运用siRNA建立稳定低表达IGF1R基因的肝癌细胞株的实验研究

目的运用siRNA技术在肝癌细胞株中建立稳定低表达人胰岛素样生长因子1类受体(IGF1R)基因的细胞株。方法构建包含封闭IGF1R基因的真核表达载体pSUPER-IGF1R-siRNA,转染SMMC7721和Hep3B细胞,G418筛选表达稳定的细胞株。通过RT-PCR、Western-blot分析与鉴定IGF1R mRNA和蛋白及cyclin D1、cyclinB1蛋白的表达,并绘制细胞生长曲线。结果在SMMC7721和Hep3B细胞中成功建立低表达IGF1R基因的细胞株,其生长明显减慢(P<0.05),且其cyclinD1表达亦明显下降(P<0.05)。结论pSUPER-IGF1R-siRNA能抑制SMMC7721和Hep3B细胞的生长。     

Recombinant glycoprotein based vaccine for Chandipura virus infection

Chandipura virus (CHPV) has emerged as an important pediatric encephalitis-causing pathogen with very high mortality in India. No specific vaccine or treatment is available till date. We attempted to prepare a candidate vaccine employing recombinant CHPV Glycoprotein (rGp). The Glycoprotein gene (G-gene) of CHPV was expressed using Baculovirus expression system. The rGp was purified by HPLC and used for mice immunization, 3 doses, and 4 weeks apart. One microgram rGp was found to be optimum. Sero-conversion was observed as early as 2nd week by detecting anti-CHPV IgG antibodies. Antibody titres were immunogen-concentration dependent. Intracerebral challenge of the immunized mice with 100 LD₅₀ of the homologous strain demonstrated 90% protection. In in vitro neutralization, antibodies from the immunized mice were able to neutralize heterologous viruses. There was 60% T cell proliferation observed against rGp in immunized mice. The study shows that rGp induces both arms of immune response and represents an ideal vaccine candidate for further evaluations.     

体外短波热疗联合前列腺灸及哈乐治疗非细菌性前列腺炎和前列腺痛疗效观察

目的 探讨慢性非细菌性前列腺炎（CNP）和前列腺痛（PD）的治疗方法。方法 应用体外短波热疗联合前列腺灸及α1A-肾上腺素能受体阻滞剂哈乐治疗CNP和PD患者26例，疗程8周。观察治疗前后前列腺炎症状评分（CPSI）与最大尿流率（MFR）变化。结果 有效率为80．8％。治疗前后CPSI和MFR均有显著改善（P〈0．05）。结论 体外短波热疗联合前列腺灸及哈乐治疗CNP和PD安全有效。     

抱茎獐牙菜中的苷类成分

目的 研究藏药抱茎獐牙菜Swertia franchetiana中的苷类成分。方法 用各种柱色谱方法进行分离纯化，根据理化性质和波谱解析鉴定其化学结构。结果 从抱茎獐牙菜的水溶性部分得到11个化合物，分别鉴定为芒果苷(mangiferin，Ⅰ)、异荭草苷(isoorlentin，Ⅱ)、当药黄素(swertisin，Ⅲ)、当药醇苷(swertianolin，Ⅳ)、1-羟基-3，5-二甲氧基汕酮-1-O-[β-D-吡喃木糖(1→6)-β-D-吡喃葡萄糖(Ⅴ)、1-羟基一3，7，8-三甲氧基汕酮-1-O-[β-D-吡喃葡萄糖(Ⅵ)、1-羟基-2，3，5-三甲氧基汕酮-1-O-[β-D-吡喃木糖(1→6)-β-D-吡喃葡萄糖(Ⅶ)、5-醛基1-异色满酮(Ⅶ)、獐牙菜苦苷(swartiamarin，Ⅸ)、龙胆苦苷(gentipicroside，Ⅹ)和獐牙菜苷(sweroside，Ⅺ)。结论 化合物Ⅵ～Ⅺ均为首次从该植物中得到，其中Ⅶ为首次从獐牙菜属植物中得到。     

Lack of Major Effects on Mouse Brain Adenosine A1 Receptors of Oral Carbamazepine and Calcium Antagonists

