中文摘要：

目的构建热休克蛋白70（HSP70）启动子调控内皮抑素（Endo）基因的重组质粒pcDNA3．1（＋）／HSP70－Endo／EGFP，观察其在HepG2细胞中的表达规律。方法用聚合酶链反应（PCR）法体外扩增HSP70启动子（350bp）；用EcoRI、SalI双酶切法切取质粒pSP—Endo／EGFP中Endo／EGFP，克隆到pcDNA3．1（＋）中获得重组质粒pcDNA3．1（＋）／HSP70-Endo／EGFP。脂质体介导转染HepG2细胞，转染24h后予以37、39、41、43、45℃加热诱导30min，荧光显微镜和流式细胞术检测EGFP的表达，逆转录（RT）－PCR法检测细胞内EndomRNA的表达；ELISA法检测转染细胞上清中Endo蛋白浓度。结果合成HSP70启动子序列测序结果与GeneBank中AL671762所提供序列完全一致。低温加热可增强HepG2细胞中Endo／EGFP表达，43℃组EGFP表达强度达37℃组的3．3倍。43℃组EndomRNA表达水平高于不加热组。43℃组上清Endo蛋白浓度为（177±28）μg/L，高于不加热组（43±11）μg/L。结论成功构建HSP70启动子调控Endo基因的重组质粒，低温加热可增强HepG2细胞中Endo基因的表达。

英文摘要：

Objective To construct the recombinant plasmid of pcDNA3.1 （ ＋ ）/HSP70-Endo/ EGFP containing gene of endostatin and promoter of heat shock protein 70 gene ,and to study its expression in HepG2 cells. Methods The HSP70 promoter （350 bp） was cloned by methods of polymerase chain reaction. Fusion gene of Endo/EGFP was cut from plasmid of pSP-Endo/EGFP using restriction endonucleases of EcoR I and Sal I , and cloned into multiple clone site of pcDNA3.1 （ ＋ ） , obtaining recombinant plasmid of pcDNA3.1 （ ＋ ）/HSP70-Endo/EGFP. HepG2 ceils were transfeeted with the recombinant plasmid using lipofectine2000. Following the thermal induction at 37,39,41,43,45℃ for 30 min ,the expression of EGFP was detected by fluorescence microscope and flow cytometry. The expression of endostatin mRNA was tested by RT-PCR. The concentration of endostatin protein in supernatant was tested by ELISA. Results The subsequence of HSP70 promoter was confirmed with that of AL671762 in GenBank. Hypothermia could induce the up-regulation of Endo/EGFP genes in HepG2 cells, especially heated at 43 ℃ ,its EGFP expression reached 3.3-fold of 37℃ group. The expression of endostatin mRNA in HepG2 ceils with thermal induction was higher than that in those without thermal induction. The concentration of endostatin protein in supernatant in 43℃ group was （ 177 ± 28 ）μg/L, higher than that of 37 ℃ group [ （43 ± 11 ） μg/L]. Conclusion Recombinant plasmid of pcDNA3. 1 （ ＋ ）/HSP70-Endo/EGFP was constructed. Hypothermia could induce HSP70 promoter to up-regulate the expression of Endo gene in HepG2 ceils.