【摘要】： 本實(shí)驗(yàn)探討了骨髓間充質(zhì)干細(xì)胞(MSCs)體外原代的培養(yǎng)方法,及MSCs傳代后在某些誘導(dǎo)條件下,分化為胰島樣細(xì)胞的實(shí)驗(yàn)方法。通過免疫細(xì)胞化學(xué)實(shí)驗(yàn)及動物實(shí)驗(yàn),驗(yàn)證了MSCs在體外成功誘導(dǎo)分化為胰島樣細(xì)胞,并且可以修復(fù)壞死胰腺細(xì)胞。 在MSCs的原代培養(yǎng)過程中,采用高速離心法及貼壁法分離純化細(xì)胞。選用含10%胎牛血清(FBS)的低糖DMEM培養(yǎng)基作為擴(kuò)增細(xì)胞的培養(yǎng)基。選擇第三代狀態(tài)良好的MSCs,進(jìn)行誘導(dǎo)實(shí)驗(yàn)。采用三種不同的誘導(dǎo)途徑,對誘導(dǎo)過程進(jìn)行比較。發(fā)現(xiàn)最佳誘導(dǎo)方法如下:向生長狀態(tài)穩(wěn)定的第三代MSCs內(nèi)加入內(nèi)含堿性成纖維細(xì)胞生長因子(bFGF)(10ng/ml),表皮細(xì)胞生長因子(EGF)(10ng/ml)的無血清的LG-DMEM培養(yǎng)基,培養(yǎng)6～7d;然后,換用無血清的高糖DMEM培養(yǎng)基,添加胰腺條件培養(yǎng)液、胰島素(25mg/ml)、地塞米松(10μg/l)、尼克酰胺(10mM/l),繼續(xù)培養(yǎng)6～7d;在顯微鏡下觀察MSCs,可以看到細(xì)胞形態(tài)漸發(fā)生改變,細(xì)胞由原來的梭形漸變?yōu)閳A形及不規(guī)則形,最后細(xì)胞聚集成團(tuán)、懸浮逐漸增多。 經(jīng)過細(xì)胞鑒定實(shí)驗(yàn),證明已成功誘導(dǎo)分化為胰島樣細(xì)胞。這一研究,為臨床胰腺類疾病(如胰腺炎及糖尿病等)的治療提供了一個(gè)新的方向。
[Abstract]:The primary culture of bone marrow mesenchymal stem cells (MSCs) in vitro and the differentiation of MSCs into islet like cells were studied. The results of immunocytochemistry and animal experiments showed that MSCs could be successfully induced to differentiate into islet like cells in vitro and could repair necrotic pancreatic cells. During the primary culture of MSCs, the cells were isolated and purified by high speed centrifugation and adherent method. The low sugar DMEM medium containing 10% fetal bovine serum (FBS) was used as the medium for cell expansion. The third generation MSCs, with good state was selected for induction experiment. Three different ways of induction were used to compare the induction process. The results showed that the optimal induction methods were as follows: adding serum-free LG-DMEM medium containing basic fibroblast growth factor (bFGF) (10ng/ml) and epidermal growth factor (EGF) (10ng/ml) into the third generation MSCs of stable growth state, and culturing for 6 to 7 days. In addition to serum-free high-glucose DMEM medium, supplemented with pancreatic conditioned medium, insulin (25mg/ml), dexamethasone (10 渭 g / 1), and nicotinamide (10mM/l), the cells were cultured for 6 to 7 days. The cells gradually changed from fusiform to round and irregular. Finally, the cells gathered into clusters and gradually increased. The differentiation into islet-like cells was successfully induced by cell identification. This study provides a new direction for the treatment of pancreatic diseases such as pancreatitis and diabetes.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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