Study Of The Effect On RNAi To Inflammatory Injury Model Of HLE-B3 Cell By Inhibiting The Expression Of NF-κB And It's Downstream Factors

Objective To observe the morphological changes of HLE-B3 cell, and to study the expression and function of NF-κB in the inflammatory injury model. Using RNAi technology, to investigate the effect of siRNA(targeting at human NF-κB P65) in inhibiting the expression of NF-κB and it's downstream factors induced by rhTNF-α.Methods 1. rhTNF-αwas added into the culture medium of HLE-B3 cell with the final concentration of 20ng/ml, and cultured for 6h, 24h and 48h to establish the inflammatory injury model. The morphological changes of HLE-B3 cells were observed with inverted microscope, the expression of NF-κB P65 protein and mRNA were detected respectively by immunohistochemical staining and real-time PCR. 2. Transfection mixtures of different amount of fluorescently-labeled siRNA with different concentration of HiperFect were used to transfect HLE-B3 cells, 12 hours later, the transfection efficiency and cytotoxicity of transfection mixtures were detected to optimize the transfection condition. 3. Three siRNAs directed against human NF-κB P65 were designed and synthesized, then were used to transfect HLE-B3 cells respectively.24 hours later,the siRNA with highest inhibiting effect was filtrated by real-time PCR. 4. HLE-B3 cells were transfected with filtrated siRNA and optimized transfection condition, 24 hours later rhTNF-αwas added. NF-κB mRNA was detected by real-time PCR, IL-1βand TGF-βwere detected by ELISA at different time.Results 1. After the stimulation of TNF-α, the bodies of HLE-B3 cells become longer and slimmer, look like fibroblast and the netting phenomenon disappear. NF-κB was detected in normal cultured HLE-B3 cells, ,the expression of NF-κB significantly increased compared with normal control group, at 6h after stimulation of TNF-α, and came to the summit at 24h. 2. The transfection efficiency was elevated with the increasing of siRNA, but there were no difference between 10nM and 25nM.When the amount of HiperFect was no more than 3.0ul,the transfection mixture was of little cytotoxicity. When the amount of HiperFect was up to 4.5ul, the cytotoxicity was siginificantly increased. 3. Among the three designed and synthesized siRNAs, two siRNAs could obviously decrease the expression of NF-κB, and one of them had an inhibiting rate of 82%. 4. In siRNA group and TNF+siRNA group, the expression of NF-κB was decreased in HLE-B3 cells at 24h after tranfection (i.e before stimulation) than that in normal control group. In TNF group, the expression of NF-κB was increased at 6h,24h,48h and 72h after stimulation of TNF-αthan that in control group. In TNF+siRNA, the expression of NF-κB was increased after stimulation but still lower than that in control group. The changes of IL-1βand TGF-βparalleled to the changes of NF-κB.Conclusion 1. The morphological changes of HLE cells in the inflammatory injury model induced by TNF-αis similar to that of LECs in the animal model of traumatic cataract, confirms the function of inflammatory injury in the the mechanism of traumatic cataract. 2. In inflammatory injury model of HLE-B3 cells, NF-κB was activated and up-regulated. NF-κB may be the key factor to start the inflammatory injury in the mechanisrfi of traumatic cataract. 3. By optimizing the parameter, HiperFect transfection reagent could transfect siRNA into HLE-B3 cells with high efficiency. 4. siRNA directed against human NF-κB P65 designed by ourselves can effectively degradate corresponding endogenous RNA. 5. Using RNAi technology can inhibit the expression of NF-κB and it's downstream factors IL-1βand TGF-βin HLE-B3 cells induced by TNF-α.