抗胃癌抗体重链可变区序列分析及空间结构模拟

目的:通过序列分析确定抗胃癌抗体重链可变区(VH)的一级结构,并借助同源模建方法模拟其三级结构.方法:从抗人胃癌噬菌体抗体库中筛选出VH基因,并进行序列测定、翻译和分析.利用计算机辅助蛋白质空间模拟技术,采用同源模建、力学优化合理模建VH的三维空间结构.结果:序列比对分析表明获得的VH序列符合鼠抗体可变区特征,通过Kabat分析确定了FR、CDR;合理搭建了抗体重链可变区的空间构象,并通过分子力学优化获得了稳定的三维结构.结论:所测得的VH一级结构和构建的三维空间结构均有较高的可靠性,为进一步的生物学实验奠定了基础.     

Isolation of drug-resistant variants of HIV-1 from patients on long-term zidovudine therapy. Canadian Zidovudine Multi-Centre Study Group

We determined whether drug-resistant variants of HIV-1 could be isolated from the peripheral blood mononuclear cells of 20 individuals with HIV infection (Centers for Disease Control groups II and III) on long-term zidovudine (AZT) therapy. Toward this end, zidovudine (10 microM) has been included in the tissue culture medium used to isolate HIV-1. Under these circumstances, virus with a zidovudine-resistant phenotype was successfully obtained in five out of 20 cases. This property of drug resistance appeared to be stable, and did not disappear upon extended replication of such virus in the absence of drug pressure. Drug-resistant virus could also be isolated from these subjects on subsequent occasions, but was not present in samples obtained prior to therapy. Replication of these zidovudine-resistant isolates in tissue culture was inhibited by each of four other nucleoside analogues. Thus, other drugs may be useful in controlling selective zidovudine-resistant variants of HIV-1.     

高灵敏度薄膜生物传感器基因芯片系统的研制

目的: 在传统基因芯片技术基础上, 应用生物传感器技术, 研制一种能将基因芯片信号原位放大后达肉眼判读灵敏度, 无需专用基因芯片检测仪器就可使用, 易于在基层医疗单位推广的薄膜生物传感器基因芯片诊断系统.方法:在薄膜生物传感器基片基础上, 经过表面化学处理, 使特定的基因捕获探针在传感器表面固定, 形成特定检测目的生物传感器基因芯片. 并以乙肝病毒YMDD区的特异序列设计为例, 通过特定的YMDD区的捕获探针, 以矩阵的形式点样于传感器芯片表面来实现本系统. 同时, 纳米金标记的检测探针取代了传统芯片中的荧光标记探针. 扩增后的目的PCR片段与捕获探针、生物素标记探针、链亲蛋白纳米金探针进行反应, 最后得到探针-生物素-链亲蛋白-纳米金复合物, 并在芯片表面经生物传感器芯片将信号原位放大, 获得肉眼观的芯片信号并进行分析, 完成芯片诊断. 以乙型肝炎病毒YMDD突变为例, 观察该芯片系统对临床诊断标本诊断的可靠性.结果: 基因芯片的检测信号经生物传感器原位放大后能肉眼判读或借助普通数码照像机或计算机扫描, 根据信号出现的特定位置即可确定突变的类型. 且该生物传感器基因芯片系统信噪比高, 在人工合成的寡核苷酸及临床血清的检测中, 均可实现生物芯片阴阳性信号完全的有或无的判读; 临床血清标本检测证实, 使用该传感器芯片系统对前期经过测序确定为YMDD突变的23份临床血清结果与测序结果完全一致. 结论:薄膜生物传感器基因芯片集纳米材料、生物传感器技术及原位放大技术为一体, 实现了信号的肉眼判读, 具有通用性高, 准确可靠, 无需大型设备, 易于在基层医疗单位推广使用.     

Suppressive oligodeoxynucleotides inhibit atherosclerosis in ApoE(-/-) mice through modulation of Th1/Th2 balance

