| ObjectiveObserve the anti-HBV activity and inhibitory secretion to HBsAg, HBeAg of compound Phyllanthus in vitro, observe the the liver protection activity of compound Phyllanthus in animal model which Hepatic injury induced by immunity. To investigate the anti-toumor activity of compound Phyllanthus in vitro and in vivo, and the protective role of compound Phyllanthus at the molecular level. The step of experiments1. Observe the anti- HBV activity and inhibitory secretion to HBsAg, HBeAg of compound Phyllanthus in vitro2. The compound Phyllanthus effect of protect the liver injury caused by ConA3. Observe the inhibit proliferation and induced apoptosis of human liver cancer HePG2 cell in vitro.4. Establishes the model of hepatocarcinoma HePG2cell xenografts in nude mice5. The anti- tumor effect' s of compound Phyllanthus in vitro6. Observe the expression of p53, c-myc in nude mice with RT-PCR, molecular cloning, gene sequence analysis which accommodate effects maybe caused by compound PhyllanthusMethods:1. HepG2. 2.15 cell line, transfected with HBV DNA, was used as an experimentmodel. MTT assay was employed to detect the toxicity of 2.2.15 cells treated by compound Phyllanthus, According to the formula to figure out the most non-toxic concentration and semi-toxic concentration(TC50) . subculturing 1.2ml 2.2.15cell suspension ( lXlOVml) in one slot of 24 cells plate, abandons the culture medium next day. the compound Phyllanthus was dilutes (series 2 time of dilutions) by culture medium on 2.2.15cell , take the most non-toxic concentration as the initial concentration and subculturing 6 slot in the same concentration, set up the control group at the same time. Substitute the medium which contains compound Phyllanthus every 3 days. Collect 250ul supernatant save at -20X? each time in order to detecting, the effects of compound Phyllanthus on the secret HBeAg, HBsAg, as well as HBVDNA , were assayed by ELISA method and FQ- PCR. To investigate the inhibitory effect according to the antigen suppression rate, half number of effective concentrations (IC50) and the treatment index (TI).2. 48 NIH mouse were divided into 6 groups at radom, respectively the blank group, model group, compound Phyllanthus large dosage group, compound Phyllanthus middle dosage group and small dosage group (40g/Kg, 20g/Kg, lOg/Kg), Bifendate control group (150mg/Kg) . All the mice injection ConA (20mg/kg) in first day morning except the blank group. Intragastric in four hours later, The blank group and model group were given the same volum normal sodium at the same time and the second intragastric in next morning. After 3 times drug administration, the blood was collected by plunk the eye ball. The ALT and AST in the blood was tested with Lais assay. The TNF-alpha and IL-8 in blood was tested by ELISA assay. After the liver takes out takes the left leaf neutral formaldehyde to be fixed, the paraffin wax embedding slice, conventional HE dyes, the light mirror observation pathological section and photographs.3. HePG2 cell was dissociated by 0.125% trypsin and counted at growing period, then raised in 96 pore plate for 24h. The different concentration of compoundPhyllanthus (50ug/ml, 100 ug/ml, 200 ug/ml, 500 ug/ml) was administered for 24h, 48h , 72h and the same volume culture medium was administered in control group. MTT assay was employed to detect the 50% inhibiting concentration (IC50), flow cytometry was adopted to measure the apoptosis rate and cell cycle , And the ultrastructure was analyzed with electronic microscope at the same time. HePG2 cell of each treatment group dyes with the Hoect33258 .apoptosis was observed by fluorescence microscope .4. HePG2 cell was dissociated by 0. 125% trypsin which contains 0. 02%EDTA and washed twice by PBS. Transfer the cell to a tube then centrifuge at lOOOrpm for 3min, adjusting cell suspension to lX108/ml with sodium chroide. The nude mice to which injected the suspension were divided into three groups namely treatment group, positive control group and negative control group at radom in next day. Compound Phyllanthus (lOml/kg) in treatment group with intragastric administration and 5-fluorouracil (20mg/kg. d) inject to intraperitoneal every 2days in positive control group. The same volume sodium chroide administer in negative control group. General state of health and tumor growth were observed every day. Draw the tumor volume time -growth curve according to measured the length(a) , width(b) and highly (c)of tumor with vernier caliperbare which on the basis of formula (V=ab2/2) every week. All the nude mices were killed after 35 days, separation blood serum-20°C to save. Strip and weigh the lump tissue then puts in the fluid nitrogen to save rapidly. AFP was tested with radio-immunity. Part of tissue was