)改變；透射電子顯微鏡觀察丹參誘導(dǎo)后細(xì)胞超微結(jié)構(gòu)改變；用共聚焦顯微鏡分析丹參誘導(dǎo)前后細(xì)胞內(nèi)DNA、RNA含量改變；用RT-PCR檢測細(xì)胞神經(jīng)相關(guān)基因pleiotrophin和神經(jīng)干細(xì)胞的標(biāo)記巢蛋白(nestin)的表達(dá)。結(jié)果：從人臍帶華爾通膠分離、培養(yǎng)的貼壁細(xì)胞，體外生長形態(tài)類似于成纖維細(xì)胞，，可以維持在未分化狀態(tài)穩(wěn)定增殖，體外增殖超過10代，細(xì)胞凍存一個月后復(fù)蘇，生長特點與凍存前基本一致。用流式細(xì)胞儀檢測，這類細(xì)胞表達(dá)MSCs的表面標(biāo)記CD29、CD44、CD59、CD90、CD105，不表達(dá)造血細(xì)胞表面標(biāo)記CD14、CD33、CD34、CD28、CD45、CD117．和與移植免疫排斥相關(guān)的表面標(biāo)記CD80(B7-1)、CD86(B7-2)、CD40。丹參和β—巰基已醇均可誘導(dǎo)人臍帶MSCs向神經(jīng)樣細(xì)胞分化，分化的細(xì)胞表達(dá)神經(jīng)干細(xì)胞的標(biāo)記巢蛋白(nestin)，神經(jīng)元的蛋白標(biāo)記β-Ⅲ類神經(jīng)微管(β-TubulinⅢ)和神經(jīng)微絲(NF)，以及神
[Abstract]:Aim: to study the biological characteristics of human umbilical cord mesenchymal stem cells (MSCs) and the possibility of inducing MSCs to differentiate into nerve cells by drugs such as Salvia miltiorrhiza (Salvia miltiorrhiza), so as to provide theoretical basis for nerve cell transplantation, establishment of umbilical cord tissue bank and Chinese medicine Danshen as nerve inducer. Methods: MSCs, was isolated and cultured from human umbilical cord (Wharton's jelly), and the cell surface markers of MSCs derived from human umbilical cord were detected, and its biological characteristics were evaluated, and the differentiation of MSCs from human umbilical cord into neural cells was induced by Salvia miltiorrhiza and 尾 -mercaptohexanol. Immunocytochemistry was used to detect the expression of neuron-associated protein, light microscope and scanning electron microscope were used to monitor the morphological changes of cells induced by Salvia miltiorrhiza, and the ultrastructure of cells induced by Salvia miltiorrhiza was observed by transmission electron microscope. The changes of DNA,RNA content in cells before and after salvia miltiorrhiza induction were analyzed by confocal microscopy, and the expression of pleiotrophin and labeled nestin (nestin) in neural stem cells were detected by RT-PCR. Results: the adherent cells were isolated from human umbilical cord and cultured in vitro. The growth morphology of adherent cells was similar to that of fibroblasts. The cells could proliferate steadily in undifferentiated state and proliferate in vitro for more than 10 generations. The cells were resuscitated after one month of cryopreservation. The growth characteristics were basically consistent with those before freezing. By flow cytometry, CD29,CD44,CD59,CD90,CD105, a surface marker expressing MSCs, did not express hematopoietic cell surface marker CD14,CD33,CD34,CD28,CD45,CD117. and CD80 (B7-1) CD86 (B7-2) CD40, which was associated with allograft immune rejection. Salvia miltiorrhiza and 尾 -mercaptohexanol can induce human umbilical cord MSCs to differentiate into neuron-like cells. Differentiated cells express neural stem cells labeled with nestin (nestin), neurons labeled with 尾-鈪

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