Atherosclerosis is a chronic inflammatory disease, which is positively and negatively regulated by T helper (Th) 1 and Th2 lymphocytes, respectively. Recent findings indicate that suppressive oligodeoxynucleotides (ODNs) expressing TTAGGG motifs selectively reduce Th1 cytokine production and have been proven effective at blocking the development of organ-specific autoimmune diseases. In the current research, we hypothesized that suppressive ODNs may alter the development of atherosclerosis. Eight-week-old homozygous ApoE^−/− male mice were injected with 300 μg ODNs A151 (TTAGGG) or nonspecific ODNs 1612. Atherosclerotic lesion sizes were dramatically reduced by ODNs A151, but not by nonspecific ODNs. MCP-1 and VCAM-1, which are the key inflammatory factors in atherogenesis, were significantly attenuated by the suppressive ODNs A151. In the splenic lymphocytes, FACS analysis showed ODNs A151 reduced the percentage of IFN-γ-producing Th1 cells and slightly increased the percentage of IL-4-producing Th2 cells, indicating that suppressive ODNs skewed the Th1/Th2 balance toward Th2 inflammation in vivo. Furthermore, ODNs A151 down-regulated the phosphorylation of STAT1 and STAT4 and suppressed up-regulation of T-bet, a signal modulator for Th1, and didn't impact GATA-3 and STAT6, which are associated with a Th2 phenotype. Consistent with this in vivo observation, ELISA analysis demonstrated that ODNs A151 suppressed Th1 cytokines IFN-γ and TNF-α, and augmented Th2 cytokines IL-4 and IL-10 in vitro. This study provides the first experimental evidence that suppressive ODNs inhibit the development of atherosclerosis through inhibition of the STAT1/4 and T-bet pathways, which further modulate the Th1/Th2 balance in vivo.     

Liver fibrosis in chronic viral hepatitis： An ultrasonographic study

AIM: To select valuable ultrasonographic predictors for the evaluation of hepatic inflammation and fibrosis degree in chronic hepatitis, and to study the value of ultrasonography in the evaluation of liver fibrosis and compensated liver cirrhosis in comparison with serology and histology. METHODS: Forty-four ultrasonographic variables were analyzed and screened using color Doppler ultrasound system in 225 patients with chronic viral hepatitis and compensated liver cirrhosis. The valuable ultrasonographic predictors were selected on the basis of a comparison with histopathological findings. The value of ultrasonography and serology in the evaluation of liver fibrosis degree and the diagnosis of compensated liver cirrhosis was also studied and compared. Meanwhile, the influencing factors on ultrasonographic diagnosis of compensated liver cirrhosis were also analyzed. RESULTS: By statistical analysis, the maximum velocity of portal vein and the degree of gall-bladder wall smoothness were selected as the valuable predictors for the inflammation grade (G), while liver surface, hepatic parenchymal echo pattern, and the wall thickness of gall-bladder were selected as the valuable predictors for the fibrosis stage (S). Three S-related independent ultrasonographyic predictors and three routine serum fibrosis markers (HA, HPCIII and CIV) were used to discriminate variables for the comparison of ultrasonography with serology. The diagnostic accuracy of ultrasonography in moderate fibrosis was higher than that of serology (P<0.01), while there were no significant differences in the general diagnostic accuracy of fibrosis as well as between mild and severe fibrosis (P<0.05). There were no significant differences between ultrasonography and serology in the diagnosis of compensated liver cirrhosis. However, the diagnostic accuracy of ultrasonography was higher in inactive liver cirrhosis and lower in active cirrhosis than that of serology (both P<0.05). False positive and false negative results where found when the diagnosis of compensated liver cirrhosis was made by ultrasonography. CONCLUSION: There are different ultrasonographic predictors for the evaluation of hepatic inflammation grade and fibrosis stage of chronic hepatitis. Both ultrasonography and serology have their own advantages and disadvantages in the evaluation of liver fibrosis and compensated liver cirrhosis. Combined application of the two methods is hopeful to improve the diagnostic accuracy.     

Expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia

AIM: To compare the difference of expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia, and to analyze the role of Bcl-2 and Bax proteins in the progression from dysplasia to carcinoma and to evaluate the correlation of Bcl-2/Bax protein expression with the biological behaviors. METHODS: Expressions of Bcl-2 and Bax were examined immunohistochemically in 27 cases of extrahepatic biliary tract carcinomas (bile duct carcinoma: n=21, carcinoma of ampulla of Vater: n=6), and 10 cases of atypical dysplasia. Five cases of normal biliary epithelial tissues were used as controls. A semiquantitative scoring system was used to assess the Bcl-2 and Bax reactivity. RESULTS: The expression of Bcl-2 was observed in 10 out of 27 (37.0%) invasive carcinomas, 1 out of 10 dysplasias, none out of 5 normal epithelial tissues. Bax expression rate was 74.1% (20/27) in invasive carcinoma, 30% (3/10) in dysplasia, and 40% (2/5) in normal biliary epithelium. Bcl-2 and Bax activities were more intense in carcinoma than in dysplasia, with no significant difference in Bcl-2 expression (P=0.110), and significant difference in Bax expression (P=0.038). Level of Bax expression was higher in invasive carcinoma than in dysplasia and normal tissue (P=0.012). Bcl-2 expression was correlated to Bax expression (P=0.0059). However, Bcl-2/Bax expression had no correlation with histological subtype, grade of differentiation, or level of invasion. CONCLUSION: Increased Bcl-2/Bax expression from dysplasia to invasive tumors supports the view that this is the usual route for the development of extrahepatic biliary tract carcinoma. Bcl-2/Bax may be involved, at least in part, in the apoptotic activity in extrahepatic biliary carcinoma.     

