慢病毒介導(dǎo)shRNA沉默乳腺癌MCF-7細(xì)胞MTDH基因?qū)ψ仙即济舾行杂绊懙难芯?/H1>
發(fā)布時(shí)間：2018-09-19 06:43
【摘要】：目的:乳腺癌已成為女性最常見的惡性腫瘤之一。化療是治療乳腺癌的主要手段之一,但由于獲得性耐藥導(dǎo)致化療失敗卻是臨床治療中的一個(gè)重大挑戰(zhàn)。MTDH原癌基因?qū)Υ龠M(jìn)腫瘤細(xì)胞的增殖、腫瘤血管生成,引起凋亡抑制有重要作用,并與腫瘤細(xì)胞侵襲、擴(kuò)散、轉(zhuǎn)移及化療耐藥密切相關(guān)。本實(shí)驗(yàn)通過研究MTDH基因沉默對乳腺癌MCF-7細(xì)胞增殖及對紫杉醇耐藥性的影響,為乳腺癌的基因治療及逆轉(zhuǎn)化療耐藥提供實(shí)驗(yàn)理論依據(jù)。方法:本實(shí)驗(yàn)成功構(gòu)建慢病毒介導(dǎo)MTDH基因沉默的MCF-7穩(wěn)轉(zhuǎn)細(xì)胞株(命名為MCF-7-MTDH/knockdown)和陰性對照病毒穩(wěn)定轉(zhuǎn)染的乳腺癌細(xì)胞株(命名為MCF-7-control)。采用Real-time PCR和Western blot法檢測空白對照組(MCF-7)、陰性對照組(MCF-7-control)、實(shí)驗(yàn)組(MCF-7-MTDH/knockdown)的MTDH mRNA和蛋白質(zhì)表達(dá)水平驗(yàn)證轉(zhuǎn)染效果。CCK-8法繪制沉默前后MCF-7細(xì)胞株的生長曲線,并通過CCK-8法檢測沉默MCF-7細(xì)胞MTDH基因?qū)ψ仙即妓幬锩舾行缘挠绊憽Ａ魇郊?xì)胞術(shù)檢測沉默前后細(xì)胞周期及紫杉醇對細(xì)胞周期和凋亡的影響。Real-time PCR和Western blot法檢測NF-κB P65,IκBα基因在MCF-7、MCF-7-control、MCF-7-MTDH/knockdown細(xì)胞中的mRNA和蛋白質(zhì)表達(dá)水平。通過建立穩(wěn)定轉(zhuǎn)染細(xì)胞的荷瘤裸鼠模型,研究MTDH沉默在體內(nèi)對紫杉醇敏感性的影響,并對成瘤標(biāo)本進(jìn)行流式細(xì)胞術(shù)檢測細(xì)胞凋亡情況。應(yīng)用SPSS21.0統(tǒng)計(jì)軟件對實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,檢驗(yàn)以P0.05為差異有顯著性意義。每組實(shí)驗(yàn)不少于3個(gè)獨(dú)立樣本的重復(fù)。結(jié)果:1慢病毒介導(dǎo)MTDH沉默的乳腺癌MCF-7穩(wěn)轉(zhuǎn)細(xì)胞株的構(gòu)建和鑒定成功構(gòu)建穩(wěn)定轉(zhuǎn)染的MCF-7-MTDH/knockdown細(xì)胞株和MCF-7-control細(xì)胞株。通過Real-time PCR檢測MCF-7細(xì)胞、mcf-7-control細(xì)胞、mcf-7-mtdh/knockdown細(xì)胞中mtdh基因mrna表達(dá)水平分別為1.001±0.010、1.014±0.018、0.285±0.036。通過westernblot檢測mtdh蛋白在三種細(xì)胞中的相對表達(dá)量分別為0.871±0.126、0.816±0.067、0.089±0.011。mcf-7-mtdh/knockdown細(xì)胞mtdh基因mrna表達(dá)和蛋白表達(dá)均低于兩對照組(p0.05)。2mtdh沉默對細(xì)胞增殖和細(xì)胞周期、細(xì)胞凋亡的影響cck-8法檢測各組細(xì)胞于0h、24h、48h、72h的增殖情況,實(shí)驗(yàn)組mcf-7-mtdh/knockdown細(xì)胞增殖速度明顯慢于mcf-7和mcf-7-control兩對照組(p0.01)。流式細(xì)胞儀檢測各組的細(xì)胞周期,結(jié)果顯示空白對照組、陰性對照組、實(shí)驗(yàn)組細(xì)胞g0/g1期比例分別為41.61±0.53%、40.27±1.22%、55.70±1.83%;實(shí)驗(yàn)組s期和g2/m期比例均減低。與對照對相比實(shí)驗(yàn)組細(xì)胞g0/g1期延長,s期和g2/m期縮短(p0.05)。流式細(xì)胞學(xué)檢測各組的細(xì)胞凋亡,結(jié)果顯示:空白對照組、陰性對照組、實(shí)驗(yàn)組細(xì)胞的凋亡率分別為4.85±0.64%、5.35±0.56%、10.21±0.29%。實(shí)驗(yàn)組mcf-7-mtdh/knockdown細(xì)胞凋亡率增加,高于對照兩組(p0.05)。3紫杉醇對mtdh基因沉默前后mcf-7細(xì)胞增殖和周期、細(xì)胞凋亡的影響0.1μg/ml紫杉醇作用48h對三組細(xì)胞的抑制率分別為40.71±0.08%、40.96±0.16%、54.25±0.06%;1μg/ml紫杉醇作用48h對三組細(xì)胞的抑制率分別為56.54±0.13%、56.49±0.04%、77.19±0.17%。0.1μg/ml、1μg/ml紫杉醇作用48h后對實(shí)驗(yàn)組mcf-7-mtdh/knockdown細(xì)胞的抑制率均高于兩對照組(p0.01)。細(xì)胞周期檢測結(jié)果顯示:紫杉醇藥物可誘導(dǎo)各組細(xì)胞g2/m期比例升高,1μg/ml用藥組較0.1μg/ml用藥組g2/m期比例升高更加明顯(p0.05);1μg/ml紫杉醇作用48h后mcf-7-mtdh/knockdown細(xì)胞的g2/m期比例明顯高于mcf-7、mcf-7-control細(xì)胞(p0.05)。細(xì)胞凋亡情況檢測結(jié)果顯示:各組細(xì)胞凋亡率隨紫杉醇濃度增加而增加。0.1μg/ml和1μg/ml紫杉醇分別作用48h后,實(shí)驗(yàn)組mcf-7-mtdh/knockdown細(xì)胞的凋亡率明顯高于兩對照組(p0.001)。4沉默前后MCF-7細(xì)胞NF-κB P65,IκBα基因的表達(dá)情況Real-time PCR和Western blot結(jié)果顯示:MTDH沉默后P65的mRNA和蛋白表達(dá)量降低,而IκBα的mRNA和蛋白表達(dá)量升高(P0.001)。與對照組相比差異有統(tǒng)計(jì)學(xué)意義。5 MTDH沉默對裸鼠成瘤的影響種植瘤的生長情況:MCF-7-MTDH/knockdown組裸鼠的腫瘤體積(374.35±16.68mm3 vs 902.7±26.53mm3 and 840.79±25.82mm3)和重量(0.459±0.051g vs 1.106±0.095g and 1.103±0.101g)明顯小于兩對照組(P0.001),生長速度較對照組明顯減慢。