间充质干细胞输注促进1型糖尿病动物模型beta细胞再生的机制研究

Mesenchymal stem cells (MSCs) are multi-potent adult stem cells with multi-lineage differentiation potential and secretory properties, which make MSCs an ideal candidate cell type for tissue function restoration and regeneration. Numerous studies have shown that infusion of MSCs can effectively lower blood glucose in diabetic animal models and give rise to regeneration of damaged beta-cells; however, the origin of the de novo regenerated beta-cells and the mechanisms involved are still elusive, hampering the widely clinical application of MSCs. The prior work of our group presented that infused MSCs can reverse hyperglycemia status mainly by ameliorating the insulin resistance of peripheral insulin target tissues in type 2 diabetic animals, but the effect of lowered blood glucose level in type 1 diabetic rats largely attributed to augmentation of beta-cell mass. Strikingly, MSC infusion resulted in a considerable appearance of insulin/glucagon double positive cells in type 1 diabetic rat islets, which were rarely observed in the normal or control animals. Herein, in this study, we focus our attention on testing the potential therapeutic effect of MSC transplantation and exploring the mechanisms involved in type 1 diabetic animal model via multiple approaches including cell transplantation, transgenic animal model, cell lineage tracing, immunofluorescence labeling and histopathology analyses. Similar to the models developed in traditional researches, the MSCs isolated from the allogeneic adult rats are cultured in vitro and are expanded to third-passage, then the MSCs or supernatantare transfused intravenously respectivelyinto diabetic rat which are induced by a single high dose of STZ (65mg/kg, i.p.). And thus the following experiments are performed to address this issue: 1) to determine whether the amelioration of the hyperglycemia in type 1 diabetic rats ascribe to restoration of insulin-producing cell mass in the pancreas, and whether the new born beta-cell derived from the heterologous source of alpha cells; 2) to recapitulate the dynamic characteristics of the cell conversion in response to infusion of MSCs, which included when the double hormone positive cells appear and reach its peak; 3)to scrutinized carefully the conversion process to de?ne the nature of intermediate cells, tracing the alteration of marked transcription factors along with the hormone changes; 4) to clarify whether there is an optimal treatment time window for alpha-cell converting into beta-cell after MSCs transplantation with respect to the duration of diabetes; 5) to estimate whether the secretory effect plays a crucial role on the regeneration of beta-cell after MSCs infusion, and whether the ef?ciency of??alpha to beta conversion with the infusion of supernatant equals that with infusion of MSCs. Of paramount importance, the data from our study will prompt widely clinical use of MSCs in future.

研究表明间充质干细胞（MSCs）输注可明显促进糖尿病动物的胰岛再生，并可有效改善高血糖状态，但其机制尚不明确。本项目组前期研究发现MSCs主要通过改善胰岛素敏感性缓解2型糖尿病大鼠的高血糖症状，而对1型糖尿病大鼠模型则主要通过促进内源beta细胞的再生发挥作用，并观察到MSCs输注后胰岛素/胰高糖素双标记细胞数量显著增加。为深入阐明MSCs输注促进胰岛再生的机制，本项目拟通过细胞移植、免疫荧光标记、转基因动物模型、细胞谱系追踪、动态病理分析等实验手段，首先明确在1型糖尿病动物模型中，MSCs输注是否主要通过重编程alpha为beta细胞促进胰岛结构重建与功能恢复，并详细研究MSCs促进该类细胞类型转变的动力学特征以及时效性特征；在此基础上进一步阐明MSCs输注促进1型糖尿病动物模型alpha细胞beta化的可能作用机制（是否依赖分泌效应），该项目的完成将为MSCs的临床应用奠定理论基础。

不同程度的Beta细胞缺失是1型糖尿病和2型糖尿病共同的关键发病机制。如何促进beta细胞的再生一直是糖尿病领域的研究热点，同时也是治愈糖尿病的希望所在。本研究通过细胞移植、活体动物实验、动态病理分析、免疫荧光标记等技术揭示在beta细胞严重受损的1 型糖尿病动物模型中，早期单次足量MSCs 输注可以促进胰腺内beta细胞数量的增加和功能的恢复，并且这种beta细胞的再生主要通过重编程胰腺内固有的alpha转变为 beta 细胞而实现。MSCs分泌的活性因子是促进胰岛内beta细胞再生的关键。这些研究结果挑战了以往传统观念（既往认为干细胞治疗的作用是基于干细胞的自身直接转化和/或胰岛内beta细胞自我复制率提高），把胰腺内beta细胞的再生归因于其他内源性非beta细胞的转变，从而指出了了实现beta细胞再生的新思路。研究中对干细胞移植的时机数量及涉及的重要机制进行了初步探讨，为后续探索干细胞移植策略奠定了基础。此外研究还提示胰腺重度毁损早期胰岛内可出现自发的beta细胞再生，给予胰岛素稳定大鼠血糖后，这些新生细胞可以存活，并且表达beta细胞特异的转录因子，新生的beta细胞同时具有糖反应性。更令人吃惊的是，在STZ注射后16小时，在胰岛中出现了一群同时表达间质细胞标志物vimentin和alpha细胞标志物MafB的类圆形中间状态细胞。而这群细胞在STZ注射后24小时获得了胰岛素的表达，是新生beta细胞的来源。这些研究结果的发现揭示了干细胞移植促进胰腺beta细胞再生的崭新机制，确定了干细胞移植的最佳时机和策略，并对胰腺受损早期出现的自身可塑性变化进行了深入，从而为未来实现beta细胞再生提供了新思路。