幽门螺杆菌尿素通道蛋白基因UreI载体的构建、融合表达及纯化

目的构建幽门螺杆菌(H.pylori,Hp)尿素通道蛋白基因UreI的融合表达载体,并在E.coliBL21中表达,为进一步研究UreI的功能奠定基础。方法利用分子克隆技术以Hp DNA染色体为模板,扩增Hp UreI基因片段,将目的基因UreI与载体pET32a( )分别经kpnⅠ和HindⅢ双酶切,纯化,连接。筛选阳性重组载体,转化大肠杆菌BL21,以IPTG诱导表达pET32a( )/UreI融合蛋白。表达产物经Ni-NTA琼脂糖树脂纯化后,用SDS-PAGE及Western blot分析。结果经酶切、测序分析表明,插入的基因片段为Hp UreI编码基因,由585 bp组成,与GenBank相应菌株HP AM417 609序列完全一致,无碱基突变。经SDS-PAGE分析显示重组质粒pET32a( )/UreI在原核细胞中得到了高效融合表达,其重组融合蛋白分子量为130 ku,经Ni-NTA琼脂糖树脂纯化后,其纯度达95%以上,Western blot分析显示,该重组融合蛋白可被6-His鼠抗单克隆抗体所识别,具有良好的反应原性。结论成功地克隆和构建了原核表达载体pET32a( )/UreI,并在原核细胞中得到了高效融合表达。     

生存素在5-氟尿嘧啶诱导人绒癌细胞株JAR凋亡中的作用

目的探讨生存素(survivin)在化疗药物5-氟尿嘧啶(5-Fu)诱导的人绒癌细胞株(JAR)凋亡中的作用。方法用JAR细胞株进行培养传代,以不同浓度5-Fu作用于JAR细胞,MTT方法观察5-Fu对JAR细胞生长的抑制情况,流式细胞仪检测细胞凋亡率,用逆转录聚合酶链(RT-PCR)和免疫印迹技术(Western-blot)方法检测survivin基因和蛋白的表达。结果①5-Fu对JAR细胞的生长具有抑制作用,并有浓度和时间的依赖性;②5-Fu诱导JAR细胞凋亡,凋亡率随浓度而增加;③5-Fu作用后survivin基因和蛋白表达减少,随浓度增加而减少。结论5-Fu可能是通过降低survivin表达水平,诱导JAR细胞凋亡。     

The changes of neuronal activity of the zone incerta in a rodent model of Parkinson＇s disease

Objective To study the changes in neuronal activity of the zone incerta (ZI) following the unilateral lesion of the nigrostriatal pathway. Methods Eiectrophysiological recordings of ZI neurons were made in normal rats and in two groups of rats at different time intervals after injection of 6-hydrodopamine (6-OHDA) into the pars compacta of snbstantia nigra by extracellular recording in vivo. Results The results showed a significant increase in the mean firing rate of ZI during the second and fourth weeks after 6-OHDA lesion [-n=32, (3.6±2. 2)Hz, P<0.001; n=35, (9.3±6.6)Hz, P<0. 001, respectively] compared to that of normal rats [n=39, (9.2±5.2)Hz]. However, no significant change was observed between two groups of 6-OHDA-lesioned rats. With regard to firing pattern, 7.7%(3/39) of ZI neurons discharged regularly, 82.1% (32/39) irregularly and 10. 3% (4/39) in bursts in normal rats. During the second week after 6-OHDA lesion, the regular, irregular and bursting firing neurons recorded in the ZI were 9.4% (3/32), 59. 4% (19/32) and 31.3% (10/32), and during fourth week, the regular, irregular and bursting firing neurons were 14.3% (5/35), 57.1% (20/35) and 28. 6% (10/35), respectively. The firing pattern of the neurons in the three different groups did not change significantly. Conclusion These results suggest that the firing rate of ZI neurons in 6-OHDA-lesioned rats is increased significantly, which may contribute to the pathophysiology of Parkinson's disease.     

Effect of ABCA1-V771M polymorphism on plasma lipid levels and its relationship with coronary atherosclerotic heart disease

Objective To explore the risk association of ABCA1-V771M polymorphism with coronary heart disease (CHD) in Hart nationality in Northwest of China. Methods With case-control study, ABCA1-V771M polymorphism was detected in 204 unrelated Hart nationality people in Northwest of China, and all the subjects by coronary angiography were grouped into 106 cases and 98 controls. The genotypes and alleles frequency distribution of ABCA1-V771M polymorphisms were analyzed by PCR-RFLP analysis, and the clinical statistics of serum lipids were compared and its effects of ABCA1-V771M polymorphism on the plasma lipid levels and coronary atherosclerotic heart disease were analyzed. Results The genotypic frequencies of ABCA1-V771M polymorphism matched well under Hardy-Weinberg equilibrium (P>0.05), V and M allelic frequencies were 33.3% and 66.7%. In comparison with VV VM genotype carriers, MM genotypes carriers had much lower plasma levels of HDL-C (P<0. 001) and much higher plasma levels of TG (P<0. 05). M allelic frequency in CHD group was significantly higher than V allelic frequency (P<0. 05). M allele was related with more severity of atherosclerosis in the coronary artery than V allele (P<0.05). However, there was no obvious difference in the incidence of AMI among carriers with three genotypes of ABCA1-V771M polymorphism (P>0.05). Conclusion ABCA1-V771M polymorphism was not only associated with the plasma levels of HDL-C and TG, but also related to the susceptibility and severity of coronary atheroselerotic heart disease. Moreover, M771 allele appeared to be atherogenie among Han population in Northwest of China.     

