论文部分内容阅读
解析比较蛋白质组学筛出的差异蛋白Annexin1的生物学功能,证实其是否在肝癌转移复发中发挥作用.分别以RT-PCR、蛋白质印迹及细胞免疫化学对差异蛋白Annexin1在转移潜能不同人肝癌细胞系中的表达情况进行再验证,然后构建Annexin1反义表达质粒,转染高转移潜能人肝癌细胞系MHCC97H,通过对MHCC97H细胞的运动、侵袭、凋亡、生长周期、MMPs分泌、克隆形成等系列检测,观察目的蛋白表达降低对其生物学行为的影响,特别是转移特性的影响.验证结果均证实Annexin1在有转移潜能人肝癌细胞系MHCC97L、MHCC97H中呈高表达.转染Annexin1反义重组表达质粒后,MHCC97H细胞中Annexin1的表达被成功抑制.依据MHCC97H/pcDNA3.1(+)ASAnnexin1,MHCC97H/pcDNA3.1(+),MHCC97H的检测排序,转染反义重组质粒后的MHCC97H细胞穿过上室底膜的细胞数(运动实验)分别为:11.13±3.31,18.88±2.03,21.86±3.38;穿过人工基底膜细胞数(侵袭实验)分别是:16.43±2.23,16.40±1.57,16.86±1.52;细胞平均集落形成率(克隆形成实验)分别为:(14.33±0.46)%,(19.35±0.49)%,(20.25±0.35)%;MHCC97H细胞凋亡比例(FCM分析)依次为22.2%,6.44%,6.97%;细胞周期各时相的比例依次为:G0-G1期79.5%/76.34%/80.5%,S期13.26%/14.4%/9.69%,G2-M期7.25%/9.26%/9.81%;细胞培养上清MMP9的定量结果依次为:26.37!g/L,28.00"g/L,31.90#g/L;MMP2定量结果依次为29.46$g/L,26.37%g/L,26.53&g/L.明胶酶谱分析细胞培养上清显示,转染Annexin1反义重组表达质粒的MHCC97H细胞分泌的MMP9活性与对照比变化不明显.综合上述结果发现,转染Annexin1反义表达质粒MHCC97H细胞运动能力及集落形成率明显降低,凋亡细胞的比例增加,而侵袭潜能,细胞周期时相,细胞分泌MMP2、MMP9的量均变化不明显.提示,差异蛋白Annexin1可能通过影响细胞凋亡和细胞运动在肝癌细胞侵袭转移过程中发挥作用.
Analyze the biological function of Annexin1, a differential protein identified by proteomic analysis, and confirm whether Annexin1 plays a role in the metastasis and recurrence of hepatocellular carcinoma.METHODS: The expression of Annexin1 in metastatic potential human hepatocarcinoma cells were detected by RT-PCR, Western blot and immunocytochemistry The expression of Annexin1 antisense plasmid was constructed and transfected into MHCC97H cell lines with high metastatic potentiality. The cell line MHCC97H was transfected into MHCC97H cells by cell cycle, invasion, apoptosis, growth cycle, secretion of MMPs, Detect and observe the effect of the decrease of the target protein on the biological behavior, especially the metastatic characteristics.The results of the verification confirmed that Annexin1 is highly expressed in the metastatic potential human hepatocellular carcinoma cell lines MHCC97L, MHCC97H.Expression of Annexin1 antisense recombinant The expression of Annexin1 was successfully inhibited in MHCC97H cells.According to the sequencing of MHCC97H / pcDNA3.1 (+) ASAnnexin1, MHCC97H / pcDNA3.1 (+) and MHCC97H, the transfected MHCC97H cells passed through the antisense recombinant plasmid The number of cells in the basement membrane (exercise test) were: 11.13 ± 3.31,18.88 ± 2.03,21.86 ± 3.38; the number of cells penetrating the artificial basement membrane (14.33 ± 0.46)%, (19.35 ± 0.49)% and (20.25 ± 0.35)%, respectively. The average colony formation rate of the cells was (16.43 ± 2.23,16.40 ± 1.57,16.86 ± 1.52) ; The proportion of apoptotic cells in MHCC97H cells was 22.2%, 6.44% and 6.97% in turn, respectively. The proportion of cells in each phase was 79.5% / 76.34% / 80.5% in G0-G1 phase and 13.26% / 14.4 in S phase % / 9.69%, G2 / M phase 7.25% / 9.26% / 9.81%. The quantitative results of MMP9 in cell culture supernatant were 26.37μg / L, 28.00mg / L and 31.90μg / L, The results were followed by 29.46 $ g / L, 26.37% g / L, 26.53 g / L. Gelatin zymography analysis of the cell culture supernatant showed that the activity of MMP9 secreted by MHCC97H cells transfected with the antisense recombinant Annexin1 plasmid did not change significantly compared with the control Obviously, we found that the expression of MMP2 and MMP9 in MHCC97H transfected with Annexin1 antisense plasmid was significantly decreased and the percentage of apoptotic cells was increased, while the invasive potential, cell cycle phase, Not obvious.It is suggested that Annexin 1 may play a role in the invasion and metastasis of hepatocellular carcinoma cells by affecting apoptosis and cell motility.