人胎脑神经干细胞的体外培养

目的从人胎脑纹状体中分离培养并鉴定神经干细胞。方法采用包含碱性成纤维细胞生长因子(bFGF)和表皮细胞生长因子(EGF)的无血清培养和单细胞克隆技术,从36周自愿水囊引产人胎脑纹状体中分离出神经干细胞,并观察神经干细胞体外培养、传代、分化潜能。结果从36周人胎脑纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈悬浮状态生长,形成神经球,该细胞具有连续克隆能力,可传代培养,呈Nestin免疫反应阳性细胞;在含血清培养时诱导神经干细胞分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论神经干细胞的存活和分裂有赖于EGF和bFGF的共同作用;36周人胎脑纹状体仍能培养出具有自我复制和分化潜能的神经干细胞。     

阻断4-1BB/4-1BBL通路在系统性红斑狼疮患者T淋巴细胞活化中的作用

目的 探讨协同刺激分子4-1BB/4-1BBL在系统性红斑狼疮(systemic lupus erythematosus,SLE)T淋巴细胞活化中的作用机制.方法 应用RT-PCR和Western blot方法检测体外培养20例SLE患者和20例正常对照者T淋巴细胞活化前后及应用抗4-1BB单抗阻断后p38 MAPK和NF-kB表达的变化.结果 SLE患者T淋巴细胞NF-kB mRNA和p38 MAPK mRNA表达及其蛋白水平明显高于正常对照组(P均＜0.01),活化后的表达进一步升高(P均＜0.01).阻断4-1BB/4-1BBL通路后SLE患者T淋巴细胞p38 MAPK mRNA及蛋白水平的表达均明显下降(P均＜0.01),但是NF-kB mRNA及蛋白水平的表达无明显变化(P＞0.05).结论 协同刺激分子4-1BB可能通过p38MAPK信号转导通路促使SLE患者T淋巴细胞的活化与增殖.     

压阻传感器在脉搏信号测试上的应用研究

脉搏是心脏和血管状态重要生理信息的外在反映，作者首先分析了在脉搏测量中传感器和放大电路设计的问题－即脉搏信号的特殊性，然后介绍膜型及囊型两种压力传感器；并给出脉搏信号放大电路。指出测量方法的进一步发展和对临床应用的意义。     

Fine Mapping Functional Sites or Regions from Case-Control Data Using Haplotypes of Multiple Linked SNPs

Previously, we reported an algorithm for scanning a large number of tightly linked single nucleotide polymorphisms (SNPs) for LD mapping of functional sites or regions from a family‐based association design. In the present study, we extend our method to a case‐control design. We first use the expectation maximization (EM) algorithm to estimate haplotype frequencies of multiple linked SNPs, and follow this by constructing a contingency table statistic S for LD analysis, based on the estimated haplotype frequencies. An empirical p‐value is obtained based on the null distribution of the maximum of S (S *) from a large number (e.g., 1,000 or more) of randomized permutations. The proposed algorithm has been implemented in a computer program in which window searching for functional SNP sites can cover any number of loci without limitation, except that of computer storage. Unlike other programs for a case‐control design that always conduct tests at a fix window width, in our program after setting a maximum size of haplotype window width, for a given maximum window width all possible widths of haplotypes are utilized to find the maximum statistic S * for each locus under investigation. The sensitivity of the proposed algorithm has been examined with simulated and real genotyping datasets. Association analyses indicate that our program is powerful enough to detect most, if not all, functional SNPs simulated in the original model or identified in the original report. Moreover, the program is very flexible and can be used in either regional or genome‐wide scanning for association analysis with SNP markers.     

Role of MyD88 in diminished tumor necrosis factor alpha production by newborn mononuclear cells in response to lipopolysaccharide

Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-alpha) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-alpha. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-alpha compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-alpha secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IkappaBalpha phosphorylation, as well as IkappaBalpha degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-alpha secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-alpha secretion in newborn monocytes.     

