联合位点突变的乙肝病毒核心启动子转染L-02及对细胞增殖能力的影响

Transfection of L-02 with combo mutant core promoter and its effect on cell proliferation ability

目的通过基因合成A1762T／G1764A／T1753A／T1768A联合突变后的核心启动子（CM—HBX），再以慢病毒感染的方式构建稳定表达核心启动子的L-02细胞株，建立肝癌发生的细胞模型。方法通过基因合成的方法，合成A1762rr／G1764A／T1753A／T1768A联合突变后的核心启动子，在N末端加上标记序列HBX—Flag，分别双酶切pLenO—RTP载体和pUC57-CM—HBX，纯化酶切产物后进行定向连接，得到重组质粒pLenO-RTP—CM—HBX。钙转法将重组质粒转入293T细胞进行病毒包装然后转染肝细胞。噻唑蓝（Mr丌）比色法检测转染后细胞增殖能力的变化，Westernblot法检测HBX—Flag在慢病毒感染后L-02中的表达以及转染后细胞内s期激酶相关蛋白2（Skp2）、增殖细胞核抗原（PCNA）、p21、p53的表达的变化。结果构建pLenO。RTP—CM—HBX核心启动子慢病毒载体可以效率较高地感染L-02（〉95％），并在细胞内转录、翻译、合成HBX—Flag，随着培养时间的延长，红色荧光强度无明显变化；同空载组和空白对照组比较，联合突变组L-02的增殖能力明显增强（P〈0．05），skp2、PCNA表达增加，p21、p53表达降低（P〈0．05）。而空载组和空白对照组的各指标差异无统计学意义（P〉0．05）。结论慢病毒载体pLenO—RTP．CM—HBX核心启动子成功转染L-02，转染后的L-02具有癌变倾向。

Objective The core promoter （CP） region overlaps with the hepatitis B virus （HBV） X gene. Clinical research has shown that A1762T/G1764A （TA）/T1753A/T1768A mutation in overlapping sequence can increase risk of hepatocellular carcinoma （HCC）. In order to establish a kind of cell model of hepatocarcinogenesis, we construct A1762T/G1764A/T1753A/T1768A combo mutant core promoter through the gene synthesis, and then infect the cell L-02. Methods In order to obtain recombinant plas-mid pLenO-RTP-CM-HBX, the pLenO-RTP carrier and pUC57-CM-HBX were connected directly after double restriction enzyme digestion. The recombinant plasmid was transfected into 293 T cells for virus packaging through Calcium turn method, and then L-02 cells were infected. The change of proliferation ability after transfection was detected through methyl thiazol tetrazolium （MTT） method. HBX-Flag, S- phase kinase-associated protein-2 （Skp2）, proliferating cell nuclear antigen （PCNA）, p21 and p53 ex- pression changes were detected by using Western blotting. Results The pLenO-RTP-CM-HBX core pro- moter lentivirus carrier could infect L-02 cells with high efficiency （ 〉 95% ） , and then HBX-Flag was transcribed, translated, synthetized and secreted in L-02 cells after transfection. The red fluorescence in- tensity did not change obviously with the extension of incubation time. The proliferation ability, and Skp2 and PCNA expression of cells in combo mutant group was increased, and p21 and p53 expression was re- duced compared to light group and blank control group （ P 〈 0. 05 ）. There was no significant difference be- tween light group and blank control group. Conclusion The pLenO-RTP-CM-HBX core promoter lentivirus carrier was transfected into Ll02 cells successfully, and the L-02 ceils after transfection had tendency to be- come cancerous.