種植瘤對紫杉醇敏感性:MCF-7-MTDH/knockdown組裸鼠腫瘤體積(91.13±16.68 mm3 vs 340.21±26.35mm3 and 322.27±25.82 mm3)和重量(0.113±0.02 vs 0.440±0.01 g and 0.397±0.03 g)明顯小于兩對照組(P0.001)。流式細(xì)胞術(shù)檢測種植瘤的細(xì)胞凋亡:未應(yīng)用紫杉醇的MCF-7、MCF-7-control、MCF-7-MTDH/knockdown三組種植瘤細(xì)胞凋亡率分別為2.477±0.055%、2.823±0.770%、9.527±0.262%。MCF-7-MTDH/knockdown組裸鼠種植瘤凋亡率增加(P0.001)。應(yīng)用紫杉醇后三組種植瘤細(xì)胞凋亡率分別為14.547±0.625%、13.320±1.529%、25.613±0.996%。MCF-7-MTDH/knockdown組裸鼠種植瘤凋亡率較兩實(shí)驗(yàn)組明顯增加(P0.001)。結(jié)論:1通過慢病毒介導(dǎo)能夠成功構(gòu)建MTDH沉默的MCF-7穩(wěn)定轉(zhuǎn)染細(xì)胞株。2 MTDH沉默可抑制乳腺癌MCF-7細(xì)胞增殖,誘導(dǎo)乳腺癌MCF-7細(xì)胞G0/G1期比例升高,促進(jìn)細(xì)胞凋亡。3 MTDH沉默可提高乳腺癌細(xì)胞對紫杉醇的敏感性,其分子機(jī)制可能與NF-κB/IκB通路抑制有關(guān)。
[Abstract]:Objective: Breast cancer has become one of the most common malignant tumors in women. Chemotherapy is one of the main methods for the treatment of breast cancer, but failure of chemotherapy due to acquired drug resistance is a major challenge in clinical treatment. Invasion, diffusion, metastasis and chemotherapeutic resistance of breast cancer cells are closely related. This study was designed to investigate the effect of MTDH gene silencing on the proliferation and paclitaxel resistance of breast cancer MCF-7 cells, and to provide experimental theoretical basis for gene therapy of breast cancer and reversal of chemotherapeutic resistance. The expression levels of MTDH mRNA and protein in MCF-7-MTDH/knockdown and MCF-7-control were detected by Real-time PCR and Western blot. CCK-8 method was used to plot the growth curve of MCF-7 cell line before and after silencing, and CCK-8 method was used to detect the effect of MTDH gene on the drug sensitivity of paclitaxel. Flow cytometry was used to detect the cell cycle before and after silencing and the effect of paclitaxel on cell cycle and apoptosis. Expression levels of mRNA and protein in MCF-7, MCF-7-control and MCF-7-MTDH/knockdown cells were measured by flow cytometry. The effect of MTDH silencing on paclitaxel sensitivity in vivo was studied by establishing a stable tumor-bearing nude mice model. Results: 1. Lentivirus-mediated MTDH silencing breast cancer MCF-7 stable transfection cell line was successfully constructed and identified. The stable transfected MCF-7-MTDH/knockdown cell line and MCF-7-control cell line were successfully constructed by Real-time PCR. The expression levels of mtdh gene mRNA in MCF-7 cells, mcf-7-mtdh/knockdown cells and mcf-7-mtdh/knockdown cells were 1.001 (+ 0.010), 1.014 (+ 0.018) and 0.285 (+ 0.036) respectively. The relative expression levels of mtdh protein in the three kinds of cells were 0.871 (+ 0.126), 0.816 (+ 0.067), 0.089 (+ 0.011) mtdh/knockdown cells by Western blot, respectively. The expression and protein expression of mcf-7-mtdh / knockdown cells were significantly slower than those of MCF-7 and mcf-7-control cells (p0.01). Cell cycle, the results showed that the blank control group, the negative control group, the experimental group G0 / G1 phase ratio was 41.61 + 0.53%, 40.27 + 1.22%, 