本文關(guān)鍵詞： 肝細胞生長因子 血管內(nèi)皮祖細胞 基因轉(zhuǎn)染 細胞培養(yǎng) 大鼠　出處：《天津醫(yī)科大學》2009年碩士論文　論文類型：學位論文

【摘要】： 目的 離體條件下從大鼠骨髓中分離出單個核細胞,使用特殊的培養(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(shù)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pIRES2-EGFP-HGF質(zhì)粒,以陽離子脂質(zhì)體(Lipofectamine~(TM)2000)介導pIRES2-EGFP-HGF質(zhì)粒轉(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。 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(shù)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,1天、2天、3天、5天、7天濃度分別為638.48±66.91、1230.33±96.35、2097.88±138.32、4017.20±169.92、1869.87±101.22(pg/ml),而空載質(zhì)粒組和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5天和7天,轉(zhuǎn)染組血管內(nèi)皮祖細胞增殖明顯加快,與空載質(zhì)粒組及陰性對照組相比均有統(tǒng)計學意義(P0.05)。 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.通過陽離子脂質(zhì)體介導,可成功的將含有HGF基因的質(zhì)粒轉(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。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(chǔ)。
[Abstract]:Purpose Mononuclear cells were isolated from rat bone marrow in vitro. The mononuclear cells were made to endothelial progenitor cells by using special culture medium. HGF gene was transfected into primary cultured rat vascular endothelial progenitor cells. To observe the expression of HGF in supernatant of transfected cells and the effect of HGF gene on the proliferation of primary cultured endothelial progenitor cells. Method 1. Bone marrow of femur and tibia of 4-6 weeks old Wistar rats were isolated by density gradient centrifugation. The endothelial progenitor cells were differentiated into vascular endothelial progenitor cells induced by EGM-2MV. The growth of cultured cells was observed by inverted phase contrast microscope. Cell uptake of DiL-acLDL-1 was observed by fluorescence microscope, and FITC-UEA-1 was used to identify the cells from a functional point of view. The cell surface antigen CD133 was determined dynamically by flow cytometry. Flk-1 VE-cadherin CD144) and CD31 further identified the cultured cells as vascular endothelial progenitor cells. 2. PIRES2-EGFP-HGF plasmid was extracted and amplified. PIRES2-EGFP-HGF plasmid was transfected into vascular endothelial progenitor cells with cationic liposome Lipofectaminetamine2 / TMN 2000 and the transfection efficiency was calculated. The expression of HGF protein after transfection was detected by ELISA method. The effect of HGF gene on the proliferation of EPCs was detected by MTT assay. Results 1. Vascular endothelial progenitor cells from rat bone marrow were isolated and cultured successfully, and identified by cell function test and flow cytometry. 2.The 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 supernatant of cell culture in HGF transfection group. The concentration of HGF was 638.48 鹵66.91 ~ 1230.33 鹵96.35 on day 1, day 2, day 3 and day 5 and day 7, respectively. 2097.88 鹵138.32 鹵4017.20 鹵169.92 鹵1869.87 鹵101.22 g / ml). The expression of HGF was not detected in the no-load plasmid group and the negative control group. After transfection, HGF gene could significantly promote the proliferation of primary cultured endothelial progenitor cells, and the proliferation of vascular endothelial progenitor cells in transfection group accelerated significantly 5 and 7 days after transfection. Compared with the blank plasmid group and the negative control group, there was significant difference (P 0.05). Conclusion 1. Mononuclear cells were isolated from rat bone marrow in vitro and vascular endothelial progenitor cells were induced. 2. The plasmid containing HGF gene could be successfully transfected into rat vascular endothelial progenitor cells by cationic liposome, and HGF protein could be detected in culture supernatant. It is proved that the transfected target gene can effectively express in the cells and promote the proliferation of EPCs, which provides the experimental basis for the next step of gene stem cell transplantation in the treatment of limb ischemic diseases.
【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】：2009
【分類號】：R346

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