甘蔗蜡提取物多廿烷醇的降血脂机制研究

采用RT-PCR、Western Blot、L-[35S]放射性标记物掺入等方法,观察甘蔗蜡提取物多廿烷醇对Hep G2细胞羟甲基戊二酸辅酶A (HMG-CoA)还原酶mRNA转录及蛋白质表达量的影响,及其对HMG-CoA 还原酶合成和降解的作用.结果表明,多廿烷醇降低HMG-CoA还原酶mRNA的转录和蛋白质的表达量,且作用效果随多廿烷醇浓度的升高而增强.此外,多廿烷醇抑制HMG-CoA还原酶的合成并加速其降解,多廿烷醇处理后HMG-CoA还原酶的半衰期明显缩短.     

丙型肝炎病毒NS4B蛋白调控Hep3B细胞非折叠蛋白质反应研究

研究表达的丙型肝炎病毒(HCV)NS4B对Hep3B细胞非折叠蛋白质反应的影响。NS4B重组真核表达质粒pcDNA3.1(-)NS4B通过脂质体转染Hep3B细胞,G418筛选和Western blot鉴定稳定转染细胞;RT-PCR检测稳定转染细胞内XBP1 mRNA剪接,Western blot鉴定ATF6蛋白切割,荧光素酶试验检测稳定转染细胞内GRP78和XBP1启动子活性。G418筛选和Western Blot鉴定证实获得稳定表达NS4B的Hep3B细胞;在该细胞内,XBP1 mRNA剪接、ATF6切割、XBP1和Grp78启动子激活均被检测到。NS4B在Hep3B的稳定表达诱导了非折叠蛋白质反应。     

Effects of NS-398 on the proliferation and apoptosis of HepG2 cells

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2(COX-2) inhibitor, on the proliferation and apoptosis in HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells was evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells; DNA ploidy and apoptotic cell percentage were examined by flow cytometry. Furthermore, the expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis in HepG2 in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with NS-398 concentration increasing. The quiescent G₀/G₁ phase was accumulated with decreasing of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, no correlations were found between COX-2 mRNA and the HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis in HepG2. Mechanisms may be involved in accumulation of quiescent G₀/G₁ phase and decrease in Bcl-2 mRNA expression, but independent to COX-2 mRNA expression.     

转人抑癌基因p53鸡的研究

【目的】探索MAR调控序列对外源人抑癌基因p53在转基因鸡体内整合稳定性的影响,为转基因禽类输卵管生物反应器的建立奠定基础。【方法】利用脂质体包埋法,将pEGFP-N1-p53/MAR重组真核表达载体转染人肝癌细胞(Hep3B),荧光显微镜和透射电镜检测转染含人野生型p53基因的Hep3B细胞形态及超微观结构的变化情况;显微注射法将脂质体介导的重组载体注入鸡X期胚盘的明区和暗区,制备G0代转基因鸡。以EGFP为报告基因,人工授精法制备G1代转基因鸡,对G0代和G1代鸡体外源基因p53进行PCR检测,并对G1代转基因鸡进行小动物活体成像检测。【结果】转染含人p53基因重组载体的Hep3B细胞明显皱缩变圆;微观结构层面,粗面内质网不明显,线粒体肿胀,细胞核中异染色质边集明显,并出现凋亡小体。G0代转基因鸡暗区组孵化率(45%)显著高于明区组孵化率(0)(P0.05),但均显著低于对照组孵化率(82.5%)(P0.05);获得4只基因组中含有外源p53基因的G0代转基因鸡,外源基因转染率为22.2%(4/18);获得2只G1代转基因鸡,转基因世代遗传率为3.08%(2/65)。【结论】重组真核表达载体pEGFP-N1-p53/MAR可以促进人肝癌细胞Hep3B的凋亡;脂质体介导的含MAR序列调控元件的pEGFP-N1-p53/MAR质粒,可以提高p53在转基因鸡基因组中的整合稳定性,并可遗传给后代。     

Construction of an eukaryotic expression vector encoding human granzyme B and it's expression in Hep2 cells

AIM: To construct pVAX1-GrB. METHODS: Lymphocytes from human laryngeal carcinoma tissue were separated from tumor tissue. The fragment of granzyme B (GrB) was amplified by RT-PCR and was recombined to the downstream of T7 promoter in the vector pVAX1. The construction was transfected into Hep2 cells with lipofectamine 2000. The expression of protein was identified by indirect immunofluorescent antibody assay. RESULTS: It has been proved that the sequence of the RT-PCR product was totally consistent with the data of GenBank by DNA sequencing analysis. The GrB cDNA fragment was cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB was maintained. The target protein was detected in the transfected Hep2 cells. CONCLUSION: The pVAX1-GrB plasmid was successfully constructed and expressed.     

甘草酸和甘草黄酮与人肝癌细胞(HepG2)作用的电化学研究

通过以人肝肿瘤细胞Hep G2为体外研究模型,采用了常见的电化学方法以及借助显微镜技术评估了甘草酸和甘草黄酮对它的影响,发现甘草酸在培养基中浓度低于2.0 g/L时,对肿瘤细胞的抑制效应比较小,当浓度扩大(大于2.0 g/L)时,抑制效应明显增大,细胞凋亡数目变化显著,并且在甘草酸浓度达到4.0 g/L之前,呈明显的浓度依赖性。而相同条件下甘草黄酮的抑制效应不大明显。通过本次研究,可以看出,甘草酸表现出了对肿瘤细胞Hep G2具有较明显的抑制作用。可以预见,作为一种比较常见的天然产物成分,它将在防癌抗癌药物的开发和研制当中得到人们的重视和应用。     

Flavonoid glycosides from Microtea debilis and their cytotoxic and anti-inflammatory effects

Two new 5-O-glucosylflavones, 5-O-β-d-glucopyranosyl cirsimaritin (1) and 5, 4′-O-β-d-diglucopyranosyl cirsimaritin (2), four known flavonoids, cirsimarin (3), cirsimaritin (4), salvigenin (5), 4′, 5-dihydroxy-7-methoxyflavone (6), and a norisoprenoid, vomifoliol (7), have been isolated from the aerial parts of Microtea debilis. All isolates were tested for cytotoxicity in human cancer cell lines (Hep G2, COLO 205, and HL-60) and anti-inflammatory activities in LPS-treated RAW264.7 macrophages. Compound 6 was found to be a potent inhibitor to nitrite production in macrophages. Compounds 2, 4, 6, and 7 showed moderate anti-proliferative activity against COLO-205 cells with IC₅₀ values of 7.1, 13.1, 6.1, and 6.8 μM, respectively.