本文關(guān)鍵詞：磷脂酶C在INS-1細(xì)胞胰島素分泌信息傳導(dǎo)途徑中的作用　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： INS-1 葡萄糖 L-谷氨酸 KCL 八肽膽囊收縮素 氯化乙酰膽堿 磷脂酶Cβ1 葡萄糖刺激胰島素分泌 過(guò)表達(dá) 胰島素 瞬時(shí)轉(zhuǎn)染

【摘要】： 目的:初步探討六類磷脂酶C(Phospholipase c)在營(yíng)養(yǎng)物質(zhì)、離子、神經(jīng)遞質(zhì)和激素刺激INS-1細(xì)胞胰島素分泌的信息傳導(dǎo)途徑中的作用。 方法:①分別設(shè)計(jì)五組刺激因素:葡萄糖、L-谷氨酸、KCL、八肽膽囊收縮素、氯化乙酰膽堿(分別屬于營(yíng)養(yǎng)物質(zhì)、離子、神經(jīng)遞質(zhì)和激素類),②設(shè)計(jì)五組刺激因素作用的時(shí)間梯度和濃度梯度,③收集刺激后的INS-1細(xì)胞培養(yǎng)上清液, ELISA法檢測(cè)細(xì)胞上清液中胰島素的含量,④最適刺激條件作用于INS-1細(xì)胞后,RT-PCR檢測(cè)六類PLC的表達(dá)。 結(jié)果:①PLCε在INS-1細(xì)胞中沒(méi)有表達(dá);②葡萄糖刺激INS-1細(xì)胞,RT-PCR檢測(cè)刺激前后表達(dá)量的變化大小順序?yàn)?PLCδPLCβPLCγPLCηPLCζ;L-谷氨酸作用后:PLCβPLCδPLCζPLCηPLCγ;KCL作用后:PLCηPLCδPLCγPLCζPLCβ;八肽膽囊收縮素作用后:PLCδPLCγPLCηPLCζPLCβ;氯化乙酰膽堿作用后:PLCδPLCβPLCγPLCηPLCζ 結(jié)論:葡萄糖、L-谷氨酸、KCL、八肽膽囊收縮素、氯化乙酰膽堿均可刺激INS-1細(xì)胞顯著分泌胰島素;六類PLC在胰島素分泌信息傳導(dǎo)途徑中表達(dá)上調(diào),但各類PLC的增加量不同。 第二章過(guò)表達(dá)PLCβ1對(duì)β細(xì)胞葡萄糖刺激胰島素分泌的影響 目的:探討磷脂酶Cβ1(phospholipase Cβ1,PLCβ1)在葡萄糖刺激胰島素分泌(glucose-stimulated insulin secretion GSIS)信息傳遞中的作用。 方法:①以最適葡萄糖濃度刺激INS-1細(xì)胞適當(dāng)時(shí)間后,RT-PCR檢測(cè)PLCβ1表達(dá)變化;②構(gòu)建PLCβ1真核表達(dá)載體(PCMV-HA-PLCβ1),轉(zhuǎn)染INS-1細(xì)胞,Western-blot檢測(cè)INS-1細(xì)胞中PLCβ1蛋白的表達(dá);③收集轉(zhuǎn)染后INS-1細(xì)胞培養(yǎng)上清液,檢測(cè)胰島素含量。 結(jié)果:①半定量RT-PCR檢測(cè)葡萄糖刺激后PLCβ1表達(dá)顯著升高。②過(guò)表達(dá)PLCβ1的INS-1細(xì)胞培養(yǎng)上清液中胰島素含量為1.906±0.080 ng/mL,較對(duì)照組(0.740±0.091 ng/mL)顯著升高(P㩳0.01)。 結(jié)論:過(guò)表達(dá)PLCβ1顯著增加INS-1細(xì)胞的胰島素分泌,提示PLCβ1可能參與GSIS信息傳遞。
[Abstract]:Objective: to study the effect of phospholipase c6 kinds of phospholipase on nutrients and ions. Role of neurotransmitters and hormones in the signaling pathway of insulin secretion in INS-1 cells. Methods five stimulation factors were designed: glucose L-glutamate KCL, cholecystokinin octapeptide, acetylcholine chloride (which belong to nutrients, ions, neurotransmitters and hormones, respectively). 2 the time gradient and concentration gradient of stimulation factors were designed to collect the culture supernatant of INS-1 cells after stimulation, and the insulin content in the supernatant was detected by ELISA method. 4 the expression of six kinds of PLC was detected by RT-PCR after the optimal stimulation condition was applied to INS-1 cells. Results: 1 PLC 蔚 was not expressed in INS-1 cells. (2) the order of the change of expression before and after glucose stimulation in INS-1 cells was: INS-1 未 PLC 尾 PLC 緯 PLC 畏 PLC 味; PLC 尾 PLC 未 PLC 味 PLC 畏 PLC 緯; After the action of KCL, KCL 畏 PLC 未 PLC 緯 PLC 味 PLC 尾; After the action of cholecystokinin octapeptide, PLC 未 PLC 緯 PLC 畏 PLC 味 PLC 尾; PLC 未 PLC 尾 PLC 緯 PLC 畏 PLC 味 after acetylcholine chloride Conclusion: glucose L-glutamate, cholecystokinin octapeptide, acetylcholine chloride can significantly stimulate the secretion of insulin in INS-1 cells. The expression of PLC was up-regulated in insulin secretory signaling pathway, but the increase of PLC was different. Effect of overexpression of PLC 尾 1 on glucose-stimulated insulin secretion in 尾 -cells Objective: to study the phospholipase C 尾 1 of phospholipase C 尾 1. PLC 尾 1 in glucose-stimulated insulin secretion. The role of GSIS in the transmission of information. Methods the expression of PLC 尾 1 was detected by RT-PCR after INS-1 cells were stimulated with the optimal glucose concentration. 2 PLC 尾 1 eukaryotic expression vector was constructed and transfected into INS-1 cells. The expression of PLC 尾 1 protein in INS-1 cells was detected by Western-blot. 3 the supernatant of INS-1 cell culture after transfection was collected and the insulin content was detected. Results:. 1 the expression of PLC 尾 1 was significantly increased by semi-quantitative RT-PCR. 2. The insulin content in the culture supernatant of INS-1 cells with overexpression of PLC 尾 1 was 1.906 鹵0.080. Ng/mL. Compared with the control group (0.740 鹵0.091 ng / mL), the P was significantly higher than that in the control group. 0.01g. Conclusion: overexpression of PLC 尾 1 significantly increases insulin secretion in INS-1 cells, suggesting that PLC 尾 1 may be involved in GSIS information transmission.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341

【共引文獻(xiàn)】

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