本文選題：骨髓間充質干細胞 + 胰島細胞再生�。� 參考：《中國醫(yī)科大學》2010年碩士論文

【摘要】： 背景及目的 糖尿病(Diabetes mellitus)是一種以高血糖為主要特征嚴重威脅人類健康的疾病。全球每年有5%死亡是由于糖尿病的原因,據(jù)WHO估計若不采取有效措施,未來10年每年因糖尿病而死亡的人數(shù)會增加50%。其中1型糖尿病是由于自身免疫病造成的Langerhans內的p細胞的缺失,而2型糖尿病是由于組織對胰島素的不敏感和β細胞分泌胰島素的減少。目前,臨床上主要是采用注射胰島素治療1型及部分2型糖尿病,但治療效果未盡如人意。最近對患者成功移植胰島或者β細胞,為攻克糖尿病提供了新的希望。但是,胰島來源不足是目前最大的難題。 干細胞作為一類具有多向分化潛能的細胞,已經(jīng)逐漸成為人們尋找胰島β細胞替代物的新資源。利用胚胎干細胞體外誘導胰島素分泌細胞為治療糖尿病提供了希望,但誘導胚胎干細胞分化比率低,誘導過程復雜,具有免疫排斥反應且易成瘤,距離臨床應用還需要克服很多技術上的困難。成體干細胞具有跨系分化的能力,如肝干細胞可以分化為胰腺p細胞,神經(jīng)干細胞能夠分化為血細胞等。骨髓間充質干細胞(Bone Marrow Mesenchymal stem cells, MSCs)是一類具有向多種細胞分化潛能的細胞,是一種理想的組織工程種子細胞,易于提取,分離及擴增,在特定的條件下,可以分化為骨、軟骨、肌肉等中胚層組織細胞,還可以跨胚層分化為神經(jīng)細胞等。體外誘導MSCs分化為胰島素分泌細胞,為治療糖尿病提供了新的策略,但仍然面臨分化過程復雜、分化比率低等困難。有研究報道,移植骨髓可以緩解糖尿病癥狀,在一定程度上降低了糖尿病的高血糖。有研究表明小鼠骨髓來源的干細胞(c-kit+)可以啟動內源的胰腺的增殖,該項研究成果認為供體的內皮細胞(PECAM+)參與了胰腺細胞的再生。同種異體移植小鼠、大鼠的MSCs可以修復多種組織損傷,如心肌缺血、腎、肺、脊髓損傷等,然而其修復機理仍不清楚。在成年小鼠與成人中胰島的增殖是通過已存在的胰島β細胞產(chǎn)生新的β細胞,而不是通過干細胞分化的途徑。移植MSCs緩解糖尿病高血糖癥狀是否與胰島的增殖有關,尚未見報道。 Cdk4基因是調節(jié)細胞的增殖一種細胞周期調節(jié)蛋白,通過與D-cyclin形成復合物指導細胞由G1期進入到S期。研究認為Cdk4基因是胰島β細胞的再生、增殖與衰老的重要的調節(jié)因子。因而MSCs是否通過Cdk4基因促進胰島細胞的再生緩解糖尿病高血糖癥狀,需要進一步的研究。 文獻報道人與小鼠的胰島在體外可以去分化成胰島前體細胞.NRSF/REST (neuron restrictive silencing factor/repressor element silencing transciption factor,NRSF／REST)基因在成熟的神經(jīng)細胞與胰島細胞中不表達,只在它們的前體細胞中表達。胰島的增殖是否通過去分化為REST表達陽性細胞來完成,MSCs是否通過這種途徑促進胰島細胞的再生緩解糖尿病高血糖癥狀尚不清楚。 本課題組前期工作發(fā)現(xiàn)體外共培養(yǎng)胰腺細胞與MSCs,可以誘導MSCs分化為胰島樣細胞。移植MSCs到STZ誘導的糖尿病大鼠體內觀察到糖尿病癥狀的改善,血糖的降低。本研究通過體內和體外實驗兩部分來探討MSCs是否促進胰島細胞的再生以及胰島中Cdk4與REST基因表達情況。 方法 1、大鼠骨髓間充質干細胞(MSCs)的分離培養(yǎng)和鑒定(1)無菌條件下取大鼠股骨骨髓細胞離心做原代培養(yǎng)(2)根據(jù)細胞的貼壁性能不同選取貼壁不牢的細胞繼續(xù)傳代培養(yǎng)(3)流式細胞鑒定CD14,CD34,CD44,CD45,CD73,CD90的表達 2、慢病毒轉染大鼠骨髓間充質干細胞(MSCs)(1)帶有GFP標簽的慢病毒質粒的包裝(2)慢病毒轉染MSCs 3、大鼠糖尿病模型的建立及移植轉染的大鼠骨髓間充質干細胞(1)一次性腹腔注射鏈脲菌素建立大鼠糖尿病模型(2)監(jiān)測大鼠血糖以確定建模是否成功(3)尾靜脈注射轉染的大鼠MSCs(4)檢測移植后糖尿病大鼠的空腹血糖的變化 4、胰島素免疫熒光染色評估胰島大小并進行軟件分析(1)獲得大鼠的胰腺冰凍切片(2)利用胰島素免疫熒光染色評估胰島的大小(3)利用軟件分析不同處理組胰島大小的差異 5、大鼠骨髓間充質干細胞(MSCs)分別與胰島素分泌細胞系(INS1)、胰島細胞共培養(yǎng)(1)大鼠胰島細胞原代培養(yǎng)(2)分隔培養(yǎng)的方法將MSCs分別和INS1細胞、胰島細胞共培養(yǎng)(3)Real-time PCR檢測細胞周期調控基因Cdk4和REST的表達情況 實驗結果 1、分離培養(yǎng)大鼠MSCs,并經(jīng)流式細胞術鑒定為CD14(-),CD34(-),CD45(-),CD44(+),CD73(+),CD90(+)。 2、移植細胞第11天檢測到移植組血糖顯著比對照組低,但此后血糖值又再次升高,與對照組無顯著差異。 3、在胰腺組織中發(fā)現(xiàn)帶有GFP標簽的MSCs,移植的MSCs植入到胰腺組織內部,并得到一定的增殖,但MSCs自身并不表達胰島素。 4、移植組胰島熒光面積顯著大比對照組,體內損傷的胰島細胞獲得再生。 5、MSC分別與INS1細胞、胰島細胞共培養(yǎng)后,INS1細胞、胰島的Cdk4基因分別高表達2.281、1.869倍,即共培養(yǎng)后促進了INS1細胞與胰島p細胞增殖。 6、MSC與胰島細胞共培養(yǎng)后,使胰島REST表達量提高1.608倍。 結論 1、移植MSCs短期可降低STZ誘導的糖尿病模型大鼠的高血糖。 2、MSC與胰島細胞共培養(yǎng)后,上調了胰島Cdk4、REST的表達。
[Abstract]:Background and purpose
Diabetes (Diabetes mellitus) is a disease that seriously threatens human health with high blood sugar. 5% deaths worldwide are due to diabetes. According to WHO estimates, if no effective measures are taken, the number of people dying of diabetes in the next 10 years is increased by 50%., of which type 1 diabetes is due to the L of autoimmune diseases. The loss of P cells in angerhans, and type 2 diabetes is due to tissue insensitivity to insulin and the decrease of insulin secretion in beta cells. Currently, the clinical use of insulin injection is mainly used to treat type 1 and type 2 diabetes, but the effect of the treatment is not satisfactory. New hope is provided. However, the insufficient source of islets is the biggest problem at present.
