發(fā)布時(shí)間：2018-11-15 09:09

【摘要】： 目的:糖尿病是一種嚴(yán)重危害人們健康的慢性疾病。胰島細(xì)胞移植可能是不依賴(lài)胰島素治愈糖尿病的最佳方法。但由于移植材料匱乏,這種治療方法的推廣受到了限制。目前研究表明多種來(lái)源干細(xì)胞可以通過(guò)體外誘導(dǎo)分化為胰島素分泌細(xì)胞,這為胰島細(xì)胞移植提供了新的來(lái)源。因骨髓干細(xì)胞具有多種分化潛能、取材容易、可以避免免疫排斥反應(yīng)等優(yōu)點(diǎn),使其成為誘導(dǎo)分化胰島細(xì)胞的最佳候選干細(xì)胞。有研究顯示骨髓干細(xì)胞在體外可以誘導(dǎo)分化為胰島素分泌細(xì)胞。各種誘導(dǎo)方法雖然不盡相同,但總體上都比較繁瑣,而且大多都應(yīng)用細(xì)胞因子。這大大地限制了骨髓干細(xì)胞作為胰島細(xì)胞移植的來(lái)源在臨床上的廣泛應(yīng)用。本實(shí)驗(yàn)嘗試在體外通過(guò)簡(jiǎn)單的非細(xì)胞因子方法誘導(dǎo)大鼠骨髓干細(xì)胞,使其分化為胰島素分泌細(xì)胞,并應(yīng)用一系列方法對(duì)誘導(dǎo)的細(xì)胞進(jìn)行檢測(cè),目的在于探索一種不應(yīng)用任何細(xì)胞因子,簡(jiǎn)單易行的誘導(dǎo)方法,從而為骨髓干細(xì)胞作為胰島細(xì)胞移植的來(lái)源在臨床上實(shí)際應(yīng)用提供進(jìn)一步的理論依據(jù)及新思路。 方法:1、制備鼠尾膠原及鋪鼠尾膠原的玻片及六孔板。2、分離大鼠骨髓細(xì)胞。3、將分離的骨髓細(xì)胞分別設(shè)為DMSO及高糖環(huán)境培養(yǎng)的誘導(dǎo)組和低糖環(huán)境培養(yǎng)的對(duì)照組。4、通過(guò)倒置顯微鏡觀察誘導(dǎo)組和對(duì)照組細(xì)胞形態(tài)的改變。5、通過(guò)雙硫腙染色的方法檢測(cè)誘導(dǎo)組及對(duì)照組細(xì)胞胰島素的合成。6、通過(guò)免疫組化的方法檢測(cè)誘導(dǎo)組及對(duì)照組細(xì)胞在蛋白質(zhì)水平上胰島素的表達(dá)。7、通過(guò)RT-PCR的方法檢測(cè)誘導(dǎo)組及對(duì)照組細(xì)胞在mRNA水平上胰島素的表達(dá)。8、通過(guò)ELISA的方法檢測(cè)誘導(dǎo)組及對(duì)照組細(xì)胞分泌到細(xì)胞外的胰島素含量。 結(jié)果:1、誘導(dǎo)組形成的細(xì)胞團(tuán)在形態(tài)上與胰島十分相似,與對(duì)照組形成的細(xì)胞團(tuán)明顯不同。2、用雙硫腙對(duì)誘導(dǎo)組和對(duì)照組的細(xì)胞團(tuán)進(jìn)行染色,誘導(dǎo)組的細(xì)胞團(tuán)呈現(xiàn)猩紅色,表明細(xì)胞中有鋅離子,提示有胰島素的合成,而對(duì)照組不顯色。3、免疫組化染色結(jié)果顯示誘導(dǎo)組除團(tuán)樣細(xì)胞胞漿中有胰島素外,圓形、橢圓形和部分梭形的分散的單個(gè)細(xì)胞中也含有胰島素。對(duì)照組的細(xì)胞中無(wú)胰島素存在。4、RT-PCR結(jié)果顯示在mRNA水平上誘導(dǎo)組細(xì)胞表達(dá)Ins1和Ins2,而對(duì)照組不表達(dá)。5、ELISA方法檢測(cè)誘導(dǎo)組細(xì)胞分泌到培養(yǎng)液上清中的胰島素的量平均為(2.01±0.77)μg/L,而對(duì)照組為(0.63±0.43)μg/L。兩者比較有顯著性差異(P0.05)。 結(jié)論:用二甲基亞砜和高糖環(huán)境的方法誘導(dǎo)骨髓干細(xì)胞,能夠形成在形態(tài)上與胰島十分相似的細(xì)胞團(tuán)。誘導(dǎo)組細(xì)胞不僅在mRNA水平及在蛋白水平表達(dá)胰島素,而且能將其分泌到細(xì)胞外,提示二甲基亞砜和高糖環(huán)境能誘導(dǎo)骨髓干細(xì)胞分化為胰島素分泌細(xì)胞。
[Abstract]:Objective: diabetes mellitus is a chronic disease that seriously endangers people's health. Islet cell transplantation may be the best way to cure diabetes without insulin dependence. However, due to the lack of transplantation materials, the promotion of this treatment is limited. Recent studies have shown that many stem cells can differentiate into insulin-secreting cells through in vitro induction, which provides a new source for islet cell transplantation. Bone marrow stem cells are the best candidate stem cells for inducing pancreatic islet cells because they have many potential of differentiation and can avoid immune rejection. Studies have shown that bone marrow stem cells can be induced to differentiate into insulin-secreting cells in vitro. Although all kinds of induction methods are not the same, they are more complicated in general, and most of them use cytokines. This greatly limits the clinical application of bone marrow stem cells as a source of islet cell transplantation. In this study, we tried to induce rat bone marrow stem cells to differentiate into insulin secreting cells by a simple non-cytokine method in vitro, and a series of methods were used to detect the induced cells. The aim of this study was to explore a simple and easy induction method without any cytokines, so as to provide further theoretical basis and new ideas for the clinical application of bone marrow stem cells as the source of islet cell transplantation. Methods: 1. The vitreous plates and six-hole plates of rat tail collagen and rat tail collagen were prepared. 