第一章臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng)鑒定及體外分化研究背景臍帶間充質(zhì)干細(xì)胞（Umbilical cord-derived mesenchymal stem cells,UC-MSCs）主要來自母體妊娠后的臍帶,具有強(qiáng)大的細(xì)胞分化潛力和細(xì)胞增殖能力,其來源豐富,安全,不存在倫理問題且分化潛力較高在臨床研究中具有很高的價(jià)值。臍帶間充質(zhì)干細(xì)胞在體外可被誘導(dǎo)分化為具有神經(jīng)功能的細(xì)胞,在臍帶干細(xì)胞移植治療腦卒中等神經(jīng)損傷疾病的研究中,給予大鼠靜脈注射臍帶間充質(zhì)干細(xì)胞后,發(fā)現(xiàn)大鼠的運(yùn)動(dòng)行為明顯改善。但臍帶間充質(zhì)干細(xì)胞治療神經(jīng)損傷類疾病的治療機(jī)理仍不明確。研究目的1.體外獲取臍帶間充質(zhì)干細(xì)胞并進(jìn)行流式鑒定。2.利用細(xì)胞因子進(jìn)行臍帶間充質(zhì)干細(xì)胞的體外成神經(jīng)誘導(dǎo)分化并進(jìn)行鑒定。3.模擬體內(nèi)環(huán)境,構(gòu)建臍帶間充質(zhì)干細(xì)胞體外炎癥模型,探討共移植細(xì)胞因子對(duì)臍帶間充質(zhì)干細(xì)胞的影響。研究方法1.臍帶間充質(zhì)干細(xì)胞的分離獲取孕鼠臍帶組織并使用PBS緩沖液洗凈血液,將組織分割后加入胰酶消化并制備單細(xì)胞懸液,進(jìn)行細(xì)胞計(jì)數(shù)。2.臍帶間充質(zhì)干細(xì)胞的培養(yǎng)將計(jì)數(shù)后的細(xì)胞,置入含有DMEM/F12的培養(yǎng)基中進(jìn)行培養(yǎng)并傳代。3.臍帶間充質(zhì)... 

【文章來源】：山東大學(xué)山東省 211工程院校 985工程院校 教育部直屬院校

【文章頁(yè)數(shù)】：128 頁(yè)

【學(xué)位級(jí)別】：博士

【部分圖文】：

圖１．分離培養(yǎng)的臍帶間充質(zhì)干細(xì)胞，分別在４倍，１０倍，２０倍鏡下細(xì)胞生長(zhǎng)??

當(dāng)細(xì)胞生長(zhǎng)至７０％？８０％時(shí)，去除培養(yǎng)基并使用ＰＢＳ洗滌細(xì)胞一次，加入誘導(dǎo)培養(yǎng)??基進(jìn)行培養(yǎng)。收集培養(yǎng)到Ｐ５代體外誘導(dǎo)分化的細(xì)胞，分別進(jìn)行固定，封閉，染色??等處理后在熒光顯微鏡下進(jìn)行觀察。如圖３所示，體外誘導(dǎo)臍帶間充質(zhì)干細(xì)胞分??化，可表達(dá)神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物ＧＦＡＰ?（紅色），神經(jīng)細(xì)胞標(biāo)志物Ｔｕｊｌ?（綠色）??以及核染色ＤＡＰＩ?（藍(lán)色）。結(jié)果表明分選的臍帶間充質(zhì)干細(xì)胞在體外可被誘導(dǎo)??分化為神經(jīng)膠質(zhì)樣細(xì)胞。??２６??

入共轉(zhuǎn)染細(xì)胞因子檢測(cè)其對(duì)臍帶間充質(zhì)干細(xì)胞增殖和凋亡的影響，ＷＢ結(jié)果顯??示，共轉(zhuǎn)染細(xì)胞因子可以促進(jìn)細(xì)胞增殖相關(guān)蛋白Ｐｉ３ｋ，?Ａｋｔ的表達(dá)（Ｐ＜０．０１），??降低凋亡相關(guān)分子Ｃａｓｐａｓｅ３的表達(dá)（Ｐ＜０．０１）。如圖５Ａ－Ｂ所示??Ｐ－ＰＢｋ??Ｈ?＾?ｍ？？??Ａ?Ｐ＜３ｋ??＝換娜??Ｐ－Ａｋｔ?—?相’ｓ?ｒ＾ｉ?＾??Ａｋｔ?－－??Ｃａｓｐａｓｅ３?｜｜ｌｊ??Ｐ－ａｃｔｉｎ?ｍｍｍ?國(guó)：：?曬．：??ｎ狀挪瓶ｍ瓶預(yù)?Ｈ；?Ｈ?�。樱�??Ｐｉ３ｋ?ＡＫ１?Ｃｎｐｎｅ３??圖５．?Ａ－Ｂ）共移植細(xì)胞因子對(duì)臍帶間充質(zhì)干細(xì)胞增殖和凋亡相關(guān)分子Ｐｉ３ｋ，??ＡＫＴ，?Ｃａｓｐａｓｅ３表達(dá)的影響。??三、結(jié)論??１．

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