Interaction with adenosine A1 receptors is a possible contributory mechanism to the anticonvulsant effects of carbamazepine (CBZ) and the dihydropyridine calcium antagonists. We measured the binding of [3H]cyclohexyladenosine to adenosine A1 receptors in mouse brain stem, cerebellum, and cortex after oral administration of nifedipine, nimodipine (NMD), and CBZ for 7 days and compared the results with binding in control mice. Equilibrium dissociation constant (Kd) and receptor numbers (Bmax) were calculated using Scatchard and saturation isotherm analyses. Mean Kds (SEM) in control brain stem, cerebellum, and cortex were 2.09 (0.31), 2.39 (0.2), and 3.12 (0.28) nM, respectively. Results of Bmax for the same areas were 188 (26), 280 (24), and 449 (54) fmol/mg protein. Nifedipine (p less than 0.005) and NMD (p less than 0.02) raised the Kd of A1 receptors only in the cerebellum, and CBZ increased cerebellar Bmax (p less than 0.05). These minor effects on A1 receptors in CF1 mice, when given in doses previously shown to have anticonvulsant properties in these animals, do not suggest that alteration in A1 receptor activity is an important mechanism for the anticonvulsant effects of these drugs.     

Quantification of the expression level of integrin receptor alpha(v)beta3 in cell lines and MR imaging with antibody-coated iron oxide particles.

Targeted imaging requires site-specific accumulation of a contrast agent (CA), and the properties of that agent must be selected according to the abundance of the target to obtain a signal above the detection limit of the instrument. However, numerical estimates of receptors per cell are rarely found in the literature. Integrin receptors would be particularly promising targets because of their accessibility from the blood stream and expression on activated neovascular endothelial cells. We systematically estimated the number of integrin receptors of cell lines and primary cells by flow cytometry analysis. Since integrin receptors are heterodimeric molecules, and alpha(v) forms complexes with various beta subunits, the numbers of alpha(v) and beta(3) subunits are therefore dissimilar. The observed values are 3 . 10(3)-1.4 . 10(4)/cell for alpha(v), and 5.3 . 10(2)-1.1 . 10(4)/cell for beta(3). Despite the low number of exposed receptors, we show that up to single-cell MR visualization can be achieved with the use of iron oxide beads complexed with antibodies as CAs.     

性别差异对脓毒症大鼠肝脏Toll样受体4和髓样分化蛋白-2基因表达的影响

目的探讨性别差异对脓毒症大鼠肝脏组织Toll样受体4(TLR4)和髓样分化蛋白- 2(MD-2)基因表达的影响。方法以脂多糖(LPS)按5 mg/kg体重由大鼠腹腔注射制作脓毒症动物模型,注射后2 h留取肝脏组织检测TLR4、MD-2和肿瘤坏死因子-α(TNF-α)基因表达,同时测定各组大鼠血浆中丙氨酸氨基转移酶(ALT)及雌二醇含量。结果正常雌雄性大鼠肝脏组织均可表达少量TLR4、MD-2、TNF-α基因,其中雌性组分别为0．175±0．034、0．211±0．044、0．201±0．068; 雄性组分别为0．205±0．061、0．243±0．049、0．243±0．063,两组数据差异无统计学意义(P> 0．05),但LPS刺激后雌性大鼠肝脏组织上述指标分别为0．615±0．089、0．708±0．181、0．730± 0．118,血浆中ALT含量为(81．07±10．72)U／L;雄性组分别为0．723±0．091、1．123±0．272、 0．881±0．156,ALT含量为(106．39±14．21)U／L,雌性组各项指标均明显低于雄性大鼠(P< 0．05)。相关分析表明雌性及雄性脓毒症大鼠肝脏组织TLR4及TNF-α基因表达与相应性别大鼠血浆中雌二醇含量呈显著负相关(P<0．05)。结论 LPS刺激后大鼠肝脏组织TLR4、MD-2及 TNF-α基因表达存在性别差异,内源性雌激素的作用可能导致雌性脓毒症大鼠肝脏组织损伤较雄性轻。     

