Suppressed proliferation of NIH3T3 cells by stable expression of perlecan shRNA lentiviral particles / 中华创伤骨科杂志

ABSTRACT

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 groupsGFP 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 perlecan shRNA lentiviral particles.