串珠素shRNA慢病毒颗粒转染对小鼠胚胎成纤维细胞增殖能力的影响

Suppressed proliferation of NIH3T3 cells by stable expression of perlecan shRNA lentiviral particles

目的通过研究串珠素shRNA慢病毒转染对小鼠胚胎成纤维细胞（NIH3T3）增殖能力的影响，探讨其在抑制硬膜外瘢痕形成中的作用和机制。方法培养小鼠NIH3T3，并分为3组：未转染的NIH3T3组（对照组）、GFP慢病毒颗粒转染组（GFP组）和串珠素shRNA慢病毒颗粒转染组（shRNA组）。GFP组和shRNA组转染1周后在荧光显微镜下观察GFP的表达情况，从而判断慢病毒载体转染目的细胞所需的合适MOI值及GFP表达时间。确定慢病毒载体转染目的细胞合适的M01值后，以此条件进行目的基因转染。用嘌呤霉素筛选获得稳定表达细胞。分别以逆转录聚合酶链反应（RT—PCR）、Westernblot及噻唑蓝（MTF）法来检测转染后细胞mRNA、蛋白质表达及细胞增殖水平的差异。结果RT-PCR检测表明，串珠素RNA在对照组与GFP组高表达，在shRNA组低表达，shRNA组分别与对照组和GFP组比较差异均有统计学意义（P〈0．05）。Westernblot检测表明，串珠素蛋白质水平在对照组与GFP组高表达，在shRNA组低表达，shRNA组分别于对照组和GFP组比较，差异均有统计学意义（P〈0．05）。1～2d各组吸光度值之间差异无统计学意义（P〉0．05）。3～6d对照组与GFP组细胞增殖接近正常，shRNA组细胞增殖能力减弱，对照组与GFP组增殖能力明显高于shRNA组，差异有统计学意义（P〈0．05）。结论串珠素shRNA慢病毒转染可以有效抑制NIH3T3的增殖能力。

Objective To investigate the impact of stable expression of perlecan shRNA lentiviral particles on proliferation of NIH3T3 cells. Methods Mouse fibroblasts were cultured. Lentiviral particles-green fluorescent protein （LV-GFP） was used to transfect the cultured NIH3T3 cells with multiplicity of infection （MOI） of 10, 30 and 50. The GFP expression was observed with fluorescence microscopy after transfection for one week to estimate the proper MOI and the time of GFP expression needed. The transfection efficiency of LV-GFP with the proper MOI by fluorescence-activated cell sorting was detected. The stably transfected cell lines were developed by puromycin screening for more than 2 weeks. The third generation HFF in good condition was randomly divided into 3 groups： GFP group, shRNA group and control group. RT-PCR, Western blot and MTT assays were used to detect the expressions of perlecan mRNA and protein and cell proliferation in the 3 groups. Results Perlecan mRNA and protein showed high expressions in the control and GFP groups but low expressions in the shRNA group, with significant differences respectively between the shRNA group and the other 2 groups （ P 〈 0. 05）. There was no significant difference between the 3 groups in the optical density at the first 2 days （ P 〉 0.05）. On 3 to 6 days the cells in the control and GFP groups grew normally while the cells in the shRNA group proliferated in a weak manner, the transfected cells in the shRNA group showed a significantly reduced proliferation rate compared with the other 2 groups （ P 〈 0. 05）. Conclusion The growth of NIH3T3 cells can be inhibited significantly by transfection with perleean shRNA lentiviral particles.