乙型肝炎病毒耐药寡核苷酸基因芯片的制备及特性分析

在构建了乙型肝炎病毒(HBV)拉米夫定抗药性相关HBV多聚酶基因突变体基础上,设计制备了HBV耐药寡核苷酸基因芯片,并对其特性进行了分析,为建立合理高效的HBV治疗方案,指导临床合理用药提供快速检测手段。一、材料与方法1.HBV耐药寡核苷酸芯片制备:因HBV拉米夫定抗药性相关突变主要位于HBV多聚酶基因的L526,A546,M550     

抗肿瘤反义寡核苷酸药物研究现状和趋势

以基因为靶的反义药物是抗肿瘤新药研究和开发的重要方向之一，已有近10种不同抗肿瘤反义寡核苷酸药物正在进行Ⅱ或Ⅲ期临床试验。基于肿瘤相关基因的多样性和复杂性，抗肿瘤反义药物的发展趋势是研究和开发靶向特定基因治疗特定类型肿瘤的“窄谱”抗肿瘤反义药物，以及由靶向多个肿瘤相关基因的反义寡核苷酸组成的“复方”式“广谱”抗肿瘤反义药物。     

脱氧核酶在转基因肝癌细胞中对HCV 5''-非编码区的抑制活性

目的　观察特异性脱氧核酶 (DRz)在丙型肝炎病毒 (HCV) 5′ 非编码区 (5′ NCR)转基因肝癌细胞株中对靶RNA的抑制活性。方法　采用 5′ GGCTAGCTACAACGA 3′基序为活性中心 ,分析HCV 5′ NCR中符合 5′…R↓Y… 3′(R =A/G ,Y =U/C)特征的位点及 5′ NCR的二级结构以确定靶位 ,设计并合成HCV靶向性脱氧核酶DRz 2 32、DRz 12 7、DRz 84、DRz1以及对应的两端被硫代修饰的DRz(TDRz)和活性中心区人工突变的硫代DRz(MDRz)。用脂质体转染法将导入HCV 5′ NCR及部分C区 (5′ NCR C)的转基因肝癌细胞株HepG2 .970 6 中 ,作用一定时间后 ,用化学发光法检测荧光素酶活性的变化 ,计算抑制率。选择抑制率较高者进行时效关系的观察和不同浓度抑制效果的比较。结果　各DRz和对应TDRz的活性无明显差别。DRz 12 7/TDRz 12 7和DRz1/TDRz1的抑制活性相对较高 ,终浓度为 0 .5 μmol/L作用 2 4h抑制率分别达 5 3.2 % / 5 0 .6 %和 4 4 .7% / 4 3.3%。各DRz/TDRz的抑制率均高于对应的MDRz ,其中DRz 12 7/TDRz 12 7与MDRz 12 7(4 1.2 % )相比抑制率有显著性差异 ,P <0 .0 5。在 0 .2 5～ 0 .75 μmol/L范围 ,抑制率随药物浓度的升高而增高。在所观察的时间点中 ,加药后 2 4h抑制率最高 ,3d后抑制率显著下降 ;DRz抑制率的下降快     

基于TLR5-NF-κB通路筛选四物汤中的活性成分

目的基于TLR5-NF-κB通路建立报告基因系统筛选四物汤中的有效成分。方法利用脂质体转染试剂Li-pofectin 2000将含有hTLR5基因和NF-κB Luc报告基因的质粒转染入人胚肾细胞(HEK293细胞),加入药物刺激后再检测细胞荧光素酶表达水平,筛选出能够激活TLR5受体及其下游NF-κB通路的有效成分。结果果糖、腺嘌呤能够显著激活此通路,且随果糖、腺嘌呤作用浓度的增加对细胞膜表面TLR5受体及其下游的NF-κB通路激活作用也随之增强,叶酸、东莨菪内酯、芍药苷、盐酸川芎嗪、β-谷甾醇、阿魏酸也能一定程度激活此通路。结论通过TLR5受体介导NF-κB通路激活剂筛选模型,筛选出四物汤内果糖、腺嘌呤、芍药苷、盐酸川芎嗪、叶酸、东莨菪内酯、β-谷甾醇、阿魏酸8种单体在一定浓度范围内能够激活TLR5受体及其下游的NF-κB通路。     

市场制度变迁中的文化约束

市场制度的变迁与人类文化的演进是一个互动的过程。文化在人类活动中产生 ,又通过人的价值观、习俗和道德等制约着人的经济行为。市场制度的演化受到文化的直接的非正式约束和间接的强制性约束。受知识和能力的限制 ,文化约束下的市场制度变迁可能是进步的 ,也可能是退步的。近代市场制度的形成与人的理性经济观、商业精神、契约文化和信用观念密切相关。在市场制度发展的初始阶段 ,特别要注重运用先进的市场经济文化引导制度的变迁。     

