The Role Of LKB1 In Maintaining AMSC Self-renewal And AMSCs In T Cell Immune Regulation

Objective:LKB1 has been well known in recent years not only for its powerful tumor suppressor function in somatic cells,but also for its role in maintaining cell homeostasis ina variety of immune cells and hematopoietic stem cells.AMSC possess the properties of pluripotent Stem cells and can inhibit the proliferation and differentiation of T cells in vitro,and successfully induce peripheral T cells to transform into treg to exert negative immune regulation,thereby inhibiting the excessive immune response of the body.The role of transcription factor LKB1 in both stem cell and immunomodulatory AMSC cells has not been reported at home and abroad.In this study,shRNA interference was successfully used to specifically knock down LKB1 gene expression in the AMSC,and the role of LKB1 in the biological characteristics and immunity of AMSC cells was investigated.Methods:AMSCs from healthy full-term cesarean section placenta amnion were obtained by tissue block culture,and their morphology,in vitro differentiation ability and surface markers were identified,which confirmed that they conformed to the definition of MSC by the International Stem Cell Research Society.Through the analysis of bioGPS gene information database,it was found that LKB1 was highly expressed in MSC.Then,the expression of LKB1 in AMSC was detected by mRNA and protein levels by Real-time PCR and Western blot,respectively.It was confirmed that LKB1 was expressed in AMSC.To investigate the role of LKB1 in the basic cell biological characteristics and immunosuppression of AMSC,we synthesized three DNA fragments of shRNA-LKB1 in the form of primers,and synthesized DNA double strands by annealing and ligated into PLKO.1-EGFP vector.On the above,after sequencing,the shRNA-LKB1 colonies successfully linked to the vector were screened,and the relevant plasmid was extracted.A four-plasmid lentiviral packaging system was used to package and concentrate the lentivirus for infection with late AMSCs.Then,AMSCs successfully transfected by flow sorting were used to amplify SK and CON.The knockdown efficiency of shRNA was detected by mRNA and protein levels by Real-time PCR and Western blot,respectively.Pick up the shRNA with the highest knockdown efficiency for later testing.The infected AMSC was identified again;and the mRNA level of the AMSC multifunctional marker gene was detected by Real-time PCR.The growth rate of the cells was observed,and the proliferation ability and apoptosis level of SK cells were analyzed by colony formation assay,proliferation-related genes,and apoptosis kits.Tcon cells were labeled with CellTrace,and SK-Tcon co-culture was used to detect the inhibition of SK on Tcon cell proliferation.Real-time PCR was used to detect the representative genes of Thl and Th2 and soluble cytokines or membrane surface molecules capable of inducing Treg production and secreted by MSC,IFN-y and IL-10,IL-6,PD-L1,PD-L2,IDO mRNA levels.Flow cytometry was used to detect the ratio of SK and CON induced by Treg at different time points and the expression of Ki67 in Treg cells.Results:Primary AMSCs isolated and cultured by tissue block conformed to the definition of MSC by the International Stem Cell Research Society.The basic biological characteristics of SK and CON cells after knockdown of LKB1 by shRNA were still consistent with MSC characteristics.The results of Real-time PCR of the three-line differentiation and differentiation-related genes confirmed that compared with CON,the ability of SK to differentiate into osteoblasts and cartilage was enhanced,while the ability to differentiate into adipogenic phase did not change significantly.At the same time,knockdown of LKB1 specifically does not affect the multifunctional marker gene of AMSC.In addition,the proliferation of SK cells was significantly reduced,and apoptosis was significantly increased,resulting in a significant slowing of SK growth.Through the Celltrace test,it was found that SK has a function of inhibiting the proliferation of Tcon cells,and knockdown of LKB1 specifically can significantly inhibit the proliferation of Tcon cells.SK can also significantly inhibit Thl expression of gene expression,increase the mRNA level of Th2-represented genes,and significantly increase the mRNA levels of soluble cytokines or membrane surface molecules that can induce Treg production and are secreted by MSCs.Specific knockdown of LKB1 enhances the ability of AMSCs to regulate T cell subsets in the anti-inflammatory direction.In T cell and SK and CON cell co-culture experiments,we again confirmed at the protein level that SK has the ability to induce Treg production in vitro.Among them,the induction ratio of Tree increased with the prolongation of co-culture time.By detecting the expression of Ki67 in induced Treg,it was found that SK can significantly promote the proliferation of induced Treg.Conclusion:1)Specific knockdown of LKB1 in AMSC does not affect its basic biological properties,but enhances its ability to differentiate into osteoblasts and cartilage.2)LKB1 maintains cell self-renewal in AMSC by promoting proliferation and inhibiting apoptosis;3)LKB1 plays a negative regulatory role in inhibiting the proliferation of Tcon cells by AMSC;4)LKB1 plays a negative regulatory role in AMSC-regulated T cell subsets in the direction of inhibition of inflammation;5)LKB1 inhibits the induction of Treg by AMSC to a certain extent,and is time-dependent;6)LKB1 negatively regulates the proliferation of induced Tregs in AMSCs.In conclusion,LKB1 maintains the self-renewal of AMSCs and participates in the immune regulation of T cells by AMSCs to maintain the immune balance of the body.