fixed by 10% neutral formalin, then paraffin section, the HE convention dyes, under the light mirror observes, photographs, observes its histo-pathology change.5. Trizol extraction was used to isolate RNA from tissue of all groups. RT-PCR has been developed to analysised the mRNA expression of p53 and c-myc . Set up the li gat ion mixture that contains p53 fragment and pTARGET? vector , then transformated ligation mixture to competent JM109. Select and culture colonies carrying recombinant plamids , The recombinants of p53 gene was send toInvitrogen Biotechnology corporation to sequencing. The sequencing results was analysed and submitted to the Genbank. Expression of p53 and c-myc protein was examined by SP immunohistochemical . Results1. In the range of TC50, HBsAg secreting inhibition of 2. 2.15 cell respectively was 54. 8%, 43. 6%, 37. 4%, 26. 7%, 14. 6% and HBeAg secreting inhibition was 78. 6%, 72.9%, 67.1%, 56.8%, 51. 5%, while the concentration of compound Phyllanthus was 250ug/ml, 125ug/ml, 62. 5ug/ml, 31.25ug/ml, 15. 6ug/ml. The treatment index was 6.1 to HBsAg and 24.6 to HBeAg .In the meantime, the inhibition effect of HBsAg , HBeAg, and HBV DNA was dose dependent. But the inhibition effect of HBV-DNA was declined when concentration of compound Phyllanthus reached lOOOug/ml, the reason was HBV-DNA released by cell death which caused by drug toxicity.2. Test of serum biochemistry showed that all model group which level of ALT, AST, TNF-a,IL-8 were higher than blank group(p<0. 01),every drug administration group contrasted to the model group, p<0. 01. That indicate all the groups of compound Phyllanthus have the effect of drop down the serum ALT, AST, TNF-a , IL-8. The results also showed that groups of compound Phyllanthus were not contrast to Bifendate group in serum ALT, AST. (p>0.05). However, groups of contrasted to Bifendate group in serum TNF- a , (p<0. 01). the results displayed compound Phyllanthus have the effect of drop dowm serum TNF-a but Bifendate have not. We find the large dosage group and the middle dosage group are superior to the small dosage group, (p<0. 05). The most serious pathological changes liver tissues were occurred in the model group. The swell of liver cell, necrosis, red blood cell and Kupffer cell accumulate in the liver blood sinus .lymphocytes soakage were find out .The Bifendate group was not so serious as the Model group, but also find out ihjured lobule structure .necrosis swell change .lymphocyte sokage , red blood cell accumulate in the liver blood sinus .The compound Phyllanthus large dosagegroup, middle dosage group are only can see slightly pathological changes , the large dosage group is the most slight .5..The tumor-growth curve showed that negative group was increased obviously , . meanwhile, the tumor growth of the compound Phyllanthus group and positive group was slowly. The tumor inhibition rate of compound Phyllanthus group was 32. 5%and 5-Fu group was 30.8% which contrast to the negative group(p<0. 05). Pathology change was different in three groups, the tumor cell was separated by the fibrous tissue which line up regularity and necrosis was not found in negative group .Necrosis and apoptosis was observed in compound Phyllanthus group. A large area Necrosis was found in the 5-Fu group but tumor cell was line up regularity.6. p53 gene expression was observed in compound Phyllanthus group , meanwhile, p53 gene was not obviously expressed in negative and positive group. The sequencing results was submitted to the Genbank showed that expression of compound Phyllanthus group was wtp53. C-myc expression was obsedved in all groups, the expression of c-myc in compound Phyllanthus group was lower than those of negative group and positive group. All samples showed positive staining of c-myc protein in tissues (plasma) . The compound Phyllanthus can signif icantaly inhibit overexpression of c-myc (p<0.01) but there was no contrast between compound Phyllanthu group and 5-FU group(p>0. 05). Conclusionl.The compound of Phyllanthus have marked anti-HBV effect in vitro and the effect have a concentration dependent.2. The compound of Phyllanthus have remarkably effect of protect the injured liver caused by ConA. the mechanism is related to the make down the level of the serum TNF-aand IL-8 .which are the major cytokine participate in liver injury induced by immune3. The compound Phyllanthus can inhibit proliferation of human hepatoma cell line HePG2, arrest cell cyecle and induced apoptosis is the mechanism4. The compound Phyllanthus have marked effect of anti-tumor in vivo and inhibit the tumor of nude mice, the mechanism is related to it' s promoting expression of wtp53 gene and inhibiting the expression of c-myc gene... |
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