Lipolysis and apoptosis of adipocytes induced by neuropeptide Y—Y5 receptor antisense oligodeoxynucleotides in obese rats

目的:探讨神经肽Y-Y5受体反义基因治疗后饮食性肥胖大鼠减肥减重效应与外周白色脂肪细胞体积、数目变化的关系。方法:建立饮食性肥胖大鼠模型,侧脑室注射Y5受体编码起始区反义、正义、错义寡脱氧核糖核酸及生理盐水,采用MPLAS-500多媒体彩色病理图文分析系统计算平均脂肪细胞面积,基因组DNA提取物凝胶电泳检测脂肪细胞凋亡,RT-PCR分析凋亡相关基因bc1-2、bax表达的改变。结果:(1)Y5受体反义基因治疗后大鼠进食量与体重显著降低,外周白色脂肪组织湿重与平均脂肪细胞面积明显减少;(2)脂肪组织基因组DNA提取物凝胶电泳出现凋亡特征性梯状条带;(3)凋亡相关基因bc1-2表达下调、bax表达上调。结论:平均脂肪细胞面积减小、脂肪细胞凋亡增加可能是Y5受体反义基因治疗减肥减重的重要原因。     

抗多药耐药基因肽核酸与反义寡脱氧核苷酸增强K562／ADM细胞的敏感性和促进凋亡

AIM: To investigate the reversal effect and apoptosis enhancement of peptide nucleic acid (PNA) and antisenseoligodeoxyribonucleotide (ASODN) targeted to multidrug resistance gene (mdrl) on human multidrug resistantleukemia K562/ADM cells. METHODS: A 15-mer PNA and the same sequence of ASODN, complementary to the5' end of the AUG initiator codon-containing region of mdrl messenger RNA (MDR1-PNA, MDR1-ASODN), weredesigned and synthesized. Proliferation and sensitivity to adriamycin of K562/ADM cells treated with MDRI-PNAand MDR1-ASODN were analyzed with a MTT colorimetric assay. Apoptotic morphologies, P-glycoprotein (P-gp)expression, intracellular adriamycin accumulation, and cell cycle were measured. RESULTS: MDRI-PNA 1 to 10μmol/L and MDR1-ASODN 2 to 20 μmol/L alone had no inhibitory effects on the proliferation of K562/ADM cells,but significantly inhibited the growth of K562/ADM cells cultured in adriamycin-containing medium. After treatment with MDRI-PNA and MDRI-ASODN, intracellular adriamycin accumulation in K562/ADM cells increasedgreatly and P-gp synthesis was strikingly reduced. The resistance to adriamycin of the drug-resistant cells waspartly reversed and the cells were induced to apoptosis by adriamycin. The reversal efficacy of MDR1-PNA was3.1-fold higher than that of the same sequence of MDR-ASODN, but neither MDRI-PNA nor MDRI-ASODNcould completely block the mdrllP-gp expression. CONCLUSION: Sequence-special PNA targeted to mdr1 genemore effectively than the same sequence of MDR1-ASODN inhibited the expression of P-glycoprotein to overcomethe drug-resistance.     

星形孢菌素诱导NG108—15细胞凋亡

目的：研究星形孢菌素是否能引起NG108—15细胞的凋亡及它对数种与凋亡相关基因蛋白表达水平的影响．方法：用相差显微镜、荧光显微镜和透射电镜观察形态学变化;琼脂糖凝胶电泳检测DNA梯带;免疫印迹法检测凋亡相关基因蛋白的表达水平．结果：经星形孢菌素0．1μmol／L处理后NG108—15细胞呈典型的凋亡形态学变化．星形孢菌素处理后6h即出现DNA凋亡梯带,可持续至24h．Bax蛋白表达在星形孢菌素处理后6h开始上升,12h至最高峰,24h后下降．Bcl-2蛋白表达在星形孢菌素处理后3h明显上升,随后逐渐下降．星形孢菌素处理后6h可见caspase-3切割产物出现,但Cdk5蛋白的表达水平未见明显变化．p53蛋白表达在星形孢菌素处理12h后下降．结论：星形孢菌素诱导了NG108—15细胞的凋亡,可能与Bax蛋白表达的增加及caspase-3切割有关,而与p53和Cdk5无明显相关性．     

Putative role of proteolysis and inflammatory response in the toxicity of nerve and blister chemical warfare agents: implications for multi-threat medical countermeasures

Despite the contrasts in chemistry and toxicity, for blister and nerve chemical warfare agents there may be some analogous proteolytic and inflammatory mediators and pathological pathways that can be pharmacological targets for a single-drug multi-threat medical countermeasure. The dermal-epidermal separation caused by proteases and bullous diseases compared with that observed following exposure to the blister agent sulfur mustard (2,2'-dichlorodiethyl sulfide) has fostered the hypothesis that sulfur mustard vesication involves proteolysis and inflammation. In conjunction with the paramount toxicological event of cholinergic crisis that causes acute toxicity and precipitates neuronal degeneration, both anaphylactoid reactions and pathological proteolytic activity have been reported in nerve-agent-intoxicated animals. Two classes of drugs already have demonstrated multi-threat activity for both nerve and blister agents. Serine protease inhibitors can prolong the survival of animals intoxicated with the nerve agent soman and can also protect against vesication caused by the blister agent sulfur mustard. Poly (ADP-ribose) polymerase (PARP) inhibitors can reduce both soman-induced neuronal degeneration and sulfur-mustard-induced epidermal necrosis. Protease and PARP inhibitors, like many of the other countermeasures for blister and nerve agents, have potent primary or secondary anti-inflammatory pharmacology. Accordingly, we hypothesize that drugs with anti-inflammatory actions against either nerve or blister agent might also display multi-threat efficacy for the inflammatory pathogenesis of both classes of chemical warfare agent.     

