本文關(guān)鍵詞： 神經(jīng)干細胞 IGFBP-4 脂質(zhì)體轉(zhuǎn)染 電轉(zhuǎn)儀轉(zhuǎn)染 增殖 分化　出處：《華北理工大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的本實驗擬通過構(gòu)建胰島素樣生長因子結(jié)合蛋白-4(IGFBP-4)高表達質(zhì)粒及干擾質(zhì)粒,并運用脂質(zhì)體和電轉(zhuǎn)儀轉(zhuǎn)染兩種方法轉(zhuǎn)染質(zhì)粒,通過對比轉(zhuǎn)染效率選取轉(zhuǎn)染效果更好的方法,用最終選擇的較高效的轉(zhuǎn)染方法將所構(gòu)建質(zhì)粒轉(zhuǎn)染至神經(jīng)干細胞內(nèi),建立IGFBP-4高表達/低表達細胞模型,繼而觀察IGFBP-4對神經(jīng)干細胞增殖、分化的影響。方法首先從大鼠腦組織中提取總RNA,RT-PCR擴增IGFBP-4基因,TA克隆擴增PCR產(chǎn)物后將目的基因片段和p EGFP-N1連接,并對陽性克隆進行測序來構(gòu)建IGFBP-4高表達質(zhì)粒,IGFBP-4干擾質(zhì)粒;體外培養(yǎng)神經(jīng)干細胞,分別用脂質(zhì)體轉(zhuǎn)染和電轉(zhuǎn)儀轉(zhuǎn)染兩種方法向神經(jīng)干細胞內(nèi)轉(zhuǎn)染所構(gòu)建質(zhì)粒,通過對比轉(zhuǎn)染效率,選取可獲得更理想效果的方法,并探尋不同質(zhì)粒的最優(yōu)轉(zhuǎn)染條件;根據(jù)最優(yōu)轉(zhuǎn)染條件,轉(zhuǎn)染各種質(zhì)粒,并運用western blot對各組細胞IGFBP-4含量進行測定;轉(zhuǎn)染后48h使用熒光免疫細胞化學(xué)法計數(shù)Nestin、GFAP、MAP-Ⅱ陽性表達的細胞數(shù),觀察神經(jīng)干細胞的分化方向;運用i CELLigence實時無標記細胞功能分析儀監(jiān)測尋找對于E-plate L8板最適的種植密度,并對比在最適種植密度下同一時間點各組細胞曲線的高度。所有數(shù)據(jù)均采用SPSS17.0軟件進行統(tǒng)計分析。結(jié)果1成功提取并擴增IGFBP-4基因,重組質(zhì)粒pEGFP-N1-IGFBP4經(jīng)酶切和序列分析鑒定與預(yù)期設(shè)計一致;2運用脂質(zhì)體Lipofectamine LTX和電轉(zhuǎn)儀CUY21EDITII兩種轉(zhuǎn)染方法均成功將質(zhì)粒導(dǎo)入神經(jīng)干細胞中,并在干細胞中有效表達,但兩種方法轉(zhuǎn)染效率差別明顯,脂質(zhì)體轉(zhuǎn)染效率為30~40%,電轉(zhuǎn)儀轉(zhuǎn)染效率可達80%以上,電轉(zhuǎn)儀轉(zhuǎn)染效率明顯高于脂質(zhì)體轉(zhuǎn)染;3選擇電轉(zhuǎn)染方法作為本實驗導(dǎo)入質(zhì)粒的方法,成功轉(zhuǎn)染4組質(zhì)粒,并獲得每種質(zhì)粒的最優(yōu)電轉(zhuǎn)染條件為:p EGFP-N1質(zhì)粒為Pp V(Poration pulse Voltage)350V,Pd V(Driving pulse Voltage)20V,脈沖時間為10ms,循環(huán)次數(shù)為20次;IGFBP-4高表達質(zhì)粒的最優(yōu)條件:Pp V為400V,Pd V為75V,脈沖時間30ms,循環(huán)次數(shù)50次;IGFBP-4干擾質(zhì)粒的對照質(zhì)粒最優(yōu)條件是:Pp V為400V,Pd V為60V,脈沖時間18ms,循環(huán)次數(shù)40次;IGFBP-4干擾質(zhì)粒的最優(yōu)條件:Pp V為400V,Pd V為35V,脈沖時間20ms,循環(huán)次數(shù)40次;4對轉(zhuǎn)染成功48h的四組細胞進行western blot實驗,發(fā)現(xiàn)轉(zhuǎn)染IGFBP-4高表達質(zhì)粒組細胞表達蛋白IGFBP-4含量明顯增高,較對照組提高1倍左右,IGFBP-4干擾質(zhì)粒組蛋白IGFBP-4含量明顯降低,干擾效率達80%,說明成功構(gòu)建IGFBP-4高/低表達細胞模型;5 i CELLigence實時無標記細胞功能分析儀監(jiān)測不同種植密度下細胞的生長曲線,最適的種植密度為每孔5×104個細胞;6選取第100h對比各組曲線高度,IGFBP-4高表達組高于其對照組,IGFBP-4干擾質(zhì)粒組低于其對照質(zhì)粒組,IGFBP-4高表達組明顯高于IGFBP-4干擾質(zhì)粒組,差別有統(tǒng)計學(xué)意義;7免疫熒光觀察各組細胞分化情況:IGFBP-4高表達對照組MAP-Ⅱ陽性細胞比例為27.2%,GFAP陽性細胞比例為32.3%;IGFBP-4高表達質(zhì)粒組神經(jīng)元細胞標志物MAP-Ⅱ陽性細胞比例為38.0%,膠質(zhì)細胞標志物GFAP陽性細胞比例為92.0%;IGFBP-4干擾質(zhì)粒對照組MAP-Ⅱ陽性細胞比例為21.7%,GFAP陽性細胞比例為86.1%;IGFBP-4干擾質(zhì)粒組MAP-Ⅱ陽性細胞比例為24.3%,GFAP陽性細胞比例為89.6%。IGFBP-4高表達組神經(jīng)元分化比例大于PEGFP-N1質(zhì)粒組(P0.05),IGFBP-4干擾質(zhì)粒組神經(jīng)元分化比例較其對照組RNAi-negative control組明顯降低(P0.05)。結(jié)論1 IGFBP-4高表達/低表達細胞模型構(gòu)建成功;2 IGFBP-4促進了神經(jīng)干細胞的增殖;3 IGFBP-4促進了神經(jīng)干細胞向神經(jīng)元方向的分化。
[Abstract]:The purpose of this experiment is to construct the insulin-like growth factor binding protein -4 (IGFBP-4) high expression plasmid and the plasmid, and the use of liposomes and electroporator transfection of two plasmids transfection method, method of selecting better transfection efficiency by comparing with the transfection method the final selection of the more efficient the constructed plasmid was transfected into neural stem cells, the establishment of IGFBP-4 high expression / low expression cell model, then observe the effect of IGFBP-4 on the proliferation of neural stem cells, in vitro. Firstly, total RNA was extracted from rat brain tissue, IGFBP-4 gene was amplified by RT-PCR, cloned TA PCR amplification products after the target gene fragment and P EGFP-N1 connection, and the positive clones sequencing to construct the high expression of IGFBP-4 plasmid, IGFBP-4 plasmid; neural stem cells cultured in vitro respectively by liposome transfection and electroporator transfection two methods to transfection of neural stem cells Through the comparison of the constructed plasmids, transfection efficiency, selection method can obtain better effect, and to explore the optimal transfection conditions of different plasmids; according to the optimal conditions of transfection, transfection plasmid and the use of Western