本文關(guān)鍵詞：優(yōu)化的小鼠STO細(xì)胞系重編程多潛能干細(xì)胞的建立　出處：《西北農(nóng)林科技大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 慢病毒載體 STO細(xì)胞 iPSCs 干細(xì)胞

【摘要】：誘導(dǎo)多功能干細(xì)胞（induced pluripotent stem cells，iPSCs）是指通過外源導(dǎo)入一些與維持胚胎干細(xì)胞（embryonic stem cell，ES細(xì)胞）多能性相關(guān)的基因，，使終末分化的成體細(xì)胞重編程為多能性干細(xì)胞，獲得多向分化潛能和自我更新能力。iPS細(xì)胞目前可以用來替代干細(xì)胞治療人類疾病，在分子細(xì)胞生物學(xué)、發(fā)育生物學(xué)、新藥物的篩選和研發(fā)、臨床細(xì)胞替代治療等方面有著廣闊的發(fā)展空間，但是在誘導(dǎo)效率和安全性方面存在一定不足，研究人員在不斷尋找高效、安全的方法來彌補(bǔ)iPS細(xì)胞的缺陷。 本研究采用連接有Oct4，Sox2，Klf4，c-Myc四個(gè)轉(zhuǎn)錄因子的慢病毒載體FUW-OSKM的誘導(dǎo)方法將鼠胚成纖維細(xì)胞系STO (SIM-6-thiogunanie-oualiain)重編程為STO-iPSCs。首先用磷酸鈣法轉(zhuǎn)染293FT細(xì)胞來包裝慢病毒，然后用慢病毒感染STO細(xì)胞，以干細(xì)胞培養(yǎng)條件培養(yǎng)，誘導(dǎo)過程中加入了小分子物質(zhì)丙戊酸（VPA，組蛋白去乙�；种苿�。誘導(dǎo)15d后，挑取陽性細(xì)胞克隆并在無飼養(yǎng)層的培養(yǎng)體系中培養(yǎng)。對獲取的STO-iPSCs進(jìn)行生物學(xué)特性分析，包括形態(tài)學(xué)觀察、堿性磷酸酶染色、免疫熒光染色、蛋白印跡檢測、實(shí)時(shí)定量PCR和維甲酸定向誘導(dǎo)分化等，同時(shí)比較了STO-iPSCs冷凍復(fù)蘇后在明膠和基質(zhì)膠包被的培養(yǎng)皿中分別飼養(yǎng)時(shí)形態(tài)上的差異。 研究結(jié)果：STO-iPSCs具有典型胚胎干細(xì)胞的形態(tài)特征；堿性磷酸酶染色呈陽性；實(shí)時(shí)定量PCR結(jié)果表明STO-iPSCs有很高的內(nèi)源性Nanog，Oct4基因表達(dá)；免疫細(xì)胞染色進(jìn)一步證明表達(dá)胚胎干細(xì)胞特異性標(biāo)志Nanog和Oct4，并且能夠體外誘導(dǎo)分化為神經(jīng)細(xì)胞；基質(zhì)膠包被的培養(yǎng)皿更適合STO-iPSCs的生長。綜上所述表明，實(shí)驗(yàn)獲得了STO-iPSCs，建立了STO-iPSCs無飼養(yǎng)層培養(yǎng)體系。 本實(shí)驗(yàn)首次采用細(xì)胞系作為重編程研究的供體細(xì)胞，并且成功誘導(dǎo)鼠胚成纖維STO細(xì)胞系為STO-iPSCs，由于細(xì)胞系可以長期在體外培養(yǎng)傳代和凍存復(fù)蘇，在研究iPS細(xì)胞信號通路和藥物檢測等試驗(yàn)中，采用細(xì)胞系作為供體細(xì)胞，可以減少每次制備供體細(xì)胞的步驟，不但豐富了供體細(xì)胞種類，還有利于iPS細(xì)胞的產(chǎn)生，有助于實(shí)驗(yàn)研究的進(jìn)展。
[Abstract]:Multifunctional stem cell induced pluripotent stem cells was induced. Pics is the introduction of some genes related to the maintenance of embryonic stem cells (es cells) by exogenous introduction. The terminal differentiation of adult cells is reprogrammed as pluripotent stem cells to obtain multidirectional differentiation potential and self-renewal ability. IPS cells can be used to replace stem cells in the treatment of human diseases in molecular cell biology. Developmental biology, screening and development of new drugs, clinical cell replacement therapy and other aspects have a broad development space, but there are certain deficiencies in the efficiency and safety of induction, researchers are constantly looking for high efficiency. A safe way to compensate for defects in iPS cells. In this study, Oct4 was connected with Sox2 and Klf4. Induction of lentivirus vector FUW-OSKM from four transcription factors of c-Myc the mouse embryo fibroblast cell line STO (. SIM-6-thiogunanie-oualiain (SIM-6-thiogunanie-oualiain) was reprogrammed as STO-iPSCs.Firstly, calcium phosphate was transfected into 293FT cells to package lentivirus. Then STO cells were infected with lentivirus and cultured in stem cell culture condition. The small molecule valproate was added in the induction process and histone deacetylation inhibitor was added after 15 days of induction. The positive cells were cloned and cultured in a culture system without feeder layer. The biological characteristics of the obtained STO-iPSCs were analyzed, including morphological observation, alkaline phosphatase staining and immunofluorescence staining. Western blotting, real-time quantitative PCR and retinoic acid induced differentiation. At the same time, the morphological differences of STO-iPSCs in culture dishes coated with gelatin and matrix were compared after freezing and resuscitation. Results\\\. Alkaline phosphatase staining was positive. The results of real-time quantitative PCR showed that STO-iPSCs had high endogenous Nanog-Oct4 gene expression. The expression of Nanog and Oct4, the specific markers of embryonic stem cells, was further demonstrated by immunocytochemistry, and differentiation into neural cells could be induced in vitro. The results showed that STO-i SCS was obtained and the STO-iPSCs culture system without feeding layer was established. In this study, the mouse embryo fibroblast STO cell line was induced to be STO-iPSCs for the first time. Because cell lines can be cultured and resuscitated in vitro for a long time, cell lines are used as donor cells in the study of iPS cell signaling pathway and drug detection. It can reduce the steps of preparing donor cells each time, which not only enriches the donor cell types, but also contributes to the production of iPS cells, which is helpful to the progress of experimental research.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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