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随着社会工业化、城市化的加速,动脉粥样硬化引起的心脑及外周血管疾病已成为全球人类第一大杀手[1]。在动脉血管内发生的一系列炎症反应,会导致血管内皮的增厚、管腔的狭窄,甚至是完全闭塞[1-2]。研究[3-4]表明,存在于骨髓和外周血中的内皮前体细胞(endothelial progenitor cells,EPCs)在损伤血管的修复与重塑中起到了重要的作用。Periostin蛋白又名骨膜蛋白,是细胞外基质蛋白家族的一种[5]。有研究[5-7]报道,Periostin蛋白对于恶性肿瘤的转移以及瘤体血管的建立起着重要作用;而用特异性抗体阻断蛋白的表达,将会导致肿瘤细胞及内皮细胞迁移能力的改变。本实验通过分离、培养和鉴定小鼠EPCs,采用CCK-8法、划痕法、Transwell小室法和流式细胞仪检测等方法,探讨Periostin蛋白过表达对EPCs生物学功能的影响,以期为后期深入研究过表达Periostin蛋白的EPCs体内实验和调控机制奠定基础。
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EPCs在EGM-2培养基中呈“克隆样”生长,3~5 d后细胞融合度超过80%。原代培养7~10 d与第1代培养2~3 d的EPCs呈“铺路石”样生长,少部分呈梭形和多边形,细胞CD34免疫荧光鉴定中红色荧光为抗原阳性,阳性率>90%,Hoechst染色呈蓝色,Merge染色呈混合色(见图 2)。
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Agarose电泳结果显示成功获取了目的基因(见图 3)。且携带目的基因的LV5/Periostin基因重组慢病毒载体构建成功。
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结果显示,未经侵染的EPCs不表达绿色荧光蛋白(GFP),经重组慢病毒LV5/Periostin侵染的EPCs,荧光倒置显微镜下可见亮绿色GFP表达(见图 4)。
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Western blotting检测显示,与阴性对照组EPCs相比,过表达慢病毒组Periostin蛋白表达显著增高(P<0.05)(见表 1、图 5)。
分组 增殖比 F P MS组内 空白对照组 1.74±0.05 阴性对照组 1.68±0.05 38.44 <0.01 0.002 过表达慢病毒组 1.98±0.03# q检验:与阴性对照组比较# P<0.05 表 1 3组EPCs表达Periostin目的蛋白的Western blotting灰度分析(ni=6;x±s)
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实验结果显示,过表达慢病毒组细胞在培养120 h后细胞增殖活性显著低于阴性对照组(P<0.05),而阴性对照组与空白对照组间差异无统计学意义(P>0.05)。在同一重复实验中过表达慢病毒组细胞迁移率、侵袭细胞数及凋亡细胞数均显著大于阴性对照组(P<0.05),且阴性对照组细胞迁移率、侵袭细胞数显著及凋亡细胞数均大于空白对照组(P<0.05)(见表 2、图 6~8)。
分组 增殖比 迁移率/% 侵袭细胞数/个 凋亡百分比/% 空白对照组 5.27±0.05 57.33±5.26 101±2.52 0.20±0.03 阴性对照组 5.18±0.03 61.33±3.51* 103±6.51* 0.20±0.01* 过表达慢病毒组 4.95±0.07# 71.00±2.65# 141±3.00# 0.50±0.02# F 29.53 9.46 79.20 192.86 P <0.01 <0.05 <0.01 <0.01 MS组内 0.003 15.670 19.244 0.001 q检验:与空白对照组比较*P<0.05;与阴性对照组比较#P<0.05 表 2 Periostin目的蛋白过表达对EPCs增殖影响的CCK-8检测分析(ni=6;x±s)
过表达Periostin蛋白对内皮前体细胞功能影响的实验研究
Effect of over-expression of Periostin protein on the biological function of endothelial progenitor cells
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摘要:
目的构建Periostin蛋白过表达慢病毒载体, 并检测其对内皮前体细胞(EPCs)功能的影响。 方法从小鼠骨髓中获取EPCs, 并进行鉴定。采用Oligos设计获得目的基因, 构建慢病毒载体LV5/Periostin。慢病毒感染EPCs, 荧光显微镜下观察被感染细胞绿色荧光蛋白(GFP)的表达情况, Western blotting检测Periostin蛋白表达情况。CCK-8法和流式细胞计数检测被感染细胞的增殖和凋亡情况, 划痕法和Transwell小室法检测被感染细胞迁移和侵袭能力的改变情况。 结果被重组慢病毒感染的EPCs明显表达GFP; 过表达慢病毒组细胞增殖活性较阴性对照组低, 而凋亡细胞较阴性对照组多(P < 0.05);过表达慢病毒组细胞的迁移率较空白对照组细胞高, 侵袭细胞数目较多(P < 0.05)。 结论可以从小鼠骨髓中成功分离和培养EPCs, 过表达Periostin蛋白会降低EPCs体外增殖的活性, 促进其凋亡; 但会增加EPCs的体外迁移和侵袭能力。 -
关键词:
- Periostin蛋白 /
- 内皮前体细胞 /
- 慢病毒载体 /
- 小鼠
Abstract:Objective To construct the over-expression of Periostin protein lentiviral vector, and investigate its effects on the biological function of endothelial progenitor cells(EPCs). Methods The mouse EPCs were isolated from bone marrow, cultured and identified.The LV5/Periostin lentiviral vector was constructed, and transferred into the EPCs.The expression of green fluorescent protein(GFP) in transfected cells was observed under fluorescence microscope.The expression level of Periostin in EPCs was verified by Western blotting, the proliferation and apoptosis of infected cells were detected using the cell counting kit-8(CCK-8) assay and flow cytometry, and the migration ability and invasion ability of cells were detected using the scratch and Transwell assgy. Results The expression of GFP in EPCs was obvious.Compared with the control group, the proliferation activity of cells was lower, and the number of apoptosis cells were more in lentiviral vector group(P < 0.05).Compared with the control group, the migration rate of cells was higher, the number of invasion cells were more in lentiviral vector group(P < 0.05). Conclusions EPCs can be isolated from bone marrow, and cultured and identified.The over-expression lentiviral vector of Periostin protein can reduce the growth and proliferation, promote the apotosis, and enhance the migration and invasion ability of EPCs. -
Key words:
- Periostin protein /
- endothelial progenitor cell /
- lentivirus vector /
- mouse
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表 1 3组EPCs表达Periostin目的蛋白的Western blotting灰度分析(ni=6;x±s)
分组 增殖比 F P MS组内 空白对照组 1.74±0.05 阴性对照组 1.68±0.05 38.44 <0.01 0.002 过表达慢病毒组 1.98±0.03# q检验:与阴性对照组比较# P<0.05 表 2 Periostin目的蛋白过表达对EPCs增殖影响的CCK-8检测分析(ni=6;x±s)
分组 增殖比 迁移率/% 侵袭细胞数/个 凋亡百分比/% 空白对照组 5.27±0.05 57.33±5.26 101±2.52 0.20±0.03 阴性对照组 5.18±0.03 61.33±3.51* 103±6.51* 0.20±0.01* 过表达慢病毒组 4.95±0.07# 71.00±2.65# 141±3.00# 0.50±0.02# F 29.53 9.46 79.20 192.86 P <0.01 <0.05 <0.01 <0.01 MS组内 0.003 15.670 19.244 0.001 q检验:与空白对照组比较*P<0.05;与阴性对照组比较#P<0.05 -
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