blot on the content of IGFBP-4 cells were measured; 48h after transfection using fluorescence count immunocytochemical method Nestin, GFAP, cell number the positive expression of MAP- II, to observe the differentiation direction of neural stem cells; real-time ncell function analyzer to find the most suitable for monitoring E-plate L8 board using I CELLigence and planting density, the optimum planting density contrast in the same time the cells were the height of the curve. All the data were analyzed by SPSS17.0 software. Results 1 the successful extraction and amplification of IGFBP-4 gene, the recombinant plasmid pEGFP-N1-IGFBP4 by restriction enzyme digestion and sequence analysis was consistent with the expected design; 2 Lipofectamine by liposome. LTX and electroporator CUY21EDITII two transfection methods were successfully plasmid into neural stem cells, and effective expression in stem cells, but the transfection efficiency of two methods have obvious differences, the transfection efficiency of liposome 30~40%, electroporator transfection efficiency can reach more than 80%, electroporator transfection efficiency was significantly higher than that of liposome transfection 3; electric transfection method as the experimental method into the plasmid, plasmid successfully transfected into 4 groups, and obtain the optimal transfection conditions for each plasmid: P EGFP-N1 plasmid Pp V (Poration pulse Voltage) 350V Pd V (Driving pulse Voltage) 20V, the pulse time is 10ms, the number of cycles for 20 optimal conditions; the high expression of IGFBP-4 plasmid: Pp V 400V, Pd V 75V, pulse duration 30ms, 50 cycles; the optimal control conditions of plasmid IGFBP-4 interference plasmid is Pp V 400V, Pd V 60V, pulse duration 18ms, 40 cycles; IGFBP-4 interference The optimal conditions: Pp V plasmid 400V, Pd V 35V, pulse duration 20ms, 40 cycles; 4 to four groups of cells transfected successfully 48h Western blot experiment, found that cells transfected with IGFBP-4 expression plasmid group and high expression of IGFBP-4 protein content was significantly higher than the control group increased 1 times, IGFBP-4 interference plasmid protein content of IGFBP-4 was decreased and the interference efficiency was 80%. The successful construction of IGFBP-4 high / low expression cell model; 5 I CELLigence real-time growth curve labeled cell function analyzer in monitoring cells under different planting densities, the optimum planting density for each hole 5 * 104 cells in 6 groups were compared; choose the 100h curve high, high expression of IGFBP-4 group is higher than that of the control group, IGFBP-4 interference plasmid was lower than that of control plasmid group, high expression of IGFBP-4 group was significantly higher than that of IGFBP-4 plasmid group, the difference was statistically significant; 7 immunofluorescence to observe the fine Cell differentiation: high expression of IGFBP-4 positive cells proportion control group MAP- II was 27.2%, the proportion of GFAP positive cells was 32.3%; the high expression of IGFBP-4 plasmid group neuron cell marker positive cells were MAP- II 38%, glial cell marker positive cells of GFAP is 92%; IGFBP-4 interference plasmid control group MAP- II positive cells ratio was 21.7% for 86.1%, the proportion of GFAP positive cells; IGFBP-4 interference plasmid MAP- II positive cells accounted for 24.3%, the proportion of GFAP positive cells in 89.6%.IGFBP-4 high expression group neuron differentiation ratio greater than PEGFP-N1 plasmid group (P0.05), IGFBP-4 plasmid group neuronal differentiation ratio significantly compared to the control group RNAi-negative control group decreased (P0.05). The low expression cell model the successful construction of the high expression of IGFBP-4 IGFBP-4 2 / 1 conclusion; promote the proliferation of neural stem cells; 3 IGFBP-4 promotes neural stem cells into neurons Differentiation.

