【摘要】： 目的：胰島細(xì)胞移植是治療Ⅰ型糖尿病最有希望的方法,由于供體細(xì)胞不足和免疫排斥反應(yīng)等問題,限制了胰島細(xì)胞移植技術(shù)的應(yīng)用。骨髓基質(zhì)干細(xì)胞(BMSCs)具有獲得容易,可體外復(fù)制和增殖,可以定向誘導(dǎo)分化為特定功能細(xì)胞的特征。因此,將BMSCs移植用于治療糖尿病具有重要的臨床應(yīng)用價(jià)值。目前,尚未獲得誘導(dǎo)干細(xì)胞向胰島細(xì)胞分化的高效誘導(dǎo)方法；取得較好治療效果的干細(xì)胞移植途徑尚未確定。本實(shí)驗(yàn)主要探索體外誘導(dǎo)BMSCs分化為胰島素分泌細(xì)胞的最佳條件；比較尾靜脈、腎被膜和胰腺內(nèi)注射三種干細(xì)胞移植途徑對糖尿病模型動(dòng)物的治療作用,以篩選出干細(xì)胞治療糖尿病的最佳移植途徑。 方法：全骨髓貼壁法分離大鼠BMSCs.體外擴(kuò)增后,部分細(xì)胞用于體外誘導(dǎo)實(shí)驗(yàn),即向培養(yǎng)體系中添加胰腺提取液、尼克酰胺、Exendin-4及不同濃度的葡萄糖；觀察分化細(xì)胞的形態(tài)變化；雙硫腙染色法(DTZ)鑒定胰島素分化細(xì)胞的生成；放射免疫法(RIA)檢測上清液中胰島素和C—肽水平。另一部分細(xì)胞進(jìn)行BrdU標(biāo)記。將標(biāo)記的BMSCs,經(jīng)尾靜脈、腎被膜和胰腺內(nèi)注射三種途徑,移植入鏈脲佐菌素(STZ)制備糖尿病模型小鼠體內(nèi)。于移植后3、7、14、28、42天測定血糖；免疫組織化學(xué)法檢測移植細(xì)胞在胰腺中的遷移和分化；放射免疫學(xué)方法監(jiān)測胰島素及C-肽水平。結(jié)果：(1)在高糖或低糖培養(yǎng)基中加入誘導(dǎo)因子尼克酰胺、Exendin-4,細(xì)胞趨向形成細(xì)胞團(tuán),高糖培養(yǎng)條件下細(xì)胞成團(tuán)趨勢更明顯。添加鼠胰腺提取液,細(xì)胞內(nèi)顆粒增多、細(xì)胞脫落、死亡。(2)誘導(dǎo)組大部分細(xì)胞雙硫腙染色呈棕紅色,對照組細(xì)胞無陽性著色。(3)誘導(dǎo)組細(xì)胞經(jīng)葡萄糖刺激后,能夠分泌胰島素和C-肽。(4)腹腔注射STZ3d后,小鼠血糖均≥16.7mmol/L,出現(xiàn)明顯多飲、多尿等癥狀,確定糖尿病模型造模成功。(5)尾靜脈、腎被膜及胰腺內(nèi)移植組與糖尿病模型對照組相比,血糖均有所降低,胰島素及C-肽均有所增加(P0.05),尤以胰腺移植組血糖下降、胰島素和C-肽升高更為顯著。(6)尾靜脈和胰腺內(nèi)移植組的胰腺內(nèi)可見BrdU陽性標(biāo)記細(xì)胞。腎被膜移植組的小鼠腎臟中有BrdU標(biāo)記的細(xì)胞,胰腺未見BrdU陽性表達(dá)的細(xì)胞。 結(jié)論：(1)在尼克酰胺、Exendin-4聯(lián)合誘導(dǎo)下,BMSCs可分化為胰島樣細(xì)胞,并且高糖培養(yǎng)基的效果要優(yōu)于低糖培養(yǎng)基。(2)三種移植途徑均可降低糖尿病模型小鼠的血糖,提高血液中胰島素和C-肽的水平。其中胰腺內(nèi)移植組的效果更明顯。
[Abstract]:Objective: islet cell transplantation is the most promising method for the treatment of type 1 diabetes mellitus. The application of islet cell transplantation is limited due to donor cell insufficiency and immune rejection. Bone marrow stromal cells (BMSCs) (BMSCs) is easy to obtain, can be replicated and proliferated in vitro, and can be induced to differentiate into specific functional cells. Therefore, BMSCs transplantation has important clinical value in the treatment of diabetes. At present, no efficient induction method has been obtained to induce stem cells to differentiate into islet cells, and the way of stem cell transplantation to obtain better therapeutic effect has not been determined. The aim of this study was to investigate the optimal conditions for inducing BMSCs to differentiate into insulin-secreting cells in vitro, and to compare the therapeutic effects of three kinds of stem cell transplantation pathways: caudal vein, renal capsule and intrapancreatic injection on diabetic model animals. In order to screen the best transplantation of stem cells to treat diabetes. Methods: rat BMSCs. was isolated by whole bone marrow adherent method. After amplification in vitro, some cells were used in the induction experiment in vitro, that is, adding pancreatic extract, nicotinamide Exendin-4 and different concentrations of glucose to the culture system to observe the morphological changes of differentiated cells. Dithizone staining (DTZ) was used to identify insulin differentiation cells and radioimmunoassay (RIA) was used to detect the levels of insulin and C-peptide in supernatant. Another part of the cells were labeled with BrdU. Streptozotocin (STZ) was transplanted into streptozotocin (STZ) into diabetic mice by injecting labeled BMSCs, through tail vein, renal capsule and pancreas. The levels of insulin and C-peptide were detected by radioimmunoassay and the migration and differentiation of transplanted cells in pancreas were detected by immunohistochemistry. Results: (1) the cells tended to form cell clusters in high sugar or low sugar medium, and the tendency of cell cluster formation was more obvious under high glucose culture condition. (2) most of the cells in the induction group showed brown red dithizone staining, while no positive staining was found in the control group. (3) the cells in the induction group were stimulated by glucose. (4) after intraperitoneal injection of STZ3d, the blood glucose of mice was more than 16.7 mmol / L, with obvious symptoms of polyhydration and polyuria. (5) the model of diabetes was successfully established. (5) the tail vein, renal capsule and intrapancreatic transplantation group were compared with the diabetic model control group. Blood glucose was decreased, insulin and C-peptide were increased (P0.05), especially in pancreas transplantation group. (6) in caudal vein and intrapancreatic transplantation group, BrdU positive labeled cells were found in pancreas. There were BrdU labeled cells in kidney and no BrdU positive cells in pancreas. Conclusion: (1) BMSCs can differentiate into islet like cells induced by nicotinamide Exendin-4, and the effect of high glucose medium is better than that of low glucose medium. Increase the levels of insulin and C-peptide in the blood. The effect of intrapancreatic transplantation was more obvious.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329.28

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 李薇;攜帶Ubc9基因的病毒表達(dá)載體的構(gòu)建及其在細(xì)胞中的表達(dá)[D];青島大學(xué);2011年

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本文編號：2214811