發(fā)布時(shí)間：2018-05-03 05:18

  本文選題：胰腺星狀細(xì)胞 + 胰石蛋白/再生蛋白��； 參考：《東南大學(xué)》2015年碩士論文

【摘要】：背景及目的病理研究顯示,2型糖尿病胰島內(nèi)存在纖維化,胰腺星狀細(xì)胞是胰島纖維化的主要始動(dòng)和效應(yīng)細(xì)胞,并且對(duì)胰島p細(xì)胞具有直接損傷作用。胰石蛋白／再生蛋白(Pancreatic stone protein/regeneration protein, PSP/reg)是再生基因家族的重要成員之一,與胰腺細(xì)胞的增殖密切相關(guān)。有研究顯示,該蛋白促進(jìn)胰島p細(xì)胞增殖及功能,并且可抑制胰腺星狀細(xì)胞活化及其致纖維化能力。本實(shí)驗(yàn)探討PSP/reg對(duì)胰腺星狀細(xì)胞(Pancreatic Stellate Cell, PSC)胰島p細(xì)胞毒性效應(yīng)的影響。方法分離培養(yǎng)美國癌癥研究所(Institute of Cancer Research, ICR)小鼠的PSC,在含或不含有PSP/reg的培養(yǎng)液中培養(yǎng)24小時(shí),收集PSC上清(PSC su pernatant, PSC-SN),將MIN-6小鼠胰島p細(xì)胞株分別培養(yǎng)在：①無血清培養(yǎng)基、②含0/30/100/200/300 ng/ml PSP/reg的無血清培養(yǎng)基、③PSC-SN、④PSP/reg干預(yù)的PSC-SN (PSP-PSC-SN)、⑤含有100 ng/ml PSP/reg 的 PSC-SN (PSP +PSC-SN)中24小時(shí)。CCK-8比色法觀察不同處理組小鼠胰島β細(xì)胞存活率；流式細(xì)胞術(shù)檢測AnnexinV-FITCPI雙染細(xì)胞比例：葡萄糖刺激的胰島素釋放(Glucose Stimulated Insulin Secretion, GSIS)試驗(yàn)；胰島素ELISA試劑盒檢測上清胰島素濃度；Western blot法檢測P62、LC3Ⅱ/Ⅰ蛋白的表達(dá)。結(jié)果1.CCK-8法檢測P SP/reg組MIN-6小鼠胰島β細(xì)胞活力百分比高于對(duì)照組,流式細(xì)胞術(shù)分析結(jié)果顯示兩組AnnexinV-FITCPI雙染細(xì)胞比例無明顯差異,GSIS試驗(yàn)結(jié)果顯示PSP/reg組葡萄糖刺激的胰島素釋放濃度高于對(duì)照組,WB檢測PSP/reg組P62蛋白含量低于對(duì)照組、LC3Ⅱ/Ⅰ比值高于對(duì)照組；2. CCK-8法檢測PSC-SN組MIN-6小鼠胰島p細(xì)胞活力百分比低于對(duì)照組,流式細(xì)胞術(shù)分析結(jié)果顯示PSC-S N組AnnexinV-FITCPI雙染細(xì)胞比例高于對(duì)照組,GSIS試驗(yàn)結(jié)果顯示PSC-SN組葡萄糖刺激的胰島素釋放濃度低于對(duì)照組,WB檢測PSC-SN組P62蛋白含量低于對(duì)照組、LC3IⅡ/Ⅰ比值高于對(duì)照組；3. CCK-8法檢測PSP-PSC-S N組MIN-6小鼠胰島p細(xì)胞活力百分比高于PSC-SN組,流式細(xì)胞術(shù)分析結(jié)果顯示PSP-PSC-SN組AnnexinV-FITCPI雙染細(xì)胞比例低于PSC-S N組,GSIS試驗(yàn)結(jié)果顯示PSP-PSC-SN組葡萄糖刺激的胰島素釋放濃度高于PSC-SN組,WB檢測PSP-PSC-SN組P62蛋白含量高于對(duì)照組、LC3Ⅱ/Ⅰ比值低于對(duì)照組；4. CCK-8法檢測PSP+PSC-SN組MIN-6小鼠胰島p細(xì)胞活力百分比高于PSC-SN組,流式細(xì)胞術(shù)分析結(jié)果顯示PSP+PSC-SN組AnnexinV-FITCPI雙染細(xì)胞比例低于PSC-SN組,GSIS試驗(yàn)結(jié)果顯示PSP+PSC-SN組葡萄糖刺激的胰島素釋放濃度高于PSC-SN組,WB檢測PSP+PSC-SN組P62蛋白含量高于對(duì)照組、LC3Ⅱ/Ⅰ比值低于對(duì)照組。結(jié)論在一定的濃度范圍內(nèi),PSP/reg濃度依賴性地促進(jìn)MIN-6細(xì)胞存活、胰島素釋放作用及MIN-6細(xì)胞的自噬水平,過高濃度的PSP/reg可導(dǎo)致MIN-6細(xì)胞的過度自噬,對(duì)MIN-6細(xì)胞的存活及胰島素釋放產(chǎn)生抑制作用；P SC-SN抑制MIN-6細(xì)胞增殖及胰島素釋放,促進(jìn)MIN-6細(xì)胞凋亡,并誘導(dǎo)MIN-6細(xì)胞的過度自噬;PSP/reg促進(jìn)PSC-SN干預(yù)的MIN-6細(xì)胞存活及胰島素釋放,減少PSC-SN誘導(dǎo)的MIN-6凋亡及過度自噬；此外,PSP/reg干預(yù)后的PSC-SN (PSP-PSC-SN)對(duì)MIN-6活力及胰島素釋放的的抑制、促進(jìn)MIN-6凋亡及過度自噬等作用減弱。綜上所述,PSP/reg本身具有增加胰島p細(xì)胞活力及功能等作用,還可減輕PSC-SN對(duì)胰島p細(xì)胞的損傷,此外,PSP/reg可作用于PSC,抑制其致胰島p細(xì)胞損傷的能力,間接發(fā)揮胰島p細(xì)胞的保護(hù)作用。
[Abstract]:Background and objective pathological studies have shown that the pancreatic islets of type 2 diabetes are fibrosis, and pancreatic stellate cells are the main initiation and effector cells of islet fibrosis and have direct damage to the islet P cells. Pancreatic stone protein/ regeneration protein (PSP/reg) is an important part of the regenerative gene family. One of the members is closely related to the proliferation of pancreatic cells. Studies have shown that the protein promotes the proliferation and function of pancreatic islet P cells and inhibits the activation and fibrosis of pancreatic stellate cells. The effect of PSP/reg on the toxic effects of pancreatic stellate cells (Pancreatic Stellate Cell, PSC) on pancreatic islet p cells. The PSC in the Institute of Cancer Research (ICR) mice was cultured for 24 hours in the culture medium containing or without PSP/reg, and collected the PSC supernatant (PSC Su pernatant, PSC-SN). (3) PSC-SN, 4 PSP/reg intervention PSC-SN (PSP-PSC-SN), and PSC-SN (PSP +PSC-SN) with 100 ng/ml PSP/reg in the 24 hour.CCK-8 colorimetric method to observe the survival rate of islet beta cells in different treatment groups, and flow cytometry to detect the proportion of AnnexinV-FITCPI double stained cells: glucose stimulated insulin release Cretion, GSIS) test, insulin ELISA kit to detect the concentration