| Objective: To investigate the effect of macrophage migration inhibitory factor(MIF)on the proliferation and differentiation of human embryonic stem cells,and to explore the regulatory mechanism,to provide the basic theory for the effective application of human embryonic stem cellsMethods:(1)The growth curve of human embryonic stem cells H9 was detected and plotted by CCK-8 method;the level of autocrine MIF in normally growing human embryonic stem cells was quantified by enzyme-linked immunosorbent assay(ELISA).(2)The effects of exogenous addition of different concentrations of MIF(30,100,300ng/m L)and MIF inhibitor ISO-1(3,7,21 μM)on the proliferation of H9 were detected by CCK-8 assay;MIF knockout H9 was constructed using CRISPR-Cas9 technology,and the knockout efficiency of MIF was detected by q PCR,and the proliferation of human embryonic stem cells after knockout of MIF was detected by CCK-8.The proliferation of human embryonic stem cells after the knockout of MIF was examined by CCK-8.(3)After exogenous addition of MIF(100ng/m L)and H9 were co-cultured for 48 h,the m RNA of self-renewal factor KLF4,c-MYC,NANOG,OCT4,SOX2 and the protein expression levels were detected by quantitative real-time fluorescence polymerase chain reaction(q PCR),immunocytochemical fluorescence(ICF)and protein blotting(Western blot),respectively.After the exogenous addition of MIF(100ng/m L)and H9 co-culture for 48 h,the m RNA and protein expression levels of neural differentiation factors FOXA2 and OTX2 were detected by q PCR,ICF and Western blot,respectively.(4)After exogenous addition of MIF(100ng/m L)and H9co-culture for 48 h,the m RNA and protein expression levels of CXCR4 were detected by q PCR and Western blot,respectively;the effects of exogenous addition of CXCR4 neutralizing antibodies at different concentrations(15,30,60ug/m L)on H9 proliferation were detected by CCK-8 method;the exogenous addition of MIF(100ng/m L)and H9 co-culture were detected by q PCR and Western blot,respectively.MIF(100ng/m L)and CXCR4(100ng/m L)were added exogenously,and the m RNA and protein expression levels of KLF4 and FOXA2 were detected by q PCR and Western blot respectively.(5)After exogenous addition of MIF(100ng/m L)and H9 were co-cultured for 48 h,the m RNA and protein expression levels of CXCR4 and β-arrestin1 were detected by q PCR and Western blot,respectively;MIF(100ng/m L)and chloroquine(10μM)were added to H9,and the protein expression levels of CXCR4 and β-arrestin1 were detected by Western blot to detect CXCR4 protein expression levels.Results:(1)The logarithmic growth period of human embryonic stem cell H9 is 3-6days,and the autocrine MIF concentration is about 20ng/m L under normal growth.(2)Exogenous addition of MIF did not affect the proliferation of H9(P>0.05)and ISO-1inhibited the proliferation of H9(P<0.05).Although the knockout of MIF in H9 was not successful,after about 50% knockout of MIF by q PCR,the proliferation of H9 after knockout was significantly inhibited by CCK-8 assay and the cells were not viable(P<0.05).(3)q PCR,ICF and Western blot showed that the m RNA and protein expression levels of KLF4 were significantly increased compared with Control group(P<0.01),while the m RNA and protein expression levels of c-MYC,NANOG,OCT4 and SOX2 were not significantly different(P> 0.05).After the exogenous addition of MIF and H9 co-culture for 48 h,q PCR,ICF and Western blot results demonstrated that the m RNA and protein expression levels of FOXA2 were significantly up-regulated compared with the Control group(P<0.01),while the m RNA and protein expression levels of OTX2 were not significantly different(P>0.05).(4)The m RNA,as well as protein expression levels of CXCR4,decreased after the addition of MIF and H9 co-culture(P<0.01);different concentrations of CXCR4 neutralizing antibodies inhibited the proliferation of H9(P<0.001);compared with the Control group,the m RNA as well as protein expression levels of KLF4 decreased in the MIF group(P<0.01),while the CXCR4 group m RNA and protein expression levels of KLF4 increased in the CXCR4 group compared with the Control group(P<0.001).Compared with the Control group,the m RNA and protein expression levels of FOXA2 increased in the MIF group(P<0.001),while the m RNA and protein expression levels of KLF4 decreased in the CXCR4 group(P<0.05).(5)The m RNA,as well as protein expression of CXCR4 and β-arrestin1,decreased after the addition of MIF and H9 coculture(P<0.01).Compared with Control group,CXCR4 protein expression decreased after adding MIF(P<0.05);CXCR4 protein expression increased after adding chloroquine(P<0.05);no significant difference was found after adding MIF and chloroquine(P>0.05).Conclusions:(1)The autocrine MIF concentration of human embryonic stem cells is about 20 ng/m L,and the physiological concentration of MIF promotes the proliferation of human embryonic stem cells and is essential for their survival.(2)Higher than physiological concentrations of MIF can inhibit the self-renewal of human embryonic stem cells and promote differentiation.(3)MIF regulates signaling to human embryonic stem cells through β-arrestin1-mediated CXCR4 internalization... |
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