本文選題：臍帶間充質(zhì)干細(xì)胞　切入點(diǎn)：分離鑒定　出處：《浙江理工大學(xué)》2010年碩士論文

【摘要】： 背景誘導(dǎo)多能干細(xì)胞(induced pluripotent stem cell,iPSC)的出現(xiàn)不僅為人體組織工程繞開了胚胎干細(xì)胞的倫理障礙,而且為干細(xì)胞的研究提供了一種新思路新材料。然而,目前iPSC技術(shù)尚不成熟,高額的費(fèi)用和極低的誘導(dǎo)成功率嚴(yán)重制約了iPSC的發(fā)展,因此尋求一種既經(jīng)濟(jì)又高效的誘導(dǎo)方法是目前亟待解決的科學(xué)難題。 間充質(zhì)干細(xì)胞(mesnchymal stem cells,MSCs)是體細(xì)胞分化較低的幾類細(xì)胞之一,因其橫向分化能力使其在成體干細(xì)胞的研究領(lǐng)域中備受矚目。相比其他成體細(xì)胞,MSCs具有更強(qiáng)的可塑性,因此本課題使用已有的逆轉(zhuǎn)錄病毒可能更容易將其誘導(dǎo)成為多能干細(xì)胞。 腺病毒載體(adenovirus vector)是一種具有高效轉(zhuǎn)染能力的基因載體,因其容量大、產(chǎn)量高、毒副作用小等優(yōu)點(diǎn)被廣泛應(yīng)用于基因治療。本課題初步探索利用腺病毒載體攜帶Oct4、Sox2、Klf4、Nanog四個(gè)轉(zhuǎn)錄因子,能否誘導(dǎo)臍帶間充質(zhì)干細(xì)胞進(jìn)行重編程,探索一條高效、經(jīng)濟(jì)的iPSC誘導(dǎo)方法,為臨床應(yīng)用和細(xì)胞治療奠定基礎(chǔ)。 實(shí)驗(yàn)內(nèi)容1)由人臍帶中分離出間充質(zhì)干細(xì)胞(UCMSCs),于體外培養(yǎng)并鑒定。2)利用已有逆轉(zhuǎn)錄病毒載體對其進(jìn)行iPSC的誘導(dǎo),并鑒定其相關(guān)指標(biāo)。3)構(gòu)建嵌合型腺病毒載體Ad5/F11b,且攜帶Oct4、Sox2、Klf4、Nanog四個(gè)轉(zhuǎn)錄因子基因。4)利用構(gòu)建好的攜帶轉(zhuǎn)錄因子基因的嵌合型腺病毒Ad5/F11b感染UCMSCs,并嘗試誘導(dǎo)出多能干細(xì)胞。 實(shí)驗(yàn)結(jié)果1)從人臍帶中分離出間質(zhì)樣細(xì)胞,經(jīng)細(xì)胞周期、表面標(biāo)記、分化潛能等指標(biāo)確定其為間充質(zhì)干細(xì)胞。2)已有的逆轉(zhuǎn)錄病毒可成功將所分離的臍帶間充質(zhì)干細(xì)胞誘導(dǎo)為人胚胎干細(xì)胞(hESCs)樣克隆團(tuán)細(xì)胞,檢測發(fā)現(xiàn)在mRNA水平上4個(gè)轉(zhuǎn)錄因子均不同程度的上調(diào)表達(dá),且與hESCs相關(guān)基因的表達(dá)水平也明顯提高�？蛇M(jìn)一步檢測其生化指標(biāo)確定是否為iPSC。3)構(gòu)建攜帶Oct4、Sox2、Klf4、Nanog四個(gè)轉(zhuǎn)錄因子基因的嵌合型腺病骨架質(zhì)粒,經(jīng)酶切鑒定確定為正確克隆,并于HEK293細(xì)胞中包裝出病毒。4)利用攜帶4個(gè)轉(zhuǎn)錄因子的嵌合型腺病毒感染UCMSCs,出現(xiàn)類似hESCs的克隆團(tuán)細(xì)胞,還需進(jìn)一步證實(shí)iPSC的誘導(dǎo)情況。 結(jié)論1)成功于人臍帶中分離出間充質(zhì)干細(xì)胞,并通過逆轉(zhuǎn)錄病毒誘導(dǎo)成hESCs樣克隆團(tuán)細(xì)胞,該細(xì)胞與hESCs相關(guān)的4個(gè)轉(zhuǎn)錄因子均不同程度的上調(diào)表達(dá),表現(xiàn)出類似hESCs特征。 2)成功構(gòu)建攜帶Oct4、Sox2、Klf4、Nanog四個(gè)轉(zhuǎn)錄因子基因的嵌合型腺病骨架質(zhì)粒,并于HEK293細(xì)胞中包裝出病毒。3)利用攜帶4個(gè)轉(zhuǎn)錄因子的嵌合型腺病毒感染UCMSCs后,出現(xiàn)類似hESCs的克隆團(tuán)細(xì)胞,還需進(jìn)一步證實(shí)iPSC的誘導(dǎo)情況。
[Abstract]:Background the emergence of induced pluripotent stem cells (pluripotent stem cell sci) has not only bypassed the ethical barriers of embryonic stem cells in human tissue engineering, but also provided a new idea and new material for the research of stem cells.However, at present, the iPSC technology is not mature, the high cost and the extremely low success rate of induction seriously restrict the development of iPSC, so it is an urgent scientific problem to seek an economic and efficient induction method.Mesenchymal stem cells (MSCs) is one of the types of cells with low somatic differentiation, which has attracted much attention in the field of adult stem cells because of its transversal differentiation ability.MSCs have stronger plasticity than other adult cells, so it may be easier to induce them into pluripotent stem cells by using existing retroviruses.Adenovirus (adenovirus) is