| BackgroundMesenchymal stem cells(MSCs)have unique low immunogenicity and immunomodulatory effects.Adipose-derived mesenchymal stem cells(ASCs)have the advantages of convenient material acquisition and abundant sources,and have become a research hotspot in the field of MSCs.Studies have shown that bone marrow mesenchymal stem cells(BMSCs)express high levels of intercellular adhesion molecule-1(ICAM-1)and vascular intercellular adhesion molecule-1(VCAM-1)in the inflammatory microenvironment,when blocking these adhesion molecules or causing these adhesion molecules could not be expressed,the immunosuppressive capacity of BMSCs was significantly decreased.However,ASCs only highly expressed ICAM-1.Whether ICAM-1 plays an important role in regulating the biological characteristics and immunosuppressive function of human ASCs has not been reported in the literature.In addition,cytokines such as IFN-?,TNF-?,and IL-1? are indispensable "authorized"factors for BMSCs exerting immunosuppressive activity,and they all can enhance the immunosuppressive capacity of BMSCs.The effect of ICAM-1 on the immunosuppressive activity of ASCs remains to be explored.The influence of ICAM-1 on the biological characteristics and immunosuppressive function of ASCs will provide a new theoretical basis for further understanding of the immunoregulatory properties and clinical application of ASCs.Objectives1.To investigate the effects of ICAM-1 on the proliferation,apoptosis,cycle,migration,and adipogenic,osteogenic and chondrogenic differentiation of ASCs.2.To investigate the role of ICAM-1 in the immune regulation of ASCs.3.To investigate the effect and mechanism of TNF-a on the expression of ICAM-1 in ASCs.4.To investigate the effects of cryopreservation on the biological characteristics and immune regulation of ASCs.Methods1.Effect of ICAM-1 on the biological characteristics of ASCsASCs were obtained from adipose tissue by collagenase digestion and observed under an microscope.Adipogenic,osteogenic and chondrogenic differentiation were induced.Surface markers were detected by flow cytometry.Constructed knockdown and overexpression plasmids of ICAM-1,knockdown and overexpression of ICAM-1 in ASCs by lentivirus infection;CCK-8 assay detected the proliferation of ASCs,and Muse cell state analyzer detected apoptosis and cell cycle;Changes in cell migration were determined by the scrap line method method and the Transwell method.Oil red O,alizarin red,and alcian blue were used to detect the adipogenic,osteogenic,and chondrogenic differentiation of ASCs.2.The role of ICAM-1 in the immune regulation of ASCsPBMC were extracted and stained with CFSE.ASCs and PBMC were inoculated at a ratio of 1:10,1:20,1:40,1:80,1:160 respectively.The effects of ASCs on the proliferation of PBMCs were observed under a microscope,and flow cytometry was used to detect PBMC proliferation.ASCs and PBMC were inoculated in a ratio of 1:10 with contact co-culture and Transwell isolation nocontact co-culture,and the effects of ASCs on the proliferation of PBMC were observed under microscope,and the effects of ASCs on the proliferation of PBMC were examined by flow cytometry.Flow cytometry was used to detect the proliferation of PBMC in shICAM-1 group,shScramble group,ICAM-1-OV group and PCDH group.3.Effect and mechanism of TNF-a on ICAM-1 expression in ASCsTNF-?,IFN,?,TGF-?1,MCP-1,IL-1?,IL,6,IL-8,IL-10,and IL-13 were added In vitro,the expression of VCAM-1 and ICAM-1 in BMSCs and ASCs were detected by flow cytometry.Flow cytometry was used to detect the expression of ICAM-1 in ASCs under the five culture conditions of stem cell culture medium,L-DMEM+FBS,L-DMEM+human serum,L-DMEM+platelet lysate,and F12 medium+FBS.The expression of ICAM-1 in generations ASCs of P2-P9 were detected by