本文關鍵詞： PKH26 大鼠骨髓間充質干細胞 生物學性狀 血管內皮細胞　出處：《山西醫(yī)科大學》2009年碩士論文　論文類型：學位論文

【摘要】： 背景 細胞標記是體內外研究間充質干細胞(mesenchymal stem cells,MSCs)的關鍵問題,研究報道PKH26標記技術操作簡單,易于檢測,標記率高,體內移植60d仍可觀察到熒光表達,國內類似報道較少。 系統(tǒng)性血管炎屬于結締組織病,易侵犯多系統(tǒng)、多臟器,發(fā)病率較高,不及時治療,常危及生命。血管內皮細胞損傷、炎性細胞浸潤是主要發(fā)病機制。研究發(fā)現(xiàn)MSCs在適宜條件下可分化為血管內皮細胞,具有免疫調節(jié)作用,參與組織重建和修復,這為調節(jié)血管炎患者機體免疫紊亂,修復受損的血管內皮細胞帶來了新的希望。 目的 1.探討大鼠骨髓間充質干細胞(rat bone marrow mesenchymal stem cells,rBMSCs)PKH26標記后生物學性狀,建立一種簡單、實用、快速的rBMSCs標記方法。 2.體外誘導rBMSCs向血管內皮細胞分化,為系統(tǒng)性血管炎及其他血管炎患者的臨床治療提供新的思路。 方法 1. rBMSCs的分離培養(yǎng)與傳代:密度梯度離心法結合貼壁法分離rBMSCs,用含10%胎牛血清的L-DMEM培養(yǎng)液培養(yǎng),細胞至80%融合時進行傳代,0.25%胰酶消化2-5min,按1:3進行傳代擴增; 2. rBMSCs的鑒定:CD45、CD31分別是造血干細胞、骨髓內皮細胞特有的表面標志,骨髓來源的MSCs上述2種抗原呈陰性,MSCs無特異性表面標志,表達CD44、CD71。我們通過流式細胞儀檢測rBMSCs表面抗原CD44、CD71、CD31及CD45的表達,通過排除法鑒定所培養(yǎng)細胞為rBMSCs; 3.檢測rBMSCs在PKH26標記后生物學性狀:PKH26標記第3代rBMSCs,熒光顯微鏡下觀察細胞形態(tài)學;流式細胞儀檢測細胞標記率;MTT法測定生長曲線,比較兩組細胞群體倍增時間;流式細胞儀檢測細胞周期,比較兩組細胞增殖指數(shù);流式細胞儀檢測兩組細胞的表面抗原CD44、CD71、CD31及CD45; 4.體外誘導rBMSCs向血管內皮細胞分化:含40ng/mlVEGF的L-DMEM誘導液誘導第3代rBMSCs向血管內皮細胞分化,于誘導后第14d流式細胞儀鑒定其表面抗原CD31表達水平,免疫組化鑒定其表面FⅧ的表達。 結果 1.經(jīng)流式細胞儀檢測表面抗原,結果顯示CD44陽性,CD71弱陽性,CD31、CD45陰性,鑒定為間充質干細胞; 2.熒光顯微鏡下觀察PKH26標記后的rBMSCs大部分呈長梭形,排列規(guī)則,呈平行、旋渦狀生長,與未標記細胞比較無明顯變化;標記后10d流式細胞儀檢測細胞標記率達91.03%,標記后40d仍可在熒光顯微鏡下觀察到熒光表達;t檢驗結果顯示兩組細胞群體倍增時間及增殖指數(shù)差異不具有統(tǒng)計學意義(P0.05);流式細胞儀檢測結果顯示兩組細胞均為CD44陽性,CD71弱陽性,CD31、CD45陰性; 3.體外誘導rBMSCs向血管內皮細胞分化,14d后流式細胞儀檢測表明CD31表達較未誘導前增加37.36%,免疫組化鑒定FⅧ表達陽性。 結論 1. PKH26標記方法操作簡單,細胞標記率高,標記時間達6周左右,未影響rBMSCs的生物學性狀,為需求大量標記后rBMSCs的科學研究提供了一種實用、簡單、快速的標記方法; 2.體外誘導條件下rBMSCs可向血管內皮細胞分化,為系統(tǒng)性血管炎及其他血管炎患者的臨床治療提供新的思路和方法。
[Abstract]:background
Cell labeling is a key issue in the study of mesenchymal stem cells (MSCs) in vivo and in vitro. The PKH26 labeling technology is simple, easy to detect, and has high labeling rate. The expression of 60d in vivo can still be observed. Similar reports are rare in China.
Systemic vasculitis belong to connective tissue disease, easy invasion of multi system, multi organ, high incidence, not timely treatment, often life-threatening. The injury of vascular endothelial cells, inflammatory cell infiltration is the main pathogenesis. MSCs was found in suitable conditions can differentiate into vascular endothelial cells, has immunomodulatory effects, participate in tissue reconstruction and restoration, the regulation of immune disorders in patients with vasculitis, repair damaged endothelial cells has brought new hope.
objective
1., we explored the biological characteristics of rat bone marrow mesenchymal stem cells (rBMSCs) PKH26 markers, and set up a simple, practical and fast rBMSCs labeling method.
2. the differentiation of rBMSCs into vascular endothelial cells in vitro provides a new way of thinking for the clinical treatment of systemic vasculitis and other vasculitis.
Method
1. rBMSCs isolation culture and passage: rBMSCs was separated by density gradient centrifugation combined with adherent method, and cultured in L-DMEM containing 10% fetal bovine serum. The cells were passaged to 80% fusion and 2-5min was digested by 0.25% trypsin, and then amplified by 1:3.
Identification of 2. rBMSCs: CD45, CD31 respectively, hematopoietic stem cells, bone marrow endothelial cell specific surface markers of bone marrow derived MSCs 2 antigens were negative, MSCs has no specific surface markers, expression of CD44 CD71., we detected by rBMSCs surface antigen CD44, CD71 flow cytometry, the expression of CD31 and CD45. By excluding identification of cultured cells is rBMSCs;
Detection of rBMSCs in biological characters of 3. PKH26 markers: PKH26 marks the third generation of rBMSCs, cell morphology was observed under the fluorescence microscope detection rate of labeled cells; flow cytometry; Determination of growth curve of MTT was compared between the two groups, the doubling time of the cells; flow cytometry cell cycle and cell proliferation index were compared between the two groups; flow cytometry cell surface antigen was detected two groups of cells in the CD44, CD71, CD31 and CD45;
4. in vitro differentiation of rBMSCs induced vascular endothelial cells: 40ng/mlVEGF containing L-DMEM was induced by the third generation of rBMSCs induced differentiation into vascular endothelial cells, the expression level of CD31 antigen on the surface of the 14d flow cytometry after induction by immunohistochemistry to identify the surface expression of F VIII.
Result
1. the surface antigen was detected by flow cytometry. The results showed that CD44 was positive, CD71 was weak positive, CD31 and CD45 were negative, which were identified as mesenchymal stem cells.
2.鑽у厜鏄懼井闀滀笅瑙傚療PKH26鏍囪鍚庣殑rBMSCs澶ч儴鍒嗗憟闀挎褰，

本文編號：1451030