MSC conditioned substrate protects islet graft function by avoiding hypoxic injury

BACKGROUND: Pancreatic islet transplantation is the most potential method for radical treatment of type 1 diabetes. The early hypoxia death after transplantation impedes the success of its treatment. In order to improve the efficacy of pancreatic islet transplantation and enhance the resistance of pancreatic islet cells to hypoxia, it is of great significance to reduce hypoxic damage before revascularization of transplantation. Mesenchymal stem cells are believed to regulate immune response and hypoxia injury through paracrine secretion. Pre treatment of pancreatic islet cells with mesenchymal stem cells (MSCs) conditioned medium (CM) enhances cell tolerance to hypoxic environments, reduces cell death caused by hypoxia, and ultimately protects pancreatic islet graft function. Method: Cultivate MIN6 cells under hypoxic conditions with 1% oxygen concentration, perform AO/PI staining at different time points, and observe their survival using fluorescence microscopy. MIN6 cells were cultured in medium containing different concentrations of MSCs under normal oxygen concentration and hypoxic conditions. Flow cytometry Annexin/PI was used to detect survival rate at different time points, and high and low glucose stimulation experiments were used to detect insulin secretion ability. The C57BL/6 type 1 diabetes mouse model was constructed. The islet cells of BALB/c mice were extracted and incubated with MSC conditioned medium for 24 hours. The islet cells were transplanted into the renal capsule of type 1 diabetes NCG mice. The weight and blood sugar of the mice were monitored. The graft function was detected by glucose tolerance test. The graft and functional survival were observed by HE and insulin immunohistochemistry. Result: Fluorescence microscopy showed that under hypoxic conditions, the death of MIN6 cells increased with the prolongation of hypoxia time. Flow cytometry showed that the survival rate of MIN6 cells after 48 hours of hypoxia was 68.07 ± 7.90%, which was significantly lower than that of the normoxic group (94.57 ± 2.12%, P<0.01). After high and low glucose stimulation under hypoxic conditions, the insulin secretion index (SI) of MIN6 decreased from 1.77 ± 0.02 to 1.43 ± 0.06. Under hypoxic conditions, both 10% and 20% conditioned medium can effectively improve cell damage caused by hypoxia, while the effect of 30% conditioned medium is weaker. The high glucose stimulation experiment detected the trend of MIN6 function consistent with survival rate. The primary islet cells of 10% conditioned medium pretreated mice were transplanted under the renal capsule to type 1 diabetes mice. The blood sugar of mice continued to decline five days after the operation. The HE and insulin immunohistochemical observation showed that the islet cells of conditioned medium group had more complete morphology and higher insulin secretion, and the glucose tolerance test showed that the graft function was better. Conclusion: It is found that hypoxia will significantly reduce the survival of MIN6 and inhibit its insulin secretion function, while MSC conditioned medium can significantly inhibit the death and dysfunction caused by hypoxia, and achieve the protective effect on the function of pancreatic islet graft in the transplantation model of type 1 diabetes mice.