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Study of the Effect on RNAi to Inflammatory Injury Model of HLE-B3 Cell by Inhibiting the Expression of IKK-α and It’s Downstream Factors

Author: ZhangJing
Tutor: ZhaoGuiQiu
School: Qingdao University
Course: Ophthalmology
Keywords: Traumatic cataract Lens epithelial cells Inflammatory injury Nuclear factor-κB kinase - α RNA interference
CLC: R776.1
Type: Master's thesis
Year: 2008
Downloads: 54
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Abstract

Purpose: the people lens epithelial cells (HLE-в3) through the establishment of tumor necrosis factor-α (TNF-α) induced inflammatory damage model, the change of the observed inflammatory injury after cell trait, to research nuclear factor-kB (NF-кB) upstream suppression factor-alpha subunit of IkB kinase (IKK-α) expression changes and role in the inflammatory lesion. Use of RNA interference (RNAi) to investigate the inhibition of the expression of IKK-α-induced inflammatory injury in human lens epithelial cells the kinase IKKα subunit small interfering RNA (siRNA) transfection HLE-B3 cells and downstream factor. Methods: 1. Recombinant human TNF-α joined HLE-B3 cells in the culture medium, the final concentration of 20ng/ml were incubated 6h, 12h, 24h and 48h inflammatory injury model preparation HLE-B3 cells. Changes in cell trait observed under an inverted microscope, using immunohistochemistry and real-time PCR method, IKK-α protein and mRNA expression were detected changes. 2. Using fluorescently labeled siRNA the transfection reagent HiperFect2000 different concentration combinations were transfected HLE-B3 cells, 12h after transfection efficiency in fluorescence observed under an inverted microscope, MTT assay was used to detect the toxicity of the transfection complexes on transfection conditions optimized. Design and in vitro transcription synthesis of three IKK-αsiRNA HLE-B3 cells were transfected detection the IKK-αmRNA expression changes by real-time quantitative PCR method, filtering out the highest siRNA inhibition efficiency. The cells were collected at different times after the screening of siRNA and optimization of transfection conditions HLE-B3 cells transfected 24 h after the TNF-α-induced, and, respectively, on the induction and culture medium, the real-time quantitative PCR The detect express IKK-αmRNA, ELISA assay IKK-α protein and downstream of IL-1β in the culture medium and the content of the TGF-β. Results: 1.TNF-α stimulation HLE-B3 cell body elongation thinner cell morphology similar to fibroblasts, the cells between the the \Immunohistochemical staining showed that: you can see a small amount of protein expression of IKK-α, TNF-α stimulation 12h, IKK-α protein expression increase the amount of normal HLE-B3 cell cytoplasm and nucleus staining, stimulated 24h 48h IKK-α protein expression further increased staining from yellow to brown. Quantitative PCR results showed that: 6h IKK-αmRNA expression level was significantly higher compared with the normal control group, TNF-α stimulation 24h than 12h after stimulation IKK-α expression levels increased further, by the IKK-αmRNA stimulus 24h expression reached peak. 2 Experimental results show that: increase the amount of siRNA can improve transfection efficiency, but the amount of siRNA transfection efficiency 10nM and 25nM no significant difference. ≤ 3.0μl HiPerFect 2000 dose of the cytotoxicity of the transfection complexes is small, the cell viability more than 80%, but when the HiperFect 2000 of the dose was increased to 5.0 μl, and a significant increase in their cytotoxicity. The composition of the mixture in transfected 10nM siRNA with 3.0μl HiPerFect 2000, the transfection efficiency of up to 70.6%, and still guarantee the survival rate of 80% of the cells. Real-time PCR confirmed: design and in vitro transcription 2 enables cells the IKK-αmRNA the expression was significantly decreased after the synthetic 3 the IKK-αsiRNA transfection HLE-B3 cells, and an inhibitory effect can be achieved 82 percent. Quantitative PCR results showed that: compared with the normal group, 24h after transfection (before adding TNF-α), siRNA group significantly reduced the expression of TNF siRNA group the IKK-αmRNA the addition of TNF-α the TNF group of IKK-αmRNA increased TNF siRNA group despite an increase in the cellular level but still below normal. Double-antibody sandwich ELISA results show: IKK-α protein in the cell culture medium for each experimental group, IL-1β, TGF-B content variation the IKK-αmRNA variation consistent. Conclusion: 1. RhTNF-α stimulation is an effective method for establishing HLE-B3 cells inflammatory injury model. Inflammatory injury model of 2.HLE-B3 IKK-α activation and increased expression of the factor showed positive expression in the cytoplasm and the nucleus, IKK-α may be the key factor for trauma impaired Open inflammatory injury. After optimization of transfection conditions the transfection reagent HiperFect 2000 can siRNA efficiently transfected into HLE-B3 cells. 4. Own design and synthesis of IKK-αsiRNA transfected into cells can be effectively achieved on the corresponding endogenous RNA degradation. 5 using RNA interference (RNAi) can effectively inhibit TNF-α-induced HLE-B3 cells of IKK-α and downstream factor IL-1β upregulation of TGF-β, thereby inhibiting injury HLE-B3 cells role.

Study of the Effect on RNAi to Inflammatory Injury Model of HLE-B3 Cell by Inhibiting the Expression of IKK-α and It’s Downstream Factors

Abstract

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