2Rat bone marrow cells were isolated. 3. The isolated bone marrow cells were divided into DMSO and high glucose culture induction group and low glucose environment culture control group respectively. The morphologic changes of cells in induction group and control group were observed by inverted microscope, and insulin synthesis in induced group and control group was detected by dithizone staining. The expression of insulin at the protein level was detected by immunohistochemical method, and the expression of insulin on the mRNA level was detected by RT-PCR method in the cells of the induced group and the control group, and the expression of insulin in the cells of the induced group and the control group at the level of mRNA was detected by the method of RT-PCR. The levels of extracellular insulin secreted into the cells of the induction group and the control group were detected by ELISA. Results: 1. The cell clusters formed in the induced group were very similar to the pancreatic islets in morphology, and were obviously different from those in the control group. 2. Dithizone was used to stain the cell clusters of the induced group and the control group, and the cells in the induced group were scarlet red. The results showed that there was zinc ion in the cells, indicating the synthesis of insulin, but not in the control group. The results of immunohistochemical staining showed that the cells in the induction group were round except for insulin in the cytoplasm of clusterlike cells. The elliptical and partially fusiform scattered individual cells also contain insulin. The results of RT-PCR showed that the expression of Ins1 and Ins2, was induced at the mRNA level in the control group, but not in the control group. The average amount of insulin secreted into the supernatant by ELISA was (2.01 鹵0.77) 渭 g / L in the induced group and (0.63 鹵0.43) 渭 g / L in the control group. There was significant difference between the two groups (P0.05). Conclusion: bone marrow stem cells induced by dimethyl sulfoxide and high glucose environment can form cell clusters similar to pancreatic islets in morphology. The induction group not only expressed insulin at the level of mRNA and protein, but also secreted it into extracellular, suggesting that dimethyl sulfoxide and high glucose environment could induce bone marrow stem cells to differentiate into insulin secreting cells.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R329.2

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