本文關(guān)鍵詞：胰腺微環(huán)境下間充質(zhì)干細(xì)胞分化為胰島樣細(xì)胞的機(jī)制初探　出處：《暨南大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 糖尿病 移植 胰腺微環(huán)境 胰島素 分布 分化 機(jī)制

【摘要】： 目的骨髓間充質(zhì)干細(xì)胞(BMSCs)植入胰腺微環(huán)境下,觀察對(duì)糖尿病模型鼠的影響,植入后細(xì)胞的再分布,初步探討植入的BMSCs分化為胰島樣細(xì)胞的機(jī)制。 方法從同種異體SD大鼠骨髓中獲取BMSCs,純化后用逆轉(zhuǎn)錄病毒(RV)轉(zhuǎn)染綠色熒光蛋白(EGFP)標(biāo)記,以多點(diǎn)注射的方法移植入糖尿病大鼠胰腺包膜下。移植后動(dòng)態(tài)監(jiān)測(cè)血糖、胰島素和C-肽變化;通過(guò)EGFP示蹤觀察植入的BMSCs的分布;移植后1、2、3、5、8及12周,取EGFP標(biāo)記的胰腺組織,Realtime-PCR檢測(cè)PDX-1、ngn 3、nestin、Nkx 2.2、pax 4和EGFP的表達(dá);移植后8周取EGFP標(biāo)記的胰腺組織,免疫熒光染色和熒光原位雜交檢測(cè)檢測(cè)EGFP和胰島素、EGFP和PDX-1的共表達(dá),流式細(xì)胞術(shù)分析EGFP標(biāo)記的胰腺組織細(xì)胞DNA倍性。 結(jié)果移植6d后糖尿病鼠血糖開(kāi)始降低,24d后接近于正常鼠水平(150±42.0mg/dL);移植14d后胰島素、C肽水平開(kāi)始升高,到56d穩(wěn)定在較高水平(1.0±0.2μg/L,1.2±0.3 nmol/L)。移植8周,植入細(xì)胞穩(wěn)定表達(dá)EGFP,植入細(xì)胞在胰腺組織分布于胰島(12.46%)、血管(4.4%)、導(dǎo)管(3.21%)、腺泡(9.24%)和組織間隙(70.69%)。免疫熒光染色發(fā)現(xiàn)有EGFP和胰島素共表達(dá)細(xì)胞(5.1696),FISH發(fā)現(xiàn)有EGFP和PDX-1 mRNA共表達(dá)細(xì)胞(0.96%);移植到胰腺微環(huán)境中的BMSCs,1周時(shí)檢測(cè)到PDX-1、ngn 3、nestin、Nkx 2.2和pax 4的表達(dá),2到3周時(shí)各基因表達(dá)水平持續(xù)上調(diào),5周左右達(dá)到最大值,以后逐漸下調(diào),12周時(shí)nestin、ngn 3、pax 4不表達(dá)。流式細(xì)胞術(shù)測(cè)定EGFP標(biāo)記的胰腺組織中細(xì)胞核型為整二倍體或四倍體,未見(jiàn)多倍體或非整倍體細(xì)胞。 結(jié)論BMSCs胰腺包膜下移植后,糖尿病大鼠血糖降低、胰島素和C肽水平升高:BMSCs植入后在胰腺中發(fā)生再分布;胰腺微環(huán)境下,BMSCs能跨胚層分化為胰島樣細(xì)胞,基因表達(dá)符合β細(xì)胞分化的時(shí)序性變化,沒(méi)有與胰腺組織細(xì)胞發(fā)生融合。
[Abstract]:Objective to observe the effect of bone marrow mesenchymal stem cells (BMSCs) implanted into pancreatic microenvironment on diabetic model rats and the redistribution of cells after implantation. To explore the mechanism of the differentiation of implanted BMSCs into islet-like cells. Methods BMSCs were obtained from allogeneic SD rat bone marrow and then transfected with green fluorescent protein (EGFP) labeled with retrovirus RV. The pancreatic capsule was transplanted into diabetic rats by multi-point injection. The changes of blood glucose, insulin and C-peptide were monitored dynamically after transplantation. The distribution of implanted BMSCs was observed by EGFP tracer. At 8 and 12 weeks after transplantation, PDX-1ngn 3 ngn 3 nestin was detected in pancreatic tissue labeled with EGFP. The expression of Nkx 2.2 pPAX4 and EGFP; The pancreatic tissues labeled with EGFP were taken 8 weeks after transplantation. The co-expression of EGFP, insulin and PDX-1 were detected by immunofluorescence staining and fluorescence in situ hybridization. DNA ploidy of pancreatic tissue cells labeled with EGFP was analyzed by flow cytometry. Results after 6 days of transplantation, the blood glucose level of diabetic rats began to decrease after 24 days and was close to that of normal mice at 150 鹵42.0 mg / d L ~ (-1). After 14 days of transplantation, the level of insulin C peptide began to increase, and at 56 days it was stable at a higher level of 1.0 鹵0.2 渭 g / L, 1.2 鹵0.3 nmol / L 路L ~ (-1) 路L ~ (-1), and was transplanted for 8 weeks. EGFP was expressed stably in the implanted cells. The implanted cells were distributed in the pancreatic islets (12.46%), and the vascular vessels were 4.4%, and the ducts were 3.21% (P < 0.05). EGFP and insulin co-expression cells were found by immunofluorescence staining. EGFP and PDX-1 mRNA co-expressed cells were found in FISH. The expression of Nkx2.2 and pax _ 4 was detected at 1 week after transplantation into pancreatic microenvironment. After 2 to 3 weeks, the expression level of each gene continued to up-regulate and reached the maximum at about 5 weeks, and then gradually down-regulated nestinine ngn 3 at 12 weeks. Pax _ 4 was not expressed. The nuclear type of pancreatic tissue labeled with EGFP was determined by flow cytometry to be diploid or tetraploid, but no polyploid or aneuploidy cells were found. Conclusion after subcapsular transplantation of BMSCs, blood glucose in diabetic rats decreased, insulin and C-peptide levels increased and redistributed in pancreas after implantation. BMSCs could differentiate into islet like cells in pancreatic microenvironment. The gene expression was consistent with the temporal changes of 尾 -cell differentiation and did not fuse with pancreatic tissue cells.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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本文編號(hào)：1380315