本文關(guān)鍵詞： 心肌營(yíng)養(yǎng)素-1(CT-1) 骨髓間充質(zhì)干細(xì)胞(BMSCs) 腺病毒 神經(jīng) 分化 凋亡 存活　出處：《遵義醫(yī)學(xué)院》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：體外培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞(rBMSCs), Adv-EGFP-CT-1(重組心肌營(yíng)養(yǎng)素-1腺病毒)轉(zhuǎn)染rBMSCs,觀察CT-1對(duì)rBMSCs向神經(jīng)方向分化的作用及對(duì)其存活的影響。 方法：(1)培養(yǎng)及鑒定rBMSCs:全骨髓貼壁篩選法分離培養(yǎng)rBMSCs,流式細(xì)胞術(shù)鑒定細(xì)胞表面標(biāo)記性蛋白。(2)Adv-EGFP-CT-1轉(zhuǎn)染rBMSCs:將Adv-EGFP-CT-1分別以感染復(fù)數(shù)(MOI)為100、200、300、400PFU/細(xì)胞轉(zhuǎn)染rBMSCs,于轉(zhuǎn)染后24h、48h、72h、96h在熒光顯微鏡下觀察轉(zhuǎn)染率,流式細(xì)胞術(shù)鑒定其最佳MOI及最佳時(shí)間點(diǎn)(熒光顯微鏡下觀察所得)轉(zhuǎn)染率。同時(shí)于轉(zhuǎn)染后24h、48h、3d、10d應(yīng)用Real-timePCR檢測(cè)轉(zhuǎn)染后rBMSCs表達(dá)CT-1mRNA的情況、應(yīng)用細(xì)胞免疫熒光檢測(cè)轉(zhuǎn)染后rBMSCs表達(dá)CT-1目的蛋白的情況,確定CT-1蛋白表達(dá)最強(qiáng)的時(shí)間點(diǎn)。(3)觀察CT-1對(duì)rBMSCs向神經(jīng)方向分化的作用：將rBMSCs分為以下4組：對(duì)照組、Adv-EGFP(重組腺病毒增強(qiáng)型綠色熒光蛋白)轉(zhuǎn)染組(Adv-EGFP轉(zhuǎn)染rBMSCs, MOI為200PFU/細(xì)胞)、Adv-EGFP-CT-1轉(zhuǎn)染組(Adv-EGFP-CT-1轉(zhuǎn)染rBMSCs, MOI為200PFU/細(xì)胞)及誘導(dǎo)劑組。除對(duì)照組外,其他各組均予神經(jīng)分化誘導(dǎo),于誘導(dǎo)后5h、3d、7d觀察各組細(xì)胞形態(tài)學(xué)變化、細(xì)胞免疫熒光檢測(cè)各組Nestin、NeuN、GFAP的表達(dá)。(4)觀察CT-1對(duì)rBMSCs神經(jīng)分化過(guò)程中細(xì)胞存活的影響：于誘導(dǎo)后5h、24h、48h DAPI染色法觀察凋亡細(xì)胞核形態(tài)；流式細(xì)胞術(shù)檢測(cè)細(xì)胞的早期凋亡率；于誘導(dǎo)后5h、3d、7d臺(tái)盼藍(lán)染色法檢測(cè)細(xì)胞存活率。 結(jié)果：(1) rBMSCs的培養(yǎng)及鑒定。rBMSCs原代細(xì)胞7天左右可以傳代,第三代(P3)得到純化,流式細(xì)胞術(shù)鑒定CD29、CD90、CD45的陽(yáng)性率分別是99.27%、93.43%、1.46%。(2)Adv-EGFP-CT-1的轉(zhuǎn)染率。其轉(zhuǎn)染率隨MOI及時(shí)間的改變而變化,MOI為200PFU/細(xì)胞、轉(zhuǎn)染后48h達(dá)高峰(轉(zhuǎn)染率為87.05%±0.95%)；Adv-EGFP-CT-1轉(zhuǎn)染rBMSCs后CT-1mRNA的表達(dá)：轉(zhuǎn)染后24hCT-1mRNA的相對(duì)表達(dá)量(423.42±11.61)均高于其他時(shí)間點(diǎn)(48h、3d、10d),差異有統(tǒng)計(jì)學(xué)意義(P0.05),48h、3d、10d之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)；Adv-EGFP-CT-1轉(zhuǎn)染rBMSCs后CT-1目的蛋白的表達(dá)：轉(zhuǎn)染后48h,CT-1熒光強(qiáng)度達(dá)到最強(qiáng)(11.44±1.44),與24h相比差異有統(tǒng)計(jì)學(xué)意義(P0.05),與3d、10d相比熒光強(qiáng)度差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(3)CT-1對(duì)rBMSCs向神經(jīng)方向分化的作用。誘導(dǎo)處理后,除對(duì)照組外,其他各組均顯示類神經(jīng)細(xì)胞樣形態(tài)改變,其中CT-1組此變化最明顯；誘導(dǎo)后5hNestin的陽(yáng)性率最高,其中CT-1組的陽(yáng)性率均高于其他各組(86.31%±4.27%),差異有統(tǒng)計(jì)學(xué)意義(P0.05)；誘導(dǎo)后5h CT-1組NeuN、GFAP即有少量表達(dá),隨后NeuN、GFAP陽(yáng)性率逐漸升高,至第7d時(shí)陽(yáng)性率最高,其中,CT-1組各時(shí)間點(diǎn)NeuN、GFAP陽(yáng)性率均高于其他各組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(4)CT-1對(duì)rBMSCs神經(jīng)分化過(guò)程中細(xì)胞存活的影響。