Study On The Role Of Osteopontin In Traumatic Brain Injury In Rat

Part one:the experimental research on effects of traumatic brain injury(TBI) by additon of osteopontin(OPN)in ratObjective:To study on effects of TBI by additon of OPN in rat.Methods:1.Established contical neuron injury model with in vitro culture in rat. Saline were added into injured cells as control,and different concentrations of OPN (0.01μg/ml,0.05μg/ml and 0.1μg/ml)were added into injured cells as OPN treatment groups.Activities of cells were detected by MTT and apoptosis were detected by flow cytometry at time point(24h,48h,72h,120h and 168h after injury).2.Established lateral fluid percussion(LFP)brain injury model in rat and designed a special respiratory mask for rat.Within 24 hours after the moderate LFP brain injury,intracerebroventricular injection was performed with 5μl saline(control group)or 5μl OPN(50ng,OPN group).Behavior and memory of rats were assessed at time point(72h,120h and 168h after injury).Apoptosis was detected in situ.Results:1.In OPN groups,cultured rat cerebral cortex cells actively proliferated and apoptosis rate decreased.Furthermore,the results were dose-dependent.2.Assisted respiration by ventilator with special mask decreased the death rate from 30%to 11.84%for LFP brain injury model in rats.3.The memory,the score of neurological function,walking and equilibrium function was improved after OPN injected intraventricular of LFP brain injury rat.Conclusion:OPN can promote the proliferation of cultured glia cell and inhibit the apoptosis of nerve cell.OPN injected intraventricular can improve the memory and neurological function after brain injured and it is dose-dependent.Part two:the experimental study on the effect of OPN which expression were blocked on injured nerve cell and injured brain in ratsObjective:To study on effect of OPN gene silencing in traumatic brain in rats.Method:1.TG005 was used as vector to construct OPN expression vector.Designed and synthesized the primer.Total RNA was extracted from myocardial of embryo rat, after that it was amplified by one-step reverse-transcription PCR.Vector and target gene of PCR product were cut by Mlul and Spel,and were purificated and dephosphorylated.Target gene and vector were connected and transferred competent E.coli and selected positive clones.The plasmid was extracted from positive E.coli and determined by enzyming.The positive clone was selected and sequenced. Sequencing identified as TG005-OPN recombinant.TG005-OPN and Lipofectamine 2000 were transferred into 293T cells,RT-PCR detection,and electrophoresis identification.2.OPN specific RNAi lentiviral vector was constructed with pRNAi-U6.2/Lenti. According to OPN genetic information,three shRNA sequences were designed.OPN -Sh1:ACGATGATGACGACGACGA,OPN-Sh2:ATGACGACGACGATGACGA, OPN-Sh3:AGCACACAAGCAGACGTTT.Three pairs of complementary chain DNA were synthesized and amplificated,pRNAi-U6.2/Lenti were cut by BamH I and Xho I,electrophoresis recovery,dephosphorylation,purification,connectivity, transferred into competent E.coli,selected positive clones and sequencing.The results indicated that the Lv-OPN-Sh was successfully contructed.Lentiviral vector were packaged by Tronolab lentiviral vector system(pRsv-REV,pMDlg-pRRE,pMD2G, Lv-OPN-Sh),and transferred into 293T cell together with TG005-OPN and Lipofectamine 2000,and then target screening,protein extranction and Western Blot detection were performed.3.Lentivirus package:the four plasmids component(packaging plasmid pRsv-REV,pMDlg-pRRE and pMD2G as well as positive plasmid Lv-OPN-Sh2)in Lentivirus system was transferred into 293T cells by Lipofectamine 2000 and then bring about lentivirus.Recombinated lentivirus plasmid infected precipited 293T cells that can express the OPN,screening the target and extracting tissue protein,Western blot detecting the effect of OPN gene silencing.Positive lenvirivus was largely produced and packed.4.Never cell apoptosis rate were detected after Lentivirus recombinated granules that included Lv-OPN-Sh2 transfect the injuried never cells.5.The rats with brain trauma were intraventricular injected Lv-OPN-Sh2 positived lenvirus recombinated plasmid 5μl or salin 5μl or TG005-OPN-Lipofecta mine 5μl or empty vector 5μl respectively.Behavior and memory function were assessed at the time of 72h,120h and 168h after injury.The apoptosis of cells were detected in situ. Results:1.TG005 linked purpose insertion element transfect the competenced Bacillus coli and positive clone was proved that about 1000bp gene fragment was expressed.From sequencing and already knowing OPN sequence GenBank comparison it was proved that gene fragment was as OPN,it is shows that OPN expression plasmid TG005-OPN was successfully prepared.2.pRNAi-U6.2/Lenti vector connected with desined composite PN-shRNA and transfect the competenced Bacillus coli.Positive clone sequencing was proved that shuttle plasmid Lv-OPN-Sh plasmid construction was successed.3.Liposome and lentivirus 4 plasmid cotransfect the incasing cells 293T and it can be seen that there was many green color.It was confirmed that there was many plasmids transfected into 293T cell.4.Viral particle infected with 293 T cell which can express the OPN gene and Western detection shows that Lv-OPN -Sh2 can obviously inhibit the expression of OPN.5.Rate of apoptosis of neuron had no increased after Lentivirus recombinated granules that included Lv-OPN-Sh2 transfect the injured neuron.6.Intraventricular injected lentiviral recombinated plasmid with Lv-OPN-Sh2 had no effect on neurological function and memory.Conclusion:OPN specific RNAi gene silencing mediated by Lentivirus vector had no affect on nerve cell proliferating and apoptosis and also no significant impact to the motor function and memory of rats with LFP brain injury.