本文選題：Neurod2基因 + Hsp70��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：研究表明大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)有益于脊髓損傷(SCI)后的功能恢復(fù),本研究利用基因編輯技術(shù)構(gòu)建人熱休克蛋白啟動(dòng)子的真核表達(dá)載體hHsp70-ND2,并探討了不同誘導(dǎo)時(shí)間對(duì)小鼠間充質(zhì)干細(xì)胞(MSCs)向神經(jīng)元樣細(xì)胞定向分化的影響。主要研究方法有,1.首先克隆了wistar大鼠Neurod2基因,對(duì)其序列進(jìn)行生物信息學(xué)分析后,構(gòu)建pIRES2-AcGFP1-ND2真核表達(dá)載體,分對(duì)照組(pIRES2-AcGFP1轉(zhuǎn)染組)與實(shí)驗(yàn)組(pIRES2-AcGFP1-ND2轉(zhuǎn)染組)轉(zhuǎn)染小鼠MSCs后,觀察AcGFP1綠色熒光蛋白表達(dá),采用real-time PCR、免疫熒光染色和western blotting技術(shù)分別檢測小鼠MSCs中Neurod2基因及蛋白的表達(dá)水平。2.替換原載體pIRES2-AcGFP1中啟動(dòng)子CMV為hHsp70啟動(dòng)子,經(jīng)雙酶切及轉(zhuǎn)染驗(yàn)證其生物活性后與Neurod2基因連接獲得重組質(zhì)粒hHsp70-ND2,將其提取純化后轉(zhuǎn)染小鼠MSCs,轉(zhuǎn)染實(shí)驗(yàn)分對(duì)照組(誘導(dǎo)0 h)與實(shí)驗(yàn)組(0.5 h、1h、2 h、3 h),在42℃分別進(jìn)行熱誘導(dǎo)后24h觀察Ac GFP1綠色熒光蛋白的表達(dá),利用real-time PCR、western blotting和免疫熒光染色技術(shù),檢測小鼠MSCs中Neurod2基因及蛋白表達(dá)水平。3.分析轉(zhuǎn)染后細(xì)胞形態(tài)的變化和神經(jīng)分化相關(guān)的髓磷脂堿蛋白(myelin basic protein,MBP)、微管相關(guān)蛋白2(microtubule associated protein 2,MAP2)、髓磷脂P0蛋白(myelin protein zero,MPZ)、表皮生長因子(epidermal growth factor,EGF)、神經(jīng)膠質(zhì)原纖維酸性蛋白質(zhì)(glial fibrillary acidic protein,GFAP)、神經(jīng)絲蛋白(neurofilament,NF)、突觸囊泡蛋白(synaptophysin,Syn)、200 kD神經(jīng)絲蛋白重鏈(200 kD neurofilament heavy,200KD NFH)等因子表達(dá)量。結(jié)果表明,1.大鼠Neurod2基因CDS區(qū)全長1 149 bp,與Norway大鼠同區(qū)域相比較,起始密碼子后672、703、747、770、794位出現(xiàn)胞嘧啶的缺失與插入,共編碼382個(gè)氨基酸,屬于bHLH家族;氨基酸序列與家鼠(99%)、羅猴(98%)、人(97.7%)同源性較高,二級(jí)結(jié)構(gòu)以α-螺旋和無規(guī)卷曲為主,空間結(jié)構(gòu)呈現(xiàn)近似“螺旋-環(huán)-螺旋(HLH)”結(jié)構(gòu),主要分布于細(xì)胞核中;轉(zhuǎn)染細(xì)胞后Neurod2基因的表達(dá)及蛋白水平顯著高于對(duì)照組(P0.05);2.替換啟動(dòng)子轉(zhuǎn)染后AcGFP1蛋白表達(dá)率為39%,插入Neurod2克隆基因后,AcGFP1綠色熒光蛋白表達(dá)從高到低依次分別為0.5 h、對(duì)照組、1 h、2 h、3 h。real-time PCR分析結(jié)果顯示,熱誘導(dǎo)0.5 h后Neurod2基因表達(dá)與對(duì)照組、2 h和3 h相比均有顯著性差異(P0.05),western blotting分析結(jié)果顯示,0.5 h熱誘導(dǎo)后NeuroD2蛋白表達(dá)與2 h有顯著性差異(P0.05)。3.細(xì)胞免疫熒光染色顯示MBP、GFAP、NF蛋白表達(dá)明顯上調(diào),同樣進(jìn)行real-time PCR和western blotting分析得出,誘導(dǎo)0.5 h和1 h后NF基因及蛋白表達(dá)量最高,并與其余誘導(dǎo)組比較差異極顯著(P0.01),誘導(dǎo)1 h后MBP基因表達(dá)與其余組比較差異顯著(P0.05),蛋白表達(dá)于2 h和3 h誘導(dǎo)組差異極顯著(P0.01);GFAP基因表達(dá)在誘導(dǎo)0.5 h和1 h后與2 h和3 h差異顯著(P0.05),蛋白表達(dá)在誘導(dǎo)2 h后表達(dá)量顯著低于其余組(P0.01);而且誘導(dǎo)后的細(xì)胞形態(tài)較轉(zhuǎn)染前有明顯變化,呈神經(jīng)樣的長梭狀細(xì)胞數(shù)目明顯增多。以上結(jié)果表明成功構(gòu)建了含人熱休克蛋白啟動(dòng)子的真核重組表達(dá)載體hHsp70-ND2,并在42℃條件下,可利用不同熱激時(shí)間實(shí)現(xiàn)對(duì)小鼠MSCs向神經(jīng)元樣細(xì)胞定向分化的調(diào)控,且得出在熱誘導(dǎo)0.5 h和1 h時(shí),其誘導(dǎo)分化效率最高。
[Abstract]:The study showed that rat bone marrow mesenchymal stem cells (MSCs) was beneficial to functional recovery after spinal cord injury (SCI). This study constructed the eukaryotic expression vector hHsp70-ND2 of human heat shock protein promoter using gene editing technique, and explored the effect of different induction time on the directional differentiation of mouse mesenchymal stem cells (MSCs) to neuron like cells. The main research methods are: 1. first, the Neurod2 gene of Wistar rat was cloned. After bioinformatics analysis of the sequence, the pIRES2-AcGFP1-ND2 eukaryotic expression vector was constructed. The expression of AcGFP1 green fluorescent protein was observed in the control group (pIRES2-AcGFP1 transfection group) and the experimental group (pIRES2-AcGFP1-ND2 transfection group) was transfected to the mouse MSCs, and real-time was observed with real-time. PCR, immunofluorescence staining and Western blotting technique were used to detect the expression level of Neurod2 gene and protein in mouse