RAI16蛋白合成肽多克隆抗体的制备及初步应用

目的: 制备RAI16蛋白的合成肽多克隆抗体, 并进行初步鉴定和应用, 为研究RAI16蛋白的功能及作用机制获得重要的实验工具.方法: 应用Fmoc法化学合成RAI16蛋白N端第44～55位氨基酸的多肽, 经C18的RP-HPLC纯化后, 通过高碘酸钠法将纯化的RAI16蛋白的多肽与KLH交联; 皮下注射抗原免疫新西兰纯种大耳白兔, 加强免疫得到抗血清, 应用蛋白G纯化获得多克隆抗体.对纯化的抗体进行ELISA、免疫组化、 Western blot等初步鉴定和应用.结果: 化学合成RAI16蛋白N端第44～55位氨基酸的多肽, 纯化后多肽纯度为96% , 达到免疫用抗原标准.多肽与KLH交联, 用于免疫动物.经纯化后的抗体效价为1∶ 125 000.该多肽抗体可特异识别人脾脏组织中相对分子质量(Mr)约为55 000的RAI16蛋白.结论: 所制备的多克隆抗体能与天然RAI16蛋白发生特异性反应, 可应用于ELISA、免疫组化、免疫沉淀和Western blot等实验, 为确定RAI16蛋白的组织分布和亚细胞定位、研究RAI16蛋白的功能及作用机制提供了重要的实验工具.     

Segregation of human immunodeficiency virus type 1 subtypes by risk factor in Australia

The aim of this study was to determine which human immunodeficiency virus type 1 (HIV-1) subtypes were circulating in Australia and to correlate the subtypes with risk factors associated with the acquisition of HIV-1 infection. DNA was extracted from peripheral blood mononuclear cells, and HIV-1 env genes were amplified and subtyped using heteroduplex mobility analysis, with selected samples sequenced and phylogenetic analysis performed. The HIV-1 env subtypes were determined for 141 samples, of which 40 were from female patients and 101 were from male patients; 13 samples were from children. Forty-seven patients were infected by homosexual or bisexual contact, 46 were infected through heterosexual contact, 21 were infected from injecting drug use (IDU), 13 were infected by vertical transmission, 8 were infected from nosocomial exposure, and 6 were infected by other modes of transmission, including exposure to blood products, ritualistic practices, and two cases of intrafamilial transmission. Five subtypes were detected; B (n = 104), A (n = 5), C (n = 17), E (CRF01_AE; n = 13), and G (n = 2). Subtype B predominated in HIV-1 acquired homosexually (94% of cases) and by IDU (100%), whereas non-subtype B infections were mostly seen in heterosexually (57%) or vertically (22%) acquired HIV-1 infections and were usually imported from Africa and Asia. Subtype B strains of group M viruses predominate in Australia in HIV-1 transmitted by homosexual or bisexual contact and IDU. However, non-B subtypes have been introduced, mostly acquired via heterosexual contact.     

鼠巨细胞病毒抑制神经干细胞分化及分化基因表达的体外研究

目的 研究鼠巨细胞病毒(MCMV)感染对体外培养神经干细胞(NSCs)分化及分化基因表达的影响,探讨CMV先天感染致神经损伤的机制.方法 体外分离培养和鉴定BALB/c胎鼠NSCs并检测其分化潜能,用感染复数(MOI)为5、1和0.1的MCMV Smith毒株感染NSCs并进行分化培养,倒置显微镜下观察细胞形态学改变,流式细胞术检测分化细胞比率,免疫荧光法观察NSCs及其分化细胞标记物Nestin、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)表达的变化,采用MCMV早期抗原(EA)示踪感染过程(MOI=1),real-time RT-PCR检测分化早期NSCs Wnt信号途径关键分化基因Wnt-3和Wnt-Ta mRNA水平的动态变化.结果 体外培养的NSCs呈球样生长,神经干细胞特异性标记Nestin表达阳性,并可进一步诱导分化为NF-200阳性的神经元和GFAP阳性的星形胶质细胞;分化培养后,感染组NSCs不能贴壁分化生长并逐渐出现肿胀,细胞Nestin表达下调缓慢并显著高于正常对照组,而GFAP和NSE表达显著低于正常对照组(P<0.05),可检测到MCMV EA的阳性表达;分化培养3-9 d,感染组Nestin阳性细胞比率显著高于正常对照组,GFAP和NSE阳性细胞比率显著低于正常对照组(P<0.05);感染组Wnt-3 mRNA水平在分化培养后第1～2天显著低于正常对照组(P<0.05),感染组Wnto-7a mRNA水平在第0.5～2天明显低于正常对照组(P<0.05);感染组和正常对照组的差异随病毒MOI的增加而更加明显.结论 MCMV感染可明显抑制NSCs向神经元和星形胶质细胞方向分化,导致分化细胞比率减少;下调或干扰NSCs wnt信号途径分化基因wnt-3和Wnt-7a的表达;抑制NSCs分化及其分化基因表达的效应与MOI大小存在一定量效依赖关系;MCMV可能通过抑制NSCs分化基因的表达来抑制其分化,这可能是CMV先天感染致脑发育异常的重要机制之一.     

