肝细胞生长因子诱导敏感非小细胞肺癌细胞对厄洛替尼耐药及机制研究
发布时间：2018-07-13 08:21
【摘要】：目的研究肝细胞生长因子(HGF)诱导敏感非小细胞肺癌(NSCLC)细胞对厄洛替尼耐药的机制,观察c-Met及其下游信号通道蛋白是否参与HGF诱导不同基因型NSCLC细胞对厄洛替尼耐药。方法 2014年1月—2015年1月,选择人NSCLC细胞株PC-9〔表皮生长因子受体(EGFR)突变型,敏感株〕、H292(EGFR野生型,敏感株)及人胚肺成纤维细胞MRC-5细胞,通过ELISA法检测PC-9、H292、MRC-5细胞培养上清液中HGF水平。用MRC-5细胞培养上清液诱导PC-9、H292细胞,采用Western blotting法检测c-Met及其下游通道蛋白表达情况。将56只雌性、SPF级BALB/c裸鼠随机分为8组,每组7只。在PC-9细胞诱导模型中,对照组(C组)和厄洛替尼处理组(E组)裸鼠皮下接种PC-9细胞悬液,MRC-5诱导组(H组)、MRC-5和厄洛替尼处理组(HE组)裸鼠皮下接种PC-9+MRC-5细胞悬液;当移植瘤直径达到4 mm时,C组和H组采用0.9%氯化钠溶液灌胃,E组和HE组采用厄洛替尼灌胃。在H292细胞诱导模型中,C组、E组裸鼠皮下接种H292细胞悬液,H组、HE组裸鼠皮下接种H292+MRC-5细胞悬液;模型建立后灌胃方式同PC-9细胞诱导模型。给药结束后处死裸鼠,比较PC-9、H292细胞诱导模型中各组移植瘤重量。采用免疫组化法检测裸鼠移植瘤组织中c-Met及其下游通道蛋白表达水平。结果 PC-9、H292细胞培养上清液中均未检测到HGF,MRC-5细胞培养上清液中HGF水平为(1 262±90)pg/ml。Western blotting法结果显示,MRC-5细胞培养上清液中HGF能活化PC-9、H292细胞中p-Met、p-Akt、p-Stat3、磷酸化细胞外调节蛋白激酶1/2(p-Erk1/2)活性。PC-9细胞诱导模型中:E组移植瘤重量小于C组(P0.05);HE组移植瘤重量小于H组,大于E组(P0.05)。H292细胞诱导模型中:E组移植瘤重量小于C组(P0.05);HE组移植瘤重量小于H组,大于E组(P0.05)。c-Met、p-Met分别定位于细胞膜和细胞质。在PC-9、H292细胞诱导模型中:C组、H组、E组、HE组c-Met表达水平比较,差异均无统计学意义(P0.05);H组、HE组p-Met表达水平分别高于C组、E组(P0.05)。Stat3定位于细胞质,p-Stat3定位于细胞核。在PC-9、H292细胞诱导模型中:C组、H组、E组、HE组Stat3表达水平比较,差异均无统计学意义(P0.05);H组、HE组p-Stat3表达水平分别高于C组、E组(P0.05)。Akt、p-Akt均定位于细胞质。在PC-9、H292细胞诱导模型中:C组、H组、E组、HE组Akt表达水平比较,差异均无统计学意义(P0.05);H组、HE组p-Akt表达水平分别高于C组、E组(P0.05)。Erk1/2定位于细胞质,p-Erk1/2定位于细胞核。在PC-9、H292细胞诱导模型中:C组、H组、E组、HE组Erk1/2表达水平比较,差异均无统计学意义(P0.05);H组、HE组p-Erk1/2表达水平分别高于C组、E组(P0.05)。结论 MRC-5细胞分泌的HGF能够在裸鼠体内诱导敏感NSCLC细胞PC-9、H292对厄洛替尼耐药,HGF通过激活c-Met及其下游通道蛋白的磷酸化可能是不同基因型NSCLC细胞对厄洛替尼耐药的重要机制。
[Abstract]:Objective to investigate the mechanism of hepatocyte growth factor (HGF) induced resistance to erlotinib in sensitive non-small cell lung cancer (NSCLC) cells, and to investigate whether c-Met and its downstream signaling channel proteins are involved in the induction of erlotinib resistance in different genotypes of NSCLC cells by HGF. Methods from January 2014 to January 2015, human NSCLC cell line PC-9 (epidermal growth factor receptor (EGFR) mutant, sensitive strain) and human embryonic lung fibroblast (MRC-5) were selected. The levels of HGF in the supernatant of PC-9 H292 MRC-5 cells were detected by Elisa. PC-9 H292 cells were induced by the supernatant of MRC-5 cell culture. The expression of c-Met and its downstream channel proteins were detected by Western blotting assay. 56 female SPF BALB / c nude mice were randomly divided into 8 groups with 7 rats in each group. In PC-9 cell induction model, nude mice in control group (C group) and erlotinib treated group (E group) were subcutaneously inoculated with PC-9 cell suspension MRC-5 (H group) and erlotinib treated group (HE group) with PC-9 MRC-5 cell suspension. When the diameter of transplanted tumor was 4 mm, group C and group H were perfused with 0.9% sodium chloride solution, group E and group