本文選題：骨髓間充質干細胞 + 產胰島素細胞�。� 參考：《第四軍醫(yī)大學》2008年碩士論文

【摘要】： 糖尿病是常見的慢性疾病,全世界患病率約6%�；颊叱Ｐ枰K身注射胰島素治療,但胰島素注射療法對血糖的控制并不理想,無法阻止糖尿病腎病、冠心病、糖尿病眼病等并發(fā)癥的發(fā)生和發(fā)展。最近幾年胰島移植的成功表明了通過補充缺乏的β細胞可以治愈糖尿病。但供體來源缺乏和移植排斥阻礙了胰島細胞移植的廣泛應用。 干細胞作為一類具有極強自我更新及多向分化潛能的細胞,已經逐漸成為人們尋找胰島β細胞替代物的新的細胞資源。由于骨髓間充質干細胞(MSCs)具有多向分化和很強的增殖能力,具有用于細胞治療的巨大潛能。它們可以分化為神經細胞、脂肪細胞、平滑肌細胞、骨骼肌細胞、心肌細胞、軟骨細胞和產胰島素細胞等。但是由骨髓間充質干細胞誘導分化而來的產胰島素細胞(IPCs),其胰島素分泌量不足正常胰腺β細胞的1%。如何促進MSCs分化為較成熟的IPCs成為亟待解決的關鍵問題。目前認為其分化機制,主要是微環(huán)境因素通過一定的信號轉導通路傳遞,轉錄因子抑制或激活,啟動關鍵基因表達的結果。 本研究的目標是:探索不同微環(huán)境下對大鼠MSCs體外分化為IPCs的影響,在糖尿病大鼠體內微環(huán)境下MSCs是否還可以分化為IPCs,為糖尿病細胞替代治療提供理想的種子細胞。 大鼠MSCs的體外分離培養(yǎng)和生物學特征:利用貼壁法分離純化大鼠MSCs,傳代培養(yǎng)于含10%胎牛血清的DMEM培養(yǎng)液中;通過倒置顯微鏡、電鏡、免疫細胞化學分析大鼠MSCs的生物學特征。結果顯示原代和傳代培養(yǎng)MSCs均保持較強的增殖能力,形態(tài)為均一的成纖維樣細胞。超微結構顯示了干細胞的幼稚特征,免疫細胞化學顯示CD34陰性,CD44陽性。結論:采用我們的方法,可以獲得生長穩(wěn)定,擴增較快和純度較高的MSCs。 不同微環(huán)境對大鼠MSCs體外分化為IPCs的影響:采用第三代MSCs,用不同的微環(huán)境進行誘導,對照組誘導劑為含有角朊細胞生長因子、胰島素鐵硒傳遞蛋白(ITS)、尼克酰胺的無血清DMEM/F12培養(yǎng)基、實驗組在對照組基礎上添加胰腺條件培養(yǎng)液;對誘導后細胞進行光、電鏡觀察,雙硫腙和免疫細胞化學等進行鑒定,并行體外葡萄糖刺激實驗,測定細胞分泌胰島素及C-肽功能。結果表明,實驗組及對照組均可誘導分化為IPCs,但實驗組分化而成的IPCs在數量及功能上均好于對照組。結論:大鼠胰腺提取物模擬的微環(huán)境能促進大鼠MSCs體外誘導分化為較高質量的IPCs。 在糖尿病大鼠體內微環(huán)境下MSCs分化為IPCs的潛能:通過腹腔注射鏈脲佐菌素(STZ)誘導健康SD大鼠建立糖尿病模型;將MSCs及由MSCs誘導分化來的IPCs注射到糖尿病模型大鼠的腎包膜下,檢測移植前后血糖、體重、尿量、生存時間變化,當移植大鼠血糖開始下降后,行荷移植物腎切除術去除移植物,繼續(xù)觀察血糖變化,看是否有血糖反跳,以評價其治療糖尿病的效果,結合標本免疫組織化學染色,觀察移植入的MSCs在糖尿病大鼠體內是否分化為IPCs。結果:將MSCs及IPCs移植到大鼠腎包膜下后,大鼠的血糖水平下降,糖尿病癥狀得到控制,生存時間明顯延長。免疫組化顯示移植入的MSCs分化為胰島素染色陽性細胞。結論:在糖尿病大鼠體內微環(huán)境下MSCs可以分化為IPCs。 綜上所述,大鼠MSCs在不同的體外及體內微環(huán)境下均可特異誘導分化為IPCs,而大鼠胰腺提取物模擬的微環(huán)境能促進大鼠MSCs體外誘導分化為IPCs;將大鼠MSCs和IPCs移植入糖尿病大鼠模型體內能夠明顯改善糖尿病癥狀,延長其生存時間。雖然骨髓間充質干細胞在安全而有效地運用于臨床治療之前還有許多基本的問題有待解決,但它為我們解決移植治療糖尿病中供體缺乏問題方面提供了一條新的途經。
[Abstract]:Diabetes is a common chronic disease, the worldwide prevalence of about 6%. patients often needs life-long insulin therapy, but insulin injection therapy is not ideal for blood glucose control, and it can not prevent diabetic nephropathy, coronary heart disease, diabetes and other complications. The lack of beta cells can cure diabetes, but the lack of donor sources and graft rejection have hindered the wide application of islet cell transplantation.
