本文選題：羊水 + 干細(xì)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(yán)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(cè)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(lái)胰腺移植和胰島細(xì)胞移植治療糖尿病取得了很大進(jìn)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(wèn)題就是胰島供體來(lái)源不足和終身使用免疫抑制劑。目前解決細(xì)胞移植供體不足最有可能的就是干細(xì)胞。利用干細(xì)胞體外誘導(dǎo)分化為胰島素分泌細(xì)胞，不僅可以從根本上解決細(xì)胞來(lái)源問(wèn)題，還可以克服免疫排斥問(wèn)題。 羊水中存在著多潛能干細(xì)胞。人羊水干細(xì)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(huì)損害母體，也不會(huì)對(duì)胚胎造成影響，也就不存在倫理和道德上的問(wèn)題。羊水中的細(xì)胞是一群處于胚胎發(fā)育早期的細(xì)胞群。從中分離的人羊水干細(xì)胞(amniotic fluid-derived stemcells, hAFS)細(xì)胞是介于胚胎干細(xì)胞(embryonic stem cells, ES)和成體干細(xì)胞之間的細(xì)胞類型，表達(dá)ES細(xì)胞和成體干細(xì)胞的標(biāo)志，具有相似的多向分化潛能，可以分化為三個(gè)胚層的細(xì)胞。hAFS細(xì)胞體外比較容易培養(yǎng)，其增殖較快，動(dòng)物體內(nèi)移植不會(huì)形成畸胎瘤，成為干細(xì)胞研究的熱點(diǎn)。 本實(shí)驗(yàn)從羊水中分離hAFS細(xì)胞，以hAFS細(xì)胞為研究對(duì)象。研究hAFS細(xì)胞的生物學(xué)特性，以及多向分化潛能，重點(diǎn)研究其分化為胰島素分泌細(xì)胞的能力。建立及優(yōu)化hAFS細(xì)胞誘導(dǎo)分化的實(shí)驗(yàn)方案，探討誘導(dǎo)后細(xì)胞的功能和活性，為其臨床應(yīng)用提供依據(jù)。 1. hAFS細(xì)胞的分離培養(yǎng)和生物學(xué)特性 從羊水中分離獲得hAFS細(xì)胞。通過(guò)流式細(xì)胞儀和免疫熒光測(cè)定，hAFS細(xì)胞具有ES細(xì)胞和間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)的標(biāo)志，同時(shí)也首次檢測(cè)到SSEA-1在hAFS細(xì)胞中的表達(dá)。hAFS在體外可以大量擴(kuò)增，具有較強(qiáng)的增殖能力。通過(guò)細(xì)胞周期和端粒酶測(cè)定，也證實(shí)細(xì)胞具有典型干細(xì)胞的特點(diǎn)。超微結(jié)構(gòu)發(fā)現(xiàn)細(xì)胞表面大量的微絨毛，胞內(nèi)富含豐富的細(xì)胞器，，說(shuō)明獲得的hAFS細(xì)胞功能活躍，具有強(qiáng)蛋白合成的能力，維持自身的增殖和分化。 2. hAFS細(xì)胞的多向分化潛能 本實(shí)驗(yàn)對(duì)hAFS向脂肪細(xì)胞、成骨細(xì)胞和神經(jīng)細(xì)胞三個(gè)方向進(jìn)行誘導(dǎo)分化。通過(guò)油紅O染色、茜素紅染色、Von Kossa’s染色、RT-PCR和免疫組化等實(shí)驗(yàn)證實(shí)了hAFS細(xì)胞在體外特定誘導(dǎo)條件下可以分化為脂肪細(xì)胞、成骨細(xì)胞和神經(jīng)細(xì)胞。 同時(shí)本實(shí)驗(yàn)也分析了hAFS細(xì)胞的致瘤性。將第3代，第5代，第8代細(xì)胞以5×10~6、1×10~7、2×10~7個(gè)細(xì)胞注射BALB/C裸鼠皮下，觀察兩個(gè)月，未發(fā)現(xiàn)腫瘤的形成。說(shuō)明hAFS細(xì)胞動(dòng)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(xì)胞分化為胰島素分泌細(xì)胞 本實(shí)驗(yàn)以胰腺體內(nèi)發(fā)育為依據(jù)，誘導(dǎo)hAFS細(xì)胞分化為胰島素分泌細(xì)胞。首先bFGF和尼克酰胺聯(lián)合誘導(dǎo)hAFS細(xì)胞分化為胰腺祖細(xì)胞。通過(guò)檢測(cè)，這些細(xì)胞表達(dá)胰腺前體細(xì)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(xì)胞分化為胰島素分泌細(xì)胞。EGF可以有效促進(jìn)Pdx-1陽(yáng)性的胰腺祖細(xì)胞增殖，exendin-4可促進(jìn)細(xì)胞內(nèi)分泌方向分化。這些胰島素分泌細(xì)胞表達(dá)胰島相關(guān)的轉(zhuǎn)錄因子，如Pdx-1、Nkx6.1等，同時(shí)表達(dá)胰島功能相關(guān)的功能基因，如胰島素、葡萄糖轉(zhuǎn)運(yùn)因子和葡萄糖激酶等。最后對(duì)這些胰島素分泌細(xì)胞進(jìn)行葡萄糖刺激試驗(yàn)，發(fā)現(xiàn)這些細(xì)胞對(duì)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(shí)驗(yàn)從羊水中分離得到一種新的hAFS細(xì)胞，hAFS細(xì)胞具有較強(qiáng)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(shí)高效誘導(dǎo)hAFS細(xì)胞分化為胰島素分泌細(xì)胞，這些細(xì)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(shí)驗(yàn)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(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

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(hào)：1959233