【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R741

【參考文獻】

相關(guān)期刊論文 前7條

1 高偉東;李曉冬;;胚胎神經(jīng)干細胞對腦損傷治療作用的實驗研究[J];解剖學(xué)研究;2010年01期

2 李紅偉;楊波;杜英;孫劍瑞;阮麗蓉;宋來君;;人胚神經(jīng)干細胞體外免疫原性測定[J];鄭州大學(xué)學(xué)報(醫(yī)學(xué)版);2006年02期

3 汪雷;宋躍明;劉立岷;李濤;龔全;楊曦;;NEP1-40基因修飾的神經(jīng)干細胞移植對脊髓損傷大鼠行為學(xué)恢復(fù)的影響[J];華西醫(yī)學(xué);2014年11期

4 嚴旺;陳慧敏;;神經(jīng)干細胞研究進展[J];現(xiàn)代實用醫(yī)學(xué);2013年01期

5 劉立新;孫凌云;郭曉紅;張騫騫;;胰島素樣生長因子結(jié)合蛋白2、4在實驗性肝細胞損傷中的表達及意義[J];中國病理生理雜志;2009年01期

6 楊坦;劉華;王肇光;高濟凡;肖東杰;汪運山;;應(yīng)用于缺血缺氧性腦病治療的神經(jīng)干細胞移植:現(xiàn)實與未來[J];中國組織工程研究;2014年01期

7 劉洋;雷旭輝;殷實;蔣傳路;;PFT-α對腦缺血后神經(jīng)干細胞移植中PUMA的調(diào)控研究[J];疑難病雜志;2014年11期

相關(guān)博士學(xué)位論文 前2條

1 唐軍;APPswe/PSldE9雙轉(zhuǎn)基因AD小鼠成年海馬神經(jīng)發(fā)生及微環(huán)境的調(diào)控機制研究[D];第三軍醫(yī)大學(xué);2009年

2 黃曉輝;MSI-H/S結(jié)直腸癌差異表達分子的篩選及其在免疫治療中的意義[D];第二軍醫(yī)大學(xué);2013年

，

本文編號：1474610