沉默树突状细胞恒定链以增强其抗肿瘤作用的体外实验研究

目的初步观察用小分子干扰RNA(siRNA)沉默树突状细胞(DCs)恒定链(Ii)后,DCs疫苗的体外抗肿瘤效果。方法从小鼠骨髓分离骨髓前体细胞,细胞经100 ng/ml GM-CSF和100 ng/ml IL-4诱导培养6 d后,转染针对DCs Ii链特异的Ii-siRNA,转染后加用50 ng/ml TNF-α继续诱导细胞成熟48 h,然后分别用Western blot检测沉默效果及CCK-8试剂盒检测DCs刺激同种异体淋巴细胞增殖的能力;此外,DCs共转染Ii-siRNA和小鼠胃癌前体细胞MFC的总RNA后,与同种异体淋巴细胞共培养,通过ELISA检测培养上清IFN-γ/和IL-4的水平,并收集致敏淋巴细胞进行体外杀伤实验。结果Ii-siRNA明显抑制DCs Ii的表达。沉默Ii链能够增强DCs的淋巴细胞增殖能力,并促使淋巴细胞向Th1的方向漂移[IFN-γ:(5107±351)pg/ml,IL-4:(65±13)pg/ml,P＜0．05]。淋巴细胞经共转染Ii-siRNA和MFC RNA的DCs激活后,明显而特异地杀伤靶肿瘤细胞(杀伤百分率: 66．94%±2．75%,P＜0．05)。结论通过siRNA沉默DCs的Ii链可能是一种行之有效的增强抗肿瘤免疫的方法。     

p38丝裂原活化蛋白激酶在糖尿病大鼠神经病理性痛中的作用

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）在链脲菌素诱导的糖尿病大鼠神经病理性痛中的作用。方法雌性Wistar大鼠31只，3月龄，体重180～220g，随机分为3组：对照组（C组，n=10）、糖尿病神经病理性痛组（D组，n=11）和p38MAPK抑制剂组（Ⅰ组，n=10）。D组、Ⅰ组单次腹腔注射链脲菌素65mg／kg制备糖尿病模型。糖尿病模型制备成功后，Ⅰ组尾静脉注射p38MAPK抑制剂SB203580 0．5mg／kg，1次／周，连续4周；C组和D组尾静脉注射等体积的生理盐水。给药4周后，测定机械缩足反应阈值（MWT）、左侧坐骨神经传导速率（NCV）、背根神经节（DRG）和脊髓的磷酸化p38MAPK水平。结果与C组比较，D组、Ⅰ组MWT下降，NCV减慢，伴有脱髓鞘现象，DRG和脊髓的磷酸化p38MAPK水平升高；与D组比较，Ⅰ组MWT升高，NCV增快，脱髓鞘程度减轻，DRG和脊髓的磷酸化p38MAPK水平下降。结论p38MAPK信号转导通路参与了糖尿病大鼠神经病理性痛的形成。     

部分背根切断对备用背根节NT-3表达的影响

目的　探讨部分去背根后备用背根节 (L6 )各类细胞NT 3及其mRNA的含量变化。　方法　对成年雄性猫行单侧部分背根切断术 (切除一侧L1 ～L5,L7～S2 DRG ,保留L6 为备用根 )。取正常组一侧和术后 3d及 7d组手术侧的L6 DRG制作 2 0 μm厚冰冻切片 ,分别用NT 3抗体及NT 3cRNA探针行免疫组织化学及原位杂交染色。观察NT 3及其mRNA在DRG各类细胞的分布 ,测定NT 3及其mRNA在神经元和卫星细胞的光密度值 ,所得数据用q检验进行统计分析。　结果　部分去背根后 ,各时相备用背根节大神经元内NT 3的光密度值较正常者进行性减少 ,(P <0 0 5 ) ,而NT 3mRNA的光密度值术后 3d减少 ,7d回升至近正常者水平。比较之 ,小神经元和卫星细胞NT 3及其mRNA的光密度值进行性增多 (P <0 0 5 )。　结论　部分背根切断对备用背根节各类细胞NT 3表达的影响不同 ,其功能意义可能与NT 3参与脊髓Ⅱ板层可塑性有关     

