本文選題：羊水 + 干細胞��； 參考：《吉林大學(xué)》2012年博士論文

【摘要】：糖尿病是由多種病因引起的以慢性高血糖為特征的代謝性疾病，嚴重危害著人類健康。目前胰島素注射是臨床上治療糖尿病的主要方法，可以短期內(nèi)控制血糖。不足之處是需要頻繁監(jiān)測血糖及胰島素注射，而且不能阻止糖尿病所引起的腎衰、心臟病變、眼底病變、壞疽等并發(fā)癥。近年來胰腺移植和胰島細胞移植治療糖尿病取得了很大進展，可有效改善患者內(nèi)源性胰島素的分泌，改善患者生活狀態(tài)。胰島移植最大的問題就是胰島供體來源不足和終身使用免疫抑制劑。目前解決細胞移植供體不足最有可能的就是干細胞。利用干細胞體外誘導(dǎo)分化為胰島素分泌細胞，不僅可以從根本上解決細胞來源問題，還可以克服免疫排斥問題。 羊水中存在著多潛能干細胞。人羊水干細胞易于獲取，可以從產(chǎn)前診斷的少量羊水中提�。灰部梢詮漠a(chǎn)后的羊水中分離。而且抽取羊水不會損害母體，也不會對胚胎造成影響，也就不存在倫理和道德上的問題。羊水中的細胞是一群處于胚胎發(fā)育早期的細胞群。從中分離的人羊水干細胞(amniotic fluid-derived stemcells, hAFS)細胞是介于胚胎干細胞(embryonic stem cells, ES)和成體干細胞之間的細胞類型，表達ES細胞和成體干細胞的標志，具有相似的多向分化潛能，可以分化為三個胚層的細胞。hAFS細胞體外比較容易培養(yǎng)，其增殖較快，動物體內(nèi)移植不會形成畸胎瘤，成為干細胞研究的熱點。 本實驗從羊水中分離hAFS細胞，以hAFS細胞為研究對象。研究hAFS細胞的生物學(xué)特性，以及多向分化潛能，重點研究其分化為胰島素分泌細胞的能力。建立及優(yōu)化hAFS細胞誘導(dǎo)分化的實驗方案，探討誘導(dǎo)后細胞的功能和活性，為其臨床應(yīng)用提供依據(jù)。 1. hAFS細胞的分離培養(yǎng)和生物學(xué)特性 從羊水中分離獲得hAFS細胞。通過流式細胞儀和免疫熒光測定，hAFS細胞具有ES細胞和間充質(zhì)干細胞(mesenchymal stem cells, MSCs)的標志，同時也首次檢測到SSEA-1在hAFS細胞中的表達。hAFS在體外可以大量擴增，具有較強的增殖能力。通過細胞周期和端粒酶測定，也證實細胞具有典型干細胞的特點。超微結(jié)構(gòu)發(fā)現(xiàn)細胞表面大量的微絨毛，胞內(nèi)富含豐富的細胞器，，說明獲得的hAFS細胞功能活躍，具有強蛋白合成的能力，維持自身的增殖和分化。 2. hAFS細胞的多向分化潛能 本實驗對hAFS向脂肪細胞、成骨細胞和神經(jīng)細胞三個方向進行誘導(dǎo)分化。通過油紅O染色、茜素紅染色、Von Kossa’s染色、RT-PCR和免疫組化等實驗證實了hAFS細胞在體外特定誘導(dǎo)條件下可以分化為脂肪細胞、成骨細胞和神經(jīng)細胞。 同時本實驗也分析了hAFS細胞的致瘤性。將第3代，第5代，第8代細胞以5×10~6、1×10~7、2×10~7個細胞注射BALB/C裸鼠皮下，觀察兩個月，未發(fā)現(xiàn)腫瘤的形成。說明hAFS細胞動物體內(nèi)移植不具有致瘤性，這也為臨床應(yīng)用提供可能。 3. hAFS細胞分化為胰島素分泌細胞 本實驗以胰腺體內(nèi)發(fā)育為依據(jù)，誘導(dǎo)hAFS細胞分化為胰島素分泌細胞。首先bFGF和尼克酰胺聯(lián)合誘導(dǎo)hAFS細胞分化為胰腺祖細胞。通過檢測，這些細胞表達胰腺前體細胞相關(guān)的轉(zhuǎn)錄因子，例如Pdx-1、Nng3、Pax4。之后加入EGF和exendin-4聯(lián)合誘導(dǎo)胰腺祖細胞分化為胰島素分泌細胞。EGF可以有效促進Pdx-1陽性的胰腺祖細胞增殖，exendin-4可促進細胞內(nèi)分泌方向分化。這些胰島素分泌細胞表達胰島相關(guān)的轉(zhuǎn)錄因子，如Pdx-1、Nkx6.1等，同時表達胰島功能相關(guān)的功能基因，如胰島素、葡萄糖轉(zhuǎn)運因子和葡萄糖激酶等。最后對這些胰島素分泌細胞進行葡萄糖刺激試驗，發(fā)現(xiàn)這些細胞對葡萄糖刺激敏感，根據(jù)葡萄糖濃度釋放相應(yīng)的胰島素。 總之，本實驗從羊水中分離得到一種新的hAFS細胞，hAFS細胞具有較強的增殖能力和多向分化潛能，體內(nèi)移植不具有致瘤性。同時高效誘導(dǎo)hAFS細胞分化為胰島素分泌細胞，這些細胞具有類似成人胰島的功能和體內(nèi)發(fā)育模式。這為糖尿病臨床治療提供重要的理論和實驗基礎(chǔ)。
[Abstract]:Diabetes is a metabolic disease characterized by a variety of causes of chronic hyperglycemia, which seriously endangers human health. At present, insulin injection is the main method for clinical treatment of diabetes, which can be used to control blood glucose in the short term. The deficiency is that frequent monitoring of blood glucose and islet injection can not be used to prevent diabetes. Renal failure, heart disease, fundus lesion, gangrene and other complications. In recent years, pancreatic transplantation and islet cell transplantation have made great progress in the treatment of diabetes, which can effectively improve the secretion of endogenous insulin and improve the patient's living condition. The biggest problem of islet transplantation is the lack of islet donor and the life-long use of immunosuppressive agents. The most likely to solve the deficiency of cell transplantation donor is stem cells. The use of stem cells to induce differentiation into insulin secreting cells in vitro can not only solve the problem of cell origin fundamentally, but also overcome the problem of immune rejection.
