The Role Of Lentiviral Sh-RNA Interferenc The Expression Of PGC-1α In DR

Objective:In order to further understand the fuction of PGC-1 alpha in the development of Diabetes Mellitus(DM), provide the certain basis for occurrence, development and treatment of DR. This experiment were designed to found the role of PGC-1 alpha in diabetic retinopathy,and the relationship of PGC-1 alpha and VEGF and EGR-1 in diabetic retinopathy in vivo and in vitro.Methods:Determination of PGC-1 alpha si-RNA sequence: sense, 5’-AAGACGGAUUGCCCUC AUUUGtt-3’; antisense,5’-CAAAUGAGGGCAAUCCGUCUUtt-3’. Synthesis of PGC-1 alpha sequence of single stranded DNA oligo, and then annealing pairing produces double chain, and then connecting directly into the RNAi lentiviral vectors which were Restriction enzyme digested through restriction sites at both ends of the double chain; connecting the product into the prepared bacterial competent cells. After PCR identification of positive recombinants. sequencing, and the results comfirmed by the comparison to be correct cloning are successful construction of PGC-1 alpha RNAi lentivirus vectors.AGEs(1g/L) cultured endothelial cells and RPE cells in good condition for 4days.Then infected lentivirus packaging PGC-1 alpha Sh RNA. After infection, cells were Divided into two groups: normal culture medium and AGEs(1g/L) continued training. Specific groups are as follows: A. common medium training; B. AGEs culture(1g/L); C. AGEs werecultured for 4 days, PGC-1α RNA interference: 1. replace the normal culture medium; 2. sustained AGEs(1g/L) culture. Test the rates of infection by lentivirus of endothelial cells and RPE cells. Detect the expression of PGC-1α and VEGF and EGR-1 in each group by immunocytochemistry. Average optical density detection, and statistical analysis. Extracting total RNA from each group, RT-PCR was used to detect the content of PGC-1 alpha and VEGF and EGR-1, induction and statistical analysis of data.Intraperitoneal injection of STZ to built diabetic rat model. Impose on rats with DM Intravitreal injection of Lentiviral packaging PGC-1 α sh RNA. Specific groups are as follows: A. Normal group B. Diabetes group(1. diabetes 4weeks 2. diabetic 8weeks) C. RNAi group(1. Intravitreal injection of PGC-1α interference RNA at DM 4 weeks, sample after 10 days. 2. Intravitreal injection of PGC-1α interference RNA at DM 8 weeks, sample after 10 days.) Detect transfection of rat vitreous: Intravitreal injection a week later, make ocular frozen sections. Observe transfection of Lentiviral sh-RNA into rat’s retina under a fluorescence microscope. Detect the expression of PGC-1α and VEGF and EGR-1 in retina of each group by immunohistochemistry. Average optical density detection, and statistical analysis. Extracting total RNA from each group, RT-PCR was used to detect the content of PGC-1 alpha and VEGF and EGR-1, induction and statistical analysis of data.Results:Part one: The sequencing results completely correct sequence alignments, Showing that the preparation of sh-RNA Lentiviral targeting pgc-1α is successful. Preparation of RNA interference showed success of PGC-1α lentivirus. Two days after lentivirus Sh RNA Targeting PGC-1α transfect into 293 T cells, observe fluorescent and visible-light pictures of the same vision. we can see Lentiviral transfection rate is over 95%.Part two: when MOI = 100 polybreen(+), endothelial cells and RPE cells infected with good effect. Infection rate is 80%-85%. Immunocytochemistry and real-time PCR assay showed that endothelial cells and RPE cells cultured in AGEs(1g/L), PGC-1α, VEGF and EGR-1 in protein and the m RNA level increased, differences between groups have statistics meaning(p<0.05). after interferencing by lentiviral Sh RNA PGC-1α then training by common culture medium, we can observe that PGC-1α, VEGF and EGR-1 levels decreased compared AGEs training(p <0.05), sustained AGEs(1g/ L) culturing after infection makesthe expression of PGC-1α, EGR-1 and VEGF was elevated compared with the replacement of common culture group, but still lower than the AGEs culture group(p <0.05).Part three: retinas showe high fluorescence under a fluorescence microscope after lentiviral Sh RNA intravitreal injection, which indicates transfection rates are high. Immunohistochemical tests and Real-time PCR showed that the PGC-1α, VEGF and EGR-1 of diabetic group increase compared with the normal group in the protein and m RNA levels, differences between groups have statistics meaning(p<0.05). PGC-1α of RNAi group decreased compared with diabetic group(p<0.05), VEGF and EGR-1 didn’t have significant changes after intravitreal injection of lentivirus Sh RNA compared with the diabetic group.Conclusions:1. A small amount of PGC-1α、EGR 1-and VEGF express in endothelial cells and RPE cells. AGEs(1g/L) can increase PGC-1α, VEGF and EGR-1 in protein and the m RNA level in RPE cells and endothelial cells.2. After transfection by PGC-1α Sh-RNA lentivirus, PGC-1α decrease to normal level in protein and m RNA level in cells intervened by AGEs. While EGR-1 and VEGF chang in the same way. In summary PGC-1α has a positive correlation between VEGF and EGR-1.3. If the endothelial cells and RPE cells were still in the AGEs environment, even after PGC-1 alpha lentiviral Sh-RNA interference, the expressions of PGC-1 alpha, VEGF and EGR-1 would be lower than the AGEs training group, but still unable to return to normal levels. We infer that this phenomenon is caused by which induced by AGEs.4. The expression of PGC-1α, VEGF and EGR-1 was significantly upregulated in DM rats’ retina. And the expression of PGC-1 and VEGF increased with the progression of DM.5. After RNA interference, PGC-1α in DM rats’ retina decreased compared with the same period of DM. But EGR-1 and VEGF had no significant changes compared with the same period of DM. So it’s infered that the environment in vivo is complicated, multiple signal pathways and "metabolic memory" were induced by DM, with the result that EGR-1 and VEGF can not back to normal level.