SMS2基因敲除对小鼠糖脂代谢及胰岛功能影响的初步研究

发布时间：2018-05-11 09:30

本文选题：鞘磷脂 + 神经酰胺　；参考：《南京医科大学》2015年硕士论文

【摘要】：第一部分SMS2敲除对小鼠胰岛内鞘脂代谢的影响目的:观察SMS2-KO小鼠胰岛内总SMS活性、SMS2m RNA水平的表达及SMS2缺失对胰岛内鞘脂代谢的影响。方法:采用PCR法鉴定SMS2-KO及同窝生WT小鼠的基因。采用薄层层析法测定SMS2-KO(n=10)及同窝生WT小鼠(n=10)胰岛内鞘磷脂合成酶(SMS)总活性;LC/MS/MS检测两组小鼠胰岛内鞘脂:鞘磷脂(SM)、神经酰胺(Cer)、一磷酸鞘氨醇(S1P)、二羟神经酰胺(DHcer)及血清中(SM和Cer)的水平(SMS2-KO,n=5;WT,n=5);Real-time PCR检测小鼠胰岛内参与鞘脂代谢相关酶:鞘磷脂合成酶(SMS1,SMS2)、葡萄糖神经酰胺合成酶(GCS)、鞘磷脂磷酸二酯酶(SMPD1,SMPD2,SMPD3,SMPD4)m RNA表达水平(SMS2-KO,n=10;WT,n=10)。结果:(1)SMS2-KO小鼠在354bp处出现条带,WT在252bp处出现条带;(2)SMS2-KO小鼠胰岛内总SMS活性下降,与WT小鼠比下降49%(P0.05);(3)两组小鼠胰岛内总SM含量下降(P0.001),总Cer含量升高(P0.001),DHcer及S1P无明显差异,血清中鞘脂的改变与胰岛内一致;(4)SMS2-KO小鼠胰岛内SMS2 m RNA表达明显低于WT(P0.001),而胰岛内SMS1、GCS、SMPD1、SMPD2、SMPD3、SMPD4 m RNA水平在两组间无明显差异(P0.05)。结论:(1)SMS2缺失导致胰岛内SMS总活性降低,几乎没有SMS2基因表达,对SMS1基因表达无明显影响。(2)SMS2缺失导致胰岛内SM含量减少,Cer水平升高,而对DHcer及S1P水平无明显影响。第二部分SMS2基因敲除对小鼠糖代谢及胰岛功能的影响目的:观察SMS2缺失对小鼠糖代谢及胰岛功能的影响,探究SMS2缺失对小鼠胰岛功能调节的可能机制。方法:腹腔注射糖耐量试验(intraperitoneal injection of glucose tolerance test,IPGTT)评估SMS2-KO小鼠及同窝生WT小鼠糖代谢情况(SMS2-KO,n=5;WT,n=4);腹腔注射胰岛素耐量试验(intraperitoneal injection of insulin tolerance test,IPITT)评估胰岛素敏感性(SMS2-KO,n=4;WT,n=4);用ELISA法检测两组小鼠空腹、葡萄糖负荷后30min及60min血清胰岛素水平(SMS2-KO,n=4;WT,n=4);分离两组小鼠胰岛,体外进行葡萄糖刺激的胰岛素分泌(glucose-stimulated insulin secreting,GSIS)试验,观察SMS2缺失对胰岛葡萄糖刺激下胰岛素分泌的影响(SMS2-KO,n=7;WT,n=7);检测两组小鼠基础及葡萄糖刺激后胰岛内ATP的含量(SMS2-KO,n=4;WT,n=6);对胰腺进行HE染色,观察两组小鼠胰岛的形态、大小及密度(SMS2-KO,n=5;WT,n=5);分离小鼠胰岛,透射电镜观察胰岛的超微结构,并评估两组小鼠胰岛β细胞内锚定的胰岛素颗粒囊泡数(SMS2-KO,n=6;WT,n=6)。结果:(1)两组小鼠体重及食物消耗无明显差异,SMS2缺失小鼠胰岛素敏感性及糖耐量均好于WT小鼠;(2)两组小鼠空腹血清胰岛素水平无明显差异,葡萄糖刺激后SMS2-KO小鼠血清胰岛素有下降的趋势,但差异没有统计学意义(P0.05);(3)两组小鼠胰岛内胰岛素含量无明显差异,分离小鼠胰岛,体外进行GSIS示,SMS2-KO小鼠葡萄糖刺激的胰岛素分泌减少,差异有统计学意义(P0.05);(4)两组小鼠胰岛内基础及葡萄糖刺激后两组小鼠胰岛内ATP的含量无明显差异(P0.05);(5)两组小鼠胰岛的形态、大小及密度无明显差异,电镜下胰岛亚细胞器高尔基体、线粒体及内质网均未见明显异常,胰岛β细胞内锚定胰岛素颗粒囊泡数无明显差异。结论:(1)SMS2缺失能增加胰岛素敏感性;(2)SMS2缺失导致胰岛素分泌减少,但其增加胰岛素敏感性的效应尚能代偿其导致的胰岛分泌功能的减少,维持机体正常的糖代谢水平。
[Abstract]:The effect of SMS2 knockout on the metabolism of sphingolipids in the islets of mice: To observe the total SMS activity in the islets of the SMS2-KO mice, the expression of SMS2m RNA and the effect of SMS2 deletion on the metabolism of sphingolipids in the islets. Methods: the PCR method was used to identify the genes of SMS2-KO and the same fossae of WT mice. The thin layer chromatography was used to determine SMS2-KO (n=10) and the same fossaic WT. The total activity of sphingomyelin synthetase (SMS) in the islets of rat (n=10) islet, and LC/MS/MS in the islets of two groups of mice: sphingolipid (SM), ceramide (Cer), sphingosine monophosphate (S1P), dihydroamide (DHcer) and serum (SM and Cer) levels (SMS2-KO, n=5; WT,). SMS1, SMS2, GCS, SMPD1, SMPD2, SMPD3, SMPD4, m RNA expression level (SMS2-KO, n=10; WT, and WT). The total SM content in the islets of the two groups decreased (P0.001), the total Cer content increased (P0.001), and there was no significant difference in DHcer and S1P, and the changes in serum sphingolipids were the same as in the islets. (4) the m RNA expression in the pancreatic islets of the SMS2-KO mice was significantly lower than that of WT (P0.001), but there was no significant difference between the two groups. Conclusion: (1) the loss of SMS2 caused the decrease of total SMS activity in the islet, almost no SMS2 