摘要
目的:构建小鼠shASPP2 H22稳转肝癌细胞系,观察ASPP2敲低对血管生成的影响。方法:针对小鼠ASPP2基因设计了3个不同的shRNA干扰序列(Y18421,Y18422,Y18423)及1个对照序列(GL427NC2),采用双酶切(AgeⅠ和EcoRⅠ)及质粒连接构建重组质粒,使用菌落PCR和测序比对进行鉴定;使用293T细胞将各重组质粒包装慢病毒并测定滴度;将shASPP2和对照慢病毒质粒转染H22细胞,采用流式细胞术测定转染效率;采用qRT-PCR、Western Blot法观察shASPP2慢病毒对H22细胞ASPP2的干扰效果;采用CCK8法观察ASPP2敲低对H22细胞增殖的影响;采用Western Blot法观察ASPP2敲低对H22细胞及上清VEGF表达和分泌的影响;采用细胞注射法建立小鼠ASPP2敲低H22细胞皮下移植瘤模型,游标卡尺法观察肿瘤体积大小,采用活体激光共聚焦观察肿瘤血管生成情况,采用Western Blot法观察肿瘤组织VEGF的表达。结果:双酶切、菌落PCR和测序鉴定结果表示各重组质粒构建成功;各重组质粒经慢病毒包装后,测定显示Y18421、Y18422、Y18423和GL427NC2慢病毒质粒的滴度分别为3.40×10^(8)TU/mL、4.08×10^(8)TU/mL、5.49×10^(8)TU/mL和1.7×10^(9)TU/mL;Y18421、Y18422、Y18423及GL427NC2慢病毒质粒转染效率分别为:86.2%、69.6%、60.8%和76.9%。与GL427NC2 H22细胞相比,Y18421 H22细胞的ASPP2 mRNA及蛋白的表达明显降低(P<0.01,P<0.05);Y18421细胞在培养24,48,72 h后增殖速率显著增加(P<0.0001,P<0.001,P<0.01);Y18421细胞及上清的VEGF表达显著升高(P<0.001,P<0.01,P<0.05)。与GL427NC2细胞移植瘤相比,Y18421细胞移植瘤体积明显增大(P<0.05),总血管长度显著增加(P<0.05),VEGF蛋白的表达明显上调(P<0.05)。结论:小鼠shASPP2 H22稳转肝癌细胞系构建成功,ASPP2敲低可能通过上调VEGF的表达促进小鼠H22细胞移植瘤血管生成。
Objective:To construct mouse shASPP2 H22 stable hepatocarcinoma cell line and observe the effect of ASPP2 knockdown on angiogenesis.Methods:Three shRNA sequences(Y18421,Y18422,Y18423)and one control sequence(GL427NC2)were designed for mouse ASPP2 gene.Recombinant plasmid was constructed by double enzyme digestion(AgeⅠand EcoRⅠ)and plasmid ligation,colony PCR and sequencing alignment were used to identified it.293T cells were used to package the lentivirus with each recombinant plasmid and determined the titers.H22 cells were transfected with shASPP2 and control lentivirus plasmids,the transfection efficiency was determined by flow cytometry.qRT-PCR,Western Blot were used to observe the interference effect of shASPP2 lentivirus on ASPP2 expression in H22 cells.CCK8 assay was used to observe the effect of ASPP2 knockdown on the proliferation of H22 cells.Western Blot was used to observe the effect of ASPP2 knockdown on the expression and secretion of VEGF in H22 cells and supernatant.The mouse subcutaneous xenograft model of ASPP2 knockdown H22 cells was established by cell injection.The tumor volume was observed by vernier caliper method,the tumor angiogenesis was observed by in vivo laser confocal microscopy,and the expression of VEGF in tumor tissue was detected by Western Blot.Results:The results of double enzyme digestion and colony PCR and sequencing alignment showed that the recombinant plasmids were successfully constructed.The titers of Y18421,Y18422,Y18423 and GL427NC2 were 3.40×10^(8)TU/mL,4.08×10^(8)TU/mL 5.49×10^(8)TU/mL and 1.7×10^(9)TU/mL,respectively.Flow cytometry showed that the transfection efficiency of lentiviral plasmid Y18421,Y18422,Y18423 and GL427NC2 were 86.2%,69.6%,60.8%and 76.9%,respectively.The expression of ASPP2 mRNA and protein in Y18421 cells was significantly lower than that in GL427NC2 cells(P<0.01,P<0.05);CCK8 results showed that the proliferation rate of Y18421 cells was significantly increased after cultured 24,48 and 72 h(P<0.0001,P<0.001,P<0.01);The expression of VEGF in Y18421 cell and its supernatant were significantly increased(P<0.001,P<0.01,P<0.05);Compared with GL427NC2 cells,Y18421 cells transplanted tumor volume was significantly increased(P<0.05),Total vessel length increased significantly(P<0.05),and the expression of VEGF protein was significantly up-regulated in Y18421 cells(P<0.05).Conclusion:The mouse shASPP2 H22 stable hepatoma cell line was successfully constructed.ASPP2 knockdown can promote the angiogenesis of mouse H22 cell transplantation tumor by promoting the expression of VEGF.
作者
高明慧
霍云飞
寇卜心
柴梦音
李全维
豆双双
庞丽君
刘晓霓
GAO Ming-hui;HUO Yun-fei;KOU Bu-xin;CHAI Meng-yin;LI Quan-wei;DOU Shuang-shuang;PANG Li-jun;LIU Xiao-ni(Beijing Institute of Hepatology,Beijing You'an Hospital,Capital Medical University,Beijing,100069,China)
出处
《现代生物医学进展》
CAS
2024年第11期2043-2051,共9页
Progress in Modern Biomedicine
基金
北京自然科学基金项目(7192084)
北京市公共医学研究所发展改革试点项目(京医研2021-10)
首都卫生发展科研专项(2020-2-1152)
国家自然科学基金项目(82003200)。