人巨细胞病毒UL136基因在临床低传代分离株中多态性分析

目的 研究人巨细胞病毒(human cytomegalovirus，HCMV)UL136基因在临床低传代分离株中的多态性，探讨其多态性与HCMV先天性感染不同致病性之间的关系。方法 对48株经荧光定量PCR方法检测HCMV DNA为阳性的临床低传代分离株进行HCMV ULl36全序列PCR扩增，对于扩增阳性的12株PCR产物进行ULl36基因全序列测定及结果分析。结果 48株临床低传代分离株ULl36 PCR扩增，12株阳性，阳性率25％，以HCMV Toledo株作为参考株，进行序列比较分析表明，12株临床分离株ULl36开放阅读框架(open reading frame，ORF)长度均与Toledo株相同，为723bp，编码241个氨基酸的蛋白。DNA序列变异均为碱基替换，不同临床分离株ULl36基因与Toledo株进行同源性比较，结果在核苷酸水平为97．7％～99．3％，氨基酸水平为96．6％～99．1％。ULl36编码蛋白的氨基酸变异率为0．83％～3．3％。二级结构预测分为两种构象。大多数HCMV ULl36蛋白翻译后修饰位点在所有分离株中均高度保守，仅几个位点在一些分离株中存在缺失或新增。Toledo株及12株临床分离株核苷酸及氨基酸序列系统进化树分析表明：45J最接近Toledo株。结论 12株临床低传代分离株HCMV ULl36基因DNA及其编码产物的氨基酸序列比较保守，但仍存在一定多态性。未发现不同临床分离株ULl36基因多态性与HCMV先天性感染的表现关系。     

人巨细胞病毒UL143基因在临床低传代分离株中的多态性研究

目的 探讨人巨细胞病毒(human eytomegalovirus,HCMV)UL143序列在临床患儿低传代分离株中的多态性及其与临床疾病的关系.方法 对19株HCMV临床低传代分离株进行HCMV-UL143 PCR扩增分析及伞序列测定分析.结果 19株HCMV感染患儿临床分离株均因碱基插入造成移码突变,开放阅读框架(ORF)比Toledo株短.根据序列变异情况可将19个序列分为2组,第1组16个序列新增一个MYRISTYL位点;缺失2个PKC磷酸化位点.未发现黄疸、小头畸形、先天性巨结肠等不同疾病类型的序列之间的差异.结论 HCMV-UL143较多存在于临床低传代分离株中,序列呈现一定多态性.     

壳聚糖纳米颗粒包裹对鼠白细胞介素2基因表达和调节小鼠免疫效应的影响

本实验制备壳聚糖纳米颗粒 (CNP) ,包裹小鼠白细胞介素 2基因 (mIL 2 )真核表达质粒 (VRMIL 2 ) ,肌注接种 2 1d昆明小鼠 ,观察mIL 2基因体内表达及其对免疫应答和免疫保护的影响。实验结果发现 :CNP包裹VRMIL 2注射小鼠血液中IgG、IgM和IgA不同程度地增多 ,均显著高于CNP包裹空白质粒组 (P <0 .0 5 ) ;其血清中IL 2、IL 4和IL 6的含量明显升高 ,与对照组差异显著 (P <0 .0 5 ) ;外周血液的白细胞和淋巴细胞数量也较对照组显著增加。免疫后 35d以大肠杆菌口服攻毒实验小鼠 ,检测发现 :CNP包裹VRMIL 2组小鼠的上述免疫指标除中性粒细胞外均显著多于对照小鼠 ,VRMIL 2接种小鼠均健康存活 ,而对照小鼠均发病 ;尽管CNP包裹VRMIL 2接种小鼠的体液和细胞免疫指标与未包裹VRMIL 2免疫鼠差异不显著 (P >0 .0 5 ) ,但剂量仅为后者的 1/ 5。这些结果表明 :壳聚糖纳米颗粒包裹VRMIL 2可显著提高外源IL 2基因体内表达水平 ,明显增强体液和细胞免疫水平的效应 ,增强对大肠杆菌的抗病力 ,提示壳聚糖纳米颗粒包裹IL 2基因可明显增强动物的体液和细胞免疫 ,可作为有效的抗感染免疫调节剂。     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。     

CO对局灶性缺血脑组织血脑屏障通透性的影响

目的:研究一氧化碳及其限速酶(血红素氧合酶-1)对局灶性缺血脑组织血脑屏障通透性的影响。方法:将SD大鼠随机分为3组(n=6),使用血红素氧合酶诱导剂及抑制剂腹腔注射,用等量生理盐水腹腔注射作为对照组,12h后复制MCAO模型。梗塞后24h后检测血液中一氧化碳浓度、血脑屏障通透性。结果:诱导剂组一氧化碳浓度明显高于对照组(P＜0.01),血脑屏障通透性明显低于对照组(P＜0.05),而抑制剂组一氧化碳浓度明显低于对照组(P＜0.01),血脑屏障通透性明显高于对照组(P＜0.05)。血红素氧合酶诱导剂、抑制剂对非梗塞侧的血脑屏障通透性没有影响(P＞0.05)。结论:一氧化碳作为一种信使分子,脑缺血时浓度升高具有保护血脑屏障的作用。