flow cytometry.Flow cytometry was used to detect the expression of ICAM-1 in P3 generation ASCs under 0.1,0.5,1,5,10,10,20,40,60,100,and 100 ng/mL of TNF-a.Real-time PCR and flow cytometry were used to detect the effects of 5 ng/mL TNF-a on ICAM-1 gene and protein expression at 1 h,3 h,6 h,12 h,24 h,and 48 h respectively.To detect the effects of mTOR pathway inhibitors PP242 and Rapamycin,JNK pathway inhibitor SP600125,MEK1/2 pathway inhibitor U0126 and NF-?B pathway inhibitor BAY11-7082 on ICAM-1 expression in ASCs,and TNF-a to cause ICAM-1 in ASCs effect of changes in expression levels.4.Effect of cryopreservation on the biological characteristics,the ICAM-1 expression and immunoregulatory function of ASCs.Different ratios of FBS and DMSO combined cryovials(100%FBS,99%FBS +1%DMSO,98%FBS + 2%DMSO,97%FBS + 3%DMSO,96%FBS + 4%DMSO,95%FBS + 5%DMSO,94%FBS + 6%DMSO,93%FBS + 7%DMSO,92%FBS +8%DMSO,91%FBS + 9%DMSO,90%FBS + 10%DMSO)were used to ASCs cryopreservation,then the cell viability of each group were dectected by a Muse cell analyzer.The CCK-8 method was used to detect the proliferation of 90%FBS and 10%DMSO,95%FBS and 5%DMSO cryopreserved and fresh ASCs.Flow cytometry was used to detect the expression of cell surface markers in 90%FBS and 10%DMSO,95%FBS and 5%DMSO cryopreserved and fresh group ASCs.Oil red O,alizarin red and alcian blue staining were used to detect the adipogenic,osteogenic and chondrogenic differentiation of 90%FBS and 10%DMSO,95%FBS and 5%DMSO cryopreserved and fresh group ASCs,respectively.The effect of 95%FBS and 5%DMSO cryopreservation group and fresh group ASCs on proliferation of PBMC in vitro was detected by flow cytometry.Results1.Effect of ICAM-1 on the biological characteristics of ASCsThe P3 generation of ASCs showed fibroblast-like adherent growth.The surface markers of mesenchymal stem cells such as CD44,CD73,CD90 and CD 105 were positively expressed,while CD45,CD34,CD11b,CD19 and HLA-DR were negatively expressed.ASCs can differentiate into adipogenesis,osteogenesis and chondrogenesis.On the third day of culture,the proliferative capacity of ASCs in the shICAM-1 group was significantly higher than that in the shScramble group,whereas the proliferation of ASCs in the ICAM-1-OV group was lower than that in the PCDH group;There was no significant change in cell death and cell cycle between knockdown and overexpression of ICAM-1 groups and the control group.The streaking results showed that the migration distances of cells in the shICAM-1 group ASCs at 6 h,12 h,and 24 h were significantly greater than those in the shScramble group,and the cell migration distance in the ICAM-1-OV group at 12 h was significantly smaller than that of PCDH group;Transwell and crystal violet staining results showed that the number of cell migration in shICAM-1 group was significantly higher than that in shScramble group at 12 h and 24 h,and the number of ASCs in ICAM-1-OV group was significantly lower than PCDH group at 24h;oil red O,alizarin red and alcian blue staining results showed that the differentiation ability of adipogenic,osteogenic and chondrogenic were similar between knockdown,overexpression of ICAM-1 ASCs group and the control group.2.The role of ICAM-1 in the immune regulation of ASCsPBMC exhibited aggregated growth and proliferation under an microscope after PHA stimulation,and PBMC became less aggregated after ASCs were added.The higher the proportion of ASCs,the weaker the aggregation ability of PBMC.The inhibitory effect of ASCs on PBMC proliferation was proportional to the proportion of ASCs and the inhibition of ASCs on proliferation of PBMCs and ASCs.When the ratio of ASCs to PBMC was 1:10 and 1:20,the difference was statistically significant compared with the group without ASCs.Both in contact