MSCs,.2. replacement of original carrier pIRES2-AcGFP1 in pIRES2-AcGFP1, pIRES2-AcGFP1 promoter CMV was a hHsp70 promoter. After double enzyme digestion and transfection, the biological activity was verified by connection with Neurod2 gene to obtain a heavy group plasmid hHsp70-ND2, then it was extracted and purified. Mice MSCs was transfected with the control group (induced 0 h) and the experimental group (0.5 h, 1H, 2 h, 3 h). The expression of Ac GFP1 green fluorescent protein was observed by 24h at 42 degrees centigrade respectively. Real-time PCR, Western enrichment and immunofluorescence staining techniques were used to detect the gene and protein expression level of the mice. Myelin basic protein (MBP), microtubule related protein 2 (microtubule associated protein 2, MAP2), myelin P0 protein (myelin protein zero), epidermal growth factor, and glial fibrillary acidic protein. GFAP), neurofilament (NF), synaptic vesicle protein (synaptophysin, Syn), 200 kD neurofilament heavy chain (200 kD neurofilament heavy, 200KD NFH). The results showed that the total length of the 1. rat Neurod2 genes was 1149. Compared with the same region of the rat, the 672703747770794 place appeared after the initial codon. Cytosine deletion and insertion, cocoding 382 amino acids, belonging to the bHLH family; the amino acid sequence and the family mouse (99%), the romaceo (98%), the human (97.7%) homology, the two structure is mainly alpha helix and random curl, the spatial structure is similar to the "spiral ring spiral (HLH)" structure, mainly distributed in the nucleus; after transfection of the cell Neurod2 gene The expression and protein level were significantly higher than that of the control group (P0.05), and the expression rate of AcGFP1 protein was 39% after transfection of the 2. promoter. After the insertion of Neurod2 gene, the expression of AcGFP1 green fluorescent protein was 0.5 h respectively from high to low. The control group, 1 h, 2 h, 3 h.real-time PCR segregation results showed that the Neurod2 gene expression and control after the heat induction of 0.5 h Compared with 2 h and 3 h, there were significant differences (P0.05). Western blotting analysis showed that the expression of NeuroD2 protein was significantly different from 2 h (P0.05) after 0.5 h heat induction (P0.05).3. cell immunofluorescence staining showed MBP, GFAP, and protein expression was up to be analyzed and induced by 0.5 and 1. The expression of gene and protein was the highest (P0.01). After 1 h induction, the expression of MBP gene was significantly different from the other groups (P0.05). The expression of protein in the 2 h and 3 h induced groups was very significant (P0.01), and the expression of GFAP gene was significantly different from 2 h and 3 h after the induction of 0.5 h and 1 h, and the protein expression was induced 2. The post expression amount was significantly lower than that of the other groups (P0.01), and the induced cell morphology changed obviously before the transfection, and the number of long spindle cells in the nerve like cells increased significantly. The above results showed that the recombinant expression vector hHsp70-ND2 containing human heat shock protein promoter was successfully constructed, and the different heat shock time could be used under the condition of 42 degrees C. The regulation of directional differentiation of mouse MSCs into neuron like cells was achieved, and it was concluded that the induction efficiency was highest when the heat was induced at 0.5 h and 1 h.

