Impact Of Phyllanthus Urinaria L And Its Compound On Expression Of Vascular Endothelial Growth Factor Receptor-3Mediated By Hepatitis B X Gene In Hepatocellular Carcinoma

Backgroud and objective:Hepatocellular carcinoma (HCC) is one of malignant tumors with highest incidence in the world.The studies on etiology of hepatology have made great improvements with the development of molecular genetics and molecular epidemiology. Among these improvements, the research on HCC patients infected with hepatitis B virus(HBV) catch more eyes.The mechanism of formation of HB or HBV-associated HCC is not yet clear. Hepatitis B virus X gene (HBx) or its encoding product, hepatitis B virus X antigene (HBxAg) play an important pole between HBV and HCC.But there is no public opinion that how HBx and HBxAg influence the formation and development of HCC.Since the growth of hepatoma cells relys on the formation of newly born blood vessel, and vascular endothelial growth factors(VEGF) is the most effective vascular growth factors, the study on VEGF has been a hot spot of HCC research. There are different influences of vascular endothelial growth factor receptors (VEGFR) in adjusting the formation HCC vessels. And VEGFR3plays a different role in different development stages.In one former research, the research group chaired by Professor Tong Guangdong, my tutor had (Data from one former research, chaired by Professor Tong Guangdong, confirmed that) confirmed that HBx from HBV influenced HCC and there was probably relationship between HBx and VEGFR3through studying VEGFR3,one of six landmarks of precancerous lesion of liver (PLL) which was found over-expressed in hepatocirrhosis (HC) patients with PLL. In addition, another research of Prof. Tong's indicated compound phyllanthus urinaria L.(CP)=suppressed the development of HCC by inhibiting the expression of HBxAg.Based on the above knowing, hepatoma cell lines stable transfected with HBx and VEGFR3were builted. And animal models prepared with these hepatoma cells were treated with the aqueous extract of phyllanthus urinaria L.(AEP). And the effect of CP in patients with PLL was observed. So as to study the effect and the mechanism of phyllanthus urinaria L(PUL) on the expressions of HBx and vascular endothelial growth factor receptor-3 (VEGFR3) genes in nude mice (Balb/c-nu mice) which transplanted by HepG2-HBx cells or HepG2-VEGFR3cells, and to explore the relationships between HBxAg and VEGFR3expressed in nude mice transplanted with hepatocellular carcinoma cells.Methods:First, to establish stable HepG2cell strains transfected by retrovirus vector carrying HBx gene and VEGFR3gene. HBx gene and VEGFR3gene be cloned from human genome and huam cDNA respectively by PCR, and proved by sequence determination and Western blot procedures. The recombinant plasmids of pBaBe-puro-HBx, pBaBe-puro-VEGFR3and pBaBe-puro-CAT(as a control group) be established by the above mentioned gene segments of HBx,VEGFR3and CAT. Human embryonic renal epithelial cell lines293T cells were transfected by recombinant plasmids together with PIK packaging plasmids.Human HepG2cells were infected by recombinant retroviruses and then HepG2cells transfected with HBx, VEGFR3and CAT were acquired.The absorbance of different HepG2cells transfected by different genes in different cell proliferation period was or were detected by Enzyme-linked immunosorbent detector, cell cycle by flow cytometry, and the expression of HBx and VEGFR3by RT-QPCR and western blot.Second, to make the aqueous extract of Phyllanthus urinaria L.(AEP) and the granular formulation of compound Phyllanthus urinaria L.(CP).Third,to make54nude mice sub-skin model transplanted by injecting given liver tumor cells strains and6mice injecting normal saline (NS) as a control group.All mice raised in SPF surroundings.Fourth,block designA. Animal experiment.10groups were divided under completely randomized digitals method by SPSS17.0. Group1st(G1), Blank control group of NS. G2, Blank control group of HepG2-HBx cell.G3,CTX-treated group of HepG2-HBx cell.G4, AEP-treated group of HepG2-HBx cell.G5,Blank control group of HepG2-CAT. G6, CTX treated group of HepG2-CAT. G7, AEP-treated of HepG2-CAT.G8. Blank control group of HepG2-VEGFR3.G9, CTX-treated group of HepG2-VEGFR3. G10,-AEP-treated group of HepG2-VEGFR3.Six times a week of AEP or NS was given by gavage administration.5times a week of CTX was given by abdominal cavity injection according to the instruction on the label.All specimens were collected at the end of experiment after30times injection of CTX.B.Clinical trial.113patients with hepatocirrhosis of precancerous lesion of liver(PLL) were separated into CP-treated group (n=59) and control group (n=54).The standard treatment of protecting liver be taken only when liver function of any group is abnormal.Fifth,A.Mice:the body mass was weighted every week.Serum was sampled from mouse eyes.The ret orbital venous plexus and the livers and tumors were drawn out by surgery operations.B.Patients:blood serum was collected respectively before treatment,12 months and24months after research.Sixth, indexes observation and detection:A.Mice:the body mass,the weight of tumors and the histomorphology were observed respectively. The histology of tumors and livers was or were examined by means of routine HE staining and light microscope.The expression of HBx, VEGFR3proteins of tumors and livers were detected by IHC and Western blot methods.Serum AFP levels were measured by ELISA methods.B.Patients:the quality of life be observed.The expression of VEGFR3,DRG2,and other four markers in blood serum were detected by ELISA methods.The count of lymphocytes in peripheral blood were detected.Seventh,Statistical evaluation:different methods be taken according to different type of given data. All data be dealed with