Effects Of Silencing Fibulin-5 On Proliferation And Apoptosis Of IgG4-ROD Lacrimal Gland Fibroblasts

Objective:To take the lacrimal gland of IgG4 related orbital disease（IgG4-ROD）confirmed by pathology as the research object,take the primary fibroblast culture and apply RNAi（RNA RNAi）silenced the Fibulin-5 gene,down regulated the mRNA and protein expression of the target gene Fibulin-5,observed the apoptosis of fibroblasts,and explored the method of inhibiting lacrimal gland fibrosis.Methods:From October 2018 to August 2019,six patients who had been hospitalized in our department and met the diagnostic criteria of IgG4-ROD were selected.The pathological lacrimal glands with partial or complete fibrosis were taken,and 0.2%type II collagenase method was used for primary culture of lacrimal gland fibroblasts.In accordance with the principle of small interference RNA（siRNA）design,the high-efficiency and specific Fibulin-5 siRNA（l1-3）was designed by Shanghai Youning biologics Co.,Ltd.according to the sequence characteristics of human Fibulin-5 gene.Three siRNAs were transfected into the fourth generation of IgG4-ROD lacrimal gland fibroblasts.Six groups were set up.Group A was blank,group B only added Lipofectamine 2000,group C was negative siRNA,group D was fibulin5-sirna-1,group E was fibulin5-sirna-2,group F was fibulin5-sirna-3.The expression of Fibulin-5 mRNA in each group was analyzed to select the best siRNA.The Fibulin-5 siRNA plasmid vector was constructed by Shanghai Youning life support Co.,Ltd.and the obtained siRNA plasmid was set at four concentrations（0,25,50 and 75 nm）.After different concentrations of siRNA were transfected into fibroblasts,CCK-8method was used to measure the inhibition rate and the best concentration of the inhibition effect was selected as the siRNA concentration for subsequent experiments.The fibroblasts of the fourth lacrimal gland were divided into four groups.Three groups of control group（blank control,empty body control,negative siRNA control）and siRNA transfection group were set up.After 72 hours,the cells were collected and Fibulin-5 mRNA was extracted from the cells.The difference of relative expression of Fibulin-5mRNA in each group was analyzed to verify the efficiency of cell silencing The expression of Fibulin-5was detected by blotting to verify the down-regulation of the target protein.Flow cytometry was used to detect the apoptosis rate at 24 h,48 h and 72 h after transfection.Results:1.Cell identification:the lacrimal gland of human igg4-rod can be cultured successfully under0.2%collagenase II method,which is proved to be fibroblast by immunofluorescence.2.Optimal siRNA screening:the relative expression of Fibulin-5 mRNA in each group of cells was expressed by 2^-△CT value.It was found that the sequence numbered Fibulin-5 siRNA-1 in the three siRNA sequences was the optimal siRNA,which had the highest efficiency of silencing Fibulin-5 gene in human lacrimal gland fibroblasts and could effectively inhibit the expression of Fibulin-5 gene.3.Cell proliferation activity:take Fibulin-5siRNA concentration of 0 nm as the blank control,24 hours after transfection,there was no difference in cell proliferation activity between each group（P>0.05）;in Fibulin-5 48h and 72h after siRNA transfection,the proliferation rate of fibroblasts in the blank control group was significantly higher than that in the groups with siRNA concentration of 25nm,50nm and 75Nm（P<0.05）,and with the increase of siRNA concentration,the proliferation rate was lower;the inhibition effect of 75Nm siRNA was the most obvious,and the cell proliferation was significantly inhibited.4.Fibulin-5 mRNA detection:there was no significant difference in the relative expression of Fibulin-5 mRNA between the three control groups（P>0.05）;however,the relative expression of Fibulin-5 mRNA in the Fibulin-5 siRNA transfected group was lower than that in the three control groups,and the expression of the target gene was significantly decreased.5.Fibulin-5 protein detection:there was no significant difference in Fibulin-5 protein expression among the three control groups（P>0.05）,but the expression of Fibulin-5 protein in the Fibulin-5 siRNA transfected group was also lower than that in the three control groups,and the expression of matrix protein Fibulin-5was significantly decreased.6.6.Apoptosis rate:24 hours,48 hours or 72 hours after transfection,there was no significant difference between the apoptosis rates of group I,II and III.The apoptosis rate of Fibulin-5siRNA transfection group was significantly higher than that of group I,II and III（P<0.05）.Conclusion:1.IgG4-ROD lacrimal gland cells were identified as fibroblasts after primary culture.2.The specific Fibulin-5 siRNA was screened out,and the Fibulin-5 siRNA recombinant plasmid was successfully constructed.At the concentration of 75 nm,it can effectively inhibit the proliferation of IgG4-ROD lacrimal gland fibroblasts.3.After transfection of IgG4-ROD lacrimal gland fibroblasts with Fibulin-5siRNA recombinant plasmid,the expression of target gene Fibulin-5 mRNA and protein can be significantly down regulated,and apoptosis can be promoted.It is speculated that Fibulin-5 can be used as the target,and RNAi technology can effectively inhibit the fibrosis of lacrimal gland tissue.