【摘要】：目的研究肝細胞生長因子(HGF)誘導(dǎo)敏感非小細胞肺癌(NSCLC)細胞對厄洛替尼耐藥的機制,觀察c-Met及其下游信號通道蛋白是否參與HGF誘導(dǎo)不同基因型NSCLC細胞對厄洛替尼耐藥。方法 2014年1月—2015年1月,選擇人NSCLC細胞株P(guān)C-9〔表皮生長因子受體(EGFR)突變型,敏感株〕、H292(EGFR野生型,敏感株)及人胚肺成纖維細胞MRC-5細胞,通過ELISA法檢測PC-9、H292、MRC-5細胞培養(yǎng)上清液中HGF水平。用MRC-5細胞培養(yǎng)上清液誘導(dǎo)PC-9、H292細胞,采用Western blotting法檢測c-Met及其下游通道蛋白表達情況。將56只雌性、SPF級BALB/c裸鼠隨機分為8組,每組7只。在PC-9細胞誘導(dǎo)模型中,對照組(C組)和厄洛替尼處理組(E組)裸鼠皮下接種PC-9細胞懸液,MRC-5誘導(dǎo)組(H組)、MRC-5和厄洛替尼處理組(HE組)裸鼠皮下接種PC-9+MRC-5細胞懸液;當(dāng)移植瘤直徑達到4 mm時,C組和H組采用0.9%氯化鈉溶液灌胃,E組和HE組采用厄洛替尼灌胃。在H292細胞誘導(dǎo)模型中,C組、E組裸鼠皮下接種H292細胞懸液,H組、HE組裸鼠皮下接種H292+MRC-5細胞懸液;模型建立后灌胃方式同PC-9細胞誘導(dǎo)模型。給藥結(jié)束后處死裸鼠,比較PC-9、H292細胞誘導(dǎo)模型中各組移植瘤重量。采用免疫組化法檢測裸鼠移植瘤組織中c-Met及其下游通道蛋白表達水平。結(jié)果 PC-9、H292細胞培養(yǎng)上清液中均未檢測到HGF,MRC-5細胞培養(yǎng)上清液中HGF水平為(1 262±90)pg/ml。Western blotting法結(jié)果顯示,MRC-5細胞培養(yǎng)上清液中HGF能活化PC-9、H292細胞中p-Met、p-Akt、p-Stat3、磷酸化細胞外調(diào)節(jié)蛋白激酶1/2(p-Erk1/2)活性。PC-9細胞誘導(dǎo)模型中:E組移植瘤重量小于C組(P0.05);HE組移植瘤重量小于H組,大于E組(P0.05)。H292細胞誘導(dǎo)模型中:E組移植瘤重量小于C組(P0.05);HE組移植瘤重量小于H組,大于E組(P0.05)。c-Met、p-Met分別定位于細胞膜和細胞質(zhì)。在PC-9、H292細胞誘導(dǎo)模型中:C組、H組、E組、HE組c-Met表達水平比較,差異均無統(tǒng)計學(xué)意義(P0.05);H組、HE組p-Met表達水平分別高于C組、E組(P0.05)。Stat3定位于細胞質(zhì),p-Stat3定位于細胞核。在PC-9、H292細胞誘導(dǎo)模型中:C組、H組、E組、HE組Stat3表達水平比較,差異均無統(tǒng)計學(xué)意義(P0.05);H組、HE組p-Stat3表達水平分別高于C組、E組(P0.05)。Akt、p-Akt均定位于細胞質(zhì)。在PC-9、H292細胞誘導(dǎo)模型中:C組、H組、E組、HE組Akt表達水平比較,差異均無統(tǒng)計學(xué)意義(P0.05);H組、HE組p-Akt表達水平分別高于C組、E組(P0.05)。Erk1/2定位于細胞質(zhì),p-Erk1/2定位于細胞核。在PC-9、H292細胞誘導(dǎo)模型中:C組、H組、E組、HE組Erk1/2表達水平比較,差異均無統(tǒng)計學(xué)意義(P0.05);H組、HE組p-Erk1/2表達水平分別高于C組、E組(P0.05)。結(jié)論 MRC-5細胞分泌的HGF能夠在裸鼠體內(nèi)誘導(dǎo)敏感NSCLC細胞PC-9、H292對厄洛替尼耐藥,HGF通過激活c-Met及其下游通道蛋白的磷酸化可能是不同基因型NSCLC細胞對厄洛替尼耐藥的重要機制。
[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 鹵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.
【作者單位】： 延邊大學(xué)附屬醫(yī)院呼吸內(nèi)科;
【基金】：國家自然科學(xué)基金資助項目(81160291)
【分類號】：R734.2

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