论文部分内容阅读
[目的]包装并鉴定表达人miR-29的慢病毒,并检测miR-29对人胃癌细胞株SGC-7901增殖的影响。[方法]化学合成miR-29前体序列,插入到慢病毒载体质粒pGCL-GFP多克隆位点后,与慢病毒包装质粒pHelper1.0及pHelper2.0共转染人胚肾HEK293T细胞包装慢病毒;分离纯化并浓缩慢病毒并检测其滴度。负载miR-29的慢病毒感染SGC-7901细胞后,采用MTT法观察增殖能力的改变。[结果]双酶切和测序结果证实miR-29前体DNA序列正确插入pGCL-GFP,包装的慢病毒滴度达2×108TU/ml;包装成功的重组慢病毒可有效增加SGC-7901细胞内miR-29的表达丰度;与未感染慢病毒的SGC-7901相比,感染负载miR29慢病毒的SGC-7901细胞增殖受到明显抑制(P<0.05),而感染空载体慢病毒的SGC-7901细胞增殖能力无明显变化;负载miR-29的慢病毒感染后对SGC-7901细胞抑制率显著高于空载体慢病毒(25.90%±7.41%vs.9.96%±4.86%,P=0.011)。[结论]成功包装并鉴定人miR-29慢病毒表达载体;miR-29可有效抑制人胃癌SGC-7901细胞株增殖能力。
[Objective] To package and identify lentivirus expressing human miR-29 and test the effect of miR-29 on the proliferation of human gastric cancer cell line SGC-7901. [Method] The miR-29 precursor sequence was chemically synthesized and inserted into the polyclone site of lentiviral vector pGCL-GFP. The recombinant plasmid was co-transfected with lentiviral packaging plasmid pHelper1.0 and pHelper2.0 into human embryonic kidney HEK293T cells for packaging lentivirus The lentivirus was isolated and purified and its titer was determined. After the SGC-7901 cells were infected with the lentivirus loaded with miR-29, MTT assay was used to observe the proliferation of SGC-7901 cells. [Results] Double enzyme digestion and sequencing confirmed that the DNA sequence of miR-29 was correctly inserted into pGCL-GFP and the titer of packaged lentivirus was 2 × 108TU / ml. The successful packaging of recombinant lentivirus could increase the number of SGC-7901 cells Compared with SGC-7901 without lentivirus, SGC-7901 cells infected with miR29 lentivirus significantly inhibited the proliferation of SGC-7901 cells (P <0.05), whereas SGC-7901 The inhibitory rate of miR-29-loaded lentivirus on SGC-7901 cells was significantly higher than that of empty vector lentivirus (25.90% ± 7.41% vs.9.96% ± 4.86%, P = 0.011). [Conclusion] The human miR-29 lentiviral vector was successfully packaged and identified. MiR-29 could effectively inhibit the proliferation of human gastric cancer cell line SGC-7901.