55.70 + 1.83%; experimental group S phase and G2 / M phase ratio were reduced. compared with the control group, the experimental group cells G0 / G1 phase prolonged, S phase and G2 / M phase shortened (p0.05). flow cytometry detection of cell apoptosis, node The results showed that the apoptosis rate of mcf-7-mtdh/knockdown cells in the experimental group was higher than that in the control group (p0.05). The inhibitory rates of paclitaxel at 48 h were 40.71 (+ 0.08%), 40.96 (+ 0.16%), 54.25 (+ 0.06%) and 56.54 (+ 0.13%), 56.49 (+ 0.04%) and 77.19 (+ 0.17%) respectively. The results of cell cycle test showed that the proportion of G2 / M phase of mcf-7-mtdh / knockdown cells was significantly higher than that of MCF-7 and mcf-7-control cells (p0.05). The percentage of G2 / M phase of mcf-7-mtdh / knockdown cells was significantly higher in 1 ug / ml group than that in 0.1 UG / ml group (p0.05). The results showed that the apoptosis rate of MCF-7 cells increased with the increase of paclitaxel concentration. After 48 hours of treatment with 0.1 ug/ml and 1 ug/ml paclitaxel respectively, the apoptosis rate of mcf-7-mtdh/knockdown cells in the experimental group was significantly higher than that in the control group (p0.001). 4 The expression of NF-kappa B P65 and I-kappa B alpha gene in MCF-7 cells before and after silencing was significantly higher than that in the control group (p0.001). After TDH silencing, the expression of P65 mRNA and protein decreased, while the expression of I-kappa Balpha mRNA and protein increased (P 0.001). The difference was statistically significant compared with the control group. 5 MTDH silencing on the growth of implanted tumors in nude mice: MCF-7-MTDH/knockdown group nude mice tumor volume (374.35+16.68mm3 vs 902.7+26.53mm3 and 840.79+25.82m) M3 and weight (0.459 + 0.051g vs 1.106 + 0.095g and 1.103 + 0.101g) were significantly lower than those of the two control groups (P 0.001), and the growth rate was significantly slower than that of the control group. Flow cytometry was used to detect the apoptosis of implant tumors: the apoptosis rates of MCF-7, MCF-7-control, MCF-7-MTDH/knockdown groups were 2.477 [0.055], 2.823 [0.770], 9.527 [0.262]. The apoptosis rates of implant tumors in MCF-7-MTDH/knockdown group were 2.477 [0.055], 2.823 [0.770], 9.527 [0.262], respectively. The apoptotic rates of the three groups were 14.547 (+ 0.625%), 13.320 (+ 1.529%) and 25.613 (+ 0.996%) respectively. The apoptotic rates of the nude mice in MCF-7-MTDH / knockdown group were significantly higher than those in the two experimental groups (P 0.001). Conclusion: 1. MTDH silencing MCF-7 stable transfected cell lines could be successfully constructed by lentivirus mediation. Proliferation of breast cancer MCF-7 cells, induction of G0/G1 phase ratio of breast cancer MCF-7 cells, and promotion of apoptosis. 3 MTDH silencing can enhance the sensitivity of breast cancer cells to paclitaxel, and its molecular mechanism may be related to the inhibition of NF-kappa B/I kappa B pathway.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;乳腺癌Metadherin基因表達(dá)的實(shí)時(shí)熒光定量方法的建立及初步應(yīng)用(英文)[J];Chinese-German Journal of Clinical Oncology;2010年06期
