白细胞介素32对人脐带来源间充质干细胞向脂肪细胞分化的调控作用OA北大核心CSTPCD

OBJEACTIVE To investigate the effect of interleukin-32(IL-32)on adipogenic differenti-ation of human umbilical cord-derived mesenchymal stem cells(HuMSCs).METHODS ① HuMSCs were activated by treatment with tumor necrosis factor-α(TNF-α)(20 μg·L-1)and interferon-γ(IFN-γ)(20 μg·L-1)for 24 h to obtain stimulated-HuMSCs(S-HuMSCs).The surface markers CD14,CD34,CD45,CD73,CD90 and CD105 of HuMSCs and S-HuMSCs were detected by flow cytometry to identify their phenotypes.After ten days of adipogenic induction,oil red O staining was performed to detect lipid droplets in HuMSCs and S-HuMSCs.Real-time quantitative PCR(RT-qPCR)was used to detect the mRNA expressions of adipogenic transcription factors peroxisome proliferator activated receptor γ(PPARγ),CCAAT enhancer binding protein α(C/EBPα)and adiponectin(ADI)to determine their adipo-genic differentiation ability.After 14 d of osteogenic induction,alkaline phosphatase(ALP)staining was performed to detect the ALP staining area.RT-qPCR was used to detect the mRNA expressions of osteo-genic related transcription factors RUNX family transcription factor 2(RUNX2),alkaline phosphatase(ALP)and distal-less homeobox 5(DLX5)to determine the osteogenic differentiation ability.②The len-tiviral vector containing the over-expressed IL-32 gene sequence with green fluorescent protein(GFP)and puromycin resistance gene and the negative control(NC)empty vector were constructed and harvested.HuMSCs were infected with these viruses to obtain IL-32highHuMSCs and NC-HuMSCs,respectively.HuMSCs,NC-HuMSCs and IL-32highHuMSCs were induced to differentiate into adipocytes.Oil red O staining was used to detect the area of lipid droplet formation in cells at 0,3,5,7,10 and 14 days after induction,and RT-qPCR was used to detect the expressions of transcription factors PPARγ,C/EBPα and ADI mRNA related to adipogenic differentiation.RESULTS ① The results of flow cytometry showed that HuMSCs and S-HuMSCs had high expressions of CD73,CD90 and CD105,low or no expressions of CD14,CD34 and CD45,and both phenotypes were consistent with the biological characteristics of MSCs.After adipogenic and osteogenic differentiation of HuMSCs and S-HuMSCs,compared with the self-differentiated group,the area of ALP staining and oil red O staining in the induced group increased(P＜0.01).The mRNA expressions of osteogenic differentiation related transcription factors RUNX2,ALP and DLX5 and adipogenic differentiation related transcription factors PPARγ,C/EBPα and ADI were also significantly increased(P＜0.01),indicating that both of them had the characteristics of MSCs.Compared with the HuMSCs induction group,the area of lipid droplet formation in the S-HuMSCs induction group was significantly reduced(P＜0.01),the mRNA expressions of adipogenic differentia-tion-related transcription factors PPARγ,C/EBPα and ADI were also significantly decreased(P＜0.05),but S-HuMSCs highly expressed IL-32(P＜0.01).②After adipogenic differentiation of HuMSCs,NC-HuMSCs and IL-32highHuMSCs in vitro,oil red O staining was performed at 0,3,5,7,10 and 14 days.From the 3rd day,the area of lipid droplet formation in the IL-32highHuMSCs induction group was significantly smaller than that in the HuMSCs and NC-HuMSCs induction groups(P＜0.01),and the mRNA expressions of adipogenic transcription factors PPARγ,C/EBPα and ADI were also significantly decreased(P＜0.05).CONCLUSION Overexpression of IL-32 can significantly reduce the adipogenic differentiation ability of HuMSCs.