本文選題：mTOR + 雷帕霉素�。� 參考：《中南大學》2008年博士論文

【摘要】： 目的觀察哺乳動物雷帕霉素靶蛋白(mammalian target ofrapamycin,mTOR)基因轉(zhuǎn)染NIH3T3成纖維細胞后細胞增殖情況及對分泌纖連蛋白(fibronectin,FN)的影響,并以雷帕霉素(rapamycin)進行干預(yù),檢測Akt-mTOR-p70S6K信號通路中各信號分子及FN的表達,以探討mTOR基因?qū)Τ衫w維細胞增殖及分泌FN的影響及機制。 方法以氯化鈣制備感受態(tài)細胞法制備感受態(tài)細胞并轉(zhuǎn)化pcDNA3.0-mTOR重組質(zhì)粒,培養(yǎng)擴增后以堿裂解法抽提質(zhì)粒,經(jīng)酶切及DNA測序鑒定,然后通過電穿孔法將質(zhì)粒轉(zhuǎn)染NIH3T3成纖維細胞,以G418篩選,擴大培養(yǎng)后獲得穩(wěn)定轉(zhuǎn)染細胞株。以雷帕霉素干預(yù)轉(zhuǎn)染細胞和非轉(zhuǎn)染細胞,采用MTT法檢測細胞增殖力,RT-PCR、Western blot等方法檢測細胞Akt、mTOR、p70S6K和FN的表達。應(yīng)用SPSS11.0統(tǒng)計軟件對數(shù)據(jù)進行統(tǒng)計學分析,計量資料以(?)±s表示,組間比較采用t檢驗和F檢驗;計數(shù)資料組間比較采用χ~2檢驗。以P＜0.05為差別有顯著性統(tǒng)計學意義。 結(jié)果 (1)提取的質(zhì)粒濃度為296ng/μL,OD260/OD280為1.88。雙酶切鑒定可見目的基因片段,DNA測序進一步證實為目的基因。 (2)mTOR基因轉(zhuǎn)染成纖維細胞后各基因及蛋白表達的檢測結(jié)果:mTOR mRNA表達,mTOR/GAPDH光密度值在轉(zhuǎn)染組、對照組分別為0.68±0.03、0.38±0.03(P＜0.01);mTOR蛋白表達,mTOR/GAPDH灰度值在轉(zhuǎn)染組、對照組分別為0.79±0.04和0.57±0.01(P＜0.01)。FN mRNA表達,FN/GAPDH光密度值在轉(zhuǎn)染組、對照組分別為0.63±0.07、0.39±0.03(P＜0.01);FN蛋白表達,FN/GAPDH灰度值在轉(zhuǎn)染組、對照組分別為0.77±0.07和0.55±0.04(P＜0.01)。Akt mRNA表達,Akt/GAPDH光密度值在轉(zhuǎn)染組、對照組分別為0.41±0.07、0.39±0.02(P＞0.05);Akt蛋白表達,Akt/GAPDH灰度值在轉(zhuǎn)染組、對照組分別為0.65±0.02和0.63±0.03(P＞0.05)。p70S6KmRNA表達,p70S6K/GAPDH光密度值在轉(zhuǎn)染組、對照組分別為0.69±0.03、0.34±0.02(P＜0.01);p70S6K蛋白表達,p70S6K/GAPDH灰度值在轉(zhuǎn)染組、對照組分別為0.75±0.06、0.53±0.05(P＜0.01)。MTT(A值)檢測結(jié)果:在各時間點A值轉(zhuǎn)染組大于對照組(P＜0.01)。 (3)雷帕霉素干預(yù)轉(zhuǎn)染前后檢測結(jié)果:Akt mRNA表達:Akt/GAPDH光密度值在轉(zhuǎn)染組、對照組、雷帕霉素干預(yù)轉(zhuǎn)染組、雷帕霉素干預(yù)對照組分別為0.4±0.06、0.39±0.02、0.41±0.05、0.39±0.04。轉(zhuǎn)染組光密度值與其他三組及其他三組之間均無顯著性差異(P＞0.05)。Akt蛋白表達:Akt/GAPDH灰度值在轉(zhuǎn)染組、對照組、雷帕霉素干預(yù)轉(zhuǎn)染組、雷帕霉素干預(yù)對照組分別為0.66±0.02、0.63±0.04、0.67±0.07、0.64±0.05,轉(zhuǎn)染組灰度值與其他三組及其他三組之間均無顯著性差異(P＞0.05)。p70S6K mRNA表達:p70S6K/GAPDH光密度值在轉(zhuǎn)染組、對照組、雷帕霉素干預(yù)轉(zhuǎn)染組、雷帕霉素干預(yù)對照組分別為0.70±0.03、0.34±0.04、0.35±0.02、0.33±0.06。轉(zhuǎn)染組光密度值大于其他三組(P＜0.01),其他三組之間無顯著性差異(P＞0.05)。p70S6K蛋白表達:p70S6K/GAPDH灰度值在轉(zhuǎn)染組、對照組、雷帕霉素干預(yù)轉(zhuǎn)染組、雷帕霉素干預(yù)對照組分別為0.76±0.05、0.54±0.04、0.52±0.01、0.53±0.03。轉(zhuǎn)染組灰度值大于其他三組(P＜0.01),其他三組之間無顯著差異(P＞0.05)。FN mRNA表達:FN/GAPDH光密度值在轉(zhuǎn)染組、對照組、雷帕霉素干預(yù)轉(zhuǎn)染組、雷帕霉素干預(yù)對照組分別為0.64±0.04、0.40±0.05、0.40±0.03、0.41±0.03。轉(zhuǎn)染組光密度值大于其他三組(P＜0.01),其他三組之間無顯著差異(P＞0.05)。FN蛋白表達:FN/GAPDH灰度值在轉(zhuǎn)染組、對照組、雷帕霉素干預(yù)轉(zhuǎn)染組、雷帕霉素干預(yù)對照組分別為0.76±0.03、0.53±0.01、0.54±0.02、0.54±0.05。轉(zhuǎn)染組灰度值大于其他三組(P＜0.01),其他三組之間無顯著性差異(P＞0.05)。MTT(A值)檢測結(jié)果:在各時間點A值轉(zhuǎn)染組大于雷帕霉素干預(yù)轉(zhuǎn)染組及對照組、雷帕霉素干預(yù)對照組(P＜0.01),后三組各時間點A值無顯著性差異(P＞0.05)。 結(jié)論 (1)mTOR基因使成纖維細胞增殖力增強,FN表達增強。 (2)mTOR基因通過Akt-mTOR-p70S6K信號通路發(fā)揮作用,不一定依賴Akt的激活。 (3)雷帕霉素可通過阻斷mTOR-p70S6K信號通路而抑制成纖維細胞增殖及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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R346

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