【目的】1.研究Snail蛋白赖氨酸残基K98、K137位点单突变对LOXL2介导的胆管癌细胞Snail蛋白、E-cadherin的影响。2.为胆管癌的靶向药物的研发提供部分实验数据。【方法】在复苏、传代的胆管癌细胞RBE中,分别瞬时转染EX-LOXL2质粒、EMS-275溶解度X-LOXL2空载质粒、EX-LOXL2+Snail K98S质粒、EX-LOXL2+Snail K98S空载质粒、EX-LOXL2+Snail K137S质粒、EX-LOXL2+Snail K137S空载质粒,通过免疫荧光技术检测各组转染效果。在瞬时转染相应质粒的胆管癌细胞RBE中,通过q PCR检测转染后各组LOXL2 m RNA、Snail m RNA水平变化。通过CO-IP检测各组LOXL2与Snail蛋白的结type 2 pathology合情况。通过Western Blot检测LOXL2、Snail蛋白、E-cadherin表达量。通过放线菌酮处理Snail蛋白,分别检测0h、1h、2h、3h各组Snail蛋白变化情况。通过分析比较各组相应指标变化,明确Snail蛋白赖氨酸残基位点K98、K137单突变对LOXL2介导的Snail蛋白、E-cadherin的影响。【结果】1.胆管癌细胞复苏、传代,培养足够细胞,瞬时转染相应质粒;通过免疫荧光技术验证相应质粒瞬时转染成功。2.通过q PCR技术检测各组LOXL2m RNA、Snail m RNA表达。与RBE组相比,EX-LOXL2组LOXL2 m RNA、Snail m RNA表达量显著升高(p<0.05);与EX-LOXL2组相比,EX-LOXL2+Snail K98S组、EX-LOXL2+Snail K137S组LOXL2 m RNA、Snail m RNA表达量无明显改变(p>0.05)。3.Western Blot检测各组LOXL2、Snail蛋白、E-cadherin表达量。与RBE组相比,EX-LOXL2组LOXL2、Snail蛋白表达量均显著升高(p<0.05),E-cadherin蛋白表达量明显下降(p<0.05)。与EX-LOXL2组相比,EXLOXL2+Snail K137S组Snail蛋白表达量明显下降(p<0.05),E-cadherin表达量明显升高(p<0.05),LOXL2表达量无明显改变(p>0.05)。与EX-LOXL2组相比,EX-LOXL2+Snail K98S组LOXL2、Snail蛋白、E-cadherin表达量均无明显改变(p>0.05)。4.CO-IP结果显示瞬时转染EX-LOXL2质粒,LOXL2与Snail蛋白相互结合,瞬时转染EX-LOXL2+Snail K98S、EX-LOXL2+Snail K137S质粒,LOXL2与Snail蛋白相互结合。5.Western Blot检测放线菌酮处理后0h、1h、2h、3h各组Snail蛋白表达量。与RBE组相比,EX-LOXL2组Snail蛋白降解速率减慢(p<0.05);与EX-LOXL2组相比,EX-LOXL2+Snail K137S组Snail蛋白降解速率加快(p<0.05);与EX-LOXL2组相比,EX-LOXL2+Snail K98S组Snail蛋白降解速率无明显改变(p>0.05)。【结论】1BMN 673临床试验.EX-LOXL2可正向调控野生型胆管癌细胞LOXL2、Snail蛋白,负性调控E-cadherin,并且能减慢Snail蛋白降解速率。2.Snail蛋白赖氨酸残基位点K98、K137单突变可能介导了LOXL2与Snail蛋白结合。3.Snail蛋白赖氨酸残基位点K137单突变可负性调控LOXL2介导的胆管癌细胞Snail蛋白;正性调控E-cadherin,并且能加快Snail蛋白降解速率。4.Snail蛋白赖氨酸残基位点K137位点有望成为胆管癌治疗的新靶点。