MicroRNA与细胞自噬调控

细胞自噬是进化过程中高度保守的物质降解再循环过程,是细胞将异常蛋白质、受损细胞器转运至溶酶体降解再利用的活动。自噬过程受到精密的调控。自噬功能障碍与神经退行性疾病、心血管疾病、肿瘤、骨代谢疾病、衰老等的发生有关。MicroRNA是一类对基因进行转录后修饰的非编码单链小RNA。越来越多的证据表明,MicroRNA可以通过调控自噬相关基因及其调节因子来影响自噬水平,是治疗自噬功能障碍所引发疾病的潜在靶点。本文对有关MicroRNA参与自噬调控的最新动态进行综述。     

基因转染的pH敏脂质体的最优质处方和制备工艺的研究

目的寻找用于基因转染的pH敏脂质体的最佳处方和制备工艺.方法采用正交设计法,考虑制备的pH值、组成摩尔比、制备方法三因素,即1个四水平因素和2个二水平因素混合状合.选用L16(41·212)正交设计表安排试验,各水平组合条件下,通过基因转染实验,以基因转染荧光测定值作为观测指标,实验重复4次.结果在基因与脂质体重量比为(18)时,组成大豆磷脂与DC-胆固醇摩尔比为(64)和在超声法制备的特定条件下,pH5.4与pH7.4基因转染荧光测定值之间差别非常显著;均值都较大,但是以pH5.4条件下基因转染荧光测定值均值最大.结论基因传染荧光测定值最高的脂质体为在pH5.4、组成大豆磷脂与DC-胆固醇摩尔比为(64)时采用超声法制备的脂质体.     

Design and activity evaluation of deoxyribozymes specifically targeting hepatitis C virus RNA

Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV)-specific deoxyribozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure of HCV 5′-noncoding region (5′-NCR) and the sites characterized with 5′…Y ↓ R…3′ ( Y = A/G, R = U/C), HCV-specific naive deoxyribozymes were designed and named DRz-232, DRz-127, DRz-84, DRzl, and the phosphorothioate deoxyribozymes (PSDRz) and mutated phosphorothioate deoxyribozymes (MPSDRz) were also designed. HCV RNA 5′-NCR was transcribed in vitro from linearized plasmid pHCV-neo and radiolabelled at its 5′-end. DRz, PSDRz or MPSDRz was respectively mixed with the substrate RNA and incubated under appropriate conditions, the cleaved products were displayed by 8% denaturated polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the optical density of each band was measured to calculate cleavage rates. After that, every kind of DRz was added respectively to the cultured transgeneic HepG2 cells containing luciferase gene controlled by HCV 5′-NCR. The ceils were lysed at intended time points and the activity of luciferase was measured with chemiluminescence method for calculating inhibition rates. Results: After incubated for 90 rain in vitro, the cleavage rates of DRz-127, PSDRz-127, DRzl and PSDRzl reached 32.6%, 30.8%, 24.3% and 21.5%, respectively. No cleavage product was observed in any MPSDRz. DRz-127, PSDRz-127, DRzl and PSDRzl had an inhibitory rate of 53.2%, 50.6%, 44.7% and 43.3% respectively in transgeneic HepG2 cells in the first 24 h when the final dose of the DRz was 0.5 la.moL/L, higher than that of the corresponding MPSDRz. There was no significant difference between the inhibitory effect of each DRz and its PSDRz in HepG2 cells, but the inhibitory rate of DRz decreased more rapidly than that of the latter with the elapse of time. The results from transfection groups were significantly better than those of non-transfection groups. Conclusion:Rationally-designed HCV-specific deoxyribozymes are able to cleave target RNA at molecular level in vitro, and efficiently inhibit the expression of luciferase gene controlled by HCV 5′-NCR in transgeneic cells. Appropriate PSDRz may be more stable, and thus more suitable than the naive DRz in the application to cells. Introduction of the deoxyribozymes with transfection is more efficient than with direct delivering ways.     

重组人生长激素基因转染细胞移植Ⅳ:转染细胞对重组生长激素基因的表达

重组人生长激素基因转染细胞移植Ⅳ：转染细胞对重组生长激素基因的表达胡志强于康震戴锁舜杨富明李呼伦辛晓敏高自强于康震陈忠斌王秀丽我们利用磷酸钙介导的方法将重组人生长激素（ｈＧＨ）基因导入培养的人胚皮肤成纤维细胞内，之后用Ｎｏｒｔｈｅｒｎ杂交证实转染的细...