HE were perfused with erlotinib. In the model of H292 cells induction, H292 MRC-5 cells were inoculated subcutaneously in H292 cell suspensions of H292 cell suspensions in H292 / E nude mice and H292 MRC-5 cell suspensions were inoculated subcutaneously in H292 cells suspension group H and H groups, and the model was established in the same way as PC-9 cells induced by gastric perfusion. At the end of administration, nude mice were killed and the tumor weight of each group in PC-9 H292 cell model was compared. The expression of c-Met and its downstream channel protein in xenografts of nude mice was detected by immunohistochemical method. Results No HGF level was detected in the supernatant of PC-9 H292 cell culture supernatant. The level of HGF in the supernatant was (1 262 卤90) PG / ml. Western blotting assay showed that HGF could activate p-Metp-Aktp-Stat3Stat3 and phosphorylated extracellular regulated protein kinase 1 / 2 (p-Erk1 / 2) in PC-9 H292 cell culture supernatant. The results showed that HGF could activate p-Metp-Aktp-Stat3 and phosphorylated extracellular regulated protein kinase 1 / 2 (p-Erk1 / 2) in PC-9 H292 cell culture supernatant. In PC-9 cell induction model, the weight of transplanted tumor in group E was lower than that in group C (P0.05) and the weight of transplanted tumor in group HE was less than that in group H, The weight of transplanted tumor in group E was lower than that in group C (P0.05). The weight of transplanted tumor in group E was lower than that in group H (P0.05), and that in group E was higher than that in group E (P0.05) .c-Metp-Met was located in cell membrane and cytoplasm, respectively. There was no significant difference in the expression of c-Met between the two groups (P0.05). The expression level of p-Met in group H was higher than that in group C (P0.05). Stat3 was located in the nucleus. There was no significant difference in the expression of Stat3 between the two groups (P0.05). The expression of p-Stat3 in group H was higher than that in group C (P0.05). The expression of p-Akt in group E was located in the cytoplasm. There was no significant difference in the expression of Akt in E group (P 0.05). The expression level of p-Akt in group H was higher than that in group C (P 0.05). Erk 1 / 2 was located in the nucleus of the cytoplasm (P 0.05). There was no significant difference in Erk 1 / 2 expression level between E group and E group (P0.05). The expression level of p-Erk 1 / 2 in H group was higher than that in E group (P 0.05). Conclusion HGF secreted by MRC-5 cells can induce sensitive NSCLC cells PC-9H292 to erlotini-resistant HGF by activating phosphorylation of c-Met and its downstream channel proteins, which may be an important mechanism of different genotypes of NSCLC cells to erlotinib resistance.
