發(fā)布時(shí)間：2018-05-11 09:30

  本文選題：鞘磷脂 + 神經(jīng)酰胺��； 參考：《南京醫(yī)科大學(xué)》2015年碩士論文

【摘要】：第一部分SMS2敲除對小鼠胰島內(nèi)鞘脂代謝的影響目的:觀察SMS2-KO小鼠胰島內(nèi)總SMS活性、SMS2m RNA水平的表達(dá)及SMS2缺失對胰島內(nèi)鞘脂代謝的影響。方法:采用PCR法鑒定SMS2-KO及同窩生WT小鼠的基因。采用薄層層析法測定SMS2-KO(n=10)及同窩生WT小鼠(n=10)胰島內(nèi)鞘磷脂合成酶(SMS)總活性;LC/MS/MS檢測兩組小鼠胰島內(nèi)鞘脂:鞘磷脂(SM)、神經(jīng)酰胺(Cer)、一磷酸鞘氨醇(S1P)、二羥神經(jīng)酰胺(DHcer)及血清中(SM和Cer)的水平(SMS2-KO,n=5;WT,n=5);Real-time PCR檢測小鼠胰島內(nèi)參與鞘脂代謝相關(guān)酶:鞘磷脂合成酶(SMS1,SMS2)、葡萄糖神經(jīng)酰胺合成酶(GCS)、鞘磷脂磷酸二酯酶(SMPD1,SMPD2,SMPD3,SMPD4)m RNA表達(dá)水平(SMS2-KO,n=10;WT,n=10)。結(jié)果:(1)SMS2-KO小鼠在354bp處出現(xiàn)條帶,WT在252bp處出現(xiàn)條帶;(2)SMS2-KO小鼠胰島內(nèi)總SMS活性下降,與WT小鼠比下降49%(P0.05);(3)兩組小鼠胰島內(nèi)總SM含量下降(P0.001),總Cer含量升高(P0.001),DHcer及S1P無明顯差異,血清中鞘脂的改變與胰島內(nèi)一致;(4)SMS2-KO小鼠胰島內(nèi)SMS2 m RNA表達(dá)明顯低于WT(P0.001),而胰島內(nèi)SMS1、GCS、SMPD1、SMPD2、SMPD3、SMPD4 m RNA水平在兩組間無明顯差異(P0.05)。結(jié)論:(1)SMS2缺失導(dǎo)致胰島內(nèi)SMS總活性降低,幾乎沒有SMS2基因表達(dá),對SMS1基因表達(dá)無明顯影響。(2)SMS2缺失導(dǎo)致胰島內(nèi)SM含量減少,Cer水平升高,而對DHcer及S1P水平無明顯影響。第二部分SMS2基因敲除對小鼠糖代謝及胰島功能的影響目的:觀察SMS2缺失對小鼠糖代謝及胰島功能的影響,探究SMS2缺失對小鼠胰島功能調(diào)節(jié)的可能機(jī)制。方法:腹腔注射糖耐量試驗(yàn)(intraperitoneal injection of glucose tolerance test,IPGTT)評估SMS2-KO小鼠及同窩生WT小鼠糖代謝情況(SMS2-KO,n=5;WT,n=4);腹腔注射胰島素耐量試驗(yàn)(intraperitoneal injection of insulin tolerance test,IPITT)評估胰島素敏感性(SMS2-KO,n=4;WT,n=4);用ELISA法檢測兩組小鼠空腹、葡萄糖負(fù)荷后30min及60min血清胰島素水平(SMS2-KO,n=4;WT,n=4);分離兩組小鼠胰島,體外進(jìn)行葡萄糖刺激的胰島素分泌(glucose-stimulated insulin secreting,GSIS)試驗(yàn),觀察SMS2缺失對胰島葡萄糖刺激下胰島素分泌的影響(SMS2-KO,n=7;WT,n=7);檢測兩組小鼠基礎(chǔ)及葡萄糖刺激后胰島內(nèi)ATP的含量(SMS2-KO,n=4;WT,n=6);對胰腺進(jìn)行HE染色,觀察兩組小鼠胰島的形態(tài)、大小及密度(SMS2-KO,n=5;WT,n=5);分離小鼠胰島,透射電鏡觀察胰島的超微結(jié)構(gòu),并評估兩組小鼠胰島β細(xì)胞內(nèi)錨定的胰島素顆粒囊泡數(shù)(SMS2-KO,n=6;WT,n=6)。結(jié)果:(1)兩組小鼠體重及食物消耗無明顯差異,SMS2缺失小鼠胰島素敏感性及糖耐量均好于WT小鼠;(2)兩組小鼠空腹血清胰島素水平無明顯差異,葡萄糖刺激后SMS2-KO小鼠血清胰島素有下降的趨勢,但差異沒有統(tǒng)計(jì)學(xué)意義(P0.05);(3)兩組小鼠胰島內(nèi)胰島素含量無明顯差異,分離小鼠胰島,體外進(jìn)行GSIS示,SMS2-KO小鼠葡萄糖刺激的胰島素分泌減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05);(4)兩組小鼠胰島內(nèi)基礎(chǔ)及葡萄糖刺激后兩組小鼠胰島內(nèi)ATP的含量無明顯差異(P0.05);(5)兩組小鼠胰島的形態(tài)、大小及密度無明顯差異,電鏡下胰島亞細(xì)胞器高爾基體、線粒體及內(nèi)質(zhì)網(wǎng)均未見明顯異常,胰島β細(xì)胞內(nèi)錨定胰島素顆粒囊泡數(shù)無明顯差異。結(jié)論:(1)SMS2缺失能增加胰島素敏感性;(2)SMS2缺失導(dǎo)致胰島素分泌減少,但其增加胰島素敏感性的效應(yīng)尚能代償其導(dǎo)致的胰島分泌功能的減少,維持機(jī)體正常的糖代謝水平。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.1

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本文編號：1873393