重组血红素加氧酶-2在大肠杆菌中表达与纯化方法的研究

目的　确定重组血红素加氧酶 2在大肠杆菌中表达与纯化方法。方法　将已构建的血红素加氧酶 2(HO 2 )的重组质粒pMW1 72a HO 2转入大肠杆菌BL 2 1 ,分析不同温度及摇床转速对HO 2表达的影响 ,确定可溶性表达最佳条件。使用超声波、超速离心、分级盐析、分子筛层析等方法纯化HO 2。检测酶活性。结果　确定了HO 2可溶性表达最佳条件为 37℃ ,(85± 5 )r min ;确定了HO 2具体纯化路线。得到蛋白纯度为 95 .0 % ,纯化倍数为 2 .9倍 ,收得率为 4 0 .9%的活性HO 2蛋白。结论　获得了纯度高、具有活性的HO 2蛋白 ,为进一步进行HO 2结构与功能之间关系的研究提供了可行的纯化路线     

大鼠肝硬化形成过程中胃黏膜微循环内皮素A受体mRNA的表达及其意义

目的观察大鼠肝硬化门静脉高压形成过程中内皮素A受体mRNA在胃黏膜微循环血管上的表达 ,探讨内皮素及其受体对肝硬化门静脉高压大鼠胃黏膜微循环的调节机制。方法应用mRNA原位杂交确定内皮素A受体的部位 ,并进行半定量分析。结果大鼠肝硬化门静脉高压形成过程中门静脉及外周血浆内皮素水平随门静脉压力升高而降低。胃黏膜内皮素A受体mR NA主要在黏膜基底部的微循环前微动脉和后微静脉血管壁表达 ,其中动脉表达明显强于静脉。其表达与门静脉压力呈正相关 (r =0 86 9,P <0 0 5 ) ,与门静脉及外周血内皮素水平呈负相关 (r =-0 797、- 0 74 1,P <0 0 5 )。结论大鼠肝硬化门静脉高压形成过程中外周及门静脉血浆内皮素水平进行性降低 ;胃黏膜微循环内皮素A受体mRNA表达量增加 ,呈现上调趋势 ;内皮素及其受体mR NA表达变化可能与胃黏膜微循环功能障碍有关。     

Global gene profiling reveals a downregulation of BMP gene expression in experimental atrophic nonunions compared to standard healing fractures.

Nonunion is a challenging problem that may occur following certain bone fractures. However, there has been little investigation of the molecular basis of nonunions. Bone morphogenetic proteins (BMPs) play a significant role in osteogenesis. However, little is known about the expression patterns of BMPs in abnormal bone healing that results in nonunion formation. These facts prompted us to investigate and compare the gene expression patterns of BMPs and their antagonists in standard healing fractures and nonunions using rat experimental models. Standard closed healing fractures and experimental atrophic nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, 10, 14, 21, and 28, total RNA was extracted from the callus of standard healing fracture and fibrous tissue of nonunion (n=4 per each time point and each group). Gene expression of BMPs, BMP antagonists, and other regulatory molecules were studied by methods including Genechip microarray and real-time quantitative RT-PCR. Gene expression of BMP-2, 3, 3B, 4, 6, 7, GDF-5, 7, and BMP antagonists noggin, drm, screlostin, and BAMBI were significantly lower in nonunions compared to standard healing fractures at several time points. Downregulation in expression of osteogenic BMPs may account for the nonunions of fracture. The balance between BMPs and their endogenous antagonists is critical for optimal fracture healing.     

重组人白细胞介素10的融合表达及鉴定

目的 研究重组人白细胞介素10(rhIL—10)载体在大肠杆菌B121(DE3)pLyse细胞中的表达，为进一步研究IL-l0在动脉粥样硬化中的作用机制奠定基础。方法 用构建成功的IL-l0—PCRT7/NT-TOPO质粒转化大肠杆菌BL21(DE3)pLyse细胞，并通过SDS-PAGE鉴定融合表达蛋白。结果 PCRT7/NT—TOPO质粒载体成功载入rhIL-l0基因；在异丙基硫代—β-D-半乳糖苷(IPTG)诱导下表达的蛋白质主要以包涵体形式存在。结论 在IPTG诱导下，重组的IL-l0—PCRT7/NT—TOPO质粒载体在大肠杆菌BL2l(DE3)pLyse细胞内成功表达。     

RNA-DNA杂交法研究乳酸脱氢酶-C基因在睾丸中特异性表达

用同位32~P标记乳酸脱氢酶-C(LDH-C)cDNA作为探针,与小鼠胸腺、脑、胰、心肌、骨骼肌、睾丸、肾、肺、肝的RNA以及人胰、皋丸、肝、骨骼肌、心肌及脑的RNA分别作点溃杂交(dot blot hybridization)及Northern印迹杂交,证实LDH-C基因只在睾丸中特异性表达。     

Construction of Shuttle Expression Plasmid and Stable Expression of Foreign Gene in Mycobacteria and E．Coli

ＣｏｎｓｔｒｕｃｔｉｏｎｏｆＳｈｕｔｔｌｅＥｘｐｒｅｓｓｉｏｎＰｌａｓｍｉｄａｎｄＳｔａｂｌｅＥｘｐｒｅｓｓｉｏｎｏｆ　ＦｏｒｅｉｇｎＧｅｎｅｉｎＭｙｃｏｂａｃｔｅｒｉａａｎｄＥ．ＣｏｌｉＨＵＡＮＧＦＵＹｏｎｇ－ｍｕ（皇甫永修）；ＺＨＡＮＧＤａ－ｊｕ...     

