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目的构建nesprin蛋白siRNA慢病毒载体(LV-siNesprin),感染骨髓间充质干细胞(MSCs),观察nesprin蛋白对MSCs的影响。方法针对nesprin靶基因序列设计并合成4对miRNA oligo,并将4对oligo退火成双链DNA,测序鉴定。将4种miRNA干扰质粒(SR-1、SR-2、SR-3、SR-4)转入大鼠血管平滑肌细胞,用反转录聚合酶链式反应(RT-PCR)和蛋白印迹法(Western blotting)检测干扰效应,筛选最佳干扰序列;将最佳干扰序列和pDONR221载体进行重组反应,获得含干扰序列的入门载体,再将入门载体和慢病毒表达的目的载体pLenti6/V5-DEST进行重组反应获得含干扰序列的LV-siNesprin+绿色荧光蛋白(GFP),包装慢病毒,测定病毒滴度。LV-siNesprin+GFP转染MSCs,Western blotting鉴定比较nesprin蛋白表达情况(分为LV-siNesprin+GFP组、GFP对照组和正常细胞组),用4,6-二脒基-2-苯基吲哚(DAPI)染色观察细胞核形态改变和四甲基偶氮唑盐(MTT)法检测MSCs感染LV-siNesprin后24 h、48 h、72 h和96 h的增殖情况(分为LV-siNesprin+GFP组、GFP对照组和正常细胞组)。结果测序证实合成的4对miRNA oligo正确,RT-PCR和Western blotting筛选出最佳干扰miRNA质粒SR-3,成功构建LV-siNesprin,包装慢病毒,病毒悬液的活性滴度为1×106ifu/ml。LV-siNesprin转染MSCs后,nesprin蛋白表达明显下降,出现细胞核融合、核碎裂等形态学改变;MTT法检测显示,LV-siNesprin+GFP组MSCs增殖速度较GFP对照组和正常细胞组减慢。结论 Nesprin蛋白对MSCs核膜稳定维持核膜空间结构起作用,并利于细胞增殖更新。
Objective To construct nesprin siRNA-lentiviral vector (LV-siNesprin) and infect bone marrow mesenchymal stem cells (MSCs) to observe the effect of nesprin protein on MSCs. Methods Four pairs of miRNA oligo were designed and synthesized according to the nesprin target gene sequence. Four pairs of oligo were annealed into double-stranded DNA and sequenced. Four miRNA interference plasmids (SR-1, SR-2, SR-3 and SR-4) were transfected into rat vascular smooth muscle cells by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting Western blotting) was used to detect the interference effect and the best interference sequence was screened. The optimal interference sequence and pDONR221 vector were recombinated to obtain the entry vector containing the interference sequence. The entry vector and lentiviral vector pLenti6 / V5-DEST Recombinant reaction containing interfering sequence of LV-siNesprin + green fluorescent protein (GFP), packaging lentivirus, determination of virus titer. MSCs were transfected with LV-siNesprin + GFP, and nesprin protein expression was analyzed by Western blotting (divided into LV-siNesprin + GFP group, GFP control group and normal cell group) (DAPI) staining and MTT assay were used to detect the proliferation of MSCs infected with LV-siNesprin at 24 h, 48 h, 72 h and 96 h (divided into LV-siNesprin + GFP Group, GFP control group and normal cell group). Results The sequencing proved that miRNA oligos were synthesized correctly, and the optimal miRNA plasmid SR-3 was screened by RT-PCR and Western blotting. LV-siNesprin was successfully constructed and packaged with lentivirus. The titer of virus suspension was 1 × 106ifu / ml. The expression of nesprin in LV-siNesprin transfected MSCs was significantly decreased, with morphological changes of nuclear fusion and nuclear fragmentation. The MTT assay showed that the proliferation of MSCs in LV-siNesprin + GFP group was slower than that in GFP control group and normal group . Conclusion The Nesprin protein plays a role in maintaining the nuclear membrane of MSCs stably and maintaining the nuclear membrane space structure, and is conducive to cell proliferation and renewal.