本文選題：全反式維甲酸　切入點(diǎn)：惡性黑色素瘤　出處：《第三軍醫(yī)大學(xué)學(xué)報(bào)》2017年22期 　論文類型：期刊論文

【摘要】：目的探討全反式維甲酸(all-trans retinoic acid,ATRA)對(duì)小鼠黑色素瘤B16F10細(xì)胞體外增殖、遷移、凋亡、致瘤性的影響及其初步作用機(jī)制。方法采用MTT法檢測(cè)ATRA對(duì)小鼠黑色素瘤細(xì)胞B16F10和小鼠成纖維細(xì)胞L929增殖能力的影響。顯微鏡下觀察給藥后兩種細(xì)胞的形態(tài)變化,應(yīng)用劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn)檢測(cè)ATRA對(duì)B16F10細(xì)胞遷移能力的影響,分別采用DAPI染色和Annexin V-FITC/PI染色觀察B16F10細(xì)胞凋亡情況,JC-1染色實(shí)驗(yàn)檢測(cè)B16F10細(xì)胞線粒體膜電位,熒光分光光度計(jì)檢測(cè)B16F10細(xì)胞中Caspase-9、Caspase-3的活化情況,利用體內(nèi)成瘤實(shí)驗(yàn)觀察ATRA對(duì)B16F10細(xì)胞致瘤性的影響。結(jié)果 MTT結(jié)果顯示ATRA呈濃度依賴性地抑制B16F10細(xì)胞和L929細(xì)胞增殖,但B16F10細(xì)胞的存活率明顯低于L929細(xì)胞的存活率(P0.05);分別以低、中、高3個(gè)濃度(6.25、16、40μmol/L)的ATRA處理細(xì)胞24 h后,顯微鏡下可見(jiàn)B16F10細(xì)胞在中濃度時(shí)呈凋亡樣改變,高濃度時(shí)細(xì)胞基本死亡,喪失正常形態(tài);而L929細(xì)胞形態(tài)僅在高濃度時(shí)發(fā)生顯著變化;劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn)提示B16F10細(xì)胞的體外遷移能力在中濃度即受到明顯抑制(P0.05);繼續(xù)以中濃度(16μmol/L)的ATRA作用B16F10細(xì)胞24 h,DAPI染色可見(jiàn)細(xì)胞核裂解,甚至可見(jiàn)凋亡小體;Annexin V-FITC/PI染色結(jié)合流式細(xì)胞儀測(cè)得細(xì)胞凋亡率為(37.27±4.42)%;JC-1染色實(shí)驗(yàn)提示線粒體膜電位明顯降低;熒光分光光度計(jì)測(cè)得給藥后Caspase-9、Caspase-3顯著被活化,活性分別增加了(1.54±0.00)、(3.09±0.01)倍(P0.05);成瘤實(shí)驗(yàn)結(jié)果顯示ATRA處理后的B16F10細(xì)胞致瘤性顯著降低(P0.01),甚至消失。結(jié)論 ATRA能顯著抑制B16F10細(xì)胞體外增殖、遷移能力,并能通過(guò)降低線粒體膜電位,活化Caspase-9、Caspase-3蛋白誘導(dǎo)細(xì)胞凋亡,進(jìn)而抑制B16F10致瘤性。
[Abstract]:Objective to investigate the proliferation, migration and apoptosis of mouse melanoma B16F10 cells induced by all-trans retinoic ATRAA. Methods the effects of ATRA on the proliferation of mouse melanoma cells B16F10 and mouse fibroblasts L929 were detected by MTT method. The morphological changes of the two cells were observed under microscope. The effect of ATRA on the migration of B16F10 cells was detected by scratch test and Transwell assay. The apoptosis of B16F10 cells was observed by DAPI staining and Annexin V-FITC / Pi staining. The mitochondrial membrane potential of B16F10 cells was detected by JC-1 staining. The activation of Caspase-9 and Caspase-3 in B16F10 cells was detected by fluorescence spectrophotometer, and the effect of ATRA on the tumorigenicity of B16F10 cells was observed by in vivo tumorigenesis. Results MTT showed that ATRA inhibited the proliferation of B16F10 cells and L929 cells in a dose-dependent manner. However, the survival rate of B16F10 cells was significantly lower than that of L929 cells (P 0.05). After treated with ATRA at low, medium and high concentrations of 6.25 ~ 1640 渭 mol / L for 24 h, it was observed that B16F10 cells showed apoptosis-like changes at medium concentration and cell death at high concentration. L929 cells lost normal morphology, while L929 cells only changed significantly at high concentration. The results of scratch test and Transwell assay showed that the migration ability of B16F10 cells in vitro was significantly inhibited at medium concentration (P 0.05), and nuclear cleavage could be observed by 24 h DAPI staining of B16F10 cells treated with ATRA at medium concentration of 16 渭 mol / L. Even the apoptotic body Annexin V-FITC / Pi staining combined with flow cytometry showed that the apoptosis rate was 37.27 鹵4.42% and the mitochondrial membrane potential was significantly decreased, and Caspase-9 caspase-3 was activated by fluorescence spectrophotometer. The results of tumorigenesis experiment showed that the tumorigenicity of B16F10 cells treated with ATRA significantly decreased or even disappeared. Conclusion ATRA can significantly inhibit the proliferation and migration of B16F10 cells in vitro, and can decrease mitochondrial membrane potential. Activation of Caspase-9 and Caspase-3 protein induces apoptosis and inhibits B 16 F 10 tumorigenicity.
【作者單位】： 重慶醫(yī)科大學(xué)附屬大學(xué)城醫(yī)院藥學(xué)部;第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所藥劑科;第三軍醫(yī)大學(xué)藥學(xué)院教學(xué)實(shí)驗(yàn)中心;
【分類號(hào)】：R965

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