The Expression Of ATM In Glioma After Having DDP Hemotherapy And Construction Of Recombinant Lentiviral Ector Of ATM-RNAi

Objective： To study the expression of ATM in pre-andpost-chemotherapeutic glioma, for determining the best time of moleculartargeted intervention to provide a basis.Methods：Ectopicly subcutaneous glioma transplantation models innude mice were established by injection of human glioma cell line U251cells． Tumor-beared nude mice were treated by the intraperitonealinjection of cisplatin(at the dose of5mg/kg/d for4-5days continuously)，chemotherapeutic and progressing glioma model after chemotherapy wereestablished．Expression of ATM in pre-and post-chemotherapeutic gliomawere detected by oligotoxicology&drug resistance microarrays、RT-PCRand immunohistochemistry.Results：Expression of ATM significantly increased in gliomasafter chemotherapy and significantly decreased when gliomas weresmallest, and increased obviously again when gliomas progressing,RT-PCR and immunohistochemistry show the same report.Conclusions：Expression of ATM changed ambulatorily in gliomas pre-and post-chemotherapy that it is significantly increased afterchemotherapy and increased obviously again when gliomas progressing. Objective: To construct a lentiviral vector expressing small—hair pin RNA (shRNA) targeting the gene of ATM, and provide the basisfor further experiments.Methods: The RNAi effective target sequence of ATM gene was tobe designed and confirmed firstly, and the synthesized cDNA containingboth sense and antisense Oligo DNA fragments of the targeting sequencewas cloned into the pGCSIL-GFP vector．The obtained lentiviral vectorcontaining Smad3shRNA which was subsequently confirmed by PCR andDNA sequencing analysis.After these identification, the293T cells werecotrans fected with lentiviral vector—shATM、pHelper1.0and pHelper2.0to produce a replication incompetent lentivirus. The titer of virus wastested according to the expression level of GFP．Finally the U87and U251cell cotrans fected with the obtained lentivirus, and the expression of ATMwere detected by RT-PCR and westen blot.Results: PCR and DNA sequencing demonstrated that lentivector carrying ATM-small interfering RNA(SiRNA) was constructedsuccessfully. The titer of concentrated virus of ATM-RNAi-LV1andATM-RNAi-LV2was3×108TU/mL and7×108TU/mL respectively, andthey could obviously inhibit the expression of ATM gene in vitro U251and U87cells, and the rate of the inhibition was more than70%(P<0.05).Conclusion: The lentivirus RNAi vector targeting ATM isconstructed successfully, and they could obviously inhibit the expressionof ATM gene in vitro U251and U87cells, and it would provide the basisfor further experiments.