| Modern medical researches have showed that cells abnormal proliferation and differentiation may result in tumor geneis. An important gene in numerous tumors geneis is P53, the wide type P53 plays an imporntant role in maintaining cell growth and inhibiting malignant hyperplasia. Many researches have showed that P53 gene is inactivated in more than half of tumors because of gene mutations or degradation of Wide type P53 (wtP53) protein. Wt P53 as a transcription factor that combine with the target genes such as p21 gene. The conformation changes of wtP53 protein because of P53gene mutations may loss its function; wtP53 protein which is degradated by MDM2 protein in ubiquitination pathway may decrease its activity.One of the most important methods for tumor therapy is to increase the activity of P53 protein.Now some strategys mainly include:(1) Recombinant vector carry wild type P53 gene(wtP53) into tumor cells for expressing P53 protein or RNAi technology reduce the antagonist protein of P53 gene. (2) Reactivation of wtP53 were used the drugs to prevent P53 from the MDM2 attack, which can inhibit the ubiquitination pathway. (3) Activation of the mtP53. Some smalll molecule drugs can change the conformation of mtP53 to regain funtions through the interaction of mtP53 and small molecule drugs. Methods of transgenic treatment have disadvantages such as immunogenicity, target-based and safety. In comparison, the activation of the wtP53 or mtP53 protein has good prospects in this aspect. At present scientists already have screened some small molecules, which have good effects in the application, some drugs have been used in clinical stage I or stage II. But the activation methods need a big compound library so works is very complex, which is one of rate-determining step for screening the small molecules.So how to construct a good compound library and build a good screening methods are the current bottlenecks.Chinese herbal medicine is well known as a very large compound library for tumor therapy, most complexity and diversity ingredients are some polarity strong material, such as flavonoids, alkaloids, saponins kind, lignanoids, terpenoids, acids, alcohols, anthraquinone class, phenols and sugar, and so on.Since chinese medicine is a very large compound library, HTS techniques used to screen antitumor activity ingredients will be a powerful tool, which include cellular and molecular level model. As a rapid sensitive screening technique, Dual-Luciferase gene reporter system is often used to screen drugs and research molecular biology mechanisms, which is usually provided by Promega Company. firefly and renilla luciferase vectors may be used together to co-transfect mammalian cells, which has some probloms that the results were more difficult to repeat; In addition, there is another weaknesses:on one hand there cannot be used to establish cell lines,it means that you would transfect everytime if you try to your study;on the other hand,some cell lines are very difficult to transfect.Compared with above methods, the lentivirus has some advandages such as high efficiency, host range, stability and so on, which is a powerful tool for establishing cell lines.Basing on the foundation of cancer pathogenesis and high throughput screening technology,we had tried to construct the Dual-Luciferase reporter vector with human P21 promoter gene and EGFP gene,which were used for Packing virus to establish lines for screening the drug that activate P53 protein. Our research mainly as follow:Part I:Constrution of the pLL3.7-Dluc plasmid.Firstly, three pairs of primers were designed by the primer designer primer5.0 for cloning gene, then amplification of P21 promoter from healthy human blood by PCR. P21 promoter was subcloned into the plasmid of pGL3-baisic and then the recombinant plasmid was digested by enzymes for P21-Fluc fragment, which was subcloned into the plasmid of pLL3.7. Amplification of Rluc gene from pRL-SV40 vector by PCR, then the Rluc gene was subcloned into the plasmid of pIRES2-EGFP. Finnally we amplificed the RLUC-IRES-EGFP fragment which was subcloned into the recombinant plasmid of pLL3.7-Fluc, just called pLL3.7-Dluc. Then pLL3.7 Dluc was transtected into 293FT cell for observing EGFP expression and detecting luciferase.We had successesfully cloned the P21 promter from human blood genome, and then successfully constructed the plasmid of pLL3.7-Dluc by series of molecular methods. The expression of EGFP and the value of Fluc (Rluc) reached to106 after transfecting into 293FT cell indicated that the construction of the pLL3.7-Dluc was successful for screening drugs.Part II:Lentivirus package for establishing cell linesThe successful constructed vectors of pLL3.7-Dluc were co-transfected with packaging plasmids pCMV-dR8.91 and envelope plasmids pCMV-VSV-G into the 293FT cells for packaging lentivirus particles.48-72h after co-transfection, the cell culture medium containing the virus particles were collected by centrifugation and subsequent filtration to obtain the virus supernatant, testing the titer of virus through to infect 293FT cells.Experimental results showed that EGFP expressed after 24 hours, and transfect efficiency was over 80%. But the titer of virus showed that there was no successful packaging virus or packaging virus titer is too low.Part III:Test of cytotoxicity and screening the components of traditional chinese medicine that activate P53 protein.The IC50 of drugs were provided by MTT experiment. The plasmid of pLL3.7-Dluc was transfected into U251 and Eca109 cells,which were treated at IC50 of drugs after 24h, and then luciferase was measured after 48h, no drugs treatment for the negative control, cells were not transfected with plasmids for blank control,the data of luciferase measurement were analyzed by SPSS 13.0 sofeware showed that curcumin,tanshinone IIA and ginsenosideRbl can enhance the activity of P53 protein.Conclusion: Sequencing, EGFP expression, measurement of luciferase after transfecting pLL3.7-Dluc into 293FT cells, we had successfully constructed the lentivirus plamid which can be used for screening drugs. But the test of virus titer showed that the experiment wasn't successful package the virus or may be the titer was too low to be tested, so we can't established stable cell lines. Drugs screening results showed that curcumin, tanshinone IIA and ginsenosideRbl can strengthen the activity of P53 protein. |
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