of insulin in the supernatant, and Western blot method to detect the expression of P62 and LC3 II / I protein. Results 1.CCK-8 method detected the percentage of islet beta cell activity in P SP/reg group MIN-6 mice higher than that of the control group. The results of flow cytometry analysis showed that the proportion of two groups of AnnexinV-FITCPI double stained cells was not obvious. The results of GSIS test showed that the insulin release concentration of glucose stimulated by PSP/reg group was higher than that of the control group. The content of P62 protein in the PSP/reg group was lower than that of the control group, and the ratio of LC3 II / I was higher than that of the control group; the percentage of P cell viability in the MIN-6 mice of PSC-SN group MIN-6 was lower than that of the control group by 2. CCK-8 method, and the result of flow cytometry analysis showed PSC-S N. The ratio of AnnexinV-FITCPI double staining cells in group PSC-SN was higher than that in the control group. The results of GSIS test showed that the concentration of insulin release in PSC-SN group was lower than that of the control group. The content of P62 protein in PSC-SN group was lower than that of the control group, and the ratio of LC3I II / I was higher than that of the control group. The percentage of LC3I II / I ratio was higher than that of the control group; the percentage of the viability of the islet P cells in the MIN-6 mice of the PSP-PSC-S N group was higher than that of the control group. C-SN group, flow cytometry analysis showed that the proportion of AnnexinV-FITCPI double staining cells in group PSP-PSC-SN was lower than that of PSC-S N group. The results of GSIS test showed that the concentration of glucose stimulated insulin release in PSP-PSC-SN group was higher than that of PSC-SN group, WB detection PSP-PSC-SN group P62 protein content was higher than that of control group, LC3 II / I ratio was lower than that of control group, and the ratio of LC3 II / I was lower than that of the control group; 4. The percentage of P cell viability of MIN-6 mice in group P+PSC-SN was higher than that in group PSC-SN. The result of flow cytometry analysis showed that the proportion of AnnexinV-FITCPI double stained cells in group PSP+PSC-SN was lower than that of PSC-SN group. The result of GSIS test showed that the concentration of insulin release in PSP+PSC-SN group was higher than that of PSC-SN group, and WB detection of PSP+PSC-SN group protein content was higher than that of control group. The ratio of LC3 II / I was lower than that in the control group. Conclusion in a certain concentration range, PSP/reg concentration depended on the survival of MIN-6 cells, insulin release and the autophagy level of MIN-6 cells. Excessive concentration of PSP/reg could lead to excessive autophagy of MIN-6 cells, inhibit the survival of MIN-6 cells and release insulin, and P SC-SN. Inhibition of MIN-6 cell proliferation and insulin release, promoting apoptosis of MIN-6 cells, and inducing hyperautophagy of MIN-6 cells; PSP/reg promotes the survival and insulin release of PSC-SN interfered MIN-6 cells, reduces PSC-SN induced MIN-6 apoptosis and excessive autophagy; furthermore, PSC-SN (PSP-PSC-SN) prognosis of PSP/reg dry to MIN-6 activity and insulin release Inhibition, promote the effect of MIN-6 apoptosis and excessive autophagy. In summary, PSP/reg itself can increase the activity and function of islet P cells, and reduce the damage of PSC-SN to islet P cells. In addition, PSP/reg can act on PSC, inhibit the energy of islet P cell damage and indirectly protect the islet P cells.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.1

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