Study on T lymphocyte subsets and NK cells in patients with Graves＇ disease combined with type 2 diabetes

Objective To investigate changes in T lymphocyte subsets and NK cells in patients with simple Graves' disease (GD)and Graves' disease combined with type 2 diabetes mellitus (GD/T2DM). Methods Fifteen cases of GD/ T2DM were selected from our hospital from November 2001 to November 2004. Before and after therapy thyroid function, thyroglobulin antibody (TGA), thyroid microsomal antibody (TMA) and blood glucose level were measured, and T lymphocyte subsets (CD3, CD4, CD8, CD4/CD8) and NK cells (CD56) were measured by immunofluorescence double labeling monoclonal antibody and flow cytometry, respectively. At the same time, comparison was made with simple GD (15 cases), T2DM (15 cases) and healthy control (20 cases). Results Before therapy, CD4/CD8, CD4 and NK cells in GD/T2DM were less than normal, and there was no significant difference in comparison with simple GD (P<0.05). In T2DM group, only CD4/CD8 and CD4 were less than those of healthy controls (P<0.05). When thyroid function recovered after 1 to 3 months of methimazole treatment in both GD/T2DM and simple GD groups, various indexes recovered, which were more obvious in simple GD. Conclusion Immune hypofunction of GD may be the key to the immune abnormality of GD/T2DM, which is more significant than that of simple GD or T2DM. The recovery of thyroid function and immune abnormality is not consistent, and the recovery of GD is more significant than that of GD/T2DM.     

2型糖尿病患者一级亲属正常糖代谢人群胰岛素抵抗分析

目的探讨2型糖尿病(T2DM)患者糖耐量正常的一级亲属胰岛β细胞功能及胰岛素抵抗情况,并分析胰岛素抵抗的相关因素。方法选取T2DM患者糖耐量正常的一级亲属63例,根据胰岛素抵抗指数分为胰岛素抵抗组及非抵抗组;选取30例健康体检者作为正常对照组。对所选的93例对象进行血压、血糖、胰岛素、血脂的测定,并计算稳态模型胰岛素抵抗指数(HOMA-IR)及胰岛β细胞功能指数(HOMA-β)。结果①糖尿病患者一级亲属组与对照组相比,胰岛素水平、HOMA-IR、HOMA-β明显升高(P<0.05);②胰岛素抵抗组体质指数、舒张压、甘油三酯、餐后血糖、HOMA-β明显高于胰岛素非抵抗组(P<0.05);③相关分析示胰岛素抵抗与体质指数、舒张压、甘油三酯、餐后胰岛素呈正相关(P<0.01,P<0.05),与空腹血糖、餐后血糖、空腹胰岛素、收缩压、胆固醇无显著相关性(P>0.05)。结论T2DM患者一级亲属存在胰岛β细胞代偿性的高分泌,早期已经出现胰岛素敏感性的下降和胰岛素抵抗。胰岛素抵抗与体重增加、舒张压升高及甘油三酯的升高有关。提示对糖尿病患者一级亲属应严格控制体重、血压及血脂,对减少T2DM具有积极意义。     

应用离心造粒法制备盐酸二甲双胍微丸的研究

目的研究离心造粒粉末层积法制备盐酸二甲双胍微丸。方法采用多功能微丸包衣造粒机制备微晶纤维素空白丸芯和盐酸二甲双胍微丸,并且通过产率、粒径和粒径分布、堆密度、表面形态和圆整度、脆碎度和释放度对盐酸二甲双胍微丸进行质量评价。结果以盐酸二甲双胍∶乳糖∶微晶纤维素=100∶5∶3的比例,主机转速150 r/min,喷浆泵转速20 r/min,供粉速度25 r/min,滚圆6 min,进行制备微丸,获得了很好的效果。结论所制备的盐酸二甲双胍微丸圆整度好,粒径均匀,硬度合格,流动性好,释放完全,无骨架结构。     

选择性垂体甲状腺激素抵抗综合征患者的临床特点与THRβ基因突变的研究

目的研究2例选择性垂体甲状腺激素抵抗综合征患者的临床特点及甲状腺激素受体β(THRβ)基因突变,探索该病的发病机制。方法收集2例患者及其父母的临床资料并留取外周血标本,提取基因组DNA,应用PCR-DNA直接测序的方法研究患者及父母的THRβ基因有无突变。结果2例患者及父母的THRβ基因第1-10外显子未发现突变。结论散发性垂体甲状腺激素抵抗综合征患者发病可能与THRβ基因突变无关。     

WWOX蛋白在非小细胞肺癌中的表达及其临床意义

目的 探讨WWOX(WW domain-containing oxidoreductase)基因在非小细胞肺癌组织中的表达及其临床意义.方法 用免疫组化方法检测50例鳞癌、31例腺癌及20例癌旁正常组织中WWOX基因蛋白的表达,并分析其与各临床病理指标之间的相关性.结果 WWOX基因在72.8%的非小细胞肺癌中失表达或表达减少,而在相邻正常肺组织中有80.0%正常表达.WWOX基因的表达与组织类型、细胞分化程度密切相关(P<0.05),在鳞癌和低分化癌中WWOX基因失表达或表达减少.结论 WWOX蛋白在非小细胞肺癌组织中呈低表达,该蛋白表达的缺失可能在不同类型非小细胞肺癌的形成中发挥了不同的作用.     

动力总成悬置支架多目标拓扑优化和强度分析

基于Optistruct,以支架的最小柔度、最大固有频率作为优化目标函数;以应力、体积作为约束函数,对一发动机后拉杆安装支架进行拓扑优化,优化结果表明:在满足悬置支架强度和模态要求下,质量减少了12%。