mTOR基因转染对NIH3T3成纤维细胞增殖的影响及其作用机制研究

发布时间：2018-06-26 11:40

本文选题：mTOR + 雷帕霉素　；参考：《中南大学》2008年博士论文

【摘要】： 目的观察哺乳动物雷帕霉素靶蛋白(mammalian target ofrapamycin,mTOR)基因转染NIH3T3成纤维细胞后细胞增殖情况及对分泌纤连蛋白(fibronectin,FN)的影响,并以雷帕霉素(rapamycin)进行干预,检测Akt-mTOR-p70S6K信号通路中各信号分子及FN的表达,以探讨mTOR基因对成纤维细胞增殖及分泌FN的影响及机制。方法以氯化钙制备感受态细胞法制备感受态细胞并转化pcDNA3.0-mTOR重组质粒,培养扩增后以碱裂解法抽提质粒,经酶切及DNA测序鉴定,然后通过电穿孔法将质粒转染NIH3T3成纤维细胞,以G418筛选,扩大培养后获得稳定转染细胞株。以雷帕霉素干预转染细胞和非转染细胞,采用MTT法检测细胞增殖力,RT-PCR、Western blot等方法检测细胞Akt、mTOR、p70S6K和FN的表达。应用SPSS11.0统计软件对数据进行统计学分析,计量资料以(?)±s表示,组间比较采用t检验和F检验;计数资料组间比较采用χ~2检验。以P＜0.05为差别有显著性统计学意义。结果 (1)提取的质粒浓度为296ng/μL,OD260/OD280为1.88。双酶切鉴定可见目的基因片段,DNA测序进一步证实为目的基因。 (2)mTOR基因转染成纤维细胞后各基因及蛋白表达的检测结果:mTOR mRNA表达,mTOR/GAPDH光密度值在转染组、对照组分别为0.68±0.03、0.38±0.03(P＜0.01);mTOR蛋白表达,mTOR/GAPDH灰度值在转染组、对照组分别为0.79±0.04和0.57±0.01(P＜0.01)。FN mRNA表达,FN/GAPDH光密度值在转染组、对照组分别为0.63±0.07、0.39±0.03(P＜0.01);FN蛋白表达,FN/GAPDH灰度值在转染组、对照组分别为0.77±0.07和0.55±0.04(P＜0.01)。Akt mRNA表达,Akt/GAPDH光密度值在转染组、对照组分别为0.41±0.07、0.39±0.02(P＞0.05);Akt蛋白表达,Akt/GAPDH灰度值在转染组、对照组分别为0.65±0.02和0.63±0.03(P＞0.05)。p70S6KmRNA表达,p70S6K/GAPDH光密度值在转染组、对照组分别为0.69±0.03、0.34±0.02(P＜0.01);p70S6K蛋白表达,p70S6K/GAPDH灰度值在转染组、对照组分别为0.75±0.06、0.53±0.05(P＜0.01)。MTT(A值)检测结果:在各时间点A值转染组大于对照组(P＜0.01)。 (3)雷帕霉素干预转染前后检测结果:Akt mRNA表达:Akt/GAPDH光密度值在转染组、对照组、雷帕霉素干预转染组、雷帕霉素干预对照组分别为0.4±0.06、0.39±0.02、0.41±0.05、0.39±0.04。转染组光密度值与其他三组及其他三组之间均无显著性差异(P＞0.05)。Akt蛋白表达:Akt/GAPDH灰度值在转染组、对照组、雷帕霉素干预转染组、雷帕霉素干预对照组分别为0.66±0.02、0.63±0.04、0.67±0.07、0.64±0.05,转染组灰度值与其他三组及其他三组之间均无显著性差异(P＞0.05)。p70S6K mRNA表达:p70S6K/GAPDH光密度值在转染组、对照组、雷帕霉素干预转染组、雷帕霉素干预对照组分别为0.70±0.03、0.34±0.04、0.35±0.02、0.33±0.06。转染组光密度值大于其他三组(P＜0.01),其他三组之间无显著性差异(P＞0.05)。p70S6K蛋白表达:p70S6K/GAPDH灰度值在转染组、对照组、雷帕霉素干预转染组、雷帕霉素干预对照组分别为0.76±0.05、0.54±0.04、0.52±0.01、0.53±0.03。转染组灰度值大于其他三组(P＜0.01),其他三组之间无显著差异(P＞0.05)。FN mRNA表达:FN/GAPDH光密度值在转染组、对照组、雷帕霉素干预转染组、雷帕霉素干预对照组分别为0.64±0.04、0.40±0.05、0.40±0.03、0.41±0.03。转染组光密度值大于其他三组(P＜0.01),其他三组之间无显著差异(P＞0.05)。FN蛋白表达:FN/GAPDH灰度值在转染组、对照组、雷帕霉素干预转染组、雷帕霉素干预对照组分别为0.76±0.03、0.53±0.01、0.54±0.02、0.54±0.05。转染组灰度值大于其他三组(P＜0.01),其他三组之间无显著性差异(P＞0.05)。MTT(A值)检测结果:在各时间点A值转染组大于雷帕霉素干预转染组及对照组、雷帕霉素干预对照组(P＜0.01),后三组各时间点A值无显著性差异(P＞0.05)。结论 (1)mTOR基因使成纤维细胞增殖力增强,FN表达增强。 (2)mTOR基因通过Akt-mTOR-p70S6K信号通路发挥作用,不一定依赖Akt的激活。 (3)雷帕霉素可通过阻断mTOR-p70S6K信号通路而抑制成纤维细胞增殖及FN的合成。
[Abstract]:Objective To observe the proliferation of mammalian target ofrapamycin (mTOR) gene transfected by rapamycin ofrapamycin (mTOR) gene and the effect on the secretion of fibronectin (fibronectin, FN) in NIH3T3 fibroblasts, and to detect the expression of each signal molecule and FN in the Akt-mTOR-p70S6K signal pathway by using rapamycin (rapamycin) to detect the expression of each signal molecule and FN in the Akt-mTOR-p70S6K signal pathway. To explore the effect and mechanism of mTOR gene on proliferation and secretion of FN in fibroblasts.
Methods using calcium chloride to prepare susceptible cells and transform pcDNA3.0-mTOR recombinant plasmid, the plasmid was extracted by alkali lysis and identified by enzyme digestion and DNA sequencing. Then transfected by electroporation, the plasmid was transfected into NIH3T3 fibroblasts, and the stable transfected cell line was obtained by G418 screening. The cells and non transfected cells were transfected by MTT, and the expression of cell Akt, mTOR, p70S6K and FN were detected by RT-PCR and Western blot. The data were statistically analyzed with SPSS11.0 statistical software. The measurement data were expressed with (?) + s, t test and F test were used in the group, and the count data were compared by chi square The difference was statistically significant between P < 0.05.
Result
(1) the plasmid concentration was 296ng/ L, OD260/OD280 was 1.88., and the target gene fragment was identified by double enzyme digestion. DNA sequencing further confirmed the target gene.
