温郁金凝集素蛋白重组及对结肠癌HCT-116细胞凋亡、迁移和侵袭的影响  

Protein recombination of Curcuma wenyujin lectin and its effects on apoptosis,migration and invasion of HCT-116 cells

作　　者：周丹 张丽萍[2] 丰梦熠 朱强[3] 任仙樱 吴志刚 姜程曦 ZHOU Dan;ZHANG Liping;FENG Mengyi;ZHU Qiang;REN Xianying;WU Zhigang;JIANG Chengxi(School of Pharmacy,Wenzhou Medical University,Wenzhou 325035,China;Institute of Life Sciences,Wenzhou University,Wenzhou 325035,China;Anhui Jiren Pharmaceutical Co.,Ltd.,Bozhou 236800,China;Wenzhou Ejin Pharmaceutical Co.,Ltd.,Yueqing 325600,China)

机构地区：[1]温州医科大学药学院,浙江温州325035 [2]温州大学生命科学研究院,浙江温州325035 [3]安徽济人药业有限公司,安徽亳州236800 [4]温州莪金医药有限公司,浙江乐清325600

出　　处：《药物评价研究》2023年第11期2366-2373,共8页Drug Evaluation Research

基　　金：安徽济人药业有限公司课题(KJHX2009);合肥未来药物开发有限公司横向课题(KJHX2008)。

摘　　要：目的 获取温郁金凝集素(CWL)重组蛋白,并研究其对结肠癌细胞凋亡、迁移和侵袭的影响。方法 根据CWL基因全长编码序列,采用大肠杆菌原核表达系统重组表达及纯化CWL;观察CWL对兔红细胞的凝血作用;CCK-8法评价CWL(10、20、40、80μg·mL^(-1))对结肠癌HCT-116细胞的毒性;流式细胞仪检测CWL(40、80μg·mL^(-1))对HCT-116细胞凋亡的影响;划痕实验检测细胞迁移能力;Transwell小室法检测细胞侵袭能力;实时荧光定量PCR(qRT-PCR)实验分析Caspase-3、 Caspase-9、 BAX、 PARPmRNA表达水平;Westernblotting检测cleaved-Caspase-3、cleaved-Caspase-9、BAX、cleaved-PARP蛋白表达水平。结果 重组纯化获得相对分子质量为3×10^(4)的CWL;高质量浓度的CWL导致红细胞出现明显的凝血情况,随着CWL质量浓度的降低,其凝血活性逐渐下降,直至消失。与对照组比较,CWL对HCT-116细胞活性具有显著抑制作用(P<0.001);40、80μg·mL^(-1) CWL可显著降低HCT-116细胞的迁移和侵袭能力(P<0.001),显著升高HCT-116细胞凋亡率(P<0.01、0.001);显著升高Caspase-3、Caspase-9、BAX、PARP的mRNA表达水平(P<0.05、0.01、0.001),同时显著增加cleaved-Caspase-3、cleaved-Caspase-9、BAX、cleaved-PARP蛋白表达水平(P<0.05、0.01)。结论 CWL可通过线粒体凋亡途径促进HCT-116细胞凋亡并抑制其迁移和侵袭,从而发挥抗肿瘤作用。Objective Curcuma wenyujin lectin(CWL)recombinant protein was obtained,and its effect on the apoptosis,migration and invasion of colon cancer cells were invesitgated.Methods According to the coding sequence of CWL gene,CWL was heterogeneously expressed and purified using Escherichia coli prokaryotic expression system.CCK-8 method was used to evaluate the toxicity of CWL to colon cancer HCT-116 cells,apoptosis was detected by flow cytometry,and cells were detected by scratch test.Migration ability,cell invasion ability was assessed using transwell chamber method.the expression levels of genes in signal pathways such as Caspase-3,Caspase-9,BAX and PARP were performed using real-time fluorescence quantitative PCR(qRT-PCR)analysis.Western blotting was used to detect the expression levels of cleaved-Casase-3,cleaved-Casase-9,BAX,and cleaved-PARP proteins.Results Recombinant purification yielded CWL of relative molecular weight of 3×10^(4).High quality concentration of CWL caused to significant coagulation of red blood cells,and as the concentration of CWL decreases,its coagulation activity gradually decreases until it disappears.Compared with control group,CWL had a significant inhibitory effect on the activity of HCT-116 cells(P<0.001),CWL of 40,80μg·mL^(-1) could significantly reduce the migration and invasion ability of HCT-116 cells(P<0.001),and significantly increased the apoptosis rate of HCT-116 cells(P<0.01,0.001).CWL of 40,80μg·mL^(-1) significantly increased mRNA expression levels of Caspase-3,Caspase-9,BAX,and PARP(P<0.05,0.01,0.001),while significantly increased protein expression levels of cleaved-Caspase-3,cleaved-Caspase-9,BAX,and cleaved-PAP(P<0.05,0.01).Conclusion CWL can promote HCT-116 cell apoptosis through the mitochondrial apoptosis pathway,inhibit HCT-116 cell migration and invasion,thereby exerting anti-tumor effects and providing a new source of drugs for the development of clinical tumor drugs.

关 键 词：温郁金凝集素 HCT-116细胞 凋亡 迁移 侵袭 线粒体凋亡 

分 类 号：R285.5[医药卫生—中药学]