Reg3α促腺泡细胞向胰岛素分泌细胞转化再生治疗糖尿病的作用和机制研究

Diabetes is one of the top serious diseases leading to great harms to people’s health and life quality. The advantage of the approach replenishing insulin-producing cells has been elucidated while effectively regeneration of insulin-producing cells in vivo is the key component of this therapy. Based on our preliminary data, diabetic mice pretreated with recombinant Reg3α protein showed a drastic increased number of the clusters containing the 1-5 insulin positive-stained cell(s) in the exacrinous acinar parts of the pancreas than other groups, indicating that recombinant Reg3α protein could drive the conversion of acinar cells to insulin-producing cells. To prove the cure effects on streptozotocin-induced (Stz) and non-obese (NOD) diabetic mice, the blood glucose, body weight and serum insulin level of each group treated with or without recombinant Reg3α protein will be detected. The co-expression of a few cell-specific markers and transcriptional factors such as insulin and Ngn-3 will further be tested to indicate the possible pathway(s) of this transdifferentiation. In the neonatal insulin-producing cells, the expressing profile of β-specific factors and functional proteins for example Mafa and glucose transport protein-2 (Glut-2) etc, and the capability of insulin secretion will also be determined to validate their β-like properties and functionality. Changes of signaling including Notch and Stat-3 will be monitored as well, to find out the possible underlying mechanism of this conversion. In summary, this study will for the first time elucidate that the administration of recombinant Reg3α protein is capable of driving pancreatic acinar cells transdifferentiate to functional insulin-producing cells in diabetic mice. Hence, recombinant Reg3α protein could serve as a potent protein candidate and further contribute to this regenerative therapy to be transferred to the clinic.

糖尿病是一种严重危害人类生命及生活质量的疾病。促进胰岛素分泌细胞再生的疗法具有先进性，但如何在体内高效地诱导胰岛素分泌细胞再生是问题的关键。我们前期研究发现，重组Reg3α蛋白预给药的糖尿病小鼠胰腺内腺泡结构区域1-5个胰岛素阳性的细胞（簇）数量显著多于其他各组，提示Reg3α可能诱导腺泡细胞向胰岛素分泌细胞转化再生。我们将在Stz和NOD小鼠中，研究Reg3α给药对血糖、体重等指标的影响，验证糖尿病的治疗作用。深入研究胰岛素、Ngn-3等细胞标志物和转录因子共表达情况，阐明转化可能经历的途径；通过Mafa、Glut-2等β细胞因子和功能蛋白的表达以及胰岛素分泌水平，判断新生细胞的β样性质和功能；研究Notch和Stat-3信号通路改变对转化过程的影响，探讨可能的作用机制。本课题将首次阐明Reg3α是促进腺泡细胞向胰岛素分泌细胞转化再生的潜在蛋白质药物，为治疗糖尿病新型药物的研发提供依据。

在1型和2型糖尿病中，自身免疫攻击和炎症引起的胰岛β细胞损伤和功能缺失将导致糖尿病加重。如何维持胰岛β细胞数量和功能成为糖尿病治疗领域的研究热点。再生蛋白（Regenerating protein，Reg）是一种类生长因子，被证实在胰腺组织再生过程中发挥重要作用。大量文献表明Reg家族蛋白与多种组织和细胞的增殖、存活以及再生密切相关。我们已制备了一种重组Reg3α蛋白（rReg3α）并验证了其抵抗胰腺炎的活性。本课题在前期实验的基础上，研究了rReg3α在糖尿病小鼠中的保护作用。具体内容包括：1）在链脲菌素（STZ）诱导的BABL/c糖尿病小鼠中，rReg3α给药后检测血糖、体重等指标以及胰岛β细胞损伤；2）在体外培养的原代胰岛和小鼠胰岛素瘤细胞（MIN6）中，通过细胞流式、Western Blotting等方法检测rReg3α给药引起的细胞增殖、凋亡及相关信号通路；3）利用麦胚凝集素（WGA）对原代腺泡细胞进行谱系示踪，通过免疫荧光染色、转录组测序、荧光定量PCR等方法验证rReg3α给药诱导的“腺泡细胞-β细胞”转化再生。结果表明，rReg3α给药能够有效缓解STZ诱导的糖尿病胰岛损伤，促进在胰腺外分泌腺区域形成体积较小、胰岛素表达较低的胰岛素阳性细胞。体外实验中，rReg3α能够上调Bcl-2和Bcl-xL保护原代胰岛和MIN6细胞抵抗凋亡，激活Erk和Akt/ATF-2通路上调cyclin-D1和CDK-4促进MIN6细胞增殖。在WGA标记的腺泡细胞中，rReg3α通过Stat3磷酸化上调Ngn3促进“腺泡细胞-β细胞”转化再生。可是，这些新生胰岛素阳性细胞的糖促胰岛素分泌（GSIS）能力极低，说明其功能性仍未成熟。本课题证明了rReg3α给药能够减轻糖尿病胰岛损伤，诱导腺泡细胞向胰岛素产生细胞转化再生，为糖尿病治疗中维持胰岛β细胞数量和胰岛素分泌功能研究提供了新方法和思路。