本文選題：胰腺干細(xì)胞　切入點(diǎn)：誘導(dǎo)分化　出處：《廣西醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:優(yōu)化胰腺干細(xì)胞原位傳代擴(kuò)增培養(yǎng)方法和技術(shù),建立并形成穩(wěn)定的胰腺干細(xì)胞系,獲得較大量胰腺干細(xì)胞并體外誘導(dǎo)分化為胰島樣結(jié)構(gòu)。 方法:優(yōu)化原位傳代擴(kuò)增培養(yǎng)的方法對(duì)胰腺干細(xì)胞進(jìn)行擴(kuò)增,建立胰腺干細(xì)胞系。選擇生長(zhǎng)狀態(tài)良好的的第二代胰腺干細(xì)胞分為四組:空白對(duì)照組、葡萄糖組、尼克酰胺組、血清尼克酰胺組,體外連續(xù)誘導(dǎo)培養(yǎng)4周,觀察胰腺干細(xì)胞的形態(tài)學(xué)特征變化規(guī)律;以DTZ染色、Mallory染色對(duì)誘導(dǎo)后獲得的胰島樣結(jié)構(gòu)行進(jìn)行初步鑒定;胰島素抗體免疫熒光組織化學(xué)染色方法,檢測(cè)所誘導(dǎo)的胰島樣結(jié)構(gòu)中的胰島素陽(yáng)性細(xì)胞。 結(jié)果:空白對(duì)照組:胰腺干細(xì)胞隨著培養(yǎng)時(shí)間的延長(zhǎng),細(xì)胞形態(tài)及生長(zhǎng)方式發(fā)生改變,由原來(lái)的圓形、立體感強(qiáng)變?yōu)楸馄�、折光性降低、立體感減弱;葡萄糖誘導(dǎo)組:誘導(dǎo)9天后,胰腺干細(xì)胞聚集,成串樣生長(zhǎng),誘導(dǎo)20天后,細(xì)胞變扁平,未形成胰島樣結(jié)構(gòu);尼克酰胺誘導(dǎo)組:誘導(dǎo)5天后,胰腺干細(xì)胞逐漸靠攏,誘導(dǎo)25天后,胰腺干細(xì)胞形成橢圓形結(jié)構(gòu);血清尼克酰胺組:誘導(dǎo)1周后,胰腺干細(xì)胞逐漸靠攏,匯集成團(tuán),誘導(dǎo)2周后,匯集的細(xì)胞團(tuán)增大,但未形成圓形或者橢圓形的結(jié)構(gòu),誘導(dǎo)3周后,細(xì)胞團(tuán)逐漸形成有完整包膜包裹的胰島樣結(jié)構(gòu);胰島樣結(jié)構(gòu)DTZ染色,呈棕紅色;Mallory染色可見(jiàn)胰島樣結(jié)構(gòu)中周?chē)募?xì)胞呈紅色,中央的細(xì)胞呈淡黃色;收集胰島樣結(jié)構(gòu),制作石蠟切片后行Mallory染色,可見(jiàn)有橘黃色、紅色兩種細(xì)胞;胰島素抗體免疫熒光組織化學(xué)染色,在紫外光線(xiàn)激發(fā)下,檢測(cè)到呈綠色熒光的胰島素陽(yáng)性細(xì)胞。 結(jié)論:(1)優(yōu)化后的原位傳代擴(kuò)增培養(yǎng)方法,可擴(kuò)增并建立胰腺干細(xì)胞系,獲得較多數(shù)量的胰腺干細(xì)胞,滿(mǎn)足后續(xù)實(shí)驗(yàn)的需要。(2)在血清尼克酰胺誘導(dǎo)條件下,胰腺干細(xì)胞可分化為有完整包膜包裹、DTZ染色陽(yáng)性、Mallory染色可見(jiàn)兩種細(xì)胞、胰島素抗體免疫熒光組織化學(xué)染色檢測(cè)到陽(yáng)性細(xì)胞的胰島樣結(jié)構(gòu)。
[Abstract]:Aim: to optimize the method and technique of in situ culture of pancreatic stem cells, to establish and form stable pancreatic stem cell lines, to obtain a large number of pancreatic stem cells and to induce and differentiate into islet like structures in vitro. Methods: pancreatic stem cell lines were established by optimizing the method of in situ passage and culture. The second generation pancreatic stem cells, which grew well, were divided into four groups: blank control group, glucose group and nicotinamide group. Serum nicotinamide group was cultured for 4 weeks in vitro to observe the morphological characteristics of pancreatic stem cells, and the islet like structure was preliminarily identified by DTZ staining. Insulin positive cells in islet-like structure were detected by immunofluorescence histochemical staining. Results: in the blank control group, with the extension of culture time, the morphology and growth pattern of pancreatic stem cells changed from round, strong stereosensory to flat, reduced refraction and reduced stereosensory. Glucose-induced group: after 9 days of induction, pancreatic stem cells gathered and grew in strands. After 20 days of induction, the cells flattened and did not form islet structure, while in nicotinamide group, pancreatic stem cells gradually converged after 5 days of induction. After 25 days of induction, pancreatic stem cells formed oval structure, serum-nicotinamide group: after 1 week of induction, pancreatic stem cells gradually converged to form clusters, and after 2 weeks of induction, the aggregation of pancreatic stem cells increased. However, the round or oval structure was not formed. After 3 weeks of induction, the islet like structure was gradually formed, and the DTZ staining of islet like structure showed that the cells around the islet like structure were red. The cells in the center were yellowish; the islet-like structures were collected, and the paraffin sections were made with Mallory staining. There were two kinds of cells, orange and red; insulin antibodies were stained by immunofluorescence histochemistry, stimulated by ultraviolet rays. Insulin positive cells with green fluorescence were detected. Conclusion the optimized in situ culture method can amplify and establish pancreatic stem cell lines and obtain a large number of pancreatic stem cells to meet the needs of subsequent experiments. Under the condition of serum nicotinamide induction, a large number of pancreatic stem cells can be obtained. Pancreatic stem cells could be differentiated into two kinds of cells with intact encapsulated DTZ positive staining. The islet like structure of the positive cells was detected by insulin antibody immunofluorescence histochemical staining.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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