摘要
目的检测柯萨奇病毒B3（CVB3）对大鼠心肌成纤维细胞骨桥蛋白（OPN）及I型胶原表达的影响，探讨OPN在病毒性心肌炎的可能发病机制。方法用重复感染率（MOI）为0．5PFU／cell的CVB3感染体外培养的大鼠心肌成纤维细胞，细胞分对照组（12h）、病毒组（感染后12h、1d,2d及3d）。用RT·PCR及WesternBlot、免疫组化检测OPN及I型胶原表达，并将OPN表达与I型胶原表达进行直线相关分析。结果（1）RT-PCR测得对照组12h、病毒组12h、1d、2d及3d5组OPN／13-actin吸光度比值分别为：0．38±0．06、0．56±0．06、0．72±0．05、0．98±0．06、0．86±0．02；WesternBlot测得上述5组的OPN／13-actin灰度值分别为：0．26±0．03、0．36±0．03、0．52±0．04、0．76±0．05、0．62±0．02；感染后12h，OPN表达即增高，于2d达到高峰，3d呈下降趋势，OPN／13-actin吸光度比值、灰度值比较差异均有统计学意义（F值分别为74．965、53．004，P均〈0．05）；（2）RT-PCR测得心肌成纤维细胞对照组12h、病毒组12h、1d、2d、3dI型胶原／13-actin吸光度比值分别为：1．12±0．03、1．184-0．01、r．22±0．02、1．33±0．02、1．28±0．03，感染后12hI型胶原表达即增高，于2d达到高峰，3d呈下降趋势，差异有统计学意义（F=38．241，P〈0．05）；（3）OPN表达与I型胶原有正相关关系（r=0．948，P〈0．001）。结论CVB3可诱导心肌成纤维细胞I型胶原及OPN表达，OPN表达与I型胶原表达呈正相关，提示OPN可能促进心肌成纤维细胞I型胶原表达，参与病毒性心肌炎心肌纤维化的发病机制。
Objective To study the expression of osteopontin （OPN） and collagen type I in the rat myocardial fibroblasts infected by coxsackievirus B3 （ CVB3 ） and to explore the possible pathogenesis of OPN on viral myocarditis. Methods Cardiac fibroblasts were isolated from neonatal rat and cultured with an traditional method. The primary cultured rat myocardial fibroblasts were infected by CVB3 which multiplicity of infection （MO1） was 0. 5 PFU/cell. The myocardial fibroblasts were divided into control group 12 h and CVB3 groups （infected after 12 h,1 d,2 d,3 d）. The expression of OPN and collagen type I were detected by RT-PCR, Western Blotting and immunohistochemistry. And the linear correlation between the expression of OPN and the expression of collagen type I was analyzed. Results （1） The gray scale values of the OPN/13-actin in control group（ 12 h）and viral groups （ 12 h,1 d,2 d,3 d） were 0. 38±0. 06,0. 56±0. 06,0. 72±0. 05,0. 98±0. 06, 0. 86±0. 02 respectively with RT-PCR, and were 0. 26±0. 03,0. 36±0. 03,0. 52±0. 04,0. 76±0. 05,0. 62±0. 02 respectively with Western Blotting. The expression of OPN was found to be increased after 12 h infection, reached to the maximum after 2 d infection and displayed a decreased tendency after 3 d infection. There was significant difference on the gray scale values of the OPN/13-actin （ F = 74. 965,53.004, respectively, P 〈 0. 05 ）. （2） The gray scale values of the collagen type I/13-actin in control group（ 12 h）and viral groups （12 h, 1 d,2 d, 3 d） were 1.12±0. 03, 1.18±0. 01,1.22±0. 02, 1.33±0.02, 1.28±0. 03, respectively with RT- PGR. These results suggested that the collagen type I expression Started to increase at 12 h when infected by C4B3, reached to the maximum at 2 d, and then decreased after 3 d infection, the difference was significant （ F = 38. 241 ,P 〈 0. 05 ）. （3） The expression of OPN was positively correlated with the expression of collagen type I （r = 0. 948 ,P 〈 0. 001 ）. Conclusion CVB3 can induce the expression of OPN and collagen type I in the rat myocardial fibroblasts and the expression of OPN and collagen type I displays positive correlation. It suggests that OPN can promote the expression of collagen type I in myocardial fibroblasts, and may play an important role in the pathogenesis of viral myocarditis.
出处
《中国综合临床》
2012年第7期704-707,共4页
Clinical Medicine of China