4-1BBL基因真核表达载体构建及在肝癌细胞中表达的研究

目的 研究含鼠源性4-lBBL真核表达载体体系的构建及其在大鼠肝细胞癌(HCC)细胞系CBRH7919中获得稳定、高效表达的方法。方法 将4-lBBL全长cDNA亚克隆到真核表达载体PCI-neo中,获得4-1BBL定向插入重组子PCI-neo-4-lBBL,采用酶切法和测序法鉴定。用阳离子脂质体将PCI-neo-4-1BBL转染CBRH7919,经G418筛选,获得阳性克隆,命名为PCI-neo-4-lBBL-CBRH7919 细胞。逆转录-聚合酶链反应(RT-PCR)检测4-1BBL在CBRH7919中的表达。结果 PCI-neo-4-1BBL经限制性内切酶切,电泳后显示有980bp的4-lBBL目的基因片段和5．4kb的线性化PCI-neo载体片段;重组子测序结果与Genbank中4-lBBL cDNA序列相符,证实构建成功。RT-PCR检测到4-lBBL在PCI-neo-4-1 BBL-CBRH7919 细胞中获得稳定表达。结论 重组4-1BBL真核表达载体构建正确,并在PCI-neo-4．1 BBL-CBRH7919 细胞中获得稳定、高效表达。     

Elevated levels of prostate-specific antigen (PSA) in prostate cancer cells expressing mutant p53 is associated with tumor metastasis

The underlying basis for rising levels of prostate-specific antigen (PSA) in prostate cancer is not fully understood, but attention has turned to the possibility that loss of normal p53 function might be directly involved. We have investigated the relationship between p53 function and PSA expression using in vitro and in vivo approaches. Three prostate cancer-derived p53 mutants (F134L, M237L, R273H) were introduced into LNCaP prostate cancer cells and stable transfectants established. Expression of mutant p53 was demonstrated by Western blot analysis, inactivation of wtp53 function, and a loss of p53-dependent responses to DNA damage induced by UV-irradiation and cisplatin. Levels of PSA mRNA and secreted protein were determined by RT-PCR and Western blotting, respectively. Serine protease activity was assessed using an esterase assay. In vivo effects of mutant p53 expression were examined after orthotopic implantation into prostates of nude mice. Expression of all p53 mutants was associated with elevated PSA mRNA and secreted PSA protein. In a representative line, mutant p53 was also associated with increased PSA protease-like activity compared with a control line expressing wildtype p53. Overall PSA levels, and PSA levels in serum from mice bearing tumors derived from cells expressing mutant p53, were increased compared with levels in mice bearing tumors derived from control cells. In addition, the tumors derived from cells with mutant p53 had increased vascularization and induced lymph node metastases. These data provide in vitro and in vivo support for the notion that p53 mutations directly contribute to increased levels of serum PSA, and are associated with more aggressive tumors.     

Epigenetic inactivation of TSLC1 gene in nasopharyngeal carcinoma

Deletion of 11q23 is a common genetic aberration in nasopharyngeal carcinoma (NPC). Multiple candidate tumor suppressor genes (TSG) were mapped to this region but few of them were investigated in NPC. TSLC1 (tumor suppressor in lung cancer) is recently reported to be a putative TSG on 11q23. This gene was found to be inactivated by promoter hypermethylation in non-small cell lung carcinoma (NSCLC), liver cancer, and breast cancer. To study the role of TSLC1 gene in NPC tumorigenesis, we screened for mutations and aberrant methylation of TSLC1 gene in 5 NPC cell lines, 3 NPC xenografts, and 38 primary NPC cases. No somatic mutations of TSLC1 were detected in the NPC samples, but a 9-bp (CCACCACCA) deletion in exon 8 was found in a primary NPC and its corresponding blood sample. Bisulfite sequencing revealed aberrant methylation of TSLC1 promoter in four NPC cell lines. Loss of TSLC1 gene expression was found in two cell lines (HK-1 and CNE-2) with dense methylation. Expression of this gene was restored in these cell lines after treatment with demethylating agent 5-aza-2'-deoxycytidine. Our results showed that silencing of TSLC1 gene expression in NPC was associated with promoter hypermethylation. Promoter hypermethylation of TSLC1 gene was further illustrated in 34.2% (13/38) of primary NPCs. No aberrant promoter methylation was found in any of the four investigated normal nasopharyngeal epithelia. Frequent epigenetic inactivation of TSLC1 gene in NPC suggested that this gene is one of the target tumor suppressor genes of this endemic cancer.