【作者】 李志远；

【导师】 翟文龙；

【作者基本信息】 郑州大学 ， 外科学（肝胆胰外科）（专业学位）， 2018， 硕士

【摘要】 目的:探讨钙网蛋白(calreticulin CRT)在胆囊癌组织中表达及其对GBC-SD细胞株生物学行为的作用。方法:利用免疫组织化学及RT-qPCR测定胆囊癌组织、癌旁组织和慢性胆囊炎组织中CRT蛋白和mRNA的表达。利用小干扰RNA转染技术沉默GBC-SD细胞株CRT表达,通过Western blotting方法检验转染效果,Cell counting kit-8(CCK-8)及平板克隆实验测定细胞的增殖能力;应用流式仪测定GBC-SD细胞株的凋亡情况及细胞周期的改变;运用划痕及Transwell小室测定GBC-SD细胞株的迁移情况;Western blotting方法检测p-Akt及MMP-9蛋白表达。结果:与肿瘤旁及慢性胆囊炎标本比较,CRT mRNA在胆囊癌标本中高表达(t=5.571,P<0.05)。siCRT-1、siCRT-2实验组24h及48h相对存活率分别为(71.5±6.3)%,(79.5±2.7)%;(62.6±8.8)%,(55.6±2.6)%。空白组,阴性对照组,siCRT-1,siCRT-2的凋亡率分别为(3.0±1.8)%,(4.7±1.3)%,(13.6±1.0)%,(20.0±4.0)%。划痕实验显示:空白组,阴性对照组,siCRT-1,siCRT-2的划痕修复率分别为(0.67±0.02),(0.58±0.02),(0.22±0.01),(0.37±0.04)。Transwell结果显示,空白组,阴性对照组,siCRT-1,siCRT-2的细胞穿膜个数分别为(301.8±10.7),(296.6±14.5),(178.0±10.1),(165.2±12.0)。与空白组及阴性对照组比较,siCRT-1,siCRT-2实验组24h,48h相对存活率明显降低,凋亡上升,迁移能力下降(F=29.310,118.618,69.651,144.515,190.145,P<0.05)。沉默CRT相关基因后,与空白组及阴性对照组相比,p-Akt及MMP-9蛋白表达程度均下降。结论:CRT在胆囊癌中高表达,CRT表达下调介导的胆囊癌生物学行为的改变可能与p-Akt/MMP-9通路相关。更多还原

【Abstract】 Objective:To explore the expression of calreticulin(CRT)in gallbladder cancer tissue and its effect on the biological behavior in gallbladder cancer GBC-SD cells.Methods:Immunohistochemistry and RT-qPCR were applied to detect the protein and mRNA levels of CRT in gallbladder cancer,adjacent cancer and chronic cholecystitis tissues.Small interfering RNA was transfected into gallbladder cancer GBC-SD cells and Western blotting were used to detect the expression of CRT.The proliferation was determined by using cell counting kit-8(CCK-8)and clone assays.Flow cytometry were applied to detect the apoptosis and cell cycle.Migration was detected by wound healing and transwell assays,respectively.The expression of p-Akt and MMP-9 were detected by using Western blotting.Results:Expression of CRT in gallbladder cancer tissues is higher than adjacent cancer tissues and chronic cholecystitis tissues(t=5.571,P<0.05).The relative growth rate in the siCRT-1 、 siCRT-2 experimental group for 24 hours,48hourrs were(71.5±6.3)%,(79.5±2.7)%;(62.6±8.8)%,(55.6±2.6)%,respectively.The apoptosis rate in the blank group,the negative control group,siCRT-1 and siCRT-2 group were(3.0±1.8)%,(4.7±1.3)%,(13.6±1.0)%,(20.0±4.0)%,respectively.Would healing assays showed that the wound closure ratio in the blank group,negative control group,siCRT-1 and siCRT-2 group were(0.67±0.02),(0.58±0.02),(0.22±0.01),(0.37±0.04),respectively.Transwell experiments showed that the numbers of migration of GBC-SD cell in the blank group,negative control group,siCRT-1 and siCRT-2 group were(301.8±10.7),(296.6±14.5),(178.0±10.1),(165.2±12.0),respectively.compared with the blank group and the negative control group,the relative growth rate for 24 hours and 48 hours was significantly lower,the apoptosis rate was higher,the numbers of migration was lower((F=29.310,118.618,69.651,144.515,190.145,P<0.05).Compared with the blank group and the negative control group,the expression of p-Akt and MMP-9 were decreased after down-regulating the expression of CRT.Conclusions:The expression of CRT in gallbladder cancer tissue was higher.CRT downregulation mediated changes of biological behaviors in gallbladder cancer may be associated with p-Akt/MMP-9 signal pathway.更多还原