，
本文編號：2249345
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2249345.html
[Abstract]:Objective: Breast cancer has become one of the most common malignant tumors in women. Chemotherapy is one of the main methods for the treatment of breast cancer, but failure of chemotherapy due to acquired drug resistance is a major challenge in clinical treatment. Invasion, diffusion, metastasis and chemotherapeutic resistance of breast cancer cells are closely related. This study was designed to investigate the effect of MTDH gene silencing on the proliferation and paclitaxel resistance of breast cancer MCF-7 cells, and to provide experimental theoretical basis for gene therapy of breast cancer and reversal of chemotherapeutic resistance. The expression levels of MTDH mRNA and protein in MCF-7-MTDH/knockdown and MCF-7-control were detected by Real-time PCR and Western blot. CCK-8 method was used to plot the growth curve of MCF-7 cell line before and after silencing, and CCK-8 method was used to detect the effect of MTDH gene on the drug sensitivity of paclitaxel. Flow cytometry was used to detect the cell cycle before and after silencing and the effect of paclitaxel on cell cycle and apoptosis. Expression levels of mRNA and protein in MCF-7, MCF-7-control and MCF-7-MTDH/knockdown cells were measured by flow cytometry. The effect of MTDH silencing on paclitaxel sensitivity in vivo was studied by establishing a stable tumor-bearing nude mice model. Results: 1. Lentivirus-mediated MTDH silencing breast cancer MCF-7 stable transfection cell line was successfully constructed and identified. The stable transfected MCF-7-MTDH/knockdown cell line and MCF-7-control cell line were successfully constructed by Real-time PCR. The expression levels of mtdh gene mRNA in MCF-7 cells, mcf-7-mtdh/knockdown cells and mcf-7-mtdh/knockdown cells were 1.001 (+ 0.010), 1.014 (+ 0.018) and 0.285 (+ 0.036) respectively. The relative expression levels of mtdh protein in the three kinds of cells were 0.871 (+ 0.126), 0.816 (+ 0.067), 0.089 (+ 0.011) mtdh/knockdown cells by Western blot, respectively. The expression and protein expression of mcf-7-mtdh / knockdown cells were significantly slower than those of MCF-7 and mcf-7-control cells (p0.01). Cell cycle, the results showed that the blank control group, the negative control group, the experimental group G0 / G1 phase ratio was 41.61 + 0.53%, 40.27 + 1.22%, 55.70 + 1.83%; experimental group S phase and G2 / M phase ratio were reduced. compared with the control group, the experimental group cells G0 / G1 phase prolonged, S phase and G2 / M phase shortened (p0.05). flow cytometry detection of cell apoptosis, node The results showed that the apoptosis rate of mcf-7-mtdh/knockdown cells in the experimental group was higher than that in the control