【摘要】：目的 體外條件下從大鼠骨髓中分離出單個核細胞,使用專用的培養(yǎng)基使其向內(nèi)皮祖細胞(endothelial progenitor cells,EPCs)分化生長,并將HGF基因轉(zhuǎn)染原代培養(yǎng)的大鼠血管內(nèi)皮祖細胞,觀察轉(zhuǎn)染后細胞上清液中HGF的表達及HGF基因?qū)υ囵B(yǎng)血管內(nèi)皮祖細胞增殖、遷移及血管形成能力的影響。 方法 1.取4-6周齡Wistar大鼠(120-150g)雙下肢股骨及脛骨骨髓,利用密度梯度離心法分離單個核細胞,并使用內(nèi)皮系專用培養(yǎng)液EGM-2MV誘導培養(yǎng),使其分化為血管內(nèi)皮祖細胞。通過倒置相差顯微鏡觀察培養(yǎng)細胞的生長情況,通過熒光顯微鏡觀察細胞攝取DiL-acLDL,結(jié)合FITC-UEA-1,從功能角度鑒定細胞,并通過流式細胞術動態(tài)測定細胞表面抗原CD133、flk-1、VE-cadherin(CD144)及CD31進一步鑒定所培養(yǎng)細胞為血管內(nèi)皮祖細胞。 2.提取和擴增pCMV-HGF質(zhì)粒,以缺陷性腺病毒(Ad-GFP)為載體介導HGF基因轉(zhuǎn)染血管內(nèi)皮祖細胞并計算轉(zhuǎn)染效率；采用ELISA法檢測轉(zhuǎn)染后HGF蛋白的表達情況；用MTT法檢測HGF基因?qū)PCs增殖的促進作用；Transwell檢測細胞的遷移能力；3D培養(yǎng)法觀察細胞體外血管形成能力。 結(jié)果 1.成功分離和培養(yǎng)出大鼠骨髓血管內(nèi)皮祖細胞,并通過細胞功能檢測和流式細胞術檢測表面抗原來鑒定。 2.成功將HGF基因轉(zhuǎn)染大鼠骨髓血管內(nèi)皮祖細胞,熒光顯微鏡下可見綠色熒光蛋白的表達；在HGF轉(zhuǎn)染組細胞培養(yǎng)上清中檢測到HGF的表達,第1天、2天、4天、7天、11天濃度分別為22.15±3.77ng/ml、39.42±7.32ng/ml、99.09±9.89ng/ml、311.87±26.56 ng/ml、224.72±20.91ng/ml,而空載腺病毒組、陰性對照組沒有檢測出HGF的表達；轉(zhuǎn)染后HGF基因?qū)υ囵B(yǎng)的血管內(nèi)皮祖細胞生長有顯著的促增殖作用,轉(zhuǎn)染4天和7天,轉(zhuǎn)染組血管內(nèi)皮祖細胞增殖明顯加快,與空載腺病毒組和陰性對照組相比均有統(tǒng)計學意義(P0.05)；轉(zhuǎn)染后細胞遷移能力增強；3D培養(yǎng)可見血管樣結(jié)構形成。 結(jié)論 1.體外條件下能夠從大鼠骨髓中成功分離出單個核細胞并誘導培養(yǎng)出血管內(nèi)皮祖細胞。 2.通過腺病毒介導,可成功的將HGF基因轉(zhuǎn)染入大鼠血管內(nèi)皮祖細胞,并且在培養(yǎng)上清液中可檢測到HGF蛋白,證實轉(zhuǎn)染后的目的基因能夠在細胞中有效的表達并促進EPCs增殖、遷移及血管形成。為下一步利用該基因進行基因-干細胞移植治療肢體缺血性疾病提供實驗基礎。
[Abstract]:Objective to isolate mononuclear cells from rat bone marrow in vitro and to differentiate and grow into endothelial progenitor cells (endothelial progenitor cells,EPCs) by using a special medium. The HGF gene was transfected into primary cultured rat vascular endothelial progenitor cells. The expression of HGF in supernatant of transfected cells and the effect of HGF gene on the proliferation, migration and angiogenesis of primary cultured endothelial progenitor cells were observed. Method 1. The bone marrow of femur and tibia of 4-6 week old Wistar rats (120-150g) was isolated by density gradient centrifugation. The mononuclear cells were induced by EGM-2MV and differentiated into endothelial progenitor cells (EPCs). The growth of cultured cells was observed by inverted phase contrast microscope, the uptake of DiL-acLDL, and FITC-UEA-1, was observed by fluorescence microscope, and the cell surface antigen CD133, was dynamically determined by flow cytometry. Flk-1,VE-cadherin (CD144) and CD31 further identified the cultured cells as vascular endothelial progenitor cells. 2. PCMV-HGF plasmid was extracted and amplified and HGF gene was transfected into vascular endothelial progenitor cells mediated by defective adenovirus (Ad-GFP) and the transfection efficiency was calculated. The expression of HGF protein after transfection was detected by ELISA method. MTT assay was used to detect the effect of HGF gene on the proliferation of EPCs; Transwell was used to detect the migration ability of cells; and 3D culture method was used to observe the ability of cells to form blood vessels in vitro. Result 1. Vascular endothelial progenitor cells from rat bone marrow were isolated and cultured successfully and identified by cell function test and flow cytometry. 2. HGF gene was successfully transfected into rat bone marrow vascular endothelial progenitor cells and the expression of green fluorescent protein was observed under fluorescence microscope. The expression of HGF was detected in the culture supernatant of HGF transfection group. The concentration of HGF was 22.15 鹵3.77ng / ml / ml 39.42 鹵7.32ng / ml / ml 99.09 鹵9.89ng / ml / ml + 311.87 鹵26.56 ng/ml, on day 1, day 2, day 4, day 7 and day 11, respectively. The expression of HGF was not detected in the control group (224.72 鹵20.91 ng / ml), while in the non-loaded adenovirus group, the expression of HGF was not detected in the negative control group. After transfection, HGF gene significantly promoted the proliferation of vascular endothelial progenitor cells in primary culture. After 4 and 7 days of transfection, the proliferation of vascular endothelial progenitor cells was significantly accelerated in the transfected group. Compared with the control group and the non-loaded adenovirus group, there was statistical significance (P0.05). After transfection, the migration ability of the cells was enhanced, and vascular like structure was found in 3D culture. Conclusion 1. Mononuclear cells were isolated from rat bone marrow in vitro and vascular endothelial progenitor cells were induced. 2. HGF gene was successfully transfected into rat vascular endothelial progenitor cells mediated by adenovirus, and HGF protein was detected in culture supernatant. It was proved that the transfected target gene could effectively express in the cells and promote the proliferation of EPCs. Migration and angiogenesis. To provide experimental basis for the treatment of limb ischemic disease by gene-stem cell transplantation.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

【引證文獻】

相關碩士學位論文 前2條

1 魯明;海拔、民族、年齡、性別對外周血EPCs數(shù)、VEGF、HIF-1α濃度的影響研究[D];蘭州大學;2012年

2 孫寧;HGF基因轉(zhuǎn)染血管內(nèi)皮祖細胞促進血管生成的動物實驗研究[D];天津醫(yī)科大學;2012年

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本文編號：2349289