ABSTRACT
AIM: To investigate the expression of connective tissue growth factor (CTGF) and α-SMA in human lens epithelium cell (HLEC) line B3 after transfection by liposome-coated siRNA targeting CTGF.METHODS: HLECs were transfected with small interfering RNA (siRNA) targeting CTGF,labeled with 5`-fluorescein isothiocyanate (5`-FITC) and coated with lipofectamine.The transfection ratio was evaluated via fluorescence intensity.Cell counting kit-8 (CCK-8) assay was performed to assess cytoviability of both non-transfected and transfected HLECs.Quantitative RT-PCR,cell immunochemistry and Western blot analysis were conducted to detect the expression changes of CTGF and α-SMA after transfection.RESULTS: A highly effective transfection ratio was observed in siRNA co-transfected with lipofectamine.The transfection ratio reached 95% at 24h.The proliferation of HLECs was inhibited by siRNA after 72h transfection.The expression of CTGF and α-SMA significantly decreased in HLECs after transfected by CTGF siRNA for 24h.This effect was not found in negative control siRNA.CONCLUSIONS: SiRNA targeting CTGF decreased CTGF and α-SMA expression in HLECs,which is a potential therapeutic strategy for posterior capsular opacification.
ABSTRACT
Background In the past few decades,the balance of Th1/Th2 is often used to explain the immune mechanisms of fungal infection and fungal disease.More recently,a novel subset of CD4+ effector Th cells has been found to participate in anti-fungal infection response.However,whether Th17 is involved in the immune response in fungal keratitis is unclear up to now.Objective Present study was to investigate the expression change of Th17 type cytokine and its specific transcription factor,retinoid-related orphan nuclear receptor gamma t (RORγt),in the cornea of Fusarium solani keratitis.Methods Ninety-six clean BALB/c mice were divided into Fusarium solani keratitis model group and control group by randomized digital table.Fusarium solani keratitis models were established by epikeratophakia-assisted corneal epithelial erasion and interlayerly injection of 5 μl (1 × 106 CFU/ml) Fusarium solani solution in the right eyes,and the equal volume of PBS was injected in the same way in the control group.10％ KOH wet film was used to examine the fungal hyphea and funga strain was identified by inoculation.The corneas were examined under the slit lamp microscope 1 day,3,5,7 days after modeling and the inflammatory response was scored based on the criteria of Wu and Hu.The histopathological examination of corneas was performed in the time points above.Real time fluorescence quantitative PCR was used to detect the expression levels of interleukin-17 (IL-17) mRNA and RORγt mRNA in the corneas.The expression of IL-17 protein in the corneas was detected by ELISA.The use and raise of the mice followed the Statement of Association for Research in Vision and Ophthalmology.Results The inflammatory scores were 3.2±0.8,6.6± 1.1,9.4± 1.1 and 6.8±0.8 in 1 day,3,5,7 days after modeling,showing a significant difference among them (F =89.786,P =0.010).The inflammatory scores were higher in the third and seventh day than that in the first day (P＜0.05),but they were significantly lower than that in the fifth day (P＜0.05).The infiltration of inflammatory cells showed a coincident tendency with the score.The expressing levels of IL-17 mRNA (2-ΔΔCt) in the corneas were 4.12±0.73,20.72±1.81 and 14.16±1.88 in 3,5,7 days after modeling,with statistically significant differences in comparison with those in the control group (P＜0.01),and the expression level was significantly higher in the fifth day than those in the first,third and seventh day in the model group(P＜0.01).The expression levels of IL-17 protein (ng/g) were significantly increased 1 day,3,5,7 days in the model group compared with the control group (P＜0.01).A similar change was found in the expression of RORγt mRNA to that of IL-17 mRNA.Conclusions Expressions of IL-17 and its transcription factor RORγt upregulate in the fungal keratitis and has an association with inflammatory degree,which suggests that Th17 subset may play an important role in the immune responses of fungal keratitis.