| For a long period of time,cancer has ruthlessly threatened human ’s life.For the tumors have many particularities,we cannot afford to find a safe,sensitive and quick method to curing them,although the since and technology is developing rapidly nowadays.During the past years,the way people treat tumors confined to surgeries,radiotherapies and chemotherapies,or combination therapies.Anyway,surgeries may increase the risk of tumor cells migration and proliferation,or the radiotherapies and chemotherapies are toxic to the organism during the treatment.What’s worse,the chemotherapy drugs may cause chemo resistance.Therefore,we must find some drugs that targets the tumors,and that be non-toxicity or low-toxicity to improve human ’s health.Fortunately,researchers found that mice can gain the ability to anti tumor and their lifespan also wasn’t affected when p53 and p19ARF level in vivo was overexpression.Another report demonstration that,mice’s lifespan was significantly extended,while it’s tumor susceptibility was the same with the control group when up-regulate the telomerase catalytic subunit-TERT in vivo.These results remind us that,if targeting the p53 and p 19ARF/p 14ARFor TERT,we can screen out drugs that resisting senescence and tumors at the same time.The establishment of GFP fluorescent cell carrier screening system that targeting p53 and p19 gene to screen plant extracts that produced in Yunnan.Then confirming the preliminary screening results of fluorescence by Western-blot,And the cell toxicity test by MTT.At last,exploring the antitumor activity and potential preliminary mechanism in the light of previous results.We constructed a cellular screening platform that based on the luciferase reporter plasmid vector.The green fluorescent tag protein’(GFP)promoter sequence is replaced by the promoter sequence of p53/p 19.Then the recombinant plasmid was transformed into E.coli colonies that were picked for amplification.Next,we extracted plasmid form E.coli colonies,and transfected into the corresponding host cells with lipofection manner,integrated into the genome.When the recombinant cell grow to cover the petri dish,adding in puromycin to screen positive cells.The host cell of p53/p 19 promoter is p53-/-MEF.We took pictures with the inverted fluorescence microscope to record those extracts that activate cell’s fluorescence.The fluorescent screening experiment was repeated three times.Following the preliminary cell’s fluorescence screening,we used Western-blot to verify the results of fluorescence screening,checking the activation of p53 and p19 gene.Then,it is necessary to test the drug toxicity,we adopted the MTT and SRB methods to examine whether the extracts was toxic to normal wild-type cells.The doses used areOμg/ml、10μg/ml、20μg/ml、40μg/ml、80μg/ml.Synchronous,we verified whether the extracts is active to TERT,the telomere reverse transcriptase,with RT-PCR method.At last,we choosed some potential extracts to test its anti-tumor activity.Fluorescent initial screening statistics of cell platform demonstrates that Eight kinds of natural extracts that includes A53、A54、A79、A89、A90、B38、B60 and B90 can stabilize activate the corresponding promoter,so we choosed these eight extracts as potential drug,MTT and SRB assays show that these extracts is low-toxic to WT-MEF cells,that they have no IC-50 values in the dose of 0-80μg/ml.The results of Western-Blot demonstrate that B60 and B90 can stabilize activate p53 and p19 on the protein level.Extracts like A54、A89、B90 et can decrease the activity of TERT through the RT-PCR assay.Tetrastigma cruciatum can inhibit the growth of colon and liver cancer cells,and also can inhibit mobility of liver and osteosarcoma cells.These results demonstrate that the GFP fluorescent cell carrier screening system is stability and credibility,can used for targeted screening of p53 and p19 genes.During the plant extracts’ screening,SRB assay is better than that of MTT assay.Western-blot assay can verify the preliminary screening results of fluorescence.The crude extract of Tetrastigma cruciatum can inhibits migration of tumor cells,the potential mechanism may targeting the p53 and p19 genes and the the activity of TERT. |