co-culture and nocontact isolation co-culture,ASCs can inhibit the proliferation of PBMC?Co-culture ASCs had a stronger inhibitory effect on the proliferation of PBMCs.The inhibitory effect of ASCs in shICAM-1 group on PBMC proliferation was stronger than that in shScramble group.The inhibitory effect of ASCs in PCDH group on PBMC proliferation was similar to ICAM-OV group.3.Effect and mechanism of TNF-a on ICAM-1 expression in ASCsTNF-? and IFN-y promoted the expression of VCAM-1 and ICAM-1 in BMSCs,but only promoted the expression of ICAM-1 in ASCs.TNF-?,IFN-? and IL-1? promoted the expression of ICAM-1 in ASCs,while TGF-?1,MCP-1,IL-6,IL-8,IL-10 and IL-13 had no effect on the expression of ICAM-1 in ASCs.The ICAM-1 in ASCs cultured in stem cell culture medium group,L-DMEM+FBS group,F12 medium+FBS group,L-DMEM+platelet lysate group were low expression,while ICAM-1 in ASCs cultured in L-DMEM+human serum was intermediate expression.As the number of passages increased,the expression of ICAM-1 in ASCs gradually increased.After ASCs were stimulated with TNF-? of 0.1 ng/mL,0.5 ng/mL,1 ng/mL,5 ng/mL,and 10 ng/mL,the expression of ICAM-1 was gradually increased.The effects of 10 ng/mL,20 ng/mL,40 ng/mL,60 ng/mL,and 100 ng/mL TNF-? on the expression of ICAM-I in ASCs were no longer significant.Flow cytometry results showed that 3h after 5 ng/mL TNF-? stimulated ASCs,the expression of ICAM-1 surface protein began to increase and lasted until 48 h.Real-time PCR results showed that ICAM-1 gene expression began to increase after ASCs were stimulated with 5 ng/mL TNF-? for 1 h,and gradually decreased after 6h.TNF-? regulated the expression of ICAM-1 in ASCs through NF-?B signaling pathway,while mTOR pathway,JNK pathway and MEK1/2 pathway have no significant effect on the expression of ICAM-1 in TNF-?-induced ASCs.4.Effects of cryopreservation on biological characteristics,ICAM-1 expression and immune regulation function of ASCsIn the cryoprotectants,the activity of ASCs increased with the increase of DMSO concentration,and the cell viability was similar in the 90%FBS and 10%DMSO groups,95%FBS and 5%DMSO groups.The cryopreserved ASCs in the 90%FBS and 10%DMSO groups,95%FBS and 5%DMSO groups showed similar proliferation rates,cell surface markers,and differentiation capabilities as fresh ASCs.There was no statistical difference in inhibition of PBMC proliferation between 95%FBS and 5%DMSO cryopreserved group ASCs and fresh group ASCs.Conclusions1.Knockdown of ICAM-1 promoted the proliferation and migration of ASCs,but had no effect on the apoptosis,cycle and differentiation of ASCs;overexpression of ICAM-1 inhibited the proliferation and migration of ASCs,but had no effect on the apoptosis,cycle and differentiation of ASCs.2.ASCs had immunosuppressive effects on PBMC,and ASCs reduced the immunosuppressive capacity of PBMC after knockdown of ICAM-1.3.TNF-? and IFN-y can promoted the expression of ICAM-1 and VCAM-1 in BMSCs;TNF-?,IFN-y and IL-1?promoted the expression of ICAM-1 in ASCs;The expression of ICAM-1 of ASCs was different in different conditions and different generations;The expression of ICAM-1 in ASCs increases with the increase of TNF-?concentration in culture conditions.When the concentration reached 10 ng/mL and above,the expression change of was no longer obvious;TNF-? first promoted the expression of ICAM-1gene in ASCs,and then increased the expression of ICAM-1 protein;TNF-a mainly regulates the expression of ICAM-1 in ASCs through NF-?B signaling pathway.4.Cryopreservation maintained the activity,proliferation capacity,surface marker,differentiation and immunoregulatory potential of ASCs;95%FBS and 5%DMSO combination were ideal cryoprotectants for cryopreservation of ASCs. |
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