灯盏花查尔酮合成酶基因表达与灯盏乙素含量关系的研究

目的: 灯盏花的有效成分灯盏乙素是一种具有显著药理活性的黄酮类化合物,目前仍缺乏对其合成途径的相关认识。查尔酮合成酶(CHS)是黄酮类生物合成的一个关键酶,该研究旨在通过研究CHS表达水平与灯盏花中各组织灯盏乙素含量的变化规律,阐明该基因的表达模式与灯盏乙素含量之间的关系。 方法: 通过RT-PCR及RACE方法从灯盏花中克隆CHS基因全长,利用荧光定量PCR方法检测该基因在灯盏花各组织中的表达量,采用HPLC分析各组织中灯盏乙素的含量。 结果: 序列全长1 270 bp,编码405氨基酸,该基因DNA序列与菊科植物的同源基因相似性在80%左右,荧光定量显示CHS在叶中表达量最高,远高于根、茎和花;HPLC发现灯盏乙素在叶中含量最高,其次是花和茎,而在根中未检测到。 结论: 相关性分析表明CHS相对表达量与灯盏花不同部位灯盏乙素含量间呈正相关关系(r=0.761,P<0.05),说明灯盏乙素的生成与CHS基因的表达密切相关。     

Honokiol up-regulates prostacyclin synthease protein expression and inhibits endothelial cell apoptosis

Honokiol is a bioactive compound extracted from the Chinese medicinal herb Magnolia officinalis. We recently demonstrated that honokiol inhibited arterial thrombosis through stimulation of prostacyclin (PGI2) generation and endothelial cell protection. The current study is designed to investigate its mechanism of stimulation of PGI2 generation and cell protection. 6-keto-PGF1alpha, the stable metabolite of PGI2, in the media of rat aortic endothelial cells was measured with radioimmunoassay kits. Indomethacin, an inhibitor of cyclooxygenase (COX) and tranylcypromine, a prostacyclin synthease inhibitor were used to ascertain the target enzyme affected by honokiol. Prostacyclin synthease protein levels in endothelial cells were determined by Western blot analysis using an anti-PGI2 synthease rabbit polyclonal antibody. Flow cytometry was used to quantify the apoptotic cells and spectrophotometry was used to test the caspase-3 activity. Honokiol (0.376-37.6 microM) increased the level of 6-keto-PGF1alpha in the media of normal endothelial cells. It counteracted the inhibitory effect of tranylcypromine on the PGI2 generation, but did not influence the effect of indomethacin; evidently, honokiol up-regulated the expression of prostacyclin synthease in the endothelial cells. These effects showed perfect concentration-dependent behavior. In addition, at lower concentration (0.376-3.76 microM), honokiol significantly decreased the percentage of apoptotic endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) and significantly lowered the activity of caspase-3 stimulated by ox-LDL. A high dose of honokiol (37.6 microM), however, failed to influence either of them. In conclusion, honokiol augments PGI2 generation in normal endothelial cells; its effect on PGI2 generation attributes to up-regulation of prostacyclin synthease expression; its cell protection may be correlated with its inhibition on apoptosis of endothelial cells. These findings have partly revealed the molecular mechanism of honokiol on inhibiting arterial thrombosis.     

诱导型一氧化氮合酶对乳腺癌细胞化学敏感性的影响

目的 分析可诱导一氧化氮合酶（iNOS）对乳腺癌细胞化学敏感性的影响，评价新型蒽醌类生物还原化合物RHl对乳腺癌细胞毒性和乏氧选择性。方法 实验以人乳腺癌细胞MDA-MB-231”和其转染空载体的对照组细胞（PEF-vector）及一氧化氮合酶基因转染的细胞克隆（iNOS10）为实验对象。噻唑蓝（MTT）法比较不同酶活性的细胞克隆对顺铂等抗肿瘤药物和丝裂霉素C（MMC）衍生物-2，5-二氮丙啶-6-羟甲基-3-甲基苯-1，4-二酮（RH1）的敏感性的差异。结果同对照组细胞相比，iNOS10细胞对顺铂等几种抗肿瘤药物呈现不同程度的耐受，而对阿霉素的敏感性有所增强（P〈0．05），用L-甲基盐酸精氨酸抑制细胞中NO的合成，则会降低iN-0S10细胞对阿霉素的敏感性，而对其他几种抗肿瘤药物的敏感性无显著影响；生物还原化合物RH1是高毒性的化合物，IC50较MMC下降577倍；乏氧条件下，细胞对RH1的化学敏感性显著增强，流式细胞术显示RH1可以引起iNOS10细胞周期的G2／M阻滞。结论 MMC衍生物RH1是高细胞毒性的化合物，可诱导一氧化氮合酶活性的增强，也可引起肿瘤细胞对部分抗肿瘤药物的敏感性降低，且对高表达iNOS的细胞具有乏氧选择性的损伤作用。     