鼠巨细胞病毒抑制神经干细胞分化及分化基因表达的体外研究

目的 研究鼠巨细胞病毒(MCMV)感染对体外培养神经干细胞(NSCs)分化及分化基因表达的影响,探讨CMV先天感染致神经损伤的机制.方法 体外分离培养和鉴定BALB/c胎鼠NSCs并检测其分化潜能,用感染复数(MOI)为5、1和0.1的MCMV Smith毒株感染NSCs并进行分化培养,倒置显微镜下观察细胞形态学改变,流式细胞术检测分化细胞比率,免疫荧光法观察NSCs及其分化细胞标记物Nestin、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)表达的变化,采用MCMV早期抗原(EA)示踪感染过程(MOI=1),real-time RT-PCR检测分化早期NSCs Wnt信号途径关键分化基因Wnt-3和Wnt-Ta mRNA水平的动态变化.结果 体外培养的NSCs呈球样生长,神经干细胞特异性标记Nestin表达阳性,并可进一步诱导分化为NF-200阳性的神经元和GFAP阳性的星形胶质细胞;分化培养后,感染组NSCs不能贴壁分化生长并逐渐出现肿胀,细胞Nestin表达下调缓慢并显著高于正常对照组,而GFAP和NSE表达显著低于正常对照组(P<0.05),可检测到MCMV EA的阳性表达;分化培养3-9 d,感染组Nestin阳性细胞比率显著高于正常对照组,GFAP和NSE阳性细胞比率显著低于正常对照组(P<0.05);感染组Wnt-3 mRNA水平在分化培养后第1～2天显著低于正常对照组(P<0.05),感染组Wnto-7a mRNA水平在第0.5～2天明显低于正常对照组(P<0.05);感染组和正常对照组的差异随病毒MOI的增加而更加明显.结论 MCMV感染可明显抑制NSCs向神经元和星形胶质细胞方向分化,导致分化细胞比率减少;下调或干扰NSCs wnt信号途径分化基因wnt-3和Wnt-7a的表达;抑制NSCs分化及其分化基因表达的效应与MOI大小存在一定量效依赖关系;MCMV可能通过抑制NSCs分化基因的表达来抑制其分化,这可能是CMV先天感染致脑发育异常的重要机制之一.     

Requirement of hydD, hydE, hypC and hypE genes for hydrogenase activity in Helicobacter pylori

Helicobacter pylori possesses a membrane-bound, nickel containing, hydrogen uptake hydrogenase enzyme; its synthesis requires structural as well as accessory proteins, the latter needed for the complete maturation of the enzyme. Our lab previously characterized mutants in the accessory hyp genes, hypA, hypB, hypD and hypF that were all severely affected for hydrogenase activity, and in some cases (hypA and hypB mutants) also affected for urease activity. This finding prompted us to disrupt the two remaining unstudied hyp genes of H. pylori, hypC and hypE, in order to see if the same pleiotropic effect would be observed. In both mutants hydrogenase activity was abolished but urease activity remained unaffected. Addition of 5 microM nickel into the growth medium partially restored the hydrogenase activity in the hypE mutant and to a lesser extent in the hypC mutant. In addition, we also disrupted the genes HP0634 (referred as hydD in the H. pylori 26695 genome database) and HP0635 (whose function was unknown, referred to here as hydE) to address their possible roles in the hydrogenase synthesis/maturation process. In both cases, hydrogenase activities were abolished and addition of nickel could not restore the activity, suggesting that these proteins are involved in the hydrogenase synthesis process rather than in nickel mobilization/insertion steps.     

Integrin alpha2beta1 affects mechano-transduction in slowly and rapidly adapting cutaneous mechanoreceptors in rat hairy skin

The role of a transmembrane protein, integrin alpha2beta1, to modulate the neural responses of cutaneous mechanoreceptors to mechanical indentation was examined using an isolated skin-nerve preparation in a rat model. Skin and its intact innervation were harvested from the medial thigh of the hindlimb and placed in a dish containing synthetic interstitial fluid. Using a standard teased nerve preparation, the neural responses of single slowly or rapidly adapting mechanoreceptors (SA or RA, respectively) were identified and the afferents categorized according to standard protocols (i.e. response to constant stimuli). The most sensitive spot of a mechanoreceptor's receptive field was identified and then stimulated using controlled compressive stress (constant or dynamic loads between threshold and saturation load for SAs and RAs, respectively). Loads were applied before, during, and after passive diffusion into the skin of a function-blocking anti-integrin alpha2 monoclonal antibody (FBmAb) or one of two types of control antibodies (immunoglobulin G or a FBmAb conjugated with a secondary antibody). The sensitivities of both SA and RA mechanoreceptors were profoundly reduced in the presence of the FBmAb, while not changing the waveforms of their action potentials or their adaptation properties. Both control antibodies had no significant effect on mechanoreceptors' sensitivities. Following removal of the FBmAb, the effects in some neurons were partially reversible. Taken together, the data from this study support the hypothesis that integrin alpha2beta1 plays a significant role in modulating mechanoreceptive response to compressive indentation.     

