本文選題：Oct4 + 胎兒骨髓間充質(zhì)干細(xì)胞　； 參考：《天津醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的 人胚胎干細(xì)胞(embryonic stem cell, ES)具有無限增殖、自我更新及多向分化的潛能,這也使得ES細(xì)胞成為了基礎(chǔ)研究和臨床應(yīng)用的種子細(xì)胞。干細(xì)胞廣泛應(yīng)用的前提是明確其自我更新和定向分化的調(diào)控機(jī)制,ES細(xì)胞多能性作用的維持與其內(nèi)部一些重要的轉(zhuǎn)錄因子的共表達(dá)及某些外源性信號分子的作用密不可分。尤其自2006年以來,誘導(dǎo)多功能干細(xì)胞(induced pluripotent stem cells, iPS)所揭起的生命科學(xué)與醫(yī)學(xué)界干細(xì)胞研究的又一個(gè)熱潮,更是證明了Sox2、Nanog等多個(gè)轉(zhuǎn)錄因子的核心調(diào)控作用。其中,Oct4是參與調(diào)控胚胎干細(xì)胞自我更新和維持其全能性的最為重要的轉(zhuǎn)錄因子之一,Oct4的作用體現(xiàn)在維持種系的持續(xù)性及各組織器官分化的精密調(diào)節(jié)上,同時(shí)它也是體外建立iPS的關(guān)鍵基因,但目前對于Oct4的具體作用機(jī)制并未完全明確。 胎兒骨髓間充質(zhì)干細(xì)胞(fetal bone marrow mesenchymal stem cells, fBMSCs)是胎兒骨髓中一種具有高度可塑性的細(xì)胞,較成人骨髓間充質(zhì)干細(xì)胞發(fā)育上更原始,fBMSCs具備良好的增殖能力,及其向三個(gè)胚層起源細(xì)胞分化的“類胚胎干細(xì)胞”的生物學(xué)特征。fBMSCs在不同的誘導(dǎo)環(huán)境下可向多種成熟體細(xì)胞分化細(xì)胞,比如,骨、軟骨、脂肪細(xì)胞、神經(jīng)細(xì)胞、胰島細(xì)胞等。同時(shí),fBMSCs可表達(dá)人ES細(xì)胞的遺傳標(biāo)志Oct4,且fBMSCs中表達(dá)的Oct4的功能與人ES細(xì)胞中表達(dá)的Oct4的功能相似。由于人ES細(xì)胞來源有限,培養(yǎng)成本昂貴,本研究選擇以人胎兒骨髓作為Oct4基因的克隆來源,以穿梭質(zhì)粒pSG5為載體,構(gòu)建真核表達(dá)質(zhì)粒pSG5-Oct4,檢測Oct4基因在胎兒胰島間質(zhì)細(xì)胞中的表達(dá)情況,并嘗試pSG5-Oct4轉(zhuǎn)染fBMSCs,探討其是否對fBMSCs的生長狀態(tài)及細(xì)胞內(nèi)部結(jié)構(gòu)產(chǎn)生影響,并為后續(xù)實(shí)驗(yàn)提供據(jù)。 方法 1、采取密度梯度離心的方法,從3～5月人工流產(chǎn)胎兒骨髓中分離出單核細(xì)胞,用于總RNA的提取。 2、根據(jù)穿梭質(zhì)粒pSG5上的酶切位點(diǎn),應(yīng)用Primer5.0引物設(shè)計(jì)軟件設(shè)計(jì)人Oct4(NM_002701.4)上下游引物,通過RT-PCR技術(shù)擴(kuò)增Oct4的開放性閱讀框。 3、在T4連接酶的作用下,將分別雙酶切后pSG5和Oct4基因連接起來,經(jīng)陽性克隆篩選、雙酶切及測序分析鑒定,確保真核表達(dá)質(zhì)粒pSG5-Oct4構(gòu)建成功。 4、采用組織塊法貼壁法原代培養(yǎng)胎兒胰島間質(zhì)細(xì)胞,倒置顯微鏡觀察胎兒胰島間質(zhì)細(xì)胞形態(tài)及生長狀況,選擇P2-P3的細(xì)胞轉(zhuǎn)染備用。 5、pSG5-Oct4轉(zhuǎn)染胎兒胰島間質(zhì)細(xì)胞,以轉(zhuǎn)染pSG5和未轉(zhuǎn)染的胰島間質(zhì)細(xì)胞為對照組,倒置顯微鏡下觀察胰島間質(zhì)細(xì)胞生長狀況及形態(tài)學(xué)是否發(fā)生發(fā)生變化。 6、利用RT-PCR技術(shù)從mRNA水平檢測真核表達(dá)質(zhì)粒pSG5-Oct4在胎兒胰島間質(zhì)細(xì)胞中表達(dá)否;免疫細(xì)胞化學(xué)檢測Oct4蛋白的表達(dá)位置。 7、嘗試真核農(nóng)達(dá)質(zhì)粒pSG5-Oct4轉(zhuǎn)染fBMSCs,觀察過表達(dá)Oct4的fBMSCs的形態(tài)學(xué)變化,并利用免疫細(xì)胞化學(xué)驗(yàn)證。 結(jié)果 1、經(jīng)RT-PCR反應(yīng),凝膠電泳見1.1kb處單一亮帶,與預(yù)計(jì)大小相符合。 2、經(jīng)RT-PCR克隆得到的Oct4基因的與Genbank數(shù)據(jù)庫中報(bào)道的人Oct4基因的同源性達(dá)到97%。 3、24h后原代培養(yǎng)的胎兒胰島間質(zhì)細(xì)胞組織塊周圍即有細(xì)胞暈形成,呈長梭形,約4d見較多的細(xì)胞游離出,匯集成束,9d左右細(xì)胞鋪滿瓶底,0.25%胰蛋白酶消化傳代。 4、pSG5-Oct4轉(zhuǎn)染胎兒胰島間質(zhì)細(xì)胞后第3天,細(xì)胞生長狀態(tài)良好,逐漸變圓,轉(zhuǎn)變?yōu)閼腋〖?xì)胞,第6天起大部分細(xì)胞變?yōu)閼腋〖?xì)胞,細(xì)胞邊界清楚,部分細(xì)胞呈團(tuán)塊狀；而對照組的細(xì)胞均未見類似變化,仍為貼壁梭形細(xì)胞。 5、經(jīng)RT-PCR反應(yīng)后,凝膠電泳見受pSG5-Oct4轉(zhuǎn)染的細(xì)胞分別在1.8kb和1.2kb左右有亮帶,與預(yù)計(jì)相符；轉(zhuǎn)變的懸浮細(xì)胞經(jīng)Oct4免疫細(xì)胞化學(xué)染色陽性 6、pSG5-Oct4轉(zhuǎn)染fBMSCs,約4周見部分fBMSCs的形態(tài)出現(xiàn)了神經(jīng)元樣變化,神經(jīng)細(xì)胞特異性標(biāo)記NSE免疫細(xì)胞化學(xué)染色陽性。 結(jié)論 1、本實(shí)驗(yàn)成功分離并培養(yǎng)了胎兒胰島間質(zhì)細(xì)胞及fBMSCs。 2、應(yīng)用基因重組技術(shù)成功構(gòu)建并鑒定了pSG5-Oct4真核表達(dá)質(zhì)粒。 3、構(gòu)建成功的pSG5-Oct4真核表達(dá)質(zhì)�？稍谔阂葝u間質(zhì)細(xì)胞中瞬時(shí)表達(dá)。 4、fBMSCs中過表達(dá)Oct4可能將fBMSCs誘導(dǎo)分化為神經(jīng)細(xì)胞,對于這一現(xiàn)象的有待進(jìn)一步探討。
[Abstract]:Purpose

