Apelin基因工程干细胞逆转胰岛素抵抗的作用与机制的研究

Insulin resistance is a Common core mechanism of pathophysiologic hallmark for coronary heart disease and type 2 diabetes mellitus (T2DM), but, up to now, it still is not restored by present medical therapies. The recently discovered peptide apelin is necessary for the maintenance of insulin sensitivity. Unfortunately, despite the clear need for novel therapies for insulin resistance, our understanding of its action for long time remains incomplete because of its half-life of no longer than 5 minutes. At present study, first time, we use combined with apelin gene and stem cells for reversing insulin resistance in vitro and vivo. First, we obseave the effect of over expression of apelin gene on glucose uptake, determining whether apelin can increase glucose uptake and increase insulin sensitivity in insulin resistant adipocytes. Furtherly, wharton's jelly-derived mesenchymal stem cells (WMSCs) are transfected with apelin-lentiviral vector in various doses, and then apelin-WMSCs-EGFP are transplanted into insulin resistant T2DM mice. After transplantation, we obseave the effect for long time of reversing insulin resistance, and action of β cell regeneration, confirm that a apelin mediate pathway and influence on inflammatory factors at the level of entirety, tissue and cells, respectively. Finally, it is confirmed the effect of reversing IR treated by the combination of apelin molecular target and WMSCs transplantation. It is thus clear that the introduction of novel therapeutic modalities for patients with insulin resistant T2DM and coronary heart disease would be greatly progress in the world .

证椐表明胰岛素抵抗(IR)是冠心病、糖尿病共同发病的核心机制，而至今还无法逆转。最近发现，apelin是维持胰岛素敏感性的必需因子，但因其体内短暂生物半衰期使其长期作用及其机制至今不清。本研究首次克服以上不足，应用apelin基因联合干细胞技术逆转IR。首先，在体外诱导的IR脂肪细胞上，观察apelin基因过表达对其葡萄糖摄取、胰岛素敏感性效应; 进而将不同剂量apelin基因转染Wharton's 源间充质干细胞(WMSCs)移植入IR 2型糖尿病小鼠模型，在整体、组织、细胞水平上观察apelin+WMSCs移植后逆转胰岛素抵抗的长期作用，对胰岛β细胞再生作用; 效应所介导的信号通路, 及对IR相关炎性因子的影响;确证apelin 分子靶与干细胞联合治疗逆转胰岛素抵抗的综合效应。这将为糖尿病与冠心病的联合治疗带来突破性进展，居国际领先。

研究背景：探索apelin-WJMSCs基因工程干细胞逆转胰岛素抵抗作用，为创新糖尿病治疗奠定理论基础。研究内容：① 体外细胞水平：在TNF-a诱导的3T3-L1胰岛素抵抗脂肪细胞模型（3T3-L1 IR）上，观察过表达apelin分子对葡萄糖摄取及胰岛素敏感性影响; ②体内整体水平：在大鼠IR T2D模型上，观察apelin-WMSCs体内移植对IR逆转及胰岛β细胞再生效应;③探讨apelin-基因逆转IR作用机制。重要结果：①在IR脂肪细胞上发现，过表达Apelin分子使IR脂肪细胞葡萄糖摄取率提高26%（P<0.05）;使胰岛素敏感性提高35%（P=0.0014）。在体外细胞水平证实了其改善IR脂肪细胞葡萄糖摄取及胰岛素敏感性。②在高脂高糖+STZ诱导的T2D大鼠模型上，将apelin-WJMSCs经静脉移植体内，未触发免疫反应，无致瘤性。改善了大鼠存活。降低了高血糖，平均空腹血糖由26.03±2.83，7天降至15.85±2.13（P<0.01）；14天降至9.41±2.05（P<0.01），降血糖持续42天（8.9±0.89，P<0.001）。Apelin-WJMSCs移植后4周末，葡萄糖耐量及胰岛素耐量明显改善(与对照组比较均P<0.01-0.05)，血浆C肽水平明显提高。病理显示：T2D+saline大鼠，胰岛中度到重度萎缩，胰岛细胞大量减少; T2D+apelin大鼠：胰岛轻度萎缩;胰岛免疫荧光染色可見Ki67与insuline共表达新生胰岛β细胞生长及增殖。③机制发现过表达apelin基因T2D大鼠血浆TNF-a，IL-6水平明显降低，脂联素水平明显升高(P<0.05-0.001)。过表达Apelin的IR脂肪细胞Akt、eNOS水平明显增高，同时伴有GLUT4表达水平从基础80.87%增加到90.83%（P<0.05），联合胰岛素共刺激GLUT4水平92.54%（P=0.02）。表明apelin通过激活PI3K/Akt及AMPK/eNOS二条信号通路，促进了GLUT4向胞膜转移。结论：本研究从分子、细胞、整体水平上证实了apelin基因过表达对糖代谢调控效应，揭示了其改善胰岛素抵抗的作用机制，首次创建了apelin-WJMSCs基因工程干细胞，为创新T2D治疗奠定了重要理论基础。