There are pluripotent stem cells in amniotic fluid. Human amniotic fluid stem cells are easy to obtain, can be extracted from a small amount of amniotic fluid diagnosed by prenatal, and can be separated from postpartum amniotic fluid. And amniotic fluid extraction does not damage the mother body, does not affect the embryo, and there is no ethical and moral problems. The cells in the amniotic fluid are in a group. Amniotic fluid-derived stemcells (hAFS) cells are the cell types between embryonic stem cells (embryonic stem cells, ES) and adult stem cells, which represent the markers of ES and adult stem cells, which have similar multidirectional differentiation potential and can be divided into three. The.HAFS cells of the germ layer are easy to culture in vitro, and their proliferation is faster. The transplantation of animals will not form teratoma, which has become the focus of stem cell research.
In this experiment, the hAFS cells were isolated from amniotic fluid, and hAFS cells were used as the research object. The biological characteristics of hAFS cells and the potential of multidifferentiation were studied. The ability to differentiate into insulin secreting cells was studied. The experimental scheme of inducing and optimizing the induction of differentiation of hAFS cells was established and optimized to explore the function and activity of the induced cells to provide the clinical application. Basis.
Isolation, culture and biological characteristics of 1. hAFS cells
HAFS cells were isolated from amniotic fluid. By flow cytometry and immunofluorescence, hAFS cells had ES and mesenchymal stem cells, MSCs, and the expression of SSEA-1 in hAFS cells was also detected for the first time. The proliferation ability of.HAFS in vitro was stronger. The cell cycle was through cell cycle. The determination of telomerase and telomerase showed that the cells have the characteristics of typical stem cells. The ultrastructure found a large number of microvilli on the surface of the cells and rich organelles in the cell, indicating that the acquired hAFS cells have active function, strong protein synthesis ability, and maintain their own proliferation and differentiation.
Multidirectional differentiation potential of 2. hAFS cells
In this experiment, hAFS was induced to differentiate into adipocytes, osteoblasts and nerve cells in three directions. Through the oil red O staining, alizarin red staining, Von Kossa 's staining, RT-PCR and immunohistochemistry, it was proved that hAFS cells could be differentiated into adipocytes, osteoblasts and nerve cells under specific induction conditions in vitro.
At the same time, the tumorigenicity of hAFS cells was also analyzed. Third, fifth, and eighth generation cells were subcutaneously injected with 5 x 10~6,1 x 10~7,2 x 10~7 cells to BALB/C nude mice. It was observed for two months and no tumor formation was found. It showed that the transplantation of hAFS cells in vivo did not have the tumorigenicity, which also provided the possibility for clinical application.
3. hAFS cells differentiate into insulin secreting cells
This experiment, based on the development of the pancreas, induces hAFS cells to differentiate into insulin secreting cells. First, bFGF and nicotinamide are combined to induce hAFS cells to differentiate into pancreatic progenitor cells. Through detection, these cells express the transcription factors related to the precursor cells of the pancreas, such as Pdx-1, Nng3, and Pax4., and join the EGF and exendin-4 to induce the pancreas. The differentiation of progenitor cells into insulin secreting cells.EGF can effectively promote the proliferation of Pdx-1 positive pancreatic progenitor cells, and exendin-4 promotes the direction differentiation of cell endocrine cells. These insulin secreting cells express islet related transcription factors, such as Pdx-1, Nkx6.1, etc., and express the functional genes related to islet function, such as insulin, glucose transport Factors and glucokinase and so on. Finally, the glucose stimulation tests on these insulin secreting cells showed that these cells were sensitive to glucose stimulation and released the corresponding insulin according to the glucose concentration.
In conclusion, a new hAFS cell was isolated from amniotic fluid. HAFS cells have strong proliferation ability and multidirectional differentiation potential. In vivo transplantation does not have tumorigenicity. At the same time, hAFS cells are highly induced to differentiate into insulin secreting cells, which are similar to adult pancreatic islets and in vivo development patterns. Bed therapy provides an important theoretical and experimental basis.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro[J];Chinese Medical Journal;2007年09期

本文編號：1959233