【作者单位】： 延边大学附属医院呼吸内科;
【基金】：国家自然科学基金资助项目(81160291)
【分类号】：R734.2
[Abstract]:Objective to investigate the mechanism of hepatocyte growth factor (HGF) induced resistance to erlotinib in sensitive non-small cell lung cancer (NSCLC) cells, and to investigate whether c-Met and its downstream signaling channel proteins are involved in the induction of erlotinib resistance in different genotypes of NSCLC cells by HGF. Methods from January 2014 to January 2015, human NSCLC cell line PC-9 (epidermal growth factor receptor (EGFR) mutant, sensitive strain) and human embryonic lung fibroblast (MRC-5) were selected. The levels of HGF in the supernatant of PC-9 H292 MRC-5 cells were detected by Elisa. PC-9 H292 cells were induced by the supernatant of MRC-5 cell culture. The expression of c-Met and its downstream channel proteins were detected by Western blotting assay. 56 female SPF BALB / c nude mice were randomly divided into 8 groups with 7 rats in each group. In PC-9 cell induction model, nude mice in control group (C group) and erlotinib treated group (E group) were subcutaneously inoculated with PC-9 cell suspension MRC-5 (H group) and erlotinib treated group (HE group) with PC-9 MRC-5 cell suspension. When the diameter of transplanted tumor was 4 mm, group C and group H were perfused with 0.9% sodium chloride solution, group E and group HE were perfused with erlotinib. In the model of H292 cells induction, H292 MRC-5 cells were inoculated subcutaneously in H292 cell suspensions of H292 cell suspensions in H292 / E nude mice and H292 MRC-5 cell suspensions were inoculated subcutaneously in H292 cells suspension group H and H groups, and the model was established in the same way as PC-9 cells induced by gastric perfusion. At the end of administration, nude mice were killed and the tumor weight of each group in PC-9 H292 cell model was compared. The expression of c-Met and its downstream channel protein in xenografts of nude mice was detected by immunohistochemical method. Results No HGF level was detected in the supernatant of PC-9 H292 cell culture supernatant. The level of HGF in the supernatant was (1 262 卤90) PG / ml. Western blotting assay showed that HGF could activate p-Metp-Aktp-Stat3Stat3 and phosphorylated extracellular regulated protein kinase 1 / 2 (p-Erk1 / 2) in PC-9 H292 cell culture supernatant. The results showed that HGF could activate p-Metp-Aktp-Stat3 and phosphorylated extracellular regulated protein kinase 1 / 2 (p-Erk1 / 2) in PC-9 H292 cell culture supernatant. In PC-9 cell induction model, the weight of transplanted tumor in group E was lower than that in group C (P0.05) and the weight of transplanted tumor in group HE was less than that in group H, The weight of transplanted tumor in group E was lower than that in group C (P0.05). The weight of transplanted tumor in group E was lower than that in group H (P0.05), and that in group E was higher than that in group E (P0.05) .c-Metp-Met was located in cell membrane and cytoplasm, respectively. There was no significant difference in the expression of c-Met between the two groups (P0.05). The expression level of p-Met in group H was higher than that in group C (P0.05). Stat3 was located in the nucleus. There was no significant difference in the expression of Stat3 between the two groups (P0.05). The expression of p-Stat3 in group H was higher than that in group C (P0.05). The expression of p-Akt in group E was located in the cytoplasm. There was no significant difference in the expression of Akt in E group (P 0.05). The expression level of p-Akt in group H was higher than that in group C (P 0.05). Erk 1 / 2 was located in the nucleus of the cytoplasm (P 0.05). There was no significant difference in Erk 1 / 2 expression level between E group and E group (P0.05). The expression level of p-Erk 1 / 2 in H group was higher than that in E group (P 0.05). Conclusion HGF secreted by MRC-5 cells can induce sensitive NSCLC cells PC-9H292 to erlotini-resistant HGF by activating phosphorylation of c-Met and its downstream channel proteins, which may be an important mechanism of different genotypes of NSCLC cells to erlotinib resistance.
【作者单位】： 延边大学附属医院呼吸内科;
【基金】：国家自然科学基金资助项目(81160291)
【分类号】：R734.2
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