(2) the detection results of gene and protein expression of mTOR gene transfected into fibroblasts: mTOR mRNA expression, mTOR/GAPDH light density value in transfection group and control group 0.68 + 0.03,0.38 + 0.03 (P < 0.01), mTOR protein expression, mTOR/GAPDH gray value in transfected group, and control group were 0.79 + 0.04 and 0.57 + 0.01 (P < 0.01).FN mRNA expression, respectively. FN/GAP The value of DH light density was 0.63 + 0.07,0.39 + 0.03 (P < 0.01) in the control group, and the expression of FN protein was expressed in the transfected group, and the control group was 0.77 + 0.07 and 0.55 + 0.04 (P < 0.01).Akt mRNA, respectively. The Akt/GAPDH light density value was in the transfected group, and the control group was 0.41 + 0.07,0.39 + 0.02 (P > 0.05), and the Akt protein was expressed. The value of gray value in the transfection group was 0.65 + 0.02 and 0.63 + 0.03 (P > 0.05).P70S6KmRNA respectively. The p70S6K/GAPDH light density value was 0.69 + 0.03,0.34 + 0.02 (P < 0.01) in the control group, and the p70S6K protein expression was expressed in the transfected group, and the p70S6K/GAPDH gray value was in the transfected group, and the group was 0.75 + 0.06,0.53 + 0.05 (P < 0.01).MTT (A), respectively. Results: at each time point, the A value transfection group was larger than that of the control group (P < 0.01).
(3) the results of rapamycin intervention before and after transfection: Akt mRNA expression: Akt/GAPDH light density value in transfection group, control group, rapamycin intervention group, the light density value of 0.4 + 0.06,0.39 + 0.02,0.41 + 0.05,0.39 + 0.04. transfection group with rapamycin control group, and no significant difference between the three groups and the other three groups (P > 0.05) The expression of.Akt protein: Akt/GAPDH gray value in transfection group, control group, rapamycin intervention group, rapamycin control group was 0.66 + 0.02,0.63 + 0.04,0.67 + 0.07,0.64 + 0.05 respectively, and there was no significant difference between the gray value of the transfected group and the other three groups and other three groups (P > 0.05).P70S6K mRNA expression: p70S6K/GAPDH light density value in the group The transfection group, control group, rapamycin intervention group, the light density value of 0.70 + 0.03,0.34 + 0.04,0.35 + 0.02,0.33 + 0.06. transfection group with rapamycin control group was greater than the other three groups (P < 0.01). There was no significant difference between the other three groups (P > 0.05).P70S6K protein expression: p70S6K/GAPDH gray value in transfection group, control group, rapamycin The gray value of 0.76 + 0.05,0.54 + 0.04,0.52 + 0.01,0.53 + 0.03. transfection group in the control group was greater than that of the other three groups (P < 0.01). There was no significant difference between the other three groups (P > 0.05).FN mRNA expression: the FN/GAPDH light density value was in the transfection group, the control group, the rapamycin intervention group, and the rapamycin intervention control The light density value of the group of 0.64 + 0.04,0.40 + 0.05,0.40 + 0.03,0.41 + 0.03. transfection group was greater than that of the other three groups (P < 0.01). There was no significant difference between the other three groups (P > 0.05).FN protein expression: the gray value of FN/GAPDH was in the transfection group, the control group, the rapamycin intervention group, the control group was 0.76 + 0.03,0.53 + 0.01,0.54 + 0., respectively. The gray value of 02,0.54 + 0.05. transfection group was greater than that of the other three groups (P < 0.01), there was no significant difference between the other three groups (P > 0.05).MTT (A value) detection results: at every time point, A value transfection group was greater than rapamycin intervention group and control group, rapamycin intervention control group (P < 0.01), and there was no significant difference between the three groups at each time point A value (P > 0.05).
conclusion
(1) mTOR gene increased the proliferation of fibroblasts and enhanced the expression of FN.
(2) mTOR gene plays a role through Akt-mTOR-p70S6K signaling pathway, and does not necessarily depend on Akt activation.
(3) rapamycin can inhibit fibroblast proliferation and FN synthesis by blocking mTOR-p70S6K signaling pathway.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

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