Study On Signal Pathway Of Lentivirus Transfected The Interfere Chk1, Chk2 Gene To Mesenchymal Stem Cell For Therapy Of Brain Tumor Radioresistence

Part I Study on Brain tumor therapy by human umbilical cord mesenchymal stem cellsObjective:Discussion of human umbilical cord mesenchymal stem cells has virtue for migration to brain tumor and it can secretion cytokins as the function for inhibition the tumor growth, so it would be a good gene vector in study of brain tumor apoptosis whatever in the domain as cyto-biological or in molecular-biology to treatment of brain tumor.Methods:Human umbilical cord was digest by collagenase and collection the cell after digested, use flow cytometry to identification the marks appear in cell, cultured for some generation then use the Transwell examination for observation the effect of MSCs migration to brain tumor; co-culture the MSCs cell with brain tumor cells and observation the result effect of tumor growth inhibited by MSCs and exam by zymography and revere zymography.Results:In exam of transwell, we can see the MSCs migration to brain tumor clearly; MSCs and U251 cell of brain tumor lineage co-cultured,we can get the observation of U251 cell became bubbled form, and apoptosis after one week in break due to MSCs intergration to U251 cells, by exam of zymography and revere zymography, we can get the result of MSCs can secretion some of the factor or cytokins as TIMP (tissue inhibitor of metalloproteinases) to inhibition matrix metalloproteinases (MMPs) who secretion by brain tumor cells.Conclusions:MSCs is a good gene vector because of it can migration to brain tumor cells as it virtue, besides it can inhibit tumor cells growth to use MSCs for treatment brain tumor. Partâ…¡Construction of lentivial vector to transfection interfere expression of the Chk1 and Chk2 gene and study in it's mechanism of radioresistence.Objective:Study the mechanism of Chk1 and Chk2 gene who use lentiviral transfection the interfered to the brain tumors and reduce it radioresistence, improved the tumor cells apoptosis after irradiationMethods:Genimic sequence of Chk1 and Chk2 gene was retrieved from Genbank and designed encording the shRNA of cDNA for Chk1 and Chk2. The cDNA was synthesized and inserted into pLk0.1 plasmid. Recombinant vectors and then transformed into E.Coli.. Selected positive colins and extracted the combinant plasmid. Use the enzyme of EcoRI and Ncol to digestion, then loading in agarose gel for electrophoresis, DNA identification, after those procedure, use lentivirus to transfection the U251 cells of brain tumor and generation. Irradiation of Ultraviolet and X-ray reverse-transcriptase-polymerase chain reaction (RT-PCR) and Western blot examination the inhibitory effect at the RNA and protein level and record the tumor cells living time.Results:After transfection the interfere gene of the Chk1 and Chk2, the both two was expression decreased in RNA and protein level exam in compare with not transfection any gene of interfered who identification by RT-PCR and Western blot exam.Conclusions:After succeed construction of lentiviral vector to interfection the brain tumor cells, the gene expression of Chk1 and Chk2 was decreased, so the way can improved the tumor cells sensitive by radiotherapy of radiation irradiation, and out of delivered the damage signal through pathway to stop the cell cycle, so it not to repaired DNA damage and led the tumor cell apoptosis or death direct. Partâ…¢Study in the signal pathway of improved the glioma cell radiosensitive by lentivial transfection interfere downregulation expression of the Chk1 and Chk2 genes.Objective:Study in Chk1 and Chk2 who use lentiviral transfection to interfered can not delivered signal for phosphorylation, Cdc25 family and other substrate, so it not work on retardant the cell cycle for repair damage of DNA after irradiation or other kinds can led damage, the tumor cells apoptosis due to not get the signal of damage by checkpoint kinases, dismatch DNA paired and increased stress of replication the DNA, and it nor work in protein function in normal biology as usual.Methods:Use ultraviolet and X-ray for irradiation the tumor cells who trasfection interfere gene of Chk1 and Chk2 by Lentivirus, then reverse-transcriptase-polymerase chain reaction (RT-PCR) and Western blot examination the inhibitory effect at the RNA and protein level and record the tumor cells living time.Results:After succeed transfection the gene of interfere, the gene of Chk1 and Chk2 was expression decreased in RNA and protein level exam in compare with not transfection any gene of interfered and not live in a long time.Conclusions:Chk1 and Chk2 is the member of (PIKKs, phosphate-dyl-inositol-3-kinase- like protein kinases), the important substrate of ATM and ATR, it active after damage by radiation can improved the tumor cells sensitive by radiotherapy of radiation irradiation, but when interfered the Chk1 and Chk2 gene expression, it can't delivered the signal through pathway of ATM/ATR-CHK1/CHK2-Cdc25 to stop the cell cycle, so it not to repaired DNA damage and led the tumor cell apoptosis.