發(fā)布時(shí)間：2018-09-02 10:06

【摘要】： 目的: 應(yīng)用血管內(nèi)皮細(xì)胞生長因子和堿性成纖維細(xì)胞生長因子在體外聯(lián)合誘導(dǎo)人外周血單個(gè)核細(xì)胞(PBMNCs)定向分化為血管內(nèi)皮細(xì)胞,為外周血干細(xì)胞在組織工程血管化及肢體缺血性疾病的細(xì)胞移植治療中的應(yīng)用提供理論依據(jù)。 方法: (1)無菌條件下,取重組人粒細(xì)胞集落刺激因子刺激后采集的PBMNCs 2ml,分別設(shè)立實(shí)驗(yàn)組1和對(duì)照組;健康人外周血20mL,作為實(shí)驗(yàn)組2。肝素抗凝。 (2)Hanks液雙倍稀釋,按1:2置于淋巴細(xì)胞分離液上層,離心后提取分液層與上層交界部位呈混濁的灰白色層,即為單個(gè)核細(xì)胞層。 (3)取離心后的細(xì)胞,向DMEM-F12培養(yǎng)基中分別加入含體積分?jǐn)?shù)為0.2的胎牛血清、10μg/L血管內(nèi)皮細(xì)胞生長因子、10μg/L堿性成纖維細(xì)胞生長因子。對(duì)照組中不加血管內(nèi)皮細(xì)胞生長因子和堿性成纖維細(xì)胞生長因子等誘導(dǎo)劑。吹打均勻并計(jì)數(shù),按1×1010 L-1接種于25cm2培養(yǎng)瓶中,于37℃、體積分?jǐn)?shù)為0.05的CO2飽和濕度培養(yǎng)箱中培養(yǎng),第3天更換培養(yǎng)基,去除未貼壁的細(xì)胞,以后每2d換液一次。 (4)倒置相差顯微鏡下觀察細(xì)胞單層排列是否呈“鋪路石”樣結(jié)構(gòu)。運(yùn)用流式細(xì)胞術(shù)檢測誘導(dǎo)后的細(xì)胞表達(dá)CD31和vWF情況。透射電鏡觀察細(xì)胞的超微結(jié)構(gòu)。 結(jié)果: (1)誘導(dǎo)分化后細(xì)胞形態(tài)學(xué)變化:經(jīng)血管內(nèi)皮細(xì)胞生長因子和堿性成纖維細(xì)胞生長因子誘導(dǎo)后的細(xì)胞形態(tài)上呈典型的“鋪路石”樣外觀,經(jīng)歷從小圓→梭形→扁平細(xì)胞的過程,符合內(nèi)皮前體細(xì)胞演變?yōu)槌墒靸?nèi)皮細(xì)胞的過程。 (2)誘導(dǎo)分化后細(xì)胞的表面標(biāo)志鑒定:誘導(dǎo)分化20d,實(shí)驗(yàn)組1中CD31+占86.9%、vWF+占82.5%、CD31+/vWF+占70.9%,實(shí)驗(yàn)組2中CD31+占62.5%、vWF+占58.2%、CD31+/vWF+占54.3%。 (3)培養(yǎng)細(xì)胞的超微結(jié)構(gòu)觀察:透射電鏡下細(xì)胞胞漿內(nèi)可見特征性W-P小體。結(jié)論: 外周血單個(gè)核細(xì)胞在血管內(nèi)皮細(xì)胞生長因子和堿性成纖維細(xì)胞生長因子聯(lián)合誘導(dǎo)下,可分化為血管內(nèi)皮細(xì)胞。
[Abstract]:Objective:
Vascular endothelial growth factor and basic fibroblast growth factor (bfgf) were combined to induce human peripheral blood mononuclear cells (PBMNCs) to differentiate into vascular endothelial cells (VECs) in vitro.
Method:
(1) PBMNCs 2ml was harvested after stimulation with recombinant human granulocyte colony-stimulating factor (rhGMCSF) under aseptic condition, and the experimental group 1 and the control group were set up respectively.
(2) Hanks solution was diluted twice and placed on the upper layer of lymphocyte separating solution at 1:2. After centrifugation, the grey-white layer, which was turbid at the junction of the lymphocyte separating layer and the upper layer, was extracted.
(3) Fetal bovine serum containing 0.2 volume fraction, 10 ug/L vascular endothelial growth factor and 10 ug/L basic fibroblast growth factor were added to the DMEM-F12 medium after centrifugation. X1010L-1 was inoculated in a 25 cm 2 culture flask and cultured in a saturated humidity incubator with a volume fraction of 0.05 and a temperature of 37 C. On the third day, the medium was replaced to remove the unadhered cells, and then the liquid was changed every 2 days.
(4) Inverted phase contrast microscope was used to observe whether the monolayer arrangement of cells was "paving stone" like structure. Flow cytometry was used to detect the expression of CD31 and vWF in the induced cells.
Result:
(1) Morphological changes after differentiation: the cells induced by vascular endothelial growth factor and basic fibroblast growth factor showed a typical "paving stone" appearance, and experienced a process from small round to spindle to flat cells, which accorded with the evolution of endothelial progenitor cells into mature endothelial cells.
(2) Identification of cell surface markers after induction of differentiation: CD31 + accounted for 86.9%, vWF + accounted for 82.5%, CD31 + / vWF + accounted for 70.9%, CD31 + accounted for 62.5%, vWF + accounted for 58.2%, CD31 + / vWF + accounted for 54.3%.
(3) ultrastructure observation of cultured cells: the characteristic W-P bodies in cytoplasm were observed under transmission electron microscope.
Peripheral blood mononuclear cells can differentiate into vascular endothelial cells induced by vascular endothelial growth factor and basic fibroblast growth factor.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

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