甲氨喋呤对胶原诱导性关节炎大鼠滑膜骨桥蛋白和整合素αVβ3表达的影响

目的 探讨甲氨喋呤对胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠滑膜骨桥蛋白(osteopontin,OPN)及其受体整合素αvβ3表达的影响及其作用机制.方法 建立CIA大鼠模型,分为模型组和甲氨喋呤(MTX)组,后者用MTX进行干预治疗;采用免疫组织化学染色技术检测滑膜组织OPN和整合素αvβ3的表达,ELISA方法检测血清TNF-α水平.结果 ① CIA模型组和MTX组大鼠的滑膜OPN、整合素αvβ3表达均明显高于正常大鼠对照组(均为P<0.01);与模型组比较,MTX组OPN和整合素αVβ3的表达减少(均为P<0.01). ②正常对照组和MTX组大鼠血清TNF-α水平显著性低于模型组大鼠(均为P<0.01).结论 滑膜OPN和整合素αvβ3异常表达在CIA发病中具有重要意义.MTX通过下调滑膜OPN和整合素αvβ3的表达,降低血清TNF-α水平从而达到治疗CIA的作用.     

肾上腺髓质素对豚鼠心室肌细胞L-型钙通道的作用及其信号转导机制

目的: 研究肾上腺髓质素( adrenomedullin, ADM)抑制豚鼠心室肌细胞L-型钙通道的信号转导机制。方法: 应用全细胞膜片钳技术,记录应用ADM(1-100 nmol·L^-1)前后L-型钙电流(I_Ca,L),以及分别记录应用ADM特异性受体拮抗剂ADM_22-52(100 nmol·L^-1)+ADM(100 nmol·L^-1)、蛋白激酶A (PKA) 特异性拮抗剂H-89(10 μmol·L^-1) + ADM(100 nmol·L^-1)、蛋白激酶C (PKC) 特异性拮抗剂PKC_19-36(10 μmol·L^-1)+ADM(100 nmol·L^-1)、PKC特异性激动剂PMA(1 μmol·L^-1)前后I_Ca,L。结果: ADM(1-100 nmol·L^-1)浓度依赖性地抑制豚鼠心室肌细胞I_Ca,L,并可被ADM_22-52(100 nmol·L^-1)完全阻断;H-89(10 μmol·L^-1)对ADM抑制I_Ca,L的作用无影响。PKC_19-36(10 μmol·L^-1)可完全阻断ADM 对I_Ca,L的抑制效应,且PMA(1 μmol·L^-1)可模拟ADM 对I_Ca,L的抑制效应。结论: ADM作用于特异性ADM受体可浓度依赖性地抑制豚鼠心室肌细胞I_Ca,L,此作用有可能与PKC激活相关。     

人核受体hLRH-1不同变异体在多种肿瘤组织和细胞系中的表达

目的:初步分析人核受体hLRH-1不同变异体在多种肿瘤组织和细胞系中的表达。方法:常规RT-PCR法分析hLRH-1v1和hLRH-1在肝癌等多种肿瘤组织和HepG2等肿瘤细胞系中的表达情况，实时定量PCR法分析hLRH-1v1，hLRH-1和hOct4在HepG2，SMMC-7721和BEL-7402三株肝癌细胞系的表达水平。结果:hLRH-1v1的表达见于所有检测的12种类型肿瘤和8种恶性肿瘤细胞系，hLRH-1的表达则仅在12种类型肿瘤中的6种肿瘤组织中可检测到，但8种恶性肿瘤细胞系均为hLRH-1阳性表达；在HepG2，SMMC-7721和BEL-7402三株肝癌细胞系中hLRH-1v1和hOct4的表达呈正相关。结论:hLRH-1v1在肿瘤中广泛表达，可能在维持肿瘤干细胞的自我更新中发挥重要作用。     

