The Effect Of Expression Of The Collagen Type â…  And â…¢after RNA Interference Of CXCL11/CXCR3Related With Silicosis Fibrosis In Human Embryo Lung Fibroblast

Objectives In this experiment, CXCL11-SiRNA and CXCR3-SiRNA interferenceplasmids were transfected into human embryonic lung fibroblasts (MRC-5) to makeCXCL11/CXCR3which were related with silicosis fibrosis keep silence. Use alveolarlavage from silicosis patients to stimulate MRC-5, then observe the expression of thecollagen type I and III and explore the function of CXCL11/CXCR3in silicosis fibrosis.Methods1Centrifuge and collect pulmonary macrophages (AM) from alveolar lavagefrom silicosis patients. Observe AM form and do HE, Antacid, Wright’s staining toidentify AM. Culture AM with non-serum medium containing SiO2for18h, thencollected AM supernatant stimulated by SiO2,filtered and saved.2Build ShRNAexpression plasmids: query for the serial number of the gene CXCL11and CXCR3onPUBMED, build the interference plasmids and the corresponding negative plasmid byGemma Company in Shanghai. The plasmids were carried by pGPU6/GFP/Neo–shGAPDH.3CXCL11-SiRNA and CXCR3-SiRNA were transfected into MRC-5. Laserscanning confocal microscope was used to observe the transfection efficiency. Extract thetotal mRNA and protein after the transfection of36~48h and the best interferencesequence was screened using RT-PCR and Western-blot method.4The experiment wasdivided into four groups. They were control group (C), in which the10%fetal bovineserum MEM medium was added; Stimulation group (S), in which10%fetal bovineserum MEM medium and AM supernatant were added; Transfection group (T), in which10%fetal bovine serum MEM medium and AM supernatant were added after transfectedFB with the best interference plasmid; Negative plasmid transfection group (N), in which10%fetal bovine serum MEM medium and AM supernatant were added after transfectedFB with the negative plasmid.5Use immunocytochemistry method and Western-blotmethod to detect the expression of collagen type I and III: test the expression of collagentype I and III in each group respectively after stimulation of12h,24h,36h,48h and72h.Results1The plasmids were transfected into the human embryo lung fibroblasts. Theexpressions of green fluorescent protein were scanned by laser scanning confocalmicroscope after36~48h. The transfection efficiency was the highest when plasmids:LipofectamineTM2000liposomes=3μg:3μl and the transfection efficiency can reach more than90%.2Extract the total mRNA and protein to screen the best interferencesequence by PT-PCR and Western blot methods: The best silent plasmid of CXCL11ispGPU6/GFP/Neo-CXCL11-homo-352and the best silent plasmid of CXCR3ispGPU6/GFP/Neo-CXCR3-203; The difference between the Non-Transfection group andthe Negative plasmid transfection group is not statistically significant.3The results of IImmunocytochemistry and Western-blot methods have shown: The Control groupexpresses a small amount of collagen type I and III; The Stimulation group comparedwith the Control group in the same time point: the result showed that the expression ofcollagen type I and III downregulated (P＜0.05); The Transfection group compared withthe Control group in the same time point: the result showed that the expression ofcollagen type I and III downregulated (P＜0.05); But the Transfection group comparedwith the Stimulation group in the same time point: the result showed that the expressionof collagen type I and III upregulated (P＜0.05); The difference of the expression ofcollagen type I and III between the Negative plasmid transfection group and theStimulation group is not statistically significant (P＞0.05).Conclusions1CXCL11-SiRNA and CXCR3-SiRNA were transfected into humanembryo lung fibroblasts respectively through technique called RNA interference, whichcan effectively inhibit the expression of CXCL11and CXCR3.2The expression ofcollagen type I and III reduced in human embryo lung fibroblasts which were stimulatedby AM supernatant after the genes of CXCL11and CXCR3which were upregulated ofsilicosis fibrosis kept silence.