As a class of cells with multiple differentiation potential, stem cells have gradually become a new resource for the search for pancreatic beta cell substitutes. The use of embryonic stem cells in vitro to induce insulin secreting cells provides a hope for the treatment of diabetes, but the induction of embryonic stem cell differentiation is low, the induction process is complex, and the immune rejection reaction is easy and easy. It is also necessary to overcome many technical difficulties. Adult stem cells have the ability to differentiate into P cells, such as liver stem cells can differentiate into pancreatic P cells, and neural stem cells can differentiate into blood cells. Bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal stem cells, MSCs) are a class of cell differentiation to multiple cell differentiation. The potential cell, an ideal tissue engineering seed cell, is easy to extract, isolate and amplify. Under specific conditions, it can differentiate into mesodermal tissue cells, such as bone, cartilage, muscle and so on. It can also differentiate into nerve cells across the embryo layer. In vitro, MSCs is induced to differentiate into Isle secreting cells, which provides a new strategy for the treatment of diabetes. However, it is reported that transplantation of bone marrow can alleviate the symptoms of diabetes and reduce the hyperglycemia of diabetes to a certain extent. Studies have shown that the bone marrow stem cells (c-kit+) of mice can initiate endogenous pancreatic proliferation. (PECAM+) participate in the regeneration of pancreatic cells. Allograft mice, rat MSCs can repair a variety of tissue damage, such as myocardial ischemia, kidney, lung, spinal cord injury and so on, but its repair mechanism is still unclear. In adult mice and adults, the proliferation of islets in adult and adult islet beta cells produce new beta cells, not through dry. The way of cell differentiation. Whether transplantation of MSCs to alleviate the symptoms of hyperglycemia in diabetes is related to islet proliferation has not been reported.
The Cdk4 gene is a cell cycle regulating protein that regulates the proliferation of cells by forming a complex with D-cyclin to guide cells from G1 to S. The study suggests that the Cdk4 gene is an important regulator of the regeneration, proliferation and senescence of islet beta cells. Therefore, MSCs has been used to promote the regeneration of islet cells by the Cdk4 basis to alleviate the high level of diabetes. The symptoms of blood sugar need further study.
It is reported that the islets of human and mouse can be differentiated into islet precursor cells.NRSF/REST (neuron restrictive silencing factor/repressor element silencing transciption factor, NRSF / REST), which are not expressed in mature nerve cells and islet cells, only in their precursor cells. The proliferation of islets is the proliferation of islet cells. It is not clear whether or not MSCs can promote the regeneration of islet cells to alleviate the symptoms of diabetic hyperglycemia by using this pathway as a REST expression positive cell.
In our previous work, we found that co culture of pancreatic cells and MSCs in vitro could induce MSCs to differentiate into islet like cells. The improvement of diabetes symptoms and the decrease of blood glucose were observed in the diabetic rats induced by MSCs to STZ. This study was to explore whether MSCs promotes the regeneration of islet cells by two parts in vivo and in vitro. The expression of Cdk4 and REST gene in islets of islets.
