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目的构建脾酪氨酸激酶(Syk)重组腺病毒并感染血管平滑肌细胞(VSMC),观察Syk对VSMC表型转换的影响。方法运用pDC315-GFP腺病毒载体系统,细菌同源重组法构建含目的基因Syk腺病毒重组质粒pDC315-GFP-Syk,经过包装、扩增、纯化后获得Syk重组腺病毒;测定病毒滴度并感染体外培养的大鼠VSMC,饥饿处理24 h,用Real-time PCR法和Western blot法对Syk、平滑肌22α蛋白(SM22α)及平滑肌α肌动蛋白(-αSM-actin)表达活性进行检测,观察Syk对平滑肌细胞表型转换的影响。结果成功构建携带Syk基因的重组腺病毒,纯化后滴度为1011pfu/mL,腺病毒感染VSMC后5 d,与空病毒组和对照组相比,Syk mRNA(741638.70±35213.53)和蛋白表达水平(2.14±0.71)增高(P<0.01);-αSM-actin(0.80±0.04)和SM22αmRNA表达水平(1.00±0.01)下降(P<0.05),同时,二者的蛋白表达水平也降低(0.61±0.10和0.18±0.06,P<0.01)。结论在VSMC中,Syk通过对其表型转换进行调控,进而影响平滑肌细胞的增殖和迁移。
Objective To construct the recombinant adenovirus of Syk and infect vascular smooth muscle cells (VSMCs) to observe the effect of Syk on the phenotype switch. Methods Recombinant adenovirus plasmid pDC315-GFP-Syk was constructed by pDC315-GFP adenovirus vector system and bacterial homologous recombination method. After packaging, amplification and purification, Syk recombinant adenovirus was obtained. The virus titer was determined and infected The VSMCs cultured in vitro were starved for 24 h. The expression of Syk, SM22α and α-actin were measured by Real-time PCR and Western blot. Syk Effect on phenotype switch of smooth muscle cells. Results The recombinant adenovirus carrying Syk gene was successfully constructed and its titer was 1011pfu / mL after purification. The expression of Syk mRNA (741638.70 ± 35213.53) and protein level ( 2.14 ± 0.71) (P <0.01). The expressions of αSM-actin (0.80 ± 0.04) and SM22αmRNA (1.00 ± 0.01) decreased (P <0.05) And 0.18 ± 0.06, P <0.01). Conclusion In VSMC, Syk affects the proliferation and migration of smooth muscle cells by regulating its phenotype.