Study On The Mechanism Of Wip1 Regulating Mesenchymal Stem Cells In The Treatment Of Type1 Diabetes In Mice

Objectives:To explore the effect and mechanism of Wild type p53-induce phosphatase1（Wip1）regulating mesenchymal stem cells（MSC）in the treatment of type 1 diabetes by using Wip1 knockout mice.This experiment can provide a theoretical and experimental basis for clarifying the mechanism of MSC in the therapy of T1DM.Methods:1.Isolation,culture,and identification of Wip1 knockout mouse derived MSC.The 1-week-old pups were isolated and analyzed by Wip1 genotype;the normal MSC(Wip1^+/+)and Wip1 knockout MSC(Wip1^-/-)were cultured respectively.Cell morphology was observed by Giemsa staining and cell phenotype was identified by flow cytometry.Then both MSCs were induced into adipocytes or osteocytes.The differentiation ability was demonstrated by oil red O staining and alkaline phosphatase staining.The expression of adipogenic key transcription factors Peroxisome proliferator-activated receptor gamma（PPAR-γ）,CCAAT enhancer binding protein alpha（CEBP/ɑ）,Runt-related transcription factor 2（RunX2）,and Osteocalcin were detected by real-time fluorescence quantitative PCR（q-PCR）.2.The effect of Wip1 in regulating MSCs for T1D treatment.Mice were given streptozotocin（STZ）to build a murine T1D model.The T1D model was treated with5×10⁵ Wip1^+/+MSC or Wip1^-/-MSC by tail vein injection to observe the efficacy.In the experiment,the models were separated into normal control group,T1DM model group,Wip1^+/+MSC treatment group,and Wip1^-/-MSC treatment group.The general physiological state,blood sugar level and body weight of mice were measured every week.After 4 weeks of treatment,HE staining and immunohistochemistry were used to detect the pathological changes of pancreatic tissue and insulin secretion in each group.Enzyme-linked immunosorbent assay（ELISA）was used to detect the expressions of inflammatory cytokines IFN-γ,IL-4,and IL-17a in the serum of mice.Flow cytometry was used to detect the intracellular inflammatory cytokines in spleen T lymphocytes.3.The mechanism of Wip1 regulating MSC in the treatment of T1DM.The target gene BST2 that contains significant expression differences and related to immune regulation was found by Wip1^+/+MSC and Wip1^-/-MSC gene chip combined with bioinformatics analysis.The expression of BST2 in Wip1^-/-MSC was analyzed by q-PCR and Western blot.The overexpression vector of Wip1-EGFP was further constructed and transfected into 293T cells.The correlation between Wip1 and BST2 expression was detected by Western blot.IFN-α,the target gene of BST2,was predicted refer to previous research results;q-PCR and ELISA was used to analyze the IFN-αexpression in Wip1^-/-MSC.Furthermore,the synthesized BST2 siRNA was transfected into Wip1^-/-MSC cells,and the expression correlation between BST2 and IFN-αwas detected by q-PCR.To demonstrate whether Wip1 regulate MSC immune function through BST2-IFN-αin vivo,we first observed Wip1^+/+and Wip1^-/-MSCs homing in the pancreas by immunofluorescence.Then,ELISA was used to inspect the expression of inflammatory factors in the pancreas and peripheral lymph nodes（PLN）of mice in each group,including IFN-α,TNF-α,IL-17a,IL-4,and IL-10;q-PCR was employed to detect the expression of IFN-α,IFN-β,and IFN-γ.Results:1.Wip1^-/-mice were successfully obtained by mouse tail genotype detection.Wip1^+/+MSC and Wip1^-/-MSC were cultured separately to P3 generation.After Giemsa staining,the nuclei of the two MSC cells can be observed to be oval,showed long fusiform and vortex-like adherent growth under an inverted microscope.Both MSCs had high expression of Sca-1,CD29 and CD90,and low expression of CD34,CD45,CD11b and MHCII.After adipogenic induced differentiation of MSCs,numerous intracellular lipid droplets can be observed by oil red O staining.The q-PCR results showed that the expressions of PPAR-γand CEBP/ɑin the induction group were significantly up-regulated.Osteogenic induced differentiation results demonstrated that the activity of alkaline phosphatase was enhanced in the induced group,and the expressions of RunX2 and Osteocalcin in the induced group also shown a significant increase in q-PCR detection.These results indicate that Wip1^+/+MSC and Wip1^-/-MSC were successfully obtained.2.In order to elucidate the therapeutic effect of Wip1^-/-MSC on mouse T1DM,we first established a mouse T1DM model and injected Wip1^+/+MSC or Wip1^-/-MSC via the tail vein.The results showed that the blood glucose of mice decreased gradually and the body weight recovered after Wip1^+/+MSC treatment.However,after Wip1^-/-MSC treatment,the blood glucose was not significantly decreased,and the body weight was not increased as much as the Wip1^+/+MSC treatment group.The results of pathological examination illustrated that the islet tissue of Wip1^+/+MSC treatment group was relatively complete and the insulin secretion increased significantly,while the islet tissue of Wip1^-/-MSC treatment group was still incomplete,and the insulin secretion was less obvious than that of Wip1^+/+MSC treatment group.The result of flow cytometry and ELISA indicated that in both Wip1^+/+MSC and Wip1^-/-MSC groups,the expression of IFN-γin Th1 subsets of spleen lymphocytes and the secretion of proinflammatory cytokines IFN-γand IL-17a were significantly up-regulated;the secretion of anti-inflammatory cytokines IL-4 were significantly down-regulated.3.To investigate the mechanism of Wip1 regulating MSC in the treatment of T1DM in mice,we first screened and obtained BST2 genes that showed obvious difference through the results of Wip1^+/+MSC and Wip1^-/-MSC gene chips,and then confirmed the high expression of BST2 in Wip1^-/-MSC by q-PCR and Western blot.Wip1-EGFP overexpression vector was constructed to transfect 293T cells to further prove that Wip1 regulates BST2 expression.In order to clarify the target gene of BST2 is IFN-α,we use q-PCR and Western blot to detect whether Wip1^-/-MSC express IFN-α.The results showed that IFN-αwas also highly expressed in Wip1^-/-MSC.Furthermore,siRNA was used to knock down the expression of BST2 in Wip1^-/-MSC,and the results showed that the expression of IFN-αwas also significantly down-regulated.These results suggested that Wip1 could regulate the immunomodulatory function of MSC by regulating BST2 and thereby affecting the expression of IFN-α.In vivo test results in mice indicated that both Wip1^+/+MSC and Wip1^-/-MSC could be homed to the damaged pancreatic tissue and play a therapeutic role.ELISA results evidenced that Wip1^-/-MSC also promoted the secretion of IFN-αin PLN,up-regulated the expression of proinflammatory cytokines TNF-αand IL-17a,and down-regulated the expression of anti-inflammatory cytokines IL-4 and IL-10.The expression of the IFN family in mouse spleen lymphocytes was detected by q-PCR,and the results revealed that Wip1^-/-MSC could obviously up-regulate the expression of IFN-α,IFN-β,and IFN-γ.Conclusion:Wip1 affects MSC immunomodulatory effect by regulating the expression of BST2-IFN-α.This study provides a new theoretical and experimental basis for elucidation of the mechanism of MSC in the therapy of T1DM.