反义皮层肌动蛋白基因对肝癌细胞功能影响的研究

Impact of antisense cortactin gene in hepatocellular carcinoma metastasis

目的:将反义的皮层肌动蛋白(cortactin)基因转染入人肝癌细胞中,观察其表达效果及其对细胞本身的影响以及该基因在肝癌细胞转移中的作用。方法:Trizol试剂法提取体外培养的人肝癌高转移细胞株MHCC97总RNA,经逆转录聚合酶链式反应(RT-PCR)扩增目的片段,PCR产物及真核表达载体分别双酶切后进行连接,并转化入大肠杆菌Ecoli.Dh5α扩增以获得重组质粒。重组质粒、空质粒分别转染入人高转移肝癌细胞株,进行扩增培养,分别绘制MHCC97/vect-cortactin(+)、MHCC97/vect和MHCC97细胞生长曲线;并进行Western-blot实验及通过小室培养模型进行细胞侵袭力实验。结果:RT-PCR获得预期大小的特异性DNA片段,经PCR、双酶切鉴定及测序证实人皮层肌动蛋白基因cDNA片段已正确的反向插入真核表达载体中。细胞生长曲线提示转染与未转染该基因的肝癌细胞生长曲线相同;Western-blot结果显示转染反义基因的肝癌细胞皮层肌动蛋白cortactin呈低表达或不表达;细胞侵袭力实验表明转染有反义基因的肝癌细胞穿膜细胞数较未转染的有非常显著差异(P<0.01)。结论:从蛋白水平和细胞水平均证实该反义基因能抑制肝癌细胞的皮层肌动蛋白的表达和游走而不影响细胞的生长特性。

Objective：To transform the human anti -sense cortical actin -associated protein （cortactin）gene into hepatocarcinoma（HCC） cells,then study the expression of this gene in the cells and the impact of the gene to the cells and HCC metastasis. Methods： Total RNA was extracted by using TRIZOL one - step method from highly metastatic cell line MHCC97. CDNA was amplified by using reversal transcriptional polymerase chain reaction （ RT - PCR）. Product of PCR,amplified by transformed into Escherichia coli DI-ISct,was inserted reversely into the eukaryotic expression vector after digestion and ligation by using restriction endonucleases and ligase. The correct eukaryotic expression vector pcDNA3. O/cortactin and the empty vector pcDNA3.0 were thansfected into MHCC97 with lipesome respectively. The cells MHCC97 ,MHCC97/vect,MHCC97/vectcortactin（ ＋ ） were cultured amplificationally ,then studied on the impact in HCC cells metastasis by using western -blotting, cell growth curve and transwell chambers culture respectively. Results：The correct cortactin cDNA clone was obtained and it was confirmed that cortactin cDNA was inserted reversely into the eukaryotic expression vector correctly by using PCR, digestion identification and se- quencing. Cell growth curves indicated that the cells MHCC97/vect - cortactin（ ＋ ） had the same curve as that of the cells MHCC97 and MHCC97/vect. Western -blotting indicated that the cortactin in the cells MHCC97/vect -cortactin（ ＋ ） expressed much lower than that in the cells MHCC97/vect and MHCC97. Transwell chambers culture indicated the quantity of traversed membrane cells of MHCC97/vect - cortactin （ ＋ ） was much less than that of the cells MHCC97/vect and MHCC97 respectively. Conclusion： The antisense cortactin gene transfected into the MHCC97 cells could inhibit the HCC cells intensively from expressing cortactin and the ability of invasion and metastasis through the levels of protein and cell,but didnt impact the HCC cell growth.