Method
1, the isolation, culture and identification of rat bone marrow mesenchymal stem cells (MSCs) (1) the bone marrow cells of the rat femur were centrifuged for the primary culture under aseptic conditions (2) to select the cells with different adherence to the cells to continue the passage culture (3) flow cytometry (3) to identify the expression of CD34, CD44, CD45, CD73, CD90.
2, lentivirus transfected rat bone marrow mesenchymal stem cells (MSCs) (1) packaged with lentiviral plasmid with GFP tag (2) lentivirus transfected MSCs.
3, establishment of rat diabetes model and transplantation of rat bone marrow mesenchymal stem cells (1) intraperitoneal injection of streptozotocin to establish diabetic rat model (2) to monitor rats' blood glucose to determine whether the model was successful (3) MSCs (4) transfected by tail vein (4) to detect the change of fasting blood glucose in diabetic rats after transplantation
4, insulin immunofluorescence staining was used to assess islet size and software analysis (1) the frozen section of the pancreas of rats was obtained (2) using insulin immunofluorescence staining to assess the size of islets (3) using software to analyze the difference of islet size in different treatment groups
5, rat bone marrow mesenchymal stem cells (MSCs) and insulin secreting cell line (INS1), islet cells co culture (1) rat islet cells primary culture (2) isolation and culture method, MSCs and INS1 cells, islet cells were co cultured (3) Real-time PCR to detect the expression of Cdk4 and REST of the cell cycle regulation gene.
experimental result
1, isolation and culture of rat MSCs and identification of CD14 (-), CD34 (-), CD45 (-), CD44 (+), CD73 (+), CD90 (+) by flow cytometry.
2, on the eleventh day after transplantation, the blood glucose level of the transplantation group was significantly lower than that of the control group, but the blood glucose level increased again thereafter, and there was no significant difference between the transplanted group and the control group.
3, in the pancreatic tissue, GFP labeled MSCs was found. The transplanted MSCs was implanted into the pancreatic tissue and proliferating, but MSCs itself did not express insulin.
4, the islet fluorescence area in the transplantation group was significantly larger than that in the control group, and the injured islet cells were regenerated.
5, after co culture of INS1 cells and islet cells, the Cdk4 genes of INS1 cells and islets were highly expressed as 2.281,1.869 times, that is, after co culture, the proliferation of INS1 cells and islet P cells was promoted.
6, the co expression of MSC and islet cells increased the expression of REST in pancreatic islets by 1.608 times.
conclusion
1, transplantation of MSCs can reduce the hyperglycemia of STZ induced diabetic rats in a short time.
2, co culture of MSC and islet cells increased the expression of Cdk4 and REST in islets.

【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329.2~8

【引證文獻】

相關期刊論文 前1條

1 吳立萍;李蒙;李瑞玉;孫艷浮;顏軍禮;;骨髓間充質干細胞及與胰島細胞共移植治療糖尿病[J];中國組織工程研究;2013年31期

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本文編號：1803010