a kind of gene vector with high efficiency of transfection. It is widely used in gene therapy because of its large capacity, high yield, low toxicity and so on.In this study, we preliminarily explored whether the adenovirus vector carrying four transcription factors of Oct4N Sox2Klf4HG Nanog could induce the reprogramming of umbilical cord mesenchymal stem cells, and explore an efficient and economical method of iPSC induction, which would lay a foundation for clinical application and cell therapy.(1) UCM SCS was isolated from human umbilical cord, cultured and identified in vitro. 2) iPSC was induced by retroviral vector.The chimeric adenovirus vector Ad5 / F11b was constructed and carried four transcription factor genes. 4) the constructed chimeric adenovirus Ad5/F11b containing transcription factor gene was used to infect UCMSCs and induce pluripotent stem cells.Results 1) Leydig cells were isolated from human umbilical cord.The differentiation potential of human embryonic stem cell like human embryonic stem cells can be induced by retrovirus, which has been identified as mesenchymal stem cell (MSCs. 2), and human embryonic stem cells (ESC)-like clone cells can be successfully induced by the isolated umbilical cord mesenchymal stem cells (UMSCs).It was found that the expression of four transcription factors was up-regulated at mRNA level, and the expression level of hESCs related genes was also significantly increased.The cytoskeleton plasmids carrying the four transcription factor genes of Oct4N, Sox2, Klf4 and Nanog could be constructed by further detecting the biochemical index of IPSC.3. The cytoskeleton plasmids of chimeric adenosis were identified by restriction endonuclease digestion and confirmed to be the correct clones.The chimeric adenovirus carrying four transcription factors was used to infect UCMSCs and clone cells similar to hESCs appeared in HEK293 cells. It is necessary to further confirm the induction of iPSC.Conclusion 1) Mesenchymal stem cells were successfully isolated from human umbilical cord and induced into hESCs like colony cells by retrovirus. The four transcription factors associated with hESCs were up-regulated in varying degrees and showed similar hESCs characteristics.2) the chimeric adenosis skeleton plasmids carrying the four transcription factor genes of Oct4G Sox2Klf4nanog were successfully constructed, and the virus was packaged in HEK293 cells. After UCMSCs was infected with the chimeric adenovirus carrying the four transcription factors, hESCs like clone cells appeared.Further confirmation of iPSC induction is needed.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊晨;顏煒群;;間充質(zhì)干細(xì)胞分離提取的優(yōu)化方法[J];內(nèi)蒙古農(nóng)業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2012年03期

，

本文編號(hào)：1719367