Human embryonic stem cells ( ES ) have the potential of unlimited proliferation , self - renewal and multi - directional differentiation , which also makes ES cells become the seed cells of basic research and clinical application .

Human fetal bone marrow mesenchymal stem cells ( FMSCs ) are a highly plastic cell in the fetal bone marrow , and the stem cells of bone marrow mesenchymal stem cells are more primitive than those of adult bone marrow mesenchymal stem cells .

method

1 . Using density gradient centrifugation , mononuclear cells were isolated from fetal bone marrow of artificial abortion from March to May , which was used for the extraction of total RNA .

2 . On the basis of the enzyme cleavage site on shuttle plasmid pSG5 , the primer designed by using the Primer5.0 primer was designed and designed by using the primer of the upstream and downstream primers of human Oct4 ( NM _ 002701 . 4 ) , and the open reading frame of Oct4 was amplified by RT - PCR .

3 . Under the action of T4 ligase , the pSG5 and Oct4 genes were linked by double enzyme digestion respectively . After screening by positive clones , double digestion and sequencing analysis , it was confirmed that the eukaryotic expression plasmid pSG5 - Oct4 was constructed successfully .

4 . The fetal islet stromal cells were cultured in primary culture by tissue block method . The morphology and growth of fetal islet cells were observed by inverted microscope , and the cells were transfected with P2 - P3 .

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本文編號：1977955