Stem cells, as a class of cells with strong self renewal and pluripotent differentiation potential, have gradually become a new cell resource for the search for substitutes for islet beta cells. Bone marrow mesenchymal stem cells (MSCs) have a multidirectional differentiation and strong proliferation ability, and have great potential for cell therapy. They can be differentiated into nerve cells. Cells, adipocytes, smooth muscle cells, skeletal myocytes, cardiomyocytes, chondrocytes, and insulin producing cells. But the insulin producing cells (IPCs) induced by bone marrow mesenchymal stem cells (MSCs), the insulin secretion of the normal pancreatic beta cells is less than the 1%. of the normal pancreatic beta cells, and it is an urgent problem to promote the differentiation of MSCs into a mature IPCs. The key problem is that the mechanism of differentiation is that the microenvironmental factors pass through a certain signal transduction pathway, the transcription factor is suppressed or activated, and the result of the key gene expression is started.
The aim of this study is to explore the effect of different microenvironment on the differentiation of rat MSCs into IPCs in vitro, and whether MSCs can also differentiate into IPCs in the microenvironment of diabetic rats and provide ideal seed cells for the replacement therapy of diabetic cells.
The isolation and culture of rat MSCs in vitro and biological characteristics: the rat MSCs was isolated and purified by adhesion method and cultured in DMEM culture solution containing 10% fetal bovine serum. The biological characteristics of MSCs were analyzed by inverted microscope, electron microscopy and immunocytochemical analysis. The results showed that both primary and subculture MSCs maintained strong proliferative ability and morphology. The ultrastructure showed the naive characteristics of the stem cells, and the immunocytochemistry showed CD34 negative and CD44 positive. Conclusion: we can obtain stable growth, faster amplification and higher purity of MSCs. by using our method.
The effect of different microenvironment on the differentiation of MSCs in vitro to IPCs: third generation MSCs was used to induce different microenvironment. The inducers in the control group were keratinocyte growth factor, insulin iron selenium transferrin (ITS), and serum free DMEM/F12 medium of nikamide, and the experimental group was added to the control group on the basis of the pancreatic condition culture solution. After induction, the cells were identified by light, electron microscopy, dithizone and immunocytochemistry, and in vitro glucose stimulation test, and the cell secretion of insulin and C- peptide were measured. The results showed that the experimental group and the control group could be induced to differentiate into IPCs, but the differentiated IPCs in the experimental group was better in quantity and function than in the control group. The simulated microenvironment of rat pancreatic extract can promote the differentiation of rat MSCs into high quality IPCs. in vitro.
The potential of MSCs to differentiate into IPCs in the microenvironment of diabetic rats: induced diabetic model in healthy SD rats by intraperitoneal injection of streptozotocin (STZ); MSCs and MSCs induced IPCs were injected into the renal capsule of diabetic rat model, and the blood glucose, weight, urine volume and survival time were detected, and the transplantation was large. After the blood glucose began to decrease, the graft nephrectomy was performed to remove the graft, continue to observe the blood sugar changes and see if there was a blood glucose rebound to evaluate the effect of the treatment of diabetes. Combined with specimen immunohistochemical staining, whether the transplanted MSCs was differentiated into IPCs. in diabetic rats: MSCs and IPCs were transplanted into the kidney of rats. After subcapsular, the blood glucose level of the rats decreased, the diabetic symptoms were controlled and the survival time was prolonged obviously. The immunohistochemical staining showed that the transplanted MSCs was differentiated into insulin positive cells. Conclusion: MSCs can be differentiated into IPCs. in the microenvironment of diabetic rats.
To sum up, the rat MSCs can differentiate into IPCs specifically in different in vitro and in vivo microenvironment, and the microenvironment simulated by the rat pancreas extract can promote the differentiation of rat MSCs into IPCs in vitro, and the transplantation of MSCs and IPCs into the rat model of diabetic rats can obviously improve the symptoms of diabetes and prolong its survival time. There are many basic problems to be solved before bone marrow mesenchymal stem cells are safely and effectively used for clinical treatment, but it provides a new way for us to solve the problem of donor deficiency in the treatment of diabetes.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R657.5;R329

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