The Mechanism Of MSCs Against Interstitial Fibrosis Induced TGF-?1 In NRK-49F And Its Correlation With Galectin-3 Gene

Objective:To observe the effects of self-made Bone marrow-derived mesenchymal stem cells conditioned medium(MSCS-CM)on NRK-49 F fibrosis induced by Transforming Growth Factor-?1(TGF-?1)in renal interstitial fibroblasts of rats,and to preliminarily explore the mechanism of the effect and the correlation with Galectin-3(Gal-3)gene expression.Methods:1.Establishment of rat NKR-49 F cell lines with different expression levels of Gal-3: NKR-49 F cell lines of rat renal interstitial fibroblasts were purchased and passed,and the lentiviral vector was used for detection and validation by Western Blot.At the same time,two types of NKR-49 F cell lines with Gal-3 knockdown and overexpression were successfully established for later use.2.Primary isolation and culture of rat MSCs and phenotype identification:First,young male SD rats were taken and the bone marrow of both femur and tibia was flushed out with normal saline under aseptic conditions,the rinsing solution was collected and centrifuged,and the supernatant was discarded.Secondly,the precipitate was resuspended with MSCS-specific medium and inoculated in T25 culture flask.Finally,according to the ratio of 1:3 in vitro,flow cytometric phenotype identification was performed at the P3 generation.The positive index was CD29,CD90,and the negative index was CD34,CD45.3.Collection of MSCS conditioned medium(MSCS-CM):When the primary MSCS cells were passed in vitro to P3 generation,the special medium without serum was used for culture.After 48 h,the supernatant was collected,the cells were removed by centrifugation,and MSCS-CM was obtained and stored in-80 refrigerator by small portion partitioning.4.Grouting and treatment of cell experiment: The cell experiment was divided into normal control group(CON),TGF-?1 model group(TGF-?1),basal medium control group(TGF-?1+MEM),MSCS-CM intervention group(TGF-?1+ MSCS-CM),and simple MSCS-CM group.TGF-?1 model group was treated with MEM complete medium containing 5 % FBS containing recombinant TGF-?1(10 ng/mL)from human source for 24 h to induce fibrosis model.MSCS-CMS intervention group treated cells with TGF-?1 for 24 h and then treated with MSCS-CM for 24 h.The basic medium control group was treated with serumfree MEM basic medium for 24 h after fibrosis induction,and the MSCS-CM group was directly treated with MSCS-CM for 24 h.5.Cell fibrosis markers and detection methods: First,after the cells of each group were collected,proteins were extracted,and the expression levels of myofibroblast marker ?-smooth muscle actin(?-SMA),the biomarker protein Galectin-3 and extracellular matrix marker protein component in early fibrosis were detected by Western Blot.Secondly,ELISA method was used to detect the level of Gal-3 in supernatant of each group.6.Detection of key protein expression in TGF-?1/Smad signaling pathway:Western Blot was used to detect the expression levels of TGF-?1 p-Smad2/3 and Smad2/3 proteins in this pathway.7.Cytokine-chip detection: Cell supernatant of each group was taken,and AAR-BLG-90(8)cytokine chip was used to detect the changes of cytokine lineage in each group.Results:1.MSCs showed fibrous swirling growth with short spindle formation.Flow cytometric phenotypic analysis showed that CD29 and CD90 were both expressed at over 95%,Osteogenesis and adipogenesis induced differentiation showed typical mineralized nodules and a large number of red lipid droplets of different sizes under the special induction medium.2.NRK-49 F cells treated with 10 ng/mL TGF-?1 for 24 h could induce ?-SMA,Gal-3,Fn,TGF-?1,and the ratio of p-Smad2/3 to Smad2/3 in NRK-49 F cells increased significantly compared with the normal group(p<0.05).After24 h treatment with serum-free MSCS-CM,the intracellular ?-SMA,Gal-3,FN,TGF-?1 and the ratio of p-Smad2/3 to Smad2/3 were significantly decreased compared with model group(p<0.05).The change of Gal-3 level in supernatant was basically consistent with the change of protein expression in cytoplasm.3.There was no significant difference between the MSCS-CM intervention group and the MSCS-CM intervention group alone.Cytokine spectrum analysis showed that TGF-?1 treatment could up-regulate interleukin family members such as IL-1?,IL-1?,IL-2,IL-3,IL-5,IL-10,IL-13,tumor necrosis factor and receptor superfamily TNF-?,FADD,chemokines such as CD106,CINC-2? /?,CINC-3,CXCR4,and transforming growth factors such as TGF-?1,TGF-?3,interferon IFN-?,matrix metalloproteinase MMP-2,MMP-8,MMP-13,as well as tissue inhibitors TIMP-2 TIMP-3 and Integrin?M,?2 of MMPs.After treatment with MSCS-CM,their expression was down-regulated to a certain extent due to insufficient sample size,which could not meet the statistical requirements.4.TGF-?1 significantly increased ?-SMA and FN protein expression in NRK-49 F cells after both knockdown and overexpression of Gal-3 compared with the control group(p<0.05).The expression of ?-SMA was decreased after24 h intervention with MSCS-CM(p<0.05).and showed no significant correlation with the expression of Gal-3 gene.Compared with the overexpression of Gal-3 cells,knockdown Gal-3 cells could promote self-repair more when the intervention of MEM in basal medium.Conclusions:1.MSCS-CM can effectively reduce the fibrosis of rat NRK-49 F cells induced by TGF-?1,and the mechanism is related to the inhibition of TGF-?1/Smad signaling pathway,the massive secretion of cytokines,and the maintenance of early self-repair ability to delay fibrosis.2.MSCS-CM can produce anti-fibrosis effect on NCR-49 F cells,whether knockdown or overexpression of Gal-3,and there is no obvious correlation with Gal-3 gene.