叶下珠提取物抗乙肝病毒及乙肝病毒X基因的研究

Inhibition of extract of phyllanthus urinaria L.on hepatitis B virus and hepatitis B X gene in vitro

目的:研究叶下珠提取物在体外抗HBV以及抑制HBX表达的作用。方法:选用2.2.15细胞以及建立HepG2X、HepG2X-H IV-1-LTR-luc iferase细胞系,用不同浓度的叶下珠药液进行细胞培养试验。确定药液对试验细胞的药物最大无毒浓度(TC0)以及半数药物有效浓度(IC50)。实验时,将细胞分空白对照组、HepG2CAT-H IV-1-LTR-lu-c iferase细胞对照组,以及不同药物浓度的试验组(15mg/m l,7.5mg/m l,3.75mg/m l),并以β-action作为内参照。用real tim e-PCR、RT-PCR,以及抑制荧光素酶(luc iferase)等方法,定量检测HBV DNA含量以及HBX表达量。结果:叶下珠提取物最大无毒剂量是15mg/m l,对HepG2.2.15细胞HBV DNA和HepG2-X细胞HBX半数有效量皆为3.75mg/m l(P<0.05),而7.5mg/m l、15mg/m l剂量与空白对照组相比较,分别为P<0.01,P<0.001。对HBX的抑制荧光素酶试验,叶下珠对细胞对照组及空白对照组抑制不明显,而高剂量用药组(15mg/m l),用药72小时与24小时比较,抑制率上升了10倍。结论:叶下珠不仅对全基因的HBV有抑制作用,还可能直接对X基因表达有抑制作用。

Objective： To study the inhibition of extract of phyllanthus urinaria L on HBV DNA and expression of hepatitis B virus X gene （HBX） in vitro. Methods： Cell line 2.2. 15, HepG2X ceils, and HepG2-HIV-1-LTR-luciferase ceils were included this study. The varied concentrations of extract of phyllanthus urinaria L. were vaccinated these cells and cultured. The maximum nontoxic concentration （ TC0 ） and 50% inhibiting concentration （ IC50 ） of the extract on cells were observed. Cell line 2. 2. 15, HepG2X cells, and HepG2X-HIV-1-LTR-luciferase cells were divided into blank group, HepG2CAT-HIV-1-LTR-luciferase cells group, and varied concentrations of extract group （ 15mg/ml, 7.5mg/ml, 3. 75mg/ml） . β-Action , a housekeeping gene was used as the internal control group. The level of HBV DNA and expression of HBX were detected by real time- PCR, RT-PCR, Luciferase assay. Results： The TC0 of extract from phyllanthus urinaria L were 15mg/ml. The IC50 of extract from phyllanthus urinaria L on HBV DNA and HBX both were 3.75mg/ml, which compared with blank control groups, they had statistical difference （P 〈 0. 05 ） . In 7. 5mg/ml and 15mg/ml doses, the difference were significance （respectivelyP 〈 0. 01, P 〈 0. 001 ） . In HBX-luciferase assay, no obviously diversity was existed among cell control group and blank groups. Whereas, treatment group that high dosage has inhibited to up 10 times at 72h with comparison of inhibited rate of themselves at 24h. Conclusion： Extract of hyllanthus urinaria can no only inhibit full length HBV DNA, and may serve as an essential mechanism in inhibit expression of HBX as well.