Association between cord blood IgE and genetic polymorphisms of interleukin-4, the β-subunit of the high-affinity receptor for IgE, lymphotoxin-α, and tumor Necrosis factor-α

High cord blood immunoglobulin E (cbIgE) is known to be associated with increased risks of atopic diseases in childhood. The relationship between genetic polymorphisms and high cbIgE has not been well documented. A cross-sectional study was conducted to assess the association between cbIgE and genetic polymorphisms of interleukin (IL)-4 -590C/T, the beta-subunit of the high-affinity receptor for IgE (FcepsilonRI-beta) E237G, lymphotoxin (LT)-alphaNcoI alleles, and tumor necrosis factor (TNF)-alpha -308G/A. A total of 320 mother-neonate pairs were recruited from four maternity hospitals from different locations of Taiwan. Cord blood was obtained and assayed for cbIgE. Polymerase chain reaction followed by restriction fragment length polymorphism was used to assess the genotypes. Three hundred pairs of mothers and neonates were included in the final analysis. Infants with IL-4 -590 C allele were found to have higher risk of elevated cbIgE (> or =0.35 IU/ml, 24.3%) (p = 0.004). After adjusting for gender, birth order, maternal age, and history of allergic disease in maternal and paternal families, odds ratios for CC and CT genotypes were 4.41 and 3.16 (95% confidence interval 0.78-22.67, and 1.66-6.13), respectively, using TT genotype as reference. The genotypes of FcepsilonRI-beta, LT-alpha, and TNF-alpha were not associated with cbIgE before or after the adjustment. Our finding suggested a significant association of cbIgE with genetic polymorphism of IL-4 -590C/T, but not with the genotypes of FcepsilonRI-beta, LT-alpha, and TNF-alpha.     

Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus

BACKGROUND: To understand the immunopathological features of oral lichen planus (OLP), we analyzed the expression of chemokines in the epithelial cell layers. Methods: Epithelia from OLP or healthy gingiva were collected by laser microdissection. The chemokine and chemokine receptor expressions in the epithelia were analyzed by DNA microarray. RESULTS: High levels of MIP-3alpha/LARC/CCL20 and its receptor CCR6 were expressed in the lesional epithelia. Furthermore, DC-CK1/CCL18, ELC/CCL19, SDF-1/CXCL12 and CXCR4 expressions were also increased. Immunohistologial analysis showed that high numbers of Langerhans cells (LCs) were present in the epithelia of OLP. Lesional epithelia also expressed high levels of the ligands specific for CXCR3 (e.g. MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) and CCR5 (e.g. RANTES/CCL5). CONCLUSIONS: Infiltration of LCs is orchestrated by CCR6. Further, LCs residing in the lesional epithelia may be a mature phenotype. Moreover, infiltration of T cells in OLP could be mediated by signaling pathways through CXCR3 and CCR5.     

The participation of adenosine receptors in the adenosine 5''-triphosphate-induced relaxation in the isolated rabbit corpus cavernosum penis

AIM: To investigate the participation of adenosine receptors in the adenosine 5'-triphosphate (ATP)-induced relaxation in the corpus cavernosum penis (CCP) of rabbits. METHODS: The ATP-induced relaxation was assessed on the noradrenaline precontracted CCP of rabbits in the presence and absence of 8-(3-chlorostyryl)caffeine (CSC); an adenosine A(2A) receptor antagonist; alloxazine and MRS1754; adenosine A(2B) receptor antagonists; and ARL67156, an inhibitor of ecto-nucleoside triphosphate diphosphohydrolases. RESULTS: Adenosine and ATP relaxed the noradrenaline precontracted CCP of rabbits in a concentration-dependent manner. The adenosine- and ATP-induced relaxations were suppressed by alloxazine and MRS1754, but not by 8-(3-chlorostyryl)caffeine. ARL67156 potentiated the ATP-induced relaxation but not the adenosine-induced one. MRS1754 suppressed the ATP-induced relaxation potentiated by ARL67156. CONCLUSIONS: The above results suggest that, in the CCP of rabbits, the adenosine receptor mediating adenosine-induced relaxation is of the A(2B) receptor and the ATP directly causes relaxation through the A(2B) receptor on the CCP.