The Role Of Wip1 In Mesenchymal Stem Cells Treating Murine Type 1 Diabetes Model

Objective: The aim of this research is to investigate the effects of two different kinds of mesenchymal stem cells（MSCs）: Wip1^+/+MSCs and Wip1^-/-MSCs therapy in Type 1 Diabetes（T1D） by using murine T1 D model. Understand the role of Wip1 during MSCs immuno-modulation and T1 D treatment. To further explain the mechanisms of MSCs in T1 D therapy and support the clinical using of MSCs.Methods:1. Created murine T1 D model. Mice were either given low does streptozotocin（STZ） constantly named as the Slow-type Model, or given STZ at high dose for one time to set up the Fast-type Model. Comparing these two models by measure the body weight, blood sugar, living status and pathological features of islets. The one with better result was chose as the model building method for this study.2. Isolation and identification of Wip1 MSCs and Wip1 MSCs. The genomic DNA were extracted from the tail of 1 week old pups to analyze the Wip1 genotype. MSCs were derived from Wip1^+/+ and Wip1^-/- mouse bone essence and identified by flowcytometry and induced into adipocytes or osteocytes, in which oil drops or the activity of alkaline phosphatase（ALP） were detected.3. The role of Wip1 in T1 D MSCs treatment therapy. The murine Type 1 Diabetes（T1D） models were treated with murine bone essence derived Wip1^+/+MSCs or Wip1^-/-MSCs. After the therapy random blood sugar change was recorded, the pathological features of islets examed with HE staining and insulin immunohistochemistry, the disorders of immune system was measured with flowcytometry and RT-PCR. The HE staining, Masson staining, PASM staining and RT-PCR were used to measure the renal pathological features changes.4. The mechanism of Wip1 in the MSC immuno-modulation. Flurence marked Wip1^+/+MSCs and Wip1^-/-MSCs were injected into the murine T1 D models diffreently. The numbers of MSCs homing to spleen were counted on the d3 and d7 after the transplantation. The Notch1 and Hes1 expression level in spleen were tested by RT-PCR and the activated NICD were detected by immunohistochemistry. Meantime DC were co-cultured with Wip1^+/+MSCs or Wip1^-/-MSCs respectively to determinethe DC maturity and Notch1, Hes1 expression level.Results:1. The pancreas of both the Slow-type Models and Fast-type Models were destroyed with fewer residual β cells and elevated blood sugar. The body weight of the Slow-type Models decreased slowly and showed gradually rising of blood sugar level with a mortality of 10%, while the body weight of the Fast-type Models decreased rapidly and showed a rapidly increased blood sugar in early stage, resulting in a higher mortality of 35%. Thus, the Slow-type Model was more acceptable method for this study due to the gradually increased blood sugar and the lower deviation caused by mortality.2. Both Wip1^+/+MSCs and Wip1^-/-MSCs grew in spindle shape during the early passages after the isolation. After passage 3 the Wip1^-/-MSCs showed a slower growth rate than the Wip1^+/+MSCs. Both those MSCs highly expressed CD29, Sca-1 and CD90, negatively expressed CD11 b, CD34, CD45, CD31 and MHCII. The Wip1^-/-MSCs had a weaker capability differentiated to the osteogenic and adipogenic than the Wip1^+/+MSCs.3. The Wip1^-/-MSCs were less capable to reduce the blood sugar level and protect β cells compare to the Wip1^+/+MSCs. The treatment of Wip1^+/+MSCs also showed better results for down regulating the Th1/Th2 ratio, reducing the production of IFN-γ, TNF-α, and increasing the production of IL4, IL10, IL17, FOXP3. Furthermore, in kidneys TGF-β1/SMAD signaling was still more activated after Wip1^-/-MSCs treatment, while the Masson and PASM staining showed much more pathological changes in Wip1^-/-MSCs treated group compared to the Wip1^+/+MSCs treated group.4. After the Wip1^+/+MSCs and Wip1^-/-MSCs were transplanted into the T1 D models, there was no significant difference between the numbers of two MSCs homing to spleen on d3 and d7. The activated NICD proportion is higher in spleens with the Wip1^+/+MSCs treatment. And also the Notch1 and Hes1 expression level in Wip1^+/+MSCs treated mice spleens was higher than that in Wip1^-/-MSCs treated group. Meanwhile DC co-cultured with Wip1^+/+MSCs exhibited higher activated Notch signaling expression compared to the Wip1^-/-MSCs co-culturing.Conclusion: The depletion of Wip1 impairs the proliferation, differentiation and the immuno-modulation abilities of MSCs and leads to the failure of T1 D treatment. The Wip1 also play an important role during the immune cells Notch1/Hes1 signal activation.