SPSS17.0.SPSS Version17.0was used for statistical analysis.Continuous variables were expressed as the mean±SD and comparisons were made by Student's test or one-way ANOVA.Categorical variables were analyzed with the chi-square test.Significance was assumed with values of P<0.05.Results:1,HBx gene and VEGFR3gene were cloned from human genome and huam cDNA respectively by PCR,and proved by sequence determination and Western blot procedures. The vector producing cell lines HepG2-HBx and HepG2-VEGFR3were established.2,The maximum non-toxic concentration effect dose of aqueous extract of PUL was15mg/mL.3,It existed an interaction between the body mass and observation periods among different groups (P<0.05).4,The comparison of weight of tumors among groups:A.In three HepG2-HBx cells transplanted groups,the tumors of G2(model control group) was biggest and the tumor of G4(AEP group) was smallest. There was a significance difference between these groups by SNK tests (P<0.05).In HepG2-CAT cells transplanted goups,the tumors of G7(AEP group) and G6(CTX) were respectively smaller than G5(model control group)(P<0.05).But there was no difference between G6and G7(P>0.05).In HepG2-VEGFR3cells transplanted goups, the tumors of G10(AEP group) and G9(CTX) were respectively smaller than G8(model control group)(P<0.05).But there was no difference between G10and G9(P>0.05)B.No difference of the weight of tumors was found in the same drug groups of different transplanted cells (P>0.05)5,No significance difference of the expression of AFP or VEGFR3in mice serum or liver tissues were found between all groups (P>0.05)6,The expression of HBx in tissue slices of Tumors in G2were stronger than that in G3by IHC,and that of G4was minimum.There was a significance difference between G2and G3,G2and G4.But there was no difference between G3and G4.The same results were observed in the expression of HBx of these groups by Western blot method.7.The expression of VEGFR3was found in tissue slices of tumors in all groups by IHC. More than75%positive staining cells were observed in G2and G8, but no significance difference between them (P>0.05).The least expression be observed in G5. And the expression in G3was less than in G4and G6(P<0.05),also less than in G9(P<0.05).the expression of VEGFR3in G9be less than in G8(P<0.05),but no difference compared with that in G10(P>0.05)8.The expression of VEGFR3was found in tissue slices of tumors in all groups by Western blot methods.A.Experimental comparision in different treatment factors of groups transplanted with the same transgenic hepatocarcinoma cells:the least expression of VEGFR3in groups administered AEP be found,but no difference between that in groups administered AEP with groups administered CTX(P>0.05). the expression of both them were less than that in groups used normal saline(P<0.05).B.Experimental comparision in the same treatment factors of groups transplanted with different transgenic hepatocarcinoma cells:in NS groups,no difference of the expression of VEGFR3be observed between groups transplanted with HepG2-VEGFR3cells and with HepG2-HBx cells group (P>0.05).A significance difference be observed in both them compared the HepG2-CAT cells group(P<0.05).In AEP treatment groups,a difference of the VEGFR3expression be observed respectively among three groups transplanted with different cells(P<0.05).The least expression be found in HepG2-HBx cells group.In CTX treatment groups, the expression of VEGFR3be found most in HepG2-VEGFR3cells group,but no difference compared with HepG2-HBx cells group(P>0.05).A significance difference be observed in both them compared the HepG2-CAT cells group(P<0.05).9.After12months treatment with CP, no difference was observed about clinical symptoms, the indexes of liver fibrosis, and HBV-DNA level between the treatment group and the control group(P>0.05),but significance differences of given indexes be found respectively after24months(P<0.05). More than60%incidence of the antibodies of URG11and VEGFR3were found in six PLL antibodies and diffierence existed in both antibodies between the treatment group and the control group(P<0.05). At the end of the oberservation,there was a difference between treatment group of2HCC developed cases with control group of9HCC cases(P<0.05).Conclusions: 1,The expression of VEGFR3can be found in all tumor tissues of Balb/c nude mice transplanted by different transgenetic HCC cells.HBx and VEGFR3promote the growth of transplanted tumor in nude mice.HBx can facilitate the high expression of VEGFR3in tumor tissues.A positive correlation can exist between genes of VEGFR3and HBx.2, AEP can suppress directly the expression of VEGFR3or and HBx, or suppress indirectly the expression of VEGFR3by suppressing directly HBx expression.The inhibitory effect of AEP can be found both in HBx positive and negative hepatocarcinoma. And CTX can also suppress the expression of VEGFR3,not by suppressing HBx expression.AEP can inhibit the development of tumor of nude mice and has no effect on the body mass of animals.3,VEGFR3is over-expressed in blood serum of PLL and HCC patients.And VEGFR3can be taken as one of predictive factors of HCC.CP can suppress directly the expression of VEGFR3.Based on the above experimental conclusion,PUL can suppress directly the expression of VEGFR3or HBx, or suppress indirectly the expression of VEGFR3by suppressing directly HBx expression.4,The levels of serum AFP, VEGFR3were not according with expected results of human clinical research.It is probably because the animal models was not the orthotopic transplantation tumor models and could bring some limitations.More samples and better models should be a good selection in further study. In order to improve reliability, the active constituent of PUL will be trying to take out by our research group in further works.