本文關(guān)鍵詞：慢病毒介導(dǎo)HCN2基因轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞的實(shí)驗(yàn)研究　出處：《瀘州醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HCN2 骨髓間充質(zhì)干細(xì)胞 慢病毒 大鼠 轉(zhuǎn)染

【摘要】：目的：探討通過超極化激活的環(huán)核苷酸門控的陽離子通道2 (hyperpolarization-activated cyclic nucleotide-gated cation channel, HCN2)基因穩(wěn)定轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞(bone mesenchymal stem cells, BMSCs)的可行性。探求HCN2轉(zhuǎn)染的最佳感染復(fù)數(shù)(multiplication of infection, MOI),為進(jìn)一步研究起搏電流的電生理學(xué)特性提供前期實(shí)驗(yàn)基礎(chǔ)。方法：1.骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)和鑒定：采用改良全骨髓貼壁培養(yǎng)的方法分離純化BMSCs,培養(yǎng)于含20%FBS的DMEM培養(yǎng)基,并傳代培養(yǎng)；觀察培養(yǎng)細(xì)胞的生長狀態(tài)；通過CD34、CD4、CD45、CD90免疫組織化學(xué)和流式細(xì)胞技術(shù)進(jìn)行細(xì)胞鑒定。應(yīng)用體外成骨誘導(dǎo),觀察BMSCs向成骨細(xì)胞分化的功能特性。2.對購得的HCN2質(zhì)粒進(jìn)行擴(kuò)增和純化,對純化質(zhì)粒行蛋白電泳和基因測序,鑒定HCN2基因。3.重組慢病毒(Feline immunodeficiency virus,FIV)載體HCN2的構(gòu)建及其鑒定：將鑒定正確的重組慢病毒進(jìn)行擴(kuò)增、純化和滴度測定。以增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein, EGFP)基因?yàn)樾盘柣?確定最佳感染復(fù)數(shù)；觀察FIV-EGFP轉(zhuǎn)染BMSCs表達(dá)強(qiáng)度隨時(shí)間的變化情況；流式細(xì)胞檢測FIV-HCN2轉(zhuǎn)染BMSCs的細(xì)胞活力的變化。結(jié)果：1.改良全骨髓貼壁分離法法獲得了高純度(90%)和高活力(90%)的BMSCs,經(jīng)形態(tài)學(xué)觀察和免疫組織化學(xué)檢測,顯示所培養(yǎng)細(xì)胞呈梭形、紡錘形和多角形,免疫化學(xué)染色CD44、CD90陽性,CD34陰性,符合BMSCs的形態(tài)和表面標(biāo)記；流式細(xì)胞儀分析,CD44、CD90陽性細(xì)胞分別占99.5%、90.9%。體外成骨誘導(dǎo)BMSCs,茜紅素染色可見大量鈣結(jié)節(jié)后,說明BMSCs具有向成骨細(xì)胞分化能力。2. HCN2的質(zhì)粒擴(kuò)增方法可靠,擴(kuò)增的質(zhì)粒經(jīng)雙酶切證實(shí)基因片段大小正常,目的基因測序結(jié)果證實(shí)與GeneBank上基本一致。3.重組慢病毒HCN2的構(gòu)建。使用包裝病毒上清液做慢病毒滴度檢測未見熒光；用上清液感染BMSCs,24h、48h、72h也未檢測出有意義的綠色熒光。結(jié)論：1.采用改良全骨髓貼壁法,可分離獲得較高純度、高活力的BMSCs。BMSCs體外增殖速度快,可短期內(nèi)達(dá)到實(shí)驗(yàn)所需要細(xì)胞數(shù)量。2.含有HCN2的質(zhì)粒擴(kuò)增方法可靠,擴(kuò)增的質(zhì)粒經(jīng)雙酶切證實(shí)基因片段大小正常,目的基因測序結(jié)果證實(shí)與GeneBank上基本一致。3.重組慢病毒HCN2轉(zhuǎn)染干細(xì)胞有待進(jìn)一步研究。
[Abstract]:Objective: to investigate the cationic channel 2 (cationic channel 2) activated by hyperpolarization-activated cyclic nucleotides (cationic acid). Hyperpolarization-activated cyclic nucleotide-gated cation channel. HCN2) gene was stably transfected into rat bone marrow mesenchymal stem cells (mesenchymal stem cells). To find out the best infection number of HCN2 transfection, multiplex of infection, moi). In order to further study the electrophysiological characteristics of pacemaker current, we provide a preliminary experimental basis. Methods: 1. Isolation of bone marrow mesenchymal stem cells. Culture and identification: BMSCs were isolated and purified by modified whole bone marrow adherent culture method and cultured on DMEM medium containing 20s. The growth state of cultured cells was observed. The cells were identified by immunohistochemistry and flow cytometry (FCM). Osteogenesis was induced by osteogenesis in vitro. To observe the functional characteristics of BMSCs differentiation into osteoblasts. 2. To amplify and purify the acquired HCN2 plasmid, and to sequence the purified plasmid by line protein electrophoresis and gene sequencing. HCN2 gene. 3.Recombinant lentivirus Feline immunodeficiency virus. Construction and identification of HCN2 vector: the correct recombinant lentivirus was amplified. Purification and titer determination. Enhanced green fluorescent protein (EGFP) gene was used as signal gene. Determine the optimal complex number of infections; The expression intensity of FIV-EGFP transfected BMSCs was observed with time. Flow cytometry was used to detect the changes of cell viability in BMSCs transfected with FIV-HCN2. Results: 1. The modified whole bone marrow adherent separation method obtained high purity and high activity. BMSCs. Morphological observation and immunohistochemical examination showed that the cultured cells were fusiform, spindle-shaped and polygonal, and immunocytochemical staining was positive for CD44 + CD90 and negative for CD34. Conformed to the morphology and surface marker of BMSCs; Flow cytometry analysis showed that CD44 + CD90 positive cells accounted for 99. 5% and 99. 9% respectively. A large number of calcium nodules were observed after osteogenesis induced by BMSCs in vitro. The results showed that BMSCs had the ability to differentiate into osteoblasts. 2. The method of plasmid amplification of HCN2 was reliable, and the amplified plasmid was confirmed to be normal by double enzyme digestion. Objective the result of gene sequencing confirmed that the construction of recombinant lentivirus HCN2 was basically consistent with that on GeneBank. No fluorescence was found in the detection of lentivirus titer using the supernatant of packaging virus. Significant green fluorescence could not be detected after infection with supernatant BMSCs1 for 24 h or 48 h. Conclusion: 1. High purity can be obtained by modified whole bone marrow adherent method. BMSCs.BMSCs with high activity can proliferate rapidly in vitro and can reach the number of cells needed in the experiment in a short time. The method of plasmid amplification containing HCN2 is reliable. The amplified plasmid was confirmed to be of normal size by double enzyme digestion. Objective Gene sequencing confirmed that it was basically the same as that on GeneBank. The recombinant lentivirus HCN2 transfected stem cells needed to be further studied.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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本文編號：1417201