【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳子興;吳偉林;謝文潔;;小鼠紅白血病細(xì)胞被誘導(dǎo)向終末期定向分化的觀察[J];細(xì)胞生物學(xué)雜志;1986年01期

2 鞏江;王征;倪士峰;吳智宇;吳一飛;趙桂仿;;干細(xì)胞培養(yǎng)定向分化的研究進(jìn)展[J];畜牧與飼料科學(xué);2009年01期

3 趙安莎;;仿生膜上納米脂筏樣生物器件構(gòu)建及其在細(xì)胞分子識(shí)別、定向分化中的應(yīng)用[J];學(xué)術(shù)動(dòng)態(tài);2011年04期

4 何丁文;徐艷杰;廖新根;殷明;;自組裝多肽納米纖維支架誘導(dǎo)骨髓間充質(zhì)干細(xì)胞定向分化[J];中國生物工程雜志;2012年02期

5 陳桃;齊暉;李富榮;;轉(zhuǎn)錄因子對(duì)干細(xì)胞高效定向分化為β細(xì)胞的作用[J];生命科學(xué);2012年02期

6 孫麗莉;心肌干細(xì)胞的定向分化和調(diào)控[J];國外醫(yī)學(xué)(生理、病理科學(xué)與臨床分冊);2004年01期

7 高波,柳川,王會(huì)信;骨髓間質(zhì)干細(xì)胞定向分化的研究進(jìn)展[J];生理科學(xué)進(jìn)展;2000年03期

8 方鑫;王芙蓉;姜亞平;;影響神經(jīng)干細(xì)胞定向分化因素的研究進(jìn)展[J];臨床神經(jīng)病學(xué)雜志;2008年01期

9 許應(yīng)星;吳巖;肖魯偉;;骨髓間充質(zhì)干細(xì)胞定向分化的經(jīng)典WNT機(jī)制[J];生命科學(xué);2010年04期

10 徐延豪;杜U，

本文編號(hào)：1780210