中文摘要：

目的构建S100A9真核表达载体pEGFP-N3-S100A9，观察其对胶质瘤细胞系U251内SIOOA9基因表达及细胞生长的影响。方法应用RT-PCR和DNA重组技术构建pEGFP-N3-S100A9融合蛋白表达载体，并用双酶切、测序进行鉴定，用脂质体转染胶质瘤细胞系U251细胞。荧光显微镜下动态观察U251细胞绿色荧光的变化；培养24h后提取转染细胞的总RNA及总蛋白。通过荧光定量PCR（qRT-PCR）观察其mRNA表达情况，Westernblot检测其蛋白表达情况，MTT法检测细胞增殖，流式细胞仪检测细胞周期分布。结果酶切鉴定与S100A9全长基因长度一致（345bp）；测序证实重组质粒中含有一与GenBank上登录的S100A9基因（NM_002965）序列完全一致的片段，表明成功构建真核表达载体；荧光显微镜下转染U251细胞可见绿色荧光蛋白表达，12h后逐渐增多，24-48h达高峰，且稳定表达较长时间；24h后通过qRT-PCR检测到mRNA表达，Westernblot检测到14×10^3目的蛋白表达（P〈0．05），其增殖能力明显增强（P〈0．01），且引起细胞G．期减少，S期增加（P〈0．05）。结论成功构建真核表达载体pEGFP-N3-S100A9，转染到U251细胞后能有效上调S100A9基因的mRNA及蛋白的表达、促进细胞增殖。

英文摘要：

Objective To construct an eukaryotic expression vector of human S100A9 full length gene pEGFP-N3-S100A9 and to observe its effect on mRNA and protein expression of SIOOA9 and the proliferation of glioma cell line U251. Methods The full length gene of S100A9 （345 bp） was obtained by RT-PCR, and was inserted into pEGFP-N3 vector by gene recombination technique. After verified by restrictive enzyme digestion and PCR, the recombinant plasmids were transferred into U251 cells by liposome. Total RNA and protein were extracted in 24 h after transfection to detect the S100A9 expression by quantitative real-time polymerase chain reaction （qRT-PCR） and Western blotting, respectively. The transfected U251 cells were continuously traced by fluorescent microscopy. The cell proliferation was analyzed by MTY assay, and the distribution of cell cycle was assessed by flow cytometry. Results The recombinant eukaryotic expression vector pEGFP-N3- S100A9 was successfully constructed, and the gene sequencing result suggested the S100A9 sequence was identical to that published in GenBank （NM-002965）. Green fluorescence could be seen by fluorescence microscopy in 8 h after transfection and a peak appeared within 24 -48 h. $100A9 mRNA was expressed and a 14 ×10^3 fusion protein was detected 24 h after transfection. The overexpression of S100A9 enhanced the cell proliferation significantly （ P 〈 0. 01 ）, and the number of cells decreased at G1 phase and increased at S phase （ P 〈 0. 05 ）. Conclusion The eukaryotic expression vector pEGFP-N3-S100A9 is constructed and expressed successfully in U251 cells, and it can effectively increase the expression of S100A9 and enhance the proliferation of U251 ceils.