叶下珠水提物对裸鼠人肝癌移植瘤HBx和VEGFR3表达的影响

目的 探讨乙型肝炎病毒x基因（hepatitis B virux x gene, HBx）和血管内皮生长因子受体3（vascular endothelial growth factor receptor 3, VEGFR3）基因与乙型肝炎相关肝癌的相关性，以及叶下株水提物（aqueous extract of phyllanthus urinaria L, AEP）对HBx和VEGFR3蛋白表达的干预作用。方法 将60只Balb/c-nu裸鼠随机分为10组。分别复制转染HBx、氯霉素乙酞转移酶（chloramphenicol acetyltransferase，CAT）和VEGFR3基因的HepG2肝癌细胞的裸鼠皮下移植瘤模型，用AEP进行干预，以生理盐水、环磷酰胺（cyclophosphamide，CTX）作为对照，共治疗6周。观察模型复制后不同时间各组裸鼠的移植瘤生长状况，采用酶联免疫吸附试验检测各组裸鼠血清甲胎蛋白（alpha fetal protein, AFP）水平，采用Western blot方法检测移植瘤组织中HBx和VEGFR3蛋白表达水平。结果 肝癌移植瘤模型复制成功。各组裸鼠体质量均随着时间显著增长。实验结束时，接种HepG2-HBx细胞的各组中，AEP处理组肿瘤质量显著低于CTX处理组（P＜0.05），其AFP水平显著高于NS处理组（P<0.05），其移植瘤组织中HBx表达水平显著低于NS组（P<0.05）。接种相同肝癌细胞的各组中，AEP处理组VEGFR3表达水平最低，但与CTX组VEGFR3表达水平比较，差异无统计学意义（P＞0.05）；AEP组和CTX组VEGFR3表达水平显著低于NS组（P＜0.05）。对于相同的处理因素，接种HepG-HBx细胞和HepG-CAT细胞的裸鼠移植瘤组织中VEGFR3表达水平均显著低于接种HepG-VEGFR3细胞的裸鼠（P＜0.01）。结论 AEP通过直接抑制HBx蛋白和VEGFR3蛋白表达，及通过抑制HBx蛋白表达而间接抑制VEGFR3蛋白表达，达到对肝癌移植瘤生长的抑制作用。

Effects of Aqueous Extract of Phyllanthus urinaria L. on Expression of Hepatitis B Virux X Gene and Vascular Endothelial Growth Factor Receptor 3 in Nude Mice Transplanted with Human Hepatoma Cells

Objective To investigate the relationship of hepatitis B virux x gene (HBx) and vascular endothelial growth factor receptor 3 (VEGFR3) gene with hepatitis B-related liver cancer and the effects of aqueous extract of Phyllanthus urinaria L. (AEP) on the expression of HBx and VEGFR3. Methods Sixty Balb/c nude mice were randomly assigned to ten groups. A subcutaneous xenograft tumor model was induced by transplanting these nude mice with HepG2-HBx cells, HepG2-chloramphenicol acetyltransferase (CAT) cells, or HepG2-VEGFR3 cells. The treated mice were given AEP, normal saline (NS), or cyclophosphamide (CTX) for six weeks. The growth of xenograft tumor was evaluated at different time points after the model was induced. Serum alpha-fetoprotein (AFP) levels were determined by enzyme-linked immunosorbent assay. The protein expression of HBx and VEGFR3 in xenograft tumor was measured by Western blot. Results The hepatoma xenograft model was successfully induced in nude mice. Each group showed a significant increase in body weight over time. At the end of the experiment, among all mice transplanted with HepG2-HBx cells, the AEP group had a significantly lower tumor weight than the CTX group (P<0.05), and it had a significantly higher AFP level (P<0.05) and a significantly lower expressionof HBx in xenograft tumor (P<0.05), as compared with the NS group. Among the mice transplanted with the same HepG2 cells, the AEP group had the lowest expression of VEGFR3, but there was no significant difference in VEGFR3 expression between the AEP group and CTX group (P>0.05); the AEP group and CTX group had significantly lower VEGFR-3 expression levels than the NS group (P<0.05). Among the mice receiving the same intervention, those transplanted with HepG2-HBx cells and HepG2-CAT cells had significantly lower expression of VEGFR3 in xenograft tumor than those transplanted with HepG2-VEGFR3 cells (P<0.01). Conclusion AEP can directly suppress the protein expression of HBx and VEGFR3 in tumor tissues or indirectly suppress the protein expression of VEGFR3 by suppressing HBx expression, thus inhibiting the growth of hepatoma xenograft.