RalB在多种肿瘤细胞中的表达及意义

目的探讨RalB(v-ral simian leukemia viral oncogene homolog B,ras related;GTP binding protein)蛋白在多种肿瘤细胞中的表达及意义。方法以人胚肾293T细胞为正常细胞对照,选用8种不同组织的肿瘤细胞,采用抗RalB的单克隆抗体通过Western blot方法检测其RalB蛋白的表达。结果RalB在293T细胞中表达明显降低,在乳腺癌细胞株MCF-7、卵巢癌细胞株HO8910、宫颈癌细胞株Hela、肺癌细胞株、结肠癌细胞株LOVO、SW480中均有表达,其中RalB蛋白在结肠癌细胞株SW480中表达明显增高。结论RalB可能以组织特异性的方式在不同肿瘤细胞中表达。

醒脑开窍液对大鼠脑缺血再灌注损伤脑组织VEGF、HIF-1α表达影响随机平行对照研究

[目的]观察醒脑开窍液对大鼠脑缺血再灌注损伤脑组织VEGF、HIF-1α表达影响。[方法]使用随机平行对照方法,将45只健康清洁级SD大鼠（体重210-230g,平均体重210±20g,鼠龄4-5周）,采用Zea longa大脑中动脉内栓线阻断方法复制脑缺血再灌注损伤模型。造模成功45只大鼠按随机数字表分为三组,15只/组,模型组、尼莫地平组、醒脑开窍液组,分别灌胃干预（剂量1m L/100g体重,模型组等量生理盐水）。连续干预7天。采用改良Bederson’s评分法,观察大鼠神经行为学改变。实时定量PCR方法检测VEGF与HIF-1αm RNA表达,Western blot方法检测蛋白表达。[结果]VEGF m RNA尼莫地平组、醒脑开窍液组3d高于模型组（P〈0.05）,不同时间（1d、3d、7d）醒脑开窍液组均与尼莫地平组无显著性差异（P〉0.05）;HIF-1αm RNA表达醒脑开窍液组7d高于模型组（P〈0.05）,不同时间（1d、3d、7d）醒脑开窍液组均与尼莫地平组无显著性差异（P〉0.05）。[结论]醒脑开窍液、尼莫地平均可提升大鼠脑缺血再灌注损伤脑组织VEGF、HIF-1α表达,干预时间越长,提升幅度越大,具有时效关系。

经HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞后HIF-1α基因的表达

常规经基因修饰腺病毒转染细胞后,即进行细胞的移植,并不对病毒转染效果进行检验。本研究采用HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞,察看基因重组腺病毒是否能够转染心肌干细胞内;研究经HIF-1α与EGFP(enhanced green fluorescence protein,EGFP)双基因重组腺病毒转染心肌干细胞后HIF-1α基因的表达情况。在荧光显微镜下,去察看病毒转染细胞的情况,确定病毒最适MOI值;采用实时定量PCR(real-time quantitative PCR,RT-qPCR)评价HIF-1α基因表达变化;应用Western blotting方法检测HIF-1α蛋白表达变化,进而获得良好的实验结果。

Soluble Expression in Escherichia Coli and Purification of Human Carboxylesterase 1

Objective: To achieve an optimized method for soluble expression of human carboxylesterase 1(hCE-1) in escherichia coil and purification by Ni2+-NTA agarose affinity chromatography, to get improved protein yield and purity for further development of hepatocellular carcinoma(HCC) diagnosis ELISA kits. Methods: The best antigen epitopes of hCE1 were predicted by comparing secondary structure, flexible regions, hydrophilicity, antigenic index surface probability of residues. Afterwards,pET-42a(+) with a His-tag and a GST-tag was applied to form recombinant plasmid pET-42a(+)/hCE1, which facilitated purification when using Ni2+-NTA agarose affinity chromatography. Protein quality was measured by SDS-PAGE and BCA protein assay.Western-blot identification was also performed to ensure the correct expression of hCE1protein. Results: The residues from 500 to 567 near C-terminal of hCE1 protein were considered the best epitopes which exhibited high hydrophilicity and high surface probability and relatively flexible secondary structure and low homology compared with hCE2 and hCE3. His-hCE1 500-567 fusion protein was achieved by IPTG-inducted expression with an expected mass of 42 kDa. After purification, the final product was specially identified, which reached over 95% purity and more than 10 mg/L of microbial culture. In Western blot, the purified fusion protein was recognized by anti-hCE1monoclonal antibody, along with previous sequencing validation, which demonstrated the correct preparation of soluble hCE1 protein. Conclusion: This is an efficacious and affordable strategy to generate fusion hCE1 of high quality in E coli, which facilitates preparation of hCE1 monoclonal antibody and further HCC diagnosis research.