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
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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。
Objective:To investigate the proliferation and secreton of fibronectin(FN)from NIH3T3 fibroblasts after tansfection with mammalian target of rapamycin(mTOR)gene,and to explore the probable mechanism of mTOR gene on proliferation and secretion of FN of NIH3T3 fibroblasts after treatment these cells with rapamycin,so that the expressions of FN gene and protein,the expressions of Akt,mTOR, p70S6K genes and proteins were checked.
Methods:The plasmids of recombinant pcDNA3/mTOR was transformed into competent cells(DH-αEscherichia coli(E.coli)) prepared with the conventional CaCl_2 method.Then it was extracted with the alkaline lysis.The recombinant vector pcDNA3/mTOR was determined by restriction enzyme digestion and sequencing analysis. Then the pcDNA3/mTOR plasmid was transfected into NIH3T3 fibroblasts with electroporation method.The positive cell clones were selected with G418.Then the transfected stable cell clones were obtained, which were treated with rapamycin.The mRNA and protein expressions of Akt、mTOR、p70S6K and FN were determined by RT-PCR and Western blotting analysis respectively.MTT assay was employed to check the proliferation of NIH3T3 fibroblasts.The SPSS11.0 software was used for the statistical analysis.The t test and F test or x~2 test was employed to analysis these date respectively in this research.The P＜0.05 was considered to be statistically significant.
Results:
(1)The recombinant pcDNA3/mTOR plasmid was identified with double digestion and nucleic acid sequencing,which proved the transfected plasmid was corrected.
(2)After transfection with mTOR gene,the results of expressions of gene and protein were as follows.The gray scale values of mTOR/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.68±0.03 and 0.38±0.03 respectively with RT-PCR(P＜0.01), and 0.79±0.04 and 0.57±0.01 respectively with Western blotting(P＜0.01). The gray scale values of FN/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.63±0.07 and 0.39±0.03 respectively with RT-PCR(P＜0.01),and 0.77±0.07 and 0.55±0.04 respectively with Western blotting(P＜0.01).The gray scale values of Akt/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.41±0.07 and 0.39±0.02 respectively with RT-PCR(P＞0.05),and 0.65±0.02 and 0.63±0.03 respectively with Western blotting(P＞0.05). The gray scale values of p7.0S6K/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.69±0.03 and 0.34±0.02 respectively with RT-PCR(P＜0.01),and 0.75±0.06 and 0.53±0.05 respectively with Western blotting(P＜0.01).The MTT analysis showed that at all time point but for the first day,the A value in transfected group was higher than that in control group(all P＜0.01).
(3)After treated with rapamycin,the results of expressions of gene and protein were as follows.The gray scale values of Akt/GAPDH of NIH3T3 fibroblasts in transfected group,control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.40±0.06,0.39±0.02,0.41±0.05 and 0.39±0.04 respectively with RT-PCR(P＞0.05),and were 0.66±0.02,0.63±0.04,0.67±0.07 and 0.64±0.05 respectively with Western blotting(P＞0.05).The gray scale values of p70S6K/GAPDH of NIH3T3 fibroblasts in transfected group, control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.70±0.03、0.34±0.04、0.35±0.02 and 0.33±0.06 respectively with RT-PCR(P＜0.01),and were 0.76±0.05、0.54±0.04、0.52±0.01、0.53±0.03 respectively with Western blotting (P＜0.01).The gray scale value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).The gray scale values of FN/GAPDH of N/H3T3 fibroblasts in transfected group,control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.64±0.04、0.40±0.05、0.40±0.03 and 0.41±0.03 respectively with RT-PCR(P＜0.01),and were 0.76±0.03、0.53±0.01、0.54±0.02 and 0.54±0.05 respectively with Western blotting (P＜0.01).The gray scale value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).The MTT analysis showed that at all time point but for the first day,the A value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).
Conclusions:
(1)Transfection of mTOR gene increases the proliferation of fibroblasts and secretion of FN.
(2)The effect of mTOR gene probable was by means of the Akt-mTOR-p70S6K signal pathway,but does not depend on the activation of Akt.
(3)The Rapamycin suppresses the proliferation of fibroblasts and secretion of FN probable by means of inhibiting mTOR-p70S6K signal pathway.
