黄芩素抑制人巨细胞病毒感染星形胶质细胞的实验研究

Experimental Study on Baicalein to Inhibit Human Cytomegalovirus Infection in Human Astrocyte

目前对人巨细胞病毒(human cytomegalovirous,HCMV)感染尚无特异有效的治疗和预防手段,研究通过观察黄芩素(baicalein,BAI)抑制HCMV体外感染人星形胶质细胞引起的IE、pp65 mRNA及蛋白表达的变化,探讨黄芩素抗HCMV感染的机制。运用MTT法检测BAI组、HCMV组、HCMV+BAI组和对照组细胞的细胞活性,观察BAI对HCMV感染致人星形胶质细胞增殖异常的抑制作用;Real-time PCR检测不同组之间IE、pp65 mRNA表达的变化;免疫荧光、Western blot技术检测IE、pp65蛋白表达的变化。MTT结果显示,20μmol/L BAI+HCMV组的吸光值在24、48、72 h均高于HCMV组(P<0.05);形态学观察病毒感染48 h时,HCMV组细胞出现明显的细胞病变效应(CPE),而20μmol/L BAI+HCMV组细胞状态较好,CPE不明显;Real-time PCR结果显示在感染48和72 h时20μmol/L BAI+HCMV组IE、pp65的mRNA表达明显低于HCMV组(P<0.05);免疫荧光、Western blot结果显示20μmol/L BAI+HCMV组细胞的IE、pp65蛋白表达明显低于HCMV组(P<0.05)。以上结果显示,适当浓度的黄芩素能抑制HCMV感染所致的细胞增殖异常,同时降低IE及pp65的mRNA和蛋白表达量。

There are still no specific effective methods yet so far to treat and prevent human cytomegalovirus （ HC- MV） infection. In this study, anti-infection mechanism of Chinese medicine baicalein （BAI） to inhibit human cyto- megalovirus （HCMV） in vitro was investigated through observation of the protein expression changes of IE and pp65 mRNA and protein expression caused by colloidal astrocyte. MTT assay were used to test cell activity in BAI group, HCMV group, HCMV ＋ BAI group, and control group and observe the inhibitory effect of BAI on HCMV infection that cause human colloidal astrocyte abnormal cell proliferation; expression changes of IE, pp65 mRNA in different groups was detected by Real-time PCR. Expression changes of IE and pp65 protein in different groups were detected by Western blot and immunofluorescence. MTY results showed that the absorbance of 20 μmol/L BAI ＋ HCMV group values were at 24,48,72 h were all higher than HCMV group （P 〈 0. 05 ）. Morphologically to observe viral infection at 48 h,in the HCMV group obvious cytopathic effects （CPE） occurred. However, in 20 μmol/L BAI ＋ HCMV group, the CPE was not clear; the results of real-time PCR showed that the expression of IE, pp65 mRNA in 20 μmol/L BAI ＋ HCMV group were obviously lower than HCMV group at 48 h, 72 h post-infection （P 〈 0. 05 ） ; the results of immuno-fluorescence and Western blot assay indicated that in 20 txmol/L BAI ＋ HCMV group the expression of IE, mRNA of pp65 protein were apparently lower than HCMV group （P 〈 0.05）. The above results revealed that baicalein at appro- priate concentration can inhibit abnormal cell proliferation and lower IE, pp65 mRNA and protein expression caused by HCMV infection.