Transient pancytopenia preceding T lineage acute lymphoblastic leukemia

Transient pancytopenia preceding acute lymphoblastic leukemia (pre-ALL) is a rare occurrence usually affecting children with subsequent development of B lineage ALL. We report a case of pre-ALL characterized by a T cell immunophenotype and abnormal karyotype t (11; 14) (q10; q10). The patient achieved a transient complete remission after initial therapy, but relapsed within a few months and died of leukemic encephalopathy. To the best of our knowledge, this is the first report of T lineage pre-ALL.     

MicroRNAs 103 and 107 link type 2 diabetes and post-menopausal breast cancer

Type 2 diabetes mellitus (T2DM) increases the incidence of post-menopausal breast cancer (PMBC). This study is intended to determine whether microRNA-103/107 (miR-103/107) should be regarded as a potential molecular link between T2DM and PMBC. Samples of serum from 90 patients with T2DM and/or PMBC were collected. Samples of serum from 20 non-diabetic post-menopausal women were used as the control. The body mass index (BMI) of patients with T2DM and PMBC was lower than the BMI of patients with only T2DM or PMBC (p?<?0.05). The expression of miR-103/107 was higher in the serum of T2DM patients compared with that in control samples (2.80?±?0.46/36.29?±?3.41 vs 0.88?±?0.25/8.59?±?1.91, p?<?0.05). The expression of miR-103/107 in the serum of PMBC patients was higher than that in T2DM patients (5.06?±?0.92/49.59?±?6.99 vs 2.80?±?0.46/36.29?±?3.41, p?<?0.05) but lower than that in patients diagnosed with both T2DM and PMBC (7.67?±?0.87/63.24?±?8.58, p?<?0.05). miR-103/107 was positively correlated with the homeostasis model assessment-insulin resistance (HOMA-IR) index (r?=?0.71, 0.685, p?<?0.01). The expression of miR-103/107 was an independent factor of the HOMA-IR index (β?=?0.638, 0.073, p?=?0.02, 0.01). There were higher levels of estradiol (E2) in patients with T2DM and/or PMBC than that in the control group. High expression of miR-103/107 results in insulin resistance and is associated with overweight or obese patients with T2DM and PMBC through elevated levels of E2. miR-103/107 may be a potential molecular link between T2DM and PMBC.     

Protection of Luteolin-7-<Emphasis Type="Italic">O</Emphasis>-Glucoside Against Doxorubicin-Induced Injury Through PTEN/Akt and ERK Pathway in H9c2 Cells

Luteolin-7-O-glucoside (LUTG) was isolated from the plants of Dracocephalum tanguticum Maxim. Previous research has showed that LUTG pretreatment had a significant protective effect against doxorubicin (DOX)-induced cardiotoxicity by reducing intracellular calcium overload and leakage of creatine kinase and lactate dehydrogenase. But the underlying mechanisms have not been completely elucidated. In the present study, we investigated the effects of LUTG on H9c2 cell morphology, viability, apoptosis, reactive oxygen species generation, and the mitochondrial transmembrane potentials. The expression of p-PTEN, p-Akt, p-ERK, p-mTOR, and p-GSK-3β were detected by Western blotting. Compared with DOX alone treatment group, the morphological injury and apoptosis of the cells in groups treated by DOX plus LUTG were alleviated, cell viability was increased, ROS generation was lowered remarkably, and mitochondrial depolarization was mitigated. In DOX group, the expression of p-PTEN was lower than normal group and the expression of p-Akt and p-ERK was higher than normal group. In the groups treated with LUTG (20 μM), the expression of p-PTEN was upregulated and the expression of p-Akt, p-ERK, p-mTOR, and p-GSK-3β was downregulated. These results indicated that the protective effects of LUTG against DOX-induced cardiotoxicity may be related to anti-apoptosis through PTEN/Akt and ERK pathway.