SARS患者TGFβ1和PDGF-BB的动态监测及其临床意义

目的动态监测严重急性呼吸综合征(SARS)患者血清转化生长因子β1(TGFβ1)和血小板衍生生长因子BB(PDGF-BB)水平的变化,以探讨细胞因子在SARS疾病中的作用。方法采用酶联免疫吸附试验 ,测定SARS患者早期、恢复期和随访时TGFβ1和PDGF -BB含量 ,并与急诊等一线未患SARS组及健康对照组比较 ,作描述性分析及方差分析。结果SARS患者早期组血清TGFβ1均值高于恢复期组和随访组 (P<0.05),SARS恢复期组、随访组TGFβ1均值均显著低于急诊等一线未患SARS组和健康对照组(P<0.01),其它组间两两比较均无显著性差异(P>0.05)。五组人群PDGF -BB均值水平总体比较无显著性差异(P>0.05)。结论TGFβ1可能与SARS患者机体调节免疫应答和免疫损伤有关 ,PDGF -BB与SARS的免疫损伤无关。     

Characterization of a gene encoding survival motor neuron (SMN)-related protein, a constituent of the spliceosome complex

Mutations in the gene encoding the Survival Motor Neuron (SMN) protein are responsible for autosomal recessive proximal spinal muscular atrophy (SMA). SMN orthologues have been identified in the nematode worm Caenorhabditis elegans and the yeast Schizosaccharomyces pombe but, to date, no human paralogues have been described. Here we describe identification and characterization of an SMN-related protein (SMNrp) gene that encodes a novel protein of 239 amino acids, which has recently been identified as a constituent of the spliceosome complex and designated SPF30. Significant similarity to the SMN protein is apparent only within a central region of SMNrp that represents a tudor domain. The SMNrp/SPF30 gene has been mapped to chromosome 10q23. It is differentially expressed, with abundant levels in skeletal muscle. An exclusively nuclear localization for SMNrp in cultured cells and muscle sections was revealed using GFP fusion constructs and thereafter confirmed with a polyclonal antibody raised against SMNrp. Overexpression of SMNrp as a fusion protein in HeLa cells in culture induced dose-dependent apoptosis with positive TUNEL staining. In addition to a possible role for this protein as a pro-apoptotic factor, SMN and its related protein share significant similarities in sequence and cellular function.      

Comparison of the structure of human intervertebral discs in the cervical,thoracic and lumbar regions of the spine

Posterior and anterior heights, cross-sectional area and shape were measured for all the intervertebral discs in four spines from elderly human cadavers. Disc height was a minimum at the T4-5 level; thoracic discs were less wedge-shaped than those in the cervical and lumbar regions. Cross-sectional area increased from the cranial to caudal extremity; at the L5-S1 level the nucleus pulposus occupied a high proportion of this area. Cervical discs tended to have an elliptical cross-sectional shape, thoracic discs were more circular and lumbar discs tended to have an elliptical cross-section which was flattened or re-entrant posteriorly. This shape distribution was quantified by defining a shape index which had a maximum value of 1 for a circular cross-section. Orientations of the reinforcing fibres in the outer lamellae of the anterior annulus fibrosus were measured from 27 discs by X-ray diffraction. For these measurements, C3-4, T7-8 and L2-3 were chosen as representative of cervical, thoracic and lumbar discs. The fibre tilt, with respect to the axis of the spine, was significantly less in the cervical discs (at 65 degrees) than in the thoracic and lumbar discs (about 70 degrees). These findings are interpreted in relation to differing functional requirements and possible mechanisms of failure in the cervical, thoracic and lumbar regions of the spine in the light of current knowledge on the biomechanics of the intervertebral disc.