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。
Objective:To investigate the proliferation and secreton of fibronectin(FN)from NIH3T3 fibroblasts after tansfection with mammalian target of rapamycin(mTOR)gene,and to explore the probable mechanism of mTOR gene on proliferation and secretion of FN of NIH3T3 fibroblasts after treatment these cells with rapamycin,so that the expressions of FN gene and protein,the expressions of Akt,mTOR, p70S6K genes and proteins were checked.
Methods:The plasmids of recombinant pcDNA3/mTOR was transformed into competent cells(DH-αEscherichia coli(E.coli)) prepared with the conventional CaCl_2 method.Then it was extracted with the alkaline lysis.The recombinant vector pcDNA3/mTOR was determined by restriction enzyme digestion and sequencing analysis. Then the pcDNA3/mTOR plasmid was transfected into NIH3T3 fibroblasts with electroporation method.The positive cell clones were selected with G418.Then the transfected stable cell clones were obtained, which were treated with rapamycin.The mRNA and protein expressions of Akt、mTOR、p70S6K and FN were determined by RT-PCR and Western blotting analysis respectively.MTT assay was employed to check the proliferation of NIH3T3 fibroblasts.The SPSS11.0 software was used for the statistical analysis.The t test and F test or x~2 test was employed to analysis these date respectively in this research.The P＜0.05 was considered to be statistically significant.
Results:
(1)The recombinant pcDNA3/mTOR plasmid was identified with double digestion and nucleic acid sequencing,which proved the transfected plasmid was corrected.
(2)After transfection with mTOR gene,the results of expressions of gene and protein were as follows.The gray scale values of mTOR/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.68±0.03 and 0.38±0.03 respectively with RT-PCR(P＜0.01), and 0.79±0.04 and 0.57±0.01 respectively with Western blotting(P＜0.01). The gray scale values of FN/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.63±0.07 and 0.39±0.03 respectively with RT-PCR(P＜0.01),and 0.77±0.07 and 0.55±0.04 respectively with Western blotting(P＜0.01).The gray scale values of Akt/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.41±0.07 and 0.39±0.02 respectively with RT-PCR(P＞0.05),and 0.65±0.02 and 0.63±0.03 respectively with Western blotting(P＞0.05). The gray scale values of p7.0S6K/GAPDH of NIH3T3 fibroblasts in transfected group,control group were 0.69±0.03 and 0.34±0.02 respectively with RT-PCR(P＜0.01),and 0.75±0.06 and 0.53±0.05 respectively with Western blotting(P＜0.01).The MTT analysis showed that at all time point but for the first day,the A value in transfected group was higher than that in control group(all P＜0.01).
(3)After treated with rapamycin,the results of expressions of gene and protein were as follows.The gray scale values of Akt/GAPDH of NIH3T3 fibroblasts in transfected group,control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.40±0.06,0.39±0.02,0.41±0.05 and 0.39±0.04 respectively with RT-PCR(P＞0.05),and were 0.66±0.02,0.63±0.04,0.67±0.07 and 0.64±0.05 respectively with Western blotting(P＞0.05).The gray scale values of p70S6K/GAPDH of NIH3T3 fibroblasts in transfected group, control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.70±0.03、0.34±0.04、0.35±0.02 and 0.33±0.06 respectively with RT-PCR(P＜0.01),and were 0.76±0.05、0.54±0.04、0.52±0.01、0.53±0.03 respectively with Western blotting (P＜0.01).The gray scale value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).The gray scale values of FN/GAPDH of N/H3T3 fibroblasts in transfected group,control group,transfected cells treated with Rapamycin group and control treated with Rapamycin group were 0.64±0.04、0.40±0.05、0.40±0.03 and 0.41±0.03 respectively with RT-PCR(P＜0.01),and were 0.76±0.03、0.53±0.01、0.54±0.02 and 0.54±0.05 respectively with Western blotting (P＜0.01).The gray scale value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).The MTT analysis showed that at all time point but for the first day,the A value in transfected group was higher than that in other three groups(P＜0.01),the values among the other three groups were no significant differences from each other(P＞0.05).
Conclusions:
(1)Transfection of mTOR gene increases the proliferation of fibroblasts and secretion of FN.
(2)The effect of mTOR gene probable was by means of the Akt-mTOR-p70S6K signal pathway,but does not depend on the activation of Akt.
(3)The Rapamycin suppresses the proliferation of fibroblasts and secretion of FN probable by means of inhibiting mTOR-p70S6K signal pathway.
引文
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