gene expression, and no obvious influence on the expression of SMS1 gene. (2) the loss of SMS2 resulted in the decrease of the content of SM in the islet, the level of Cer increased, but no significant effect on the level of DHcer and S1P. The effect of the second part SMS2 based on the effect of knockout on the glucose metabolism and islet function of mice was observed. The effect of SMS2 deletion on the glucose metabolism and islet function in mice, and to explore the possible mechanism of SMS2 deletion on the function regulation of islet in mice. Methods: intraperitoneal injection of glucose tolerance test (intraperitoneal injection of glucose tolerance test, IPGTT) to evaluate the glucose metabolism of SMS2-KO mice and the same fossaic WT mice. Intraperitoneal injection of insulin tolerance test (IPITT) was used to evaluate the insulin sensitivity (SMS2-KO, n=4; WT, n=4), and two groups of mice were tested with ELISA method. Glucose-stimulated insulin secreting, GSIS) tests were conducted to observe the effects of SMS2 deletion on insulin secretion under islet glucose stimulation (SMS2-KO, n=7; WT, n=7), and the basis of two groups of mice and the content of ATP in the islets of pancreas after glucose stimulation (SMS2-KO, n=4), and the morphology, size and density of the islets of the two groups of mice were observed. MS2-KO, n=5; WT, n=5); the islets of mice were separated and the ultrastructure of the islets was observed by transmission electron microscopy. The number of insulin particle vesicles anchored in the islet beta cells of the two groups of mice (SMS2-KO, n=6; WT, n=6) were evaluated. Results: (1) there was no significant difference in weight and food consumption between the two groups, and the insulin sensitivity and glucose tolerance in the SMS2 deficient mice were better than those of WT mice; (2) two There was no significant difference in serum insulin level in the fasting serum of mice. There was no significant difference in serum insulin in SMS2-KO mice after glucose stimulation, but the difference was not statistically significant (P0.05). (3) there was no significant difference in insulin content in the islets of the two groups, isolated mouse islets, GSIS in vitro, and glucose stimulated insulin secretion in SMS2-KO mice. The difference was statistically significant (P0.05); (4) there was no significant difference in the content of ATP in the islets of the two groups of mice in the two groups of islets and glucose stimulation (P0.05); (5) there was no significant difference in the shape, size and density of the islets of the two groups, and the islet subcellular apparatus, the Golgi bodies, the mitochondria and the endoplasmic reticulum, were not obviously abnormal, and the islet beta was thin. There is no significant difference in the number of intracellular anchored insulin particles. Conclusion: (1) SMS2 deletion can increase insulin sensitivity; (2) the loss of SMS2 leads to the decrease of insulin secretion, but the effect of increasing insulin sensitivity can still compensate for the decrease of islet secretory function and maintain normal glucose metabolism.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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