本文關(guān)鍵詞： PKH26 大鼠骨髓間充質(zhì)干細(xì)胞 生物學(xué)性狀 血管內(nèi)皮細(xì)胞　出處：《山西醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 背景 細(xì)胞標(biāo)記是體內(nèi)外研究間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)的關(guān)鍵問(wèn)題,研究報(bào)道PKH26標(biāo)記技術(shù)操作簡(jiǎn)單,易于檢測(cè),標(biāo)記率高,體內(nèi)移植60d仍可觀察到熒光表達(dá),國(guó)內(nèi)類似報(bào)道較少。 系統(tǒng)性血管炎屬于結(jié)締組織病,易侵犯多系統(tǒng)、多臟器,發(fā)病率較高,不及時(shí)治療,常危及生命。血管內(nèi)皮細(xì)胞損傷、炎性細(xì)胞浸潤(rùn)是主要發(fā)病機(jī)制。研究發(fā)現(xiàn)MSCs在適宜條件下可分化為血管內(nèi)皮細(xì)胞,具有免疫調(diào)節(jié)作用,參與組織重建和修復(fù),這為調(diào)節(jié)血管炎患者機(jī)體免疫紊亂,修復(fù)受損的血管內(nèi)皮細(xì)胞帶來(lái)了新的希望。 目的 1.探討大鼠骨髓間充質(zhì)干細(xì)胞(rat bone marrow mesenchymal stem cells,rBMSCs)PKH26標(biāo)記后生物學(xué)性狀,建立一種簡(jiǎn)單、實(shí)用、快速的rBMSCs標(biāo)記方法。 2.體外誘導(dǎo)rBMSCs向血管內(nèi)皮細(xì)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(jié)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(xì)胞至80%融合時(shí)進(jìn)行傳代,0.25%胰酶消化2-5min,按1:3進(jìn)行傳代擴(kuò)增; 2. rBMSCs的鑒定:CD45、CD31分別是造血干細(xì)胞、骨髓內(nèi)皮細(xì)胞特有的表面標(biāo)志,骨髓來(lái)源的MSCs上述2種抗原呈陰性,MSCs無(wú)特異性表面標(biāo)志,表達(dá)CD44、CD71。我們通過(guò)流式細(xì)胞儀檢測(cè)rBMSCs表面抗原CD44、CD71、CD31及CD45的表達(dá),通過(guò)排除法鑒定所培養(yǎng)細(xì)胞為rBMSCs; 3.檢測(cè)rBMSCs在PKH26標(biāo)記后生物學(xué)性狀:PKH26標(biāo)記第3代rBMSCs,熒光顯微鏡下觀察細(xì)胞形態(tài)學(xué);流式細(xì)胞儀檢測(cè)細(xì)胞標(biāo)記率;MTT法測(cè)定生長(zhǎng)曲線,比較兩組細(xì)胞群體倍增時(shí)間;流式細(xì)胞儀檢測(cè)細(xì)胞周期,比較兩組細(xì)胞增殖指數(shù);流式細(xì)胞儀檢測(cè)兩組細(xì)胞的表面抗原CD44、CD71、CD31及CD45; 4.體外誘導(dǎo)rBMSCs向血管內(nèi)皮細(xì)胞分化:含40ng/mlVEGF的L-DMEM誘導(dǎo)液誘導(dǎo)第3代rBMSCs向血管內(nèi)皮細(xì)胞分化,于誘導(dǎo)后第14d流式細(xì)胞儀鑒定其表面抗原CD31表達(dá)水平,免疫組化鑒定其表面FⅧ的表達(dá)。 結(jié)果 1.經(jīng)流式細(xì)胞儀檢測(cè)表面抗原,結(jié)果顯示CD44陽(yáng)性,CD71弱陽(yáng)性,CD31、CD45陰性,鑒定為間充質(zhì)干細(xì)胞; 2.熒光顯微鏡下觀察PKH26標(biāo)記后的rBMSCs大部分呈長(zhǎng)梭形,排列規(guī)則,呈平行、旋渦狀生長(zhǎng),與未標(biāo)記細(xì)胞比較無(wú)明顯變化;標(biāo)記后10d流式細(xì)胞儀檢測(cè)細(xì)胞標(biāo)記率達(dá)91.03%,標(biāo)記后40d仍可在熒光顯微鏡下觀察到熒光表達(dá);t檢驗(yàn)結(jié)果顯示兩組細(xì)胞群體倍增時(shí)間及增殖指數(shù)差異不具有統(tǒng)計(jì)學(xué)意義(P0.05);流式細(xì)胞儀檢測(cè)結(jié)果顯示兩組細(xì)胞均為CD44陽(yáng)性,CD71弱陽(yáng)性,CD31、CD45陰性; 3.體外誘導(dǎo)rBMSCs向血管內(nèi)皮細(xì)胞分化,14d后流式細(xì)胞儀檢測(cè)表明CD31表達(dá)較未誘導(dǎo)前增加37.36%,免疫組化鑒定FⅧ表達(dá)陽(yáng)性。 結(jié)論 1. PKH26標(biāo)記方法操作簡(jiǎn)單,細(xì)胞標(biāo)記率高,標(biāo)記時(shí)間達(dá)6周左右,未影響rBMSCs的生物學(xué)性狀,為需求大量標(biāo)記后rBMSCs的科學(xué)研究提供了一種實(shí)用、簡(jiǎn)單、快速的標(biāo)記方法; 2.體外誘導(dǎo)條件下rBMSCs可向血管內(nèi)皮細(xì)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，

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