胰腺干细胞及转分化过程的免疫学特性研究

发布时间：2018-09-09 13:22

【摘要】： 目的：分离、纯化、扩增大鼠胰腺导管上皮细胞，在体外诱导转分化为胰岛样细胞，分离、纯化大鼠胰岛，比较胰腺干细胞、转分化胰岛样细胞与天然胰岛的免疫学的特性，探讨胰腺干细胞在临床应用中免疫学问题，为干细胞临床应用打下基础。方法：(1)分离消化SD大鼠胰腺，用差异性贴壁、低血清培养等方法纯化出大鼠胰腺导管上皮细胞，通过含KGF、EGF、ITS等细胞因子的基础培养基使其有效扩增，观察细胞增殖能力，并绘制生长曲线，免疫细胞化学染色法鉴定其表面标志CK-19的表达。当细胞扩增、融合至80％—90％时，改变细胞培养方案，通过含有exendin-4、KGF、NIC等细胞因子和葡萄糖的培养基，使胰腺导管上皮细胞转分化胰岛样细胞。(2)观察细胞生长状态并摄片。对转分化4周后的胰岛样细胞进行DTZ染色鉴定。(3)分离纯化SD大鼠天然胰岛，DTZ染色鉴定。(4)分离SD大鼠外周血淋巴细胞，分别地与胰腺干细胞、转分化的胰岛样细胞、天然胰岛细胞混合培养，用ELISA检测培养上清中IL-2的水平，Elispot检测IFN-γ的分泌水平，流式细胞术检测共培养后的淋巴细胞的MHC-Ⅰ和MHC-Ⅱ的表达。(5)把胰腺干细胞、转分化的胰岛样细胞、天然胰岛细胞分别注入大鼠大腿内侧肌，5d后处死，做病理切片，HE染色，观其炎症反应。(6)制备1型糖尿病大鼠模型，将胰腺干细胞、转分化的胰岛样细胞、天然胰岛细胞移植到糖尿病大鼠模型皮下，分别在1d、3d、5d、7d采取大鼠未静脉血，流式细胞术检测MHC-Ⅱ的表达和ELISA检测血清中IFN-γ的水平。结果：(1)成功分离、纯化了大鼠胰腺导管上皮细胞，使其有效扩增，并有效抑制了胰岛细胞和成纤维细胞的污染，纯化后大部分细胞CK-19(胰腺导管上皮细胞的标志)表达阳性。(2)诱导后细胞逐渐增大、变形，并出现类圆形小细胞，聚集成团，呈圆形或椭圆形的胰岛样团状结构，细胞团慢慢增大、增多，4周后细胞团DTZ染色阳性。(3)分离纯化的大鼠天然胰岛，DTZ染色阳性。(4)胰腺干细胞、转分化的胰岛样细胞、天然胰岛细胞分别与淋巴细胞共培养5d，上清中IL-2的水平分别为44.1pg／ml、230.7pg／ml、317.6pg／ml，Elispot检测IFN-γ斑点依次增多，MHC-Ⅰ的表达分别为15.0％、16.7％、17.9％，MHC-Ⅱ的表达分别为0.8％、转分化胰岛样细胞和天然胰岛细胞周围炎症反应依次加强。(6)糖尿病大鼠模型造模成功，移植后胰腺导管上皮细胞组MHC-Ⅱ表达未见差异，，而转分化的胰岛样细胞组和天然胰岛组，MHC-Ⅱ的表达随时间延长而增加。血清中IFN-γ的水平，转分化的胰岛样细胞组和天然胰岛细胞组较高胰腺干细胞组显著升高(P＜0.05)。结论：大鼠胰腺导管上皮细胞在体外可得到有效扩增并具有干细胞潜能，与天然胰岛细胞相比较，大鼠胰腺干细胞免疫原性较低，转分化的胰岛样细胞免疫原性逐渐升高，表明胰腺干细胞在转分化为胰岛细胞过程中，免疫原性逐渐增强，为探讨治疗性干细胞免疫应答提供了依据。
[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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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