Construction And Identification Of MiR RNAi Lentiviral Vector Targeting For PTEN Which Has The Repairing Potential Of Damages In Central Nervous System

With the development of molecular biology and genetics,genetic tools have became a favorable means of studying the relationship between genes and proteins,exploring the intracellular signaling pathways,decrypting damaged cell repair mechanisms,and developing neuroprotective drugs.Previous studies have shown that the using chemically synthesized si RNA transfectes cultured neuronal cells to silence PTEN gene can promote the growth of neuronal processes and accelerate the growth of neuronal growth cone even in oxidative stress circumistances by activating m TOR signaling pathway.However,there are some limitations when use the si RNA to explore the relationship between genes and proteins.For example,we can not replace the promoter and can not achieve the long-term inhibition of genes.In order to further study the relationship between PTEN gene and central nervous system injury,we are focusing our attention on the micro RNA-mediated gene silencing.The lentiviral vector p WPXL(12257)containing the enhanced green fluorescent protein(e GFP)marker was selected as the vector backbone,and the micro RNA targeting PTEN m RNA was selected as the target gene to construct the recombinant lentiviral vector and realize the specific silencing of PTEN m RNA.Then the recombinant lentiviral vector was packaged into lentiviral particles and lentivirus particles were used to infect the cells and then played a role in gene silencing,which achieved efficient,stable and long-term interference of PTEN gene in target cells.Constructing the rats spinal cord semi-transected model,injecting the lentiviral particles in the injured site,obsering whether the virus particles can retrograde along the neuronal axons to the soma and silencing the PTEN gene by activating the m TOR signaling pathway.Methods:The recombinant lentiviral vectors were constructed by primer design(transcription product can target to PTEN m RNA),PCR,gel recovery,T-vector biding,digestion,transformation,plasmid extraction,target fragment and vector framework connecting and named as 12257-PTEN-micro RNA1,12257-PTEN-micro RNA2 and 12257-PTEN-micro RNA3.The recombinant lentiviral vectors were sequenced to determine whether the target fragment was linked to the vector framework and transfected with PC12 cells to observe whether the recombinant vectors could activate the m TOR signal pathway and compare the gene silencing efficiency of the three recombinant lentiviral vectors.The recombinant lentiviral vector with the highest interference efficiency was packaged into lentivirus particles.The experiment was divided into three groups:(1)blank control group(2)empty vector infection group(3)PTEN RNAi group.lentivirus particle titer was detected,RT-PCR method was used to verify lentivirus particle gene silencing efficiency.The rats were infected with lentiviral particles.This experiment was divided into four groups,6 rats in each group:(1)sham group was labled sham(2)Sample injured group.(3)Empty vector virus infection group was labeled NC(4)PTEN gene silencing group was Labeled PTEN RNAi.Sham group only removed T9 and T10 segment spinal lamina,the rats spinal cord in the sample injured group were injected with 0.9% Na Cl after semi-transected,NC group and PTEN RNAi group were injected with empty vector virus particles and PTEN interference group virus particles respectively after semi-transected injury.Paraffin sections were prepared and immunohistochemical staining was performed to observe the gene silencing efficiency of virus particles in experimental animals.Results:The sequencing results showed that the interference fragment was successfully linked to the vector framework.PC12 cells was transfected with recombinant lentiviral vectors followed with immunohistochemical staining,the results showedp S6 K positive expression in these three vectors and the expression of p S6 K is highest in the vector 12257-PTEN-micro RNA2(in the follwing experiments was named as 12257-PTEN-micro RNA).The recombinant lentiviral vector 12257-PTENmicro RNA with the highest gene interference was packaged into lentivirus particles.The results of titer analysis showed that BT=4×108TU / ml in empty vector infection group and BT=7×108TU / ml in PTEN RNAi group.72 hours later RT-PCR results showed that compared with the empty vector group the PTEN m RNA expression level been decreased obvirously(P < 0.05)in the PTEN RNAi group.Immunohistochemical staining of sensory cortical motor area in brain tissue after 14 days of infection with lentiviral granule in rats,the results showed that the e GFP negative expression in the sham operation group and sample injured group,while the e GFP positive expression in the empty vector virus infection group and PTEN RNAi group.The p S6 K is negative expression in the sham operation group,the simple injury group and the empty vector virus group,but it is positive expression in the PTEN gene silencing group.Conclusion:We successfully constructed the mi R RNAi lentiviral vector for PTEN12257-PTEN-Micro RNA,which has gene silencing efficiency.Then the lentiviral vector 12257-PTEN-micro RNA was packaged into lentiviral particles not only has a high concentration but also has a stable interference effect on the target gene in vivo and in vitro.