诱导型一氧化氮合酶影响吲哚醌类化合物抗肿瘤活性的实验研究

观察诱导型一氧化氮合酶(iducible nitric oxide synthase,iNOS)对新型吲哚醌类生物还原化合物630和630Ac的抗肿瘤活性和乏氧选择性的影响。以人纤维肉瘤细胞HT1080及其iNOS基因转染的细胞克隆为研究对象,噻唑蓝法检测不同iNOS活性的细胞克隆对化合物630和630Ac化学敏感性的差异;比较乏氧和有氧条件下半数抑制浓度(IC50)的差异;流式细胞术观察化合物对细胞周期的影响。630Ac和630在实验中均显示出较强的抗肿瘤活性,且630Ac的细胞毒性强于630;而iNOS转染的细胞对630和630Ac敏感性较亲本细胞HT1080有所下降,IC50分别高1~7倍左右。氧对630的细胞毒性无显著影响,而630Ac则具有较好的乏氧选择性细胞毒作用,尤其是对iNOS转基因细胞,有氧和乏氧条件下IC50相差4~7倍。提示iNOS活性的增强会引起肿瘤细胞对化合物630和630Ac敏感性的降低。     

胸苷酸合成酶基因多态性与氟尿嘧啶治疗晚期胃癌疗效相关性的研究

目的：研究胸营酸合成酶（TS）基因的5’-UTR28bp核苷酸片段重复多态和3’-UTR1494修饰处存在的6bp核苷酸片段缺失或插入多态与应用以氟尿嘧啶（5-FU）为主的联合化疗方案治疗胃癌疗效的相关性。方法：收集经病理学确诊的晚期胃癌74例，所有患者使用以5-Fu为主的化疗方案治疗，化疗前抽静脉血，提取白细胞DNA用PCR—DHPLC技术检测TS5’-UTR1R28bp及3’-UTR6bp基因型：结果：（1）74例胃癌患者的TS5’-UTR28bp31t／3R、2R／2R＋21t／3R基因型分别为21．6％（16／74）和78．4％（58／74），而相应的化疗客观有效率分别为6．3％（1／16）和36．2％（21／58），3R／3R组的化疗疗效比2R／2R＋2R／31／绀低（X^2=4．05，P〈0．05）；（2）74例胃癌患者的TS3’-UTR＋／＋6bp、＋／-6bp、-／-6bp基因型分别为5．4％（4／74）、52．7％（39／74）和41．9％（31／74），相应各组的化疗有效率分别为0（0／4）、25．6％（10／39）和41．9％（13／31），不同基因型与化疗疗效之间无相天性（P=0．076）。结论：TS5’-UTR28bp多态性与5-FU疗效之间存在相关性，其检测有助于指导晚期胃癌选择5-FU化疗，而TS3’-UTR多态性与5-FU疗效之间没有显示相关性，     

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive human cancer with increasing incidence worldwide. Multiple efforts have been made to explore pharmaceutical therapies to treat HCC, such as targeted tyrosine kinase inhibitors, immune based therapies and combination of chemotherapy. However, limitations exist in current strategies including chemoresistance for instance. Tumor initiation and progression is driven by reprogramming of metabolism, in particular during HCC development. Recently, metabolic associated fatty liver disease (MAFLD), a reappraisal of new nomenclature for non-alcoholic fatty liver disease (NAFLD), indicates growing appreciation of metabolism in the pathogenesis of liver disease, including HCC, thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment. In this review, we introduce directions by highlighting the metabolic targets in glucose, fatty acid, amino acid and glutamine metabolism, which are suitable for HCC pharmaceutical intervention. We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment. Furthermore, opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.