CT-1組及對(duì)照組,細(xì)胞核大部分為淡藍(lán)色圓形或橢圓形,而空病毒組及誘導(dǎo)劑組出現(xiàn)較多凋亡細(xì)胞核；誘導(dǎo)后各時(shí)間點(diǎn),CT-1組及對(duì)照組早期凋亡率較空病毒組及誘導(dǎo)劑組明顯減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；誘導(dǎo)后不同時(shí)間點(diǎn),CT-1組及對(duì)照組存活率均高于空病毒組及誘導(dǎo)劑組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：(1)全骨髓貼壁篩選法可以獲得純化的rBMSCs;(2) Adv-EGFP-CT-1可以有效地轉(zhuǎn)染rBMSCs, CT-1可在rBMSCs中高效長(zhǎng)期表達(dá)；(3)CT-1可以促進(jìn)rBMSCs向神經(jīng)方向分化；(4)CT-1修飾的rBMSCs,聯(lián)合神經(jīng)誘導(dǎo)劑,為rBMSCs向神經(jīng)方向分化提供可行的誘導(dǎo)方法；(5)CT-1可以維持rBMSCs神經(jīng)分化過(guò)程中細(xì)胞的存活。
[Abstract]:Objective : To observe the effect of CT - 1 on neural differentiation and its survival in vitro . Methods : ( 1 ) To investigate the effect of CT - 1 on the expression of CT - 1 mRNA and to determine the expression of CT - 1 mRNA . The expression of CT - 1 mRNA was observed at 24 h , 48 h , 72 h and 96 h after transfection . Results : ( 1 ) The expression of CT - 1 mRNA was higher in CT - 1 group than in other groups ( P0.05 ) . The positive rate of CT - 1 mRNA was higher than that in other groups ( P0.05 ) . ( 4 ) The effect of CT - 1 on the survival of the cells in the course of neurodifferentiation of rMSCs . The majority of the nuclei in CT - 1 group and the control group were pale blue circle or oval , while the empty virions and the inducing agent group appeared more apoptotic nuclei . After induction , the early apoptosis rate of CT - 1 and control group were significantly decreased ( P0.05 ) ; the survival rates of CT - 1 and control groups were higher than those of the empty virus group and the inducer group after induction ( P0.05 ) . Conclusion : ( 1 ) The recombinant rMSCs can be efficiently transfected by the whole marrow adherent screening method ; ( 2 ) the high - efficiency and long - term expression of rMSCs can be promoted by CT - 1 . ( 3 ) CT - 1 can promote the differentiation of rMSCs into the neural direction ; ( 4 ) CT - 1 modified rMSCs and combined nerve inducing agents provide a feasible induction method for the differentiation of rMSCs into the neural direction ; and ( 5 ) CT - 1 can maintain the survival of the cells during the neurodifferentiation of rMSCs .

【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：

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