中药复方对辐射损伤小鼠脾细胞凋亡及mRNA表达的影响

目的探讨中药复方地甘口服液(DGOL)对辐射损伤小鼠脾细胞凋亡及凋亡相关基因bcl-2和bax mRNA表达的作用.方法建立辐射损伤模型(一次性7.0Gy的X射线照射),喂饲100%地甘口服液每次0.2 ml/10 g小鼠,每日2次.第10天用原位末端标记的TUNEL技术检测脾组织细胞凋亡和原位杂交法检测凋亡相关基因bcl-2和bax mRNA的表达.结果地甘口服液组与对照组比脾脏的细胞凋亡率和bax mRNA表达显著减少(P＜0.01),bcl-2 mRNA的表达显著增强(P＜0.01).结论中药复方地甘口服液可能通过促进bcl-2和降低bax mRNA的表达,促进造血细胞的增生,提高机体免疫力.     

神经肽P物质对肉芽组织成纤维细胞增殖及表皮生长因子基因表达的作用

目的：探讨感觉神经肽P物质（SP）对离体培养的肉芽组织成纤维细胞的促增殖作用及其对表皮生长因子（EGF）基因表达的调控作用。方法：采用MTT法测定SP对肉芽组织成纤维细胞的促增殖作用；采用逆转录－聚合酶链反应（RT－PCR）方法检测SP对成纤维细胞EGF基因表达的调控作用，观察时间及剂量－效应关系。结果：10^-9～10^-5mol/L的SP在体外对肉芽组织成纤维细胞均具有明显的促增殖作用（P＜0．01），且具有明显的剂量依赖性（γ=0.594,P＜0．01），EGF抗体只能部分抑制这一作用（与对照组比较，P＜0．01；与10^-7mol/L SP组比较，P＜0．05）。10^-7mol/L SP可诱导成纤维细胞EGF mRNA的表达，在作用后6h与对照组比较，差异有非常显著性意义（P＜0．01）；SP在10^-8～10^-6mol/L范围内可以显著促进成纤维细胞EGF mRNA表达，在10^-7mol/L达到峰值，当浓度＞10^-7mol/L时，其促EGF mRNA表达的效应强度随浓度升高而有所降低，至10^-5mol/L时与对照组比较，差异无显著性意义。结论：SP对肉芽组织成纤维细胞具有明显的促增殖作用，这种作用与其诱导成纤维细胞EGF表达有关。     

The epidemiology and surveillance of blood lead in Taiwan (R.O.C.): A report on the PRESS-BLL project

 To monitor the lead hazards in industries and to investigate the prevalence of elevated blood lead levels (BLLs) in lead-exposed workers, a lead surveillance system (PRESS-BLLs) has been established and operated in Taiwan, Republic of China, since July 1993. A cohort of lead-exposed workers who received a periodic annual health examination at 55 accredited hospital laboratories was constructed. A total of 9807 separate BLL measurements were reported to the system in 1994. The mean BLL was 15.8 μg/dl in male workers and 11.6 μg/dl in female workers. The mean BLL of lead-exposed workers was significantly (P<0.05, z-test) higher than that of the general Taiwanese population (8.6 μg/dl for males and 6.7 μg/dl for females). In addition, the BLLs of 983 (10.0%) workers exceeded the regulatory action level (40 μg/dl for males; 30 μg/dl for females). The workplaces and homes of 57% of the workers with elevated BLLs were thoroughly investigated to determine the sources of lead contamination. These actions identified the causes of elevated BLLs and set up strategies to reduce workers’ lead exposure. The establishment of this occupational lead surveillance system represents a method for monitoring of lead hazards from occupational and environmental settings to prevent lead poisoning. The information acquired from the system can help in the setting up of a priority of prevention and the development of control measures. It is also useful for further monitoring of changes in the BLLs of the lead-exposed-worker cohort. The Health Department of Taiwan can use this information to evaluate the effectiveness of current industrial hygiene practice. Subjects with elevated BLLs have been medically treated and placed on long-term follow-up for sequelae. Received: 2 September 1996/Accepted: 29 November 1996     

The effect of sodium lauryl sulphate on the expression of cytokeratin mRNA in hamster cheek pouch epithelium

The effect of sodium lauryl sulphate (SLS) on cytokeratin (CK) gene expression in hamster cheek pouch epithelium was studied with a hybridohistochemical technique. Using specific human anti-sense RNA probes, the plausible hamster mRNA counterparts for these human CK mRNAs were localized by detection of heterologous hybrids. In comparison with normal epithelium, the expression and distribution pattern of CK mRNAs in the hamster cheek pouch were obviously changed after application of SLS. There was a decreased expression of CK mRNAs in the hyperplastic basal layer, and increased expression in the hypertrophic granular layer. Strikingly, hybridization with the human CK 18 cRNA probe revealed an additionally expressed CK mRNA in the SLS-treated epithelium that was not found in the untreated epithelium. The present study indicates that cRNA probes for human CK mRNAs can be used successfully, not only to distinguish between different hamster CK mRNAs but also to investigate changes in CK gene expression upon the induction of non-neoplastic and neoplastic alterations in the hamster cheek pouch model. This may help elucidate the molecular changes involved in epithelial pathologies.