摘要
目的:采用NF-κB p65 ASODN技术特异性阻断目的基因,探讨ASODN干扰前后胃癌细胞生长增殖及凋亡、细胞形态的变化,初步研究NF-κB p65下调对胃癌SGC-7901细胞凋亡的影响,探讨诱导凋亡可能的机制。
方法:人工合成NF-κB p65 ASODN,通过脂质体转染法转染人胃癌细胞系SGC-7901,实验分为对照组、Lip组、ASODN+Lip组和MSODN+Lip组,核苷酸浓度为10μM、20μM、40μM。分别在药物转染后24h、48h、72h收集细胞,采用MTT法测定NF-κB p65 ASODN对胃癌细胞增殖及凋亡的影响;流式细胞仪检测胃癌细胞凋亡率及细胞周期;光镜观察药物作用后细胞形态学变化;通过免疫细胞化学技术检测NF-κB p65,Bcl-2蛋白的表达。
结果:①MTT法检测显示:不同浓度的NF-κB p65 ASODN均可抑制胃癌SGC-7901细胞的增殖,呈时间-剂量依赖性;②流式细胞仪检测显示:NF-κBp65 ASODN可诱导细胞凋亡,使细胞阻滞于G0/G1期,NF-κB p65 ASODN处理SGC7901细胞48 h后凋亡率为(50.51±3.35)%,错义寡核苷酸(MSODN)处理组为(20.34±2.11)%,两组相比,差异有显著性(P<0.05)。③HE染色显示:NF-κB p65 ASODN处理SGC-7901细胞48h后,光镜下可见细胞缩小变圆,核浓缩、核碎裂、边聚等。④免疫细胞化学结果显示:NF-κB p65 ASODN处理SGC-7901细胞48小时后, NF-κB p65蛋白及Bcl-2蛋白的表达水平较对照组显著下降(P<0.05)。
结论:①NF-κB p65 ASODN与阳离子脂质体形成的复合物可以成功转染进入SGC-7901细胞内;②NF-κBp 65 ASODN在体外可以较明显的抑制SGC-7901细胞的增殖、诱导SGC-7901细胞凋亡,作用强度呈剂量-时间依赖性;③N F-κB p65 ASODN可以下调NF-κB p65,Bcl-2蛋白的表达;④NF-κB p65 ASODN诱导SGC-7901细胞凋亡的机制可能是其经脂质体转染入细胞后与细胞核及胞浆内NF-κB p65 mRNA特异性结合,抑制或封闭NF-κB p65 mRNA的表达,下调NF-κB p65蛋白及其下游产物Bcl-2等相关抑凋亡基因蛋白的表达,从而抑制细胞增殖,诱导细胞凋亡。
Objective:The investigation used NF-κB p65 ASODN technique to block objective genes specificity, approached the the effect of NF-κB p65 ASODN on proliferation and apoptosis and change in form of SGC-7901, invested the effect of apotosis in SGC-7901 by down regulatiing the expression of NF-κB p65, approached the Mechanism of action of inducing apotosis.
Methods:Man-made synthesis NF-κBp65 ASODN, Transfecting SGC-7901 trough liposome infection protocol. The experiment can be divided into groups: control group, Lip group, ASODN+Lip group, MSODN+Lip group. ASODN was divided with different concentrations (10μM,20μM,40μM) and collecting cells in different times(24h,48h,72h). The effect of NF-κB p65 ASODN on cell proliferation was observed by MTT colormetric assay. The rate of SGC-7901apoptosis and cell cycle was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed by light microscopy. And then the expression of apoptosis-regulating protein NF-κB p65, Bcl-2 in SGC-7901 after apoptosis induced by NF-κBp65 ASODN were examined by immunocytochemical staining assay .
Result:①MTT analysis: NF-κB p65 ASODN with different concentrations incubated with SGC-7901 for 24h, 48h and 72h could significantly inhibit SGC-7901 proliferation in dose-dependent and time-dependent manner compared with the control group(p<0.05);②Flow cytometry quantitation: NF-κB p65 ASODN could induce cells apoptosis and block cells in G0/G1 time. The apoptosis ratio of ASODN+Lip group was (50.51±3.35)%, which was significantly higher than that of MSODN+Lip group (20.34±2.11)% (P<0.05).③HE staining: Treatment with 40μM concentrations of Ursolic acid for 48h resulted in morphologic changes of SGC-7901, including karyorrhexis and cytoplasm vacuolization.④Immunocytochemistry: NF-κB p65 ASODN incubated with SGC-7901 for 48h, the expressions of NF-κB p65, bcl-2 protein in SGC-7901 were significantly up-regulated(P<0.05).
Conclusion:①The compounds with NF-κB p65 ASODN and liposome could transfected into SGC-7901 successfully.②NF-κB p65 ASODN could significantly inhibit SGC-7901 proliferation and induce apoptosis in dose-dependent and time-dependent manner.③NF-κB p65 ASODN could down regulated the expressions of NF-κB p65 and Bcl-2 protein in SGC-7901.④Our results suggested that SGC-7901 cells apoptosis induced by NF-κB p65 ASODN through mechanisms was that it could transfected into SGC-7901 with liposome, then specific bind with NF-κB p65 mRNA, inhibit and block the expression of NF-κB p65 mRNA, reduce the expressions of NF-κB p65 and Bcl-2 protein, thus inhibit proliferation and induce cells apoptosis at last.
引文
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