【摘要】： 目的：分離、純化、擴(kuò)增大鼠胰腺導(dǎo)管上皮細(xì)胞，在體外誘導(dǎo)轉(zhuǎn)分化為胰島樣細(xì)胞，分離、純化大鼠胰島，比較胰腺干細(xì)胞、轉(zhuǎn)分化胰島樣細(xì)胞與天然胰島的免疫學(xué)的特性，探討胰腺干細(xì)胞在臨床應(yīng)用中免疫學(xué)問(wèn)題，為干細(xì)胞臨床應(yīng)用打下基礎(chǔ)。 方法：(1)分離消化SD大鼠胰腺，用差異性貼壁、低血清培養(yǎng)等方法純化出大鼠胰腺導(dǎo)管上皮細(xì)胞，通過(guò)含KGF、EGF、ITS等細(xì)胞因子的基礎(chǔ)培養(yǎng)基使其有效擴(kuò)增，觀察細(xì)胞增殖能力，并繪制生長(zhǎng)曲線，免疫細(xì)胞化學(xué)染色法鑒定其表面標(biāo)志CK-19的表達(dá)。當(dāng)細(xì)胞擴(kuò)增、融合至80％—90％時(shí)，改變細(xì)胞培養(yǎng)方案，通過(guò)含有exendin-4、KGF、NIC等細(xì)胞因子和葡萄糖的培養(yǎng)基，使胰腺導(dǎo)管上皮細(xì)胞轉(zhuǎn)分化胰島樣細(xì)胞。(2)觀察細(xì)胞生長(zhǎng)狀態(tài)并攝片。對(duì)轉(zhuǎn)分化4周后的胰島樣細(xì)胞進(jìn)行DTZ染色鑒定。(3)分離純化SD大鼠天然胰島，DTZ染色鑒定。(4)分離SD大鼠外周血淋巴細(xì)胞，分別地與胰腺干細(xì)胞、轉(zhuǎn)分化的胰島樣細(xì)胞、天然胰島細(xì)胞混合培養(yǎng)，用ELISA檢測(cè)培養(yǎng)上清中IL-2的水平，Elispot檢測(cè)IFN-γ的分泌水平，流式細(xì)胞術(shù)檢測(cè)共培養(yǎng)后的淋巴細(xì)胞的MHC-Ⅰ和MHC-Ⅱ的表達(dá)。(5)把胰腺干細(xì)胞、轉(zhuǎn)分化的胰島樣細(xì)胞、天然胰島細(xì)胞分別注入大鼠大腿內(nèi)側(cè)肌，5d后處死，做病理切片，HE染色，觀其炎癥反應(yīng)。(6)制備1型糖尿病大鼠模型，將胰腺干細(xì)胞、轉(zhuǎn)分化的胰島樣細(xì)胞、天然胰島細(xì)胞移植到糖尿病大鼠模型皮下，分別在1d、3d、5d、7d采取大鼠未靜脈血，流式細(xì)胞術(shù)檢測(cè)MHC-Ⅱ的表達(dá)和ELISA檢測(cè)血清中IFN-γ的水平。 結(jié)果：(1)成功分離、純化了大鼠胰腺導(dǎo)管上皮細(xì)胞，使其有效擴(kuò)增，并有效抑制了胰島細(xì)胞和成纖維細(xì)胞的污染，純化后大部分細(xì)胞CK-19(胰腺導(dǎo)管上皮細(xì)胞的標(biāo)志)表達(dá)陽(yáng)性。(2)誘導(dǎo)后細(xì)胞逐漸增大、變形，并出現(xiàn)類圓形小細(xì)胞，聚集成團(tuán)，呈圓形或橢圓形的胰島樣團(tuán)狀結(jié)構(gòu)，細(xì)胞團(tuán)慢慢增大、增多，4周后細(xì)胞團(tuán)DTZ染色陽(yáng)性。(3)分離純化的大鼠天然胰島，DTZ染色陽(yáng)性。(4)胰腺干細(xì)胞、轉(zhuǎn)分化的胰島樣細(xì)胞、天然胰島細(xì)胞分別與淋巴細(xì)胞共培養(yǎng)5d，上清中IL-2的水平分別為44.1pg／ml、230.7pg／ml、317.6pg／ml，Elispot檢測(cè)IFN-γ斑點(diǎn)依次增多，MHC-Ⅰ的表達(dá)分別為15.0％、16.7％、17.9％，MHC-Ⅱ的表達(dá)分別為0.8％、轉(zhuǎn)分化胰島樣細(xì)胞和天然胰島細(xì)胞周圍炎癥反應(yīng)依次加強(qiáng)。(6)糖尿病大鼠模型造模成功，移植后胰腺導(dǎo)管上皮細(xì)胞組MHC-Ⅱ表達(dá)未見(jiàn)差異，，而轉(zhuǎn)分化的胰島樣細(xì)胞組和天然胰島組，MHC-Ⅱ的表達(dá)隨時(shí)間延長(zhǎng)而增加。血清中IFN-γ的水平，轉(zhuǎn)分化的胰島樣細(xì)胞組和天然胰島細(xì)胞組較高胰腺干細(xì)胞組顯著升高(P＜0.05)。 結(jié)論：大鼠胰腺導(dǎo)管上皮細(xì)胞在體外可得到有效擴(kuò)增并具有干細(xì)胞潛能，與天然胰島細(xì)胞相比較，大鼠胰腺干細(xì)胞免疫原性較低，轉(zhuǎn)分化的胰島樣細(xì)胞免疫原性逐漸升高，表明胰腺干細(xì)胞在轉(zhuǎn)分化為胰島細(xì)胞過(guò)程中，免疫原性逐漸增強(qiáng)，為探討治療性干細(xì)胞免疫應(yīng)答提供了依據(jù)。
[Abstract]:AIM: To isolate, purify, enlarge rat pancreatic ductal epithelial cells, induce and transdifferentiate into islet-like cells in vitro, isolate and purify rat islets, compare the immunological characteristics of pancreatic stem cells, transdifferentiated islet-like cells and natural islets, and explore the immunological problems of pancreatic stem cells in clinical application. Basics.
Methods: (1) SD rat pancreas was isolated and digested, and the pancreatic ductal epithelial cells were purified by differential adherence and low serum culture. The cell proliferation was observed and the growth curve was drawn. The surface marker of CK-19 was identified by immunocytochemical staining. When the cells proliferated and fused to 80%-90%, the cell culture scheme was changed, and the pancreatic ductal epithelial cells were transdifferentiated into islet-like cells by means of the medium containing exendin-4, KGF, NIC and glucose. (2) The cell growth status was observed and the cells were taken. The islet-like