| Backgrounds:Diabetes mellitus (DM) is a serious threat to human health which continues deepening all over the world. DM patients have no choices but to rely on insulin productions to control their blood glucose levels, at the risk of insulin resistance and fatal hypoglycemia. With the rapid development of transplantation technique and immunotherapy, whole pancreas transplantation and islet transplantation has become an accessible method to treat DM. Compared with whole pancreas transplantation, islet transplantation has the advantages in operation, meanwhile it has a higher safety and validity. However, islets transplantation still has problems with grafts supply and long survival rate at present. Mesenchymal stem cell, isolated from fresh human umbilical cord, whose functions of immunoregulation and paracrining protective cytokines to promote tissues repair has been universally accepted, can be treated as support agent to facilitate islets survive and function maintain. As blood supply can’t be established in time at early stage, grafts suffer serious ischemia and anoxia. And then cells occur apoptosis quickly. The outcome of islets-MSC coculture system is also dissatisfactory. Therefore, inhibition the occurrence of apoptosis of β cells and protecting islets functions are the keys of islets transplantation. Hardikar et al first discovered after being excised80%-90%pancreatic tissues of STZ induced diabetes rats, rats regained a normal blood level, which indicated that residual pancreatic tissues may exert quite strong regenerative ability by secreting protective cytokines to promote β cells reproduce. Fu YS et al also found regenerative pancreatic extract could improve the paracrine function of MSC.Objective:In the present study, we try to build a islets-mesenchymal stem cells coculture system in a simulative microenvironment in vitro to assess whether regenerative pancreatic extract will benefit the coculture system to protect islets from damage and maintain their functions.Experiment groups:Group one:Normal rat islets coculture with human umbilical MSC (HUMSC) and meanwhile added with RPE.Group two:HUMSC coculture with rat islets isolated from partial pancreatectomy rats.Control:Normal rat islets coculture with HUMSC.Materials and methods:1Combine collagenase V digestion with discontinuous ficoll-paque density gradient centrifugation to isolate rat islets. Residual pancreatic tissues proteins were harvested at72hours after60%pancreatectomy, followed by being added into islets-MSC coculture system at simulative microenvironment in vitro,β cells of all groups primarily cultured in5%O2for14days and then transferred in18%O2until60days. Observe the changes in morphology and functions of β cells of all groups under such environment.2After culture in hypoxia environment for3days, the expression of caspase-3in β cells were assessed by Western blotting.3On day3, correlated cytokines(human derived VEGF, b-FGF and rats derived IL-1β) were measured by Elisa.4Collect all groups medium on day3、7、14、21、30、45、60, insulin quantification assay and glucose stimulation experiments were conducted to further assess β cells functions.Results:Islets and mesenchymal stem cells cocultured in simulative environment in vitro for60days, the morphology of islets of all groups changed for a certain degree. Especially in hypoxia culture period, apoptosis and necrosis occurred in the centre and periphery of islets. Even β cells appeared to crash and die. The islets of group one had the least extent in β cells injury, and began to improve their status since culture in normoxia condition. Cytokines levels were measured on day3, compared with islets of control group, human derived VEGF(P<0.01) and b-FGF(P<0.05) were extremely higher than that in group one and group two, meanwhile rat derived IL-1β was much lower(P<0.01). Basic insulin levels and high glucose stimulated insulin levels had obvious difference among three groups. On day3after culturing in vitro, islets of control group were of the highest basic insulin levels. On day7, the basic insulin levels began to lower than that in group one. From then on, the basic insulin levels in group one is much higher than that in control group(P<0.01). The results of high glucose stimulation test on day14and60revealed that stimulated insulin level of group one is about high to4folds of that before high glucose stimulation. By contrast, change of insulin levels between before and after stimulation only up to1.5folds in group two and control group.Conclusion:Residual pancreatic tissues after60%pancreatectomy can produce anti-apoptosis and tissues repairing factors to facilitate cells self-repair, meanwhile improve MSC paracrine function and inhibit β cells from secreting inflammatory factor like IL-1β. RPE can benefit the islets-MSC coculture system to protect β cells from damage and maintain their survival and functions. Islets and mesenchymal stem cells coculture system added with RPE may have significant potential to be a more suitable method in β cell preservation before and after transplantation. |
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