Construction Of Tissue-engineered Islets And The Study On The Anti-apoptotic Effect Of HUC-MSC In Tissue-engineered Islets

Objective:Early islet cell apoptosis after pancreatic transplantation is a key factor affecting the effectiveness of transplantation.Pseudo-islets（primary islet isolation and re-aggregation）and heterospheroids（primary islet isolation and re-aggregation with other types of cells）are both tissue-engineered islets,and can serve as tools for islet research.The aim of this study is to:1.Optimize the construction method of pseudo-islets to improve construction efficiency;2.Perform functional activity evaluation of pseudo-islets and heterospheroids,and investigate whether pancreatic islet heterospheroids constructed with human umbilical cord mesenchymal stem cells（hUC-MSC）can improve the effectiveness of pancreatic transplantation;3.Explore for the first time the anti-apoptotic effect of hUC-MSC in pancreatic islet heterospheroids,and provide a reference for maintaining islet survival and improving the effectiveness of pancreatic transplantation.Methods:hUC-MSC were extracted by tissue adhesion method,and cell surface antigens were identified using flow cytometry.The tri-lineage differentiation potential of hUC-MSC was evaluated by inducing adipogenic,osteogenic,and chondrogenic differentiation.The hanging drop culture system(Akura^TMPLUS droplet plate combined with ultra-low binding96-well plate as the collection plate)was used to construct pseudo-islets and heterospheroids of INS-1 and/or hUC-MSC.The efficiency of spheroid formation using hanging drop system and culture dish,the effect of different cell numbers on the size of pseudo-islets and heterospheroids,the morphological changes during the evolution of pseudo-islets and heterospheroids,and the cell distribution in heterospheroids were compared.The functional activity of the prepared（pre-transplantation）pseudo-islets and heterospheroids was evaluated by glucose stimulation test,fluorescein diacetate（FDA）and propidium iodide（PI）staining.Subsequently,the pseudo-islets and heterospheroids were transplanted into the portal vein of diabetic mice induced by streptozotocin（STZ）,and the hypoglycemic effect was observed.Meanwhile,to verify the anti-apoptotic effect of hUC-MSC in pancreatic islet heterospheroids,a pancreatic apoptosis model was constructed by inducing H₂O₂.After spheroid formation,the samples were divided into three groups:blank group（pseudo-islets constructed by INS-1）,model group（pseudo-islets constructed by INS-1 induced by 50μmol/L H₂O₂）,and experimental group（heterospheroids constructed by INS-1:hUC-MSC=1:1induced by 50μmol/L H₂O₂）.After being cultured for 72 hours under the same conditions,the apoptosis index of pancreatic islet cells was detected by Annexin V-FITC/PI double staining,the relative m RNA expression levels of Bcl-2,Bax,and Caspase-3 were detected by RT-q PCR,and the relative protein expression levels of Bcl-2,Bax,and Caspase-3 were detected by Western blotting.Results:1.Primary hUC-MSC extracted by the tissue block attachment method crawled out at the edge of the tissue block after 3-7 days of culture.When the density of hUC-MSC was low,they appeared relatively loose,with large cell spacing,flattened morphology,and high stretchability.As the density of hUC-MSC increased,the distance between cells became closer,and the cells showed a more compact arrangement and a rounder morphology.In addition,INS-1 cells cultured in the same conditions proliferated quickly,and their cell morphology appeared as polygonal or irregular polygons,with tight cell adhesion.2.Surface antigen identification of hUC-MSC revealed that CD90（99.79%）,CD105（99.71%）,and CD73（99.89%）were positive,while HLA-DR（0.07%）was negative.The presence of calcium deposits indicates the potential of hUC-MSC to differentiate into osteogenic cells,while the production of intracellular lipid droplets is a prominent feature of adipocytes.Alcian blue staining of the specimen shows the acidic mucopolysaccharides present in cartilage tissue.3.Pseudo-islets and heterospheroids constructed by the hanging drop system were spherical in shape,and compared with human islets extracted in the control group,their morphology was similar.4.By constructing pseudo-islets of INS-1 cells with different cell numbers,it was found that the diameter of the pseudo-islets gradually increased with an increase in cell number.When the initial cell number of INS-1 cells was 300,the diameter of the pseudo-islets was approximately150.17±6.47μm.The diameters of the pseudo-islets of INS-1 cells with cell numbers of 200,250 and 350 were 119.01±4.36μm,130.86±5.21μm,and166.35±5.84μm,respectively.The difference between them was statistically significant（P<0.05）.The diameter of heterospheroids was 172.65±6.15μm,and compared to INS-1 cells with a cell number of 350,there was no significant difference（P>0.05）.5.When comparing the spheroid formation between the hanging drop system and the dish hanging drop method,we found that the construction time of pseudo-islets was faster in the hanging drop system（52.44±13.37 h）than in the dish hanging drop method（113.54±24.99 h）.The difference between the two methods was statistically significant（P<0.01）.6.During the study of the formation process of pseudo-islets and heterospheroids,it was observed that both pseudo-islets and heterospheroids were relatively dispersed and unevenly distributed when injected into the hanging drop culture plate at 0 hours.At the same time,the number of heterospheroids was significantly higher than that of pseudo-islet cells.At approximately 12 hours,both groups