Inhibitory Effect On LPS-induced Retinal Microglial Activation Of Downregulation Of T-PA Expression By SiRNA Interference

Part one:The expression of tissue plasminogen activator(t-PA) in primary cultured rat retinal microgliaThe primary cultured microglia cells were isolated from retina of S-D rats and activated with different concentration of LPS(0,3,10,30,100,300ng/ml).Double immunofluorescence(OX42 and t-PA) of microglia were performed and western blot were used to detect t-PA expression.The results showed that the isolated microglia could adhere 24 hours later,and mostly had small round cell body.As time went by,the microglia cells gradully grew short thin processes and became multi or bipolar ramified cells on the 7th day.The silent microglia had small cell body,little processes,and weak OX42 stain;the activated microglia had larger cell body,more processes,and stronger OX42 stain. Unstimulated microglia only had weak t-PA expression,with carmoisine t-PA stain; Under the stimulation of LPS,the t-PA expression became strong,and the t-PA stain appeared crimson in colour.The result of Western blot showed that all groups of microglia had a band of t-PA expression,and the gray scale increased gradually with increasing concentrations of LPS.In this part of experiment,we demonstrated a successful method of primary culture and isolation of retinal microglia.The conclusions are that microglia can be activated by LPS,and the activated microglia can express t-PA in a dose related pattern.Part two:Knockdown of the expression of t-PA of microglia by RNA interferenceAccording to literature and RNAi design software(oligoengine RNAi),we selected a highly effective RNAi sequence,and got the siDNA with chemical synthesis method. Then the siDNA was inserted into the lentivirus RNAi vector,the production was used to transfect the bacterium competent cells,then the positive clone was identified by sequencing to confirm that the preparation of the vetor was successful.The successfully prepared vector was ultrapure extracted and cotransfected the 293T cells with liposome method.The expression of EGFP was observed using a fluorescent microscope,then the virus titer of lentivirus vector was analyzed.Microglia was transfected with t-PA siRNA lentivirus or blank control lentivirus.The efficiency of transfection was determined by flow cytometry,and the efficiency of the objective gene t-PA supression was identified by Real Time PCR.The results showed that the sequence of positive clone which was analyzed by seguencing was extremely same to the objective gene and it proved that the preparation of vetor was successful.The efficiency of transfection of lentivirus to 293T cells was as high as 85%,and this ensured the success of lentivirus package. The lentivirus titer was about 5×10~8 TU/ml.The efficiency of transfection of lentivirus to microglia was 88%which was determined by flow cytometry.The results of Real Time PCR showed that the inhibitory efficiency of t-PA mRNA expression was 80%.In this part of investigation,the system of t-PA siRNA lentivirus expressive vector was successfully synthesized.The lentivirus system could transfect microglia and downregulate the expression of t-PA with a high efficacy.All of these results built a good foundation of further investigation of the role of t-PA during process of the activation of microglia.Part three:The inhibitory effect on microglia activation when t-PA expression was downregulated by RNA interferenceMicroglia cells were transfected with t-PA siRNA lentivirus(RNA interference group) or blank control lentivirus(control group)(MOI=10),two days later stimulated by 30ng/ml LPS.Then 100μl culture media was acquired every 1,3,6,12,24 hours later.The concentration of IL-1βand TNF-αin the media was assessed by Elisa.The microglia cells were harvested 24 hours later for OX42 and Iba-1 immuno-fluorescence.The results showed that OX42 and Iba-1 stain of both groups were positive,there was no difference of OX42 expression between two groups,but the Iba-1 expression of RNA interference group was downregulated markedly.The concentration of TNF-αin RNA interference group was significant lower than control group at all times,and the IL-1βconcentration was significant lower than control group at all times except the collection one hour after LPS treatment.After downregulation of t-PA expression by RNA interference,the Iba-1 expression was weakened and the secretion of IL-1βand TNF-αdecreased significantly.It hint that the activation of microglia induced by LPS is inhibited when t-PA expression is knocked down by RNA interference.