Study On Germplasm Identification,Suspension Culture And Effects On Apoptosis Of Human Hepatocarcinoma HepG₂ Cells Induced By The Active Components Of Tetrastigma Hemsleyanum

Tetrastigma hemsleyanum Diels et Gilg,native to China,is a rare and endangered perennial herb of the family Vitaceae.Root tubers or the whole herbs are used as the raw material in Chinese medicine.In recent years,wild T.hemsleyanum plants are on the verge of extinction,there are many kinds of counterfeits on the market,which would severely influence the safety and effectiveness for its clinical use.Extracts of total flavonoids from T.hemsleyanum have been used as a broad spectrum antibiotic agent.Ethylacetate or chloroform of T.hemsleyanum has a potent antitumor activity in vitro and in vivo.However,the molecular mechanism remains elusive.In this study,inter-simple sequence repeat(ISSR),cleaved amplified polymorphic sequence(CAPS),and the internal transcribed spacer region II(ITS2)barcode were used for the first time for the authentication of T.hemsleyanum from its five most commonly counterfeits.T.hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production.The effects of calcium ions(Ca2+)on induction were studied.The apoptosis mechanism of HepG2 cells exposed to different concentrations of EET in vitro was also investigated.The results of this study will provide theory basis for the rational exploitation of T.hemsleyanum.The main contents and results of this study are as follow:(1)The proper ISSR-PCR reaction system and amplification process for T.hemsleyanum were optimized,11 stable and repeatable ISSR primers were also screened.ISSR analysis suggested that it was a useful method for distinguishing T.hemsleyanum from its adulterants of different genus.However,the cluster dendrograms derived from ISSR profiles proved insufficient to distinguish T.hemsleyanum from those adulterants of the same genus.(2)ITS2 sequences of all the six taxa were amplified and sequenced.The Neighbor-Joining tree constructed from the ITS2 sequences showed that T.hemsleyanum species were clearly differentiated from all counterfeits samples.Different T.hemsleyanum samples from Zhejiang province were clustered in the same clade,however,the other samples had not significant correlation to geographical distance.(3)A mutation site in the ITS2 region among T.hemsleyanum had been found which could be recognized by the restriction endonuclease Ncol.T.hemsleyanum could be readily distinguished from counterfeits as the PCR products from T.hemsleyanum could be digested sufficiently by Ncol,while the ITS2 fragments from 5 counterfeits could not be digested basically.It was much easier and less time-consuming than DNA sequencing.The false negative results were less likely to occur as the target fragment was very short.(4)The effect of ethyl acetate extract from T.hemsleyanum(EET)on cell cycle and apoptosis in human hepatoma HepG2 cells were investigated.The morphology of EET-treated HepG2 cells showed evidence of apoptosis that included blebbing and chromatin condensation,nucleic fragmentation,and so on.The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis.The cell-cycle analysis indicated that EET was able to induce S phase arrest and typical subdiploid peak in a dose-and time-dependent manner.In conclusion,the cytotoxicity on HepG2 cells induced by EET is a result of both cell-cycle arrest and apoptosis.(5)Flow cytometer and inverted fluorescence microscope were used to detect the mitochondrial membrane potential and cytosolic Ca2+ level.Western blotting analysis was used to evaluate the expression of the apoptosis-related proteins.Annexin V/PI staining was used to investigate cell apoptosis.Spectrophotometry was used to detect the activity of caspase family.The results showed that distinct apoptotic morphological changes occurred in HepG2 cells treated by EET.EET caused collapse of mitochondrial membrane potential,elevation of cytosolic Ca2+ level,and evoked release of cytochrome c from mitochondria in a concentration-dependent manner.The apoptosis was accompanied by a significant activation of caspase-3,caspase-9,and the cleavage.(6)T.hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production.The effects of calcium ions(Ca2+)on induction were also studied.It was found that the most effective elicitors were 50μM of the heavy metal ion copper(Cu2+)and 100 μM of the rare earth element cerium(Ce3+).The decrease in exogenous Ca2+ concentration could stimulate H2O2 bursts and peroxidase activity caused by metal elicitors.Therefore,reducing the Ca2+concentration in culture medium,or adding Ca2+ antagonists could be used to improve flavonoid production and cell growth in combination with induction by metal elicitors during in vitro culture of T.hemsleyanum suspension cells.