showed obvious cell aggregation,forming clusters that were slightly dispersed and had varying degrees of tightness.At 24 hours,the heterospheroid group showed obvious cell aggregation,forming a nearly spherical shape,while the pseudo-islet group exhibited cell aggregation visible to the naked eye,but with low density.The heterospheroid group was significantly better than the pseudo-islet group in terms of spheroid formation.After collecting the pseudo-islets and heterospheroids into the collection plate,both groups showed obvious spheroid formation after approximately 24 hours.Both groups were spherical and had a more regular shape,with the heterospheroids being slightly larger in diameter than the pseudo-islets.7.After preparation of the heterospheroids,INS-1 cells were labeled in red and hUC-MSC cells were labeled in green.After combining the two,it was observed that INS-1 and hUC-MSC cells were heterogeneously distributed.8.The overall cell survival rate of prepared heterospheroids was higher than that of pseudo-islets,and the difference was statistically significant（P<0.05）.9.Under low glucose stimulation,the insulin release of heterospheroids was higher than that of pseudo-islets,and the difference was statistically significant（P<0.01）.Under high glucose stimulation,the insulin release of heterospheroids was also higher than that of pseudo-islets,with a statistically significant difference（P<0.05）.However,there was no significant difference in insulin release index（SI）between heterospheroids and pseudo-islets（P>0.05）.10.One week before establishing the diabetic model,the blood glucose and body weight of the experimental animals remained relatively stable.On the day after injection with STZ（day 8）,it was found that the blood glucose levels of all three groups of mice had significantly increased compared to the previous day（day 7）（P<0.01）.By day 10,only one mouse had a blood glucose level below 20 mmol/L,and from day 11-13,all mice maintained a stable blood glucose level above 20 mmol/L.On day 12,12 mice had a blood glucose level above 30 mmol/L,and on day 13,16 mice had a blood glucose level above 30 mmol/L.After transplantation,it was found on the next day（day 14）that the decrease in blood glucose of the heterospheroid group was8.05±1.45 mmol/L,the pseudo-islets group was 6.7±0.72 mmol/L,and the control group（physiological saline group）was 3.9±1.34 mmol/L.Both pseudo-islet and heterospheroid groups had significant statistical differences in glucose reduction compared to the control group（P<0.01）,but there was no statistically significant difference between the heterospheroid and pseudo-islet groups（P>0.05）.Within one week after transplantation,the blood glucose levels of the control group showed a slight rebound and approached the pre-transplantation level,while the heterospheroid group showed a trend of initially decreasing and then slowly rising,but still remained lower than the other two groups（P<0.05）.After injection of STZ（day 7）,the body weights of all three groups of animals showed a continuously decreasing trend,and there was no statistically significant difference between the groups（P>0.05）.Within one week after transplantation,there was no obvious increase in body weight of mice in all three groups when compared with the weight reduction between day 13 and day 20,and there was no statistically significant difference among the three groups（P>0.05）.11.After 72 hours of apoptosis induction,the flow cytometry was used to measure the percentage of apoptotic cells.After performing analysis of variance（ANOVA）,it was found that the apoptosis rate in the model group was significantly increased compared to the blank group,with a statistically significant difference（P<0.01）.The apoptosis rate in the experimental group was significantly decreased compared to the model group,with a statistically significant difference（P<0.01）.12.After apoptosis induction in the control group,model group and experimental group,using ANOVA analysis,it was found that compared with the control group,the relative expression levels of Bax and Caspase-3 m RNA increased（P<0.01）,while the relative expression level of Bcl-2 m RNA decreased（P<0.01）and the Bcl-2/Bax ratio decreased（P<0.05）in the model group.Compared with the model group,the relative expression levels of Bax and Caspase-3 m RNA decreased（P<0.01）and the relative expression level of Bcl-2 m RNA and the Bcl-2/Bax ratio increased（P<0.01）in the experimental group.13.After inducing apoptosis in the control group,model group and experimental group,using ANOVA analysis,it was found that compared with the control group,the relative expression level of Bcl-2 protein decreased（P<0.05）,while the relative expression levels of Bax and Caspase-3 protein increased（P<0.01）and the Bcl-2/Bax ratio decreased（P<0.01）in the model group.Compared with the model group,the relative expression level of Bcl-2protein and the Bcl-2/Bax ratio increased（P<0.01）,and the relative expression levels of Bax protein decreased（P<0.05）and Caspase-3 protein decreased（P<0.01）in the experimental group.Conclusions:1.The hanging drop system culture method can improve the efficiency of constructing pseudo-islets;2.Islet heterospheroids constructed with hUC-MSC can improve the transplantation effect of islets;3.Islet heterospheroids constructed with hUC-MSC may significantly inhibit apoptosis of islet cells;4.The anti-apoptotic effect of hUC-MSC in islet heterospheroids may involve increasing the m RNA and protein expression of Bcl-2,decreasing the m RNA and protein expression of Bax and Caspase-3,and increasing the ratio of Bcl-2/Bax m RNA and protein.