cells were identified by DTZ staining after 4 weeks of transdifferentiation. Isolation and purification of natural islets of SD rats and identification by DTZ staining. (4) Isolation of peripheral blood lymphocytes from SD rats and mixed culture with pancreatic stem cells, transdifferentiated islet-like cells and natural islet cells respectively. The level of IL-2 in culture supernatant was detected by ELISA, the level of IFN-gamma secretion was detected by Elispot, and the co-cultured lymphocytes were detected by flow cytometry. Expression of MHC-I and MHC-II. (5) Pancreatic stem cells, transdifferentiated islet-like cells and natural islet cells were injected into the medial thigh muscles of rats respectively, and then sacrificed 5 days later. Pathological sections were made, HE staining was used to observe the inflammatory reaction. (6) The model of type 1 diabetic rats was established, and pancreatic stem cells, transdifferentiated islet-like cells and natural islet cells were transplanted into the rats. The expression of MHC-II and the level of IFN-gamma in serum were detected by flow cytometry and ELISA respectively.
Results: (1) The pancreatic ductal epithelial cells of rats were successfully isolated and purified, which effectively expanded and inhibited the contamination of islet cells and fibroblasts. Most of the purified cells were positive for CK-19 (a marker of pancreatic ductal epithelial cells). (3) The isolated and purified rat islets were positive for DTZ staining. (4) Pancreatic stem cells, transdifferentiated islet-like cells, natural islet cells and lymphocytes were co-cultured for 5 days, and the levels of IL-2 in the supernatant were 44.1 pg/m, respectively. The expression of MHC-I was 15.0%, 16.7%, 17.9%, and the expression of MHC-II was 0.8% respectively. The inflammation around the transdifferentiated islet-like cells and the natural islet cells was enhanced in turn. (6) The model of diabetic rats was successfully established. After transplantation, the expression of MHC-I in pancreatic ductal epithelial cells was increased in turn. The expression of MHC-II in the transdifferentiated islet-like cells and the natural islets increased with time. The levels of IFN-gamma in serum, the transdifferentiated islet-like cells and the natural islets were significantly higher than those in the pancreatic stem cells (P<0.05).
CONCLUSION: Rat pancreatic ductal epithelial cells can be effectively expanded in vitro and possess stem cell potential. Compared with natural pancreatic islet cells, the immunogenicity of rat pancreatic stem cells is lower, and the immunogenicity of transdifferentiated islet-like cells is gradually increased, which indicates that the immunogenicity of pancreatic stem cells is gradually enhanced during the process of transdifferentiation into islet cells. It provides a basis for exploring the therapeutic stem cell immune response.
【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】：2007
【分類號(hào)】：R392

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