Quasi-living surface graft polymerization with phosphorylcholine group(s) at the terminal end

A surface graft polymer with one or two phosphorylcholine (PC) polarheads at the terminus of the growing chain end was prepared by sequential reactions on a glass substrate. The dithiocarbamate group covalently bound to glass surfaces was derivatized with one or two PC groups and then irradiated with ultraviolet light in the presence of N,N-dimethylacrylamide (DMAAm). X-ray photoelectron spectroscopy, wettability measurements and dye staining experiment for the PC group showed that the resultant graft copolymers were produced via iniferter-based quasi-living radical polymerization, in which the polyDMAAm graft chain contains one or two PC groups at the terminal end of the graft chain. These polymer surface grafts may help provide biocompatibility.     

双相气道正压无创机械通气上呼吸道影像分析

目的应用多层螺旋CT对患者不同通气状态下上呼吸道放射性成像,证实在全身麻醉无自主呼吸的条件下,双相气道正压(bi-level positive airway pressure,BiPAP)无创机械通气能克服上呼吸道阻力,实施有效的机械通气。方法选择拟实施全身麻醉的择期手术患者10例,分别对患者清醒自主呼吸、麻醉诱导后自主呼吸停止、BiPAP无创通气时头颈部正位和侧位作螺旋CT扫描。监测扫描过程的无创血压(NIBP)、脉搏氧饱和度(SpO2)、心率(HR)、自主呼吸频率(RR)。测量上呼吸道各软组织区(软腭后区RP、舌根后区RG、会厌区EPG)的最窄气道横截面左右径、前后径线长度及相应横截面积。结果头部正位麻醉诱导后各软组织区的最窄横截面左右径、前后径线长度及相应横截面积均比清醒时缩小(P<0.05),BiPAP通气时各截面径线和面积与清醒期比较差异仍有统计学意义(P<0.05,P<0.01)。头部侧位BiPAP通气时各径线和截面积与清醒时比较,差异无统计学意义。EPG区和RG区在BiPAP通气期的侧位截面积明显比正位时增大(P<0.05,P<0.01)。诱导期正、侧位SpO2均明显下降(P<0.01);头部正位BiPAP通气时与诱导期的SpO2比较虽有改善,但差异无统计学意义(P>0.05);头部侧位BiPAP通气时SpO2较诱导期明显升高(P<0.01),基本恢复到清醒期水平(P>0.05)。结论麻醉诱导后上呼吸道的通气面积明显减少,气道通畅度下降;头颈部侧位时上呼吸道各软组织区最狭窄处的通气截面积比正位时显著改善,以会厌区最明显。无明显上呼吸道梗阻性病史的成年患者全身麻醉时,头部侧位BiPAP无创通气能克服上呼吸道阻力,实施有效的机械通气,保证通气和氧合正常。     

高压氧治疗重度颅脑损伤后临床、脑电地形图及预后的探讨

目的:探讨高压氧治疗重度颅脑损伤的疗效。方法:重度颅脑损伤患者35例为治疗组,20例为对照组,观察高压的氧治疗前后临床、脑电地形图的变化及预后。结果:治疗组临床(GCS)、脑电地形图及预后经高压氧治疗后均明显改善,而对照组改善不明显。结论:高压氧能够明显改善重度颅脑损伤患者的临床,脑电地形图及预后。     

组蛋白去乙酰化酶抑制剂与5-FU合用对肝癌细胞HepG2的影响

目的：探讨MS-275和5-Fu联用对HepG2细胞周期和凋亡的影响，并对其机制进行初步探讨。方法：将细胞分成对照组、MS-275组、5-FU组和联合用药组。流式细胞仪检测各组细胞凋亡和周期变化；Westernblot分析各组Bcl-2、Bax、CyclinD1、P21蛋白表达。结果：MS-275和5-Fu联用能抑制肝癌细胞生长，诱导G0—G1期阻滞，促进细胞凋亡。上述效应具有时间和剂量依赖性。二者联用可以使P21蛋白上调，Bcl-2、CyclinD1蛋白下调，Bax蛋白无明显变化。结论：5-Fu和MS-275联用能提高HepG2细胞凋亡率和周期阻滞，其机制与下调CyclinD1、Bcl-2蛋白的表达、上调P21蛋白表达有关。