group (p0.05). The inhibitory rates of paclitaxel at 48 h were 40.71 (+ 0.08%), 40.96 (+ 0.16%), 54.25 (+ 0.06%) and 56.54 (+ 0.13%), 56.49 (+ 0.04%) and 77.19 (+ 0.17%) respectively. The results of cell cycle test showed that the proportion of G2 / M phase of mcf-7-mtdh / knockdown cells was significantly higher than that of MCF-7 and mcf-7-control cells (p0.05). The percentage of G2 / M phase of mcf-7-mtdh / knockdown cells was significantly higher in 1 ug / ml group than that in 0.1 UG / ml group (p0.05). The results showed that the apoptosis rate of MCF-7 cells increased with the increase of paclitaxel concentration. After 48 hours of treatment with 0.1 ug/ml and 1 ug/ml paclitaxel respectively, the apoptosis rate of mcf-7-mtdh/knockdown cells in the experimental group was significantly higher than that in the control group (p0.001). 4 The expression of NF-kappa B P65 and I-kappa B alpha gene in MCF-7 cells before and after silencing was significantly higher than that in the control group (p0.001). After TDH silencing, the expression of P65 mRNA and protein decreased, while the expression of I-kappa Balpha mRNA and protein increased (P 0.001). The difference was statistically significant compared with the control group. 5 MTDH silencing on the growth of implanted tumors in nude mice: MCF-7-MTDH/knockdown group nude mice tumor volume (374.35+16.68mm3 vs 902.7+26.53mm3 and 840.79+25.82m) M3 and weight (0.459 + 0.051g vs 1.106 + 0.095g and 1.103 + 0.101g) were significantly lower than those of the two control groups (P 0.001), and the growth rate was significantly slower than that of the control group. Flow cytometry was used to detect the apoptosis of implant tumors: the apoptosis rates of MCF-7, MCF-7-control, MCF-7-MTDH/knockdown groups were 2.477 [0.055], 2.823 [0.770], 9.527 [0.262]. The apoptosis rates of implant tumors in MCF-7-MTDH/knockdown group were 2.477 [0.055], 2.823 [0.770], 9.527 [0.262], respectively. The apoptotic rates of the three groups were 14.547 (+ 0.625%), 13.320 (+ 1.529%) and 25.613 (+ 0.996%) respectively. The apoptotic rates of the nude mice in MCF-7-MTDH / knockdown group were significantly higher than those in the two experimental groups (P 0.001). Conclusion: 1. MTDH silencing MCF-7 stable transfected cell lines could be successfully constructed by lentivirus mediation. Proliferation of breast cancer MCF-7 cells, induction of G0/G1 phase ratio of breast cancer MCF-7 cells, and promotion of apoptosis. 3 MTDH silencing can enhance the sensitivity of breast cancer cells to paclitaxel, and its molecular mechanism may be related to the inhibition of NF-kappa B/I kappa B pathway.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.9
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;乳腺癌Metadherin基因表達(dá)的實(shí)時(shí)熒光定量方法的建立及初步應(yīng)用(英文)[J];Chinese-German Journal of Clinical Oncology;2010年06期
，本文編號：2249345
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2249345.html
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