联合利用嵌套缺失和酚筛选法构建和鉴定目的基因的缺失突变体

目的 介绍一种快捷、无突变的目的基因缺失体构建和鉴定方法.方法 用Mlu I 和 Sac I酶将GCLC/pGL3在GCLC基因与pGL3连接处切开,形成以GCLC端为3'凹端而pGL3载体端为3'凸端的线型载体.利用外切核酸酶Ⅲ从GCLC基因端(3'凹端)单向逐一切除GCLC基因上的核苷酸,并在不同的时间终止其反应以获得一系列的GCLC基因缺失体.将GCLC基因缺失体与pGL3载体自身环化构建成GCLC基因序列缺失体的pGL3报道载体.最后用酚抽提和凝胶电泳法粗略挑选出不同长度的GCLC/pGL3缺失突变体.结果 利用嵌套缺失法构建出各种长度的GCLC基因的缺失突变体,且不存在基因突变的现象.利用酚筛选法快速鉴定出29种不同长度的缺失突变体.结论 联合利用嵌套缺失和酚筛选法可以高保真而快速地构建和鉴定目的基因的缺失突变体.     

妊娠17～37周胎儿可溶性白介素2受体和NK细胞含量的变化

目的：探讨妊娠17～37周正常和宫内感染时胎儿外周血可溶性白介素2受体（Soluble interleuldn-2 receptor，sIL-2R）和自然杀伤细胞（Natural killer，NK）含量的变化。方法：超声引导下行脐带穿刺术，收集129例胎儿外周血，包括97例正常对照组胎儿血，32例宫内感染组（单纯疱疹病毒感染、弓形虫感染、风疹病毒感染）胎儿血，采用双色免疫荧光标记流式细胞仪技术测定胎JLPF周血NK细胞百分率，双抗体夹心酶联免疫吸附试验测定胎JLPF周血中sIL-2R的含量，分析生理状态下胎儿外周血NK细胞、sIL-2R的状况和宫内感染时NK细胞和sIL-2R含量的变化。结果：妊娠17—37周胎儿外周血NK细胞、sIL-2R含量不随孕周改变，r（NK）=-0．03，P〉0．05；r（sIL-2R）=0．167，P〉0．05，宫内感染时NK细胞含量减少，sIL-2R含量增多，与正常对照组相比差异有显著意义；t（NK）=4．29，P〈0．01；t（sIL-2R）=-5．833，P〈0．01。结论：妊娠17—37周胎儿外周血有一定量的NK细胞和sIL-2R存在，但机体免疫功能仍不完善，宫内感染时机体容易出现免疫抑制状态。     

The influence of the herpes simplex virus-1 DNA template environment on the regulation of gene expression

To determine the role of the HSV-1 genome structure and environment on the regulation of gene expression, we constructed recombinant viruses containing a heterologous gene inserted into either the immediate early ICP0 or late glycoprotein C (gC) genes of HSV-1. The heterologous gene consisted of the SV40 early promoter (without enhancer sequences) linked to the coding sequences for the bacterial chloramphenicol acetyl transferase (CAT). The expression of CAT was examined in Vero cells infected with either virus (named ICP0-CAT and Sph 6). For both recombinants, expression of CAT was not dependent upon prior viral protein synthesis. The kinetics of expression of CAT-specific mRNA resembled that of the HSV-1 genes into which CAT was inserted. Primer extension analysis revealed that the SV40 promoter is recognized and used when placed in cis in two different HSV-1 genome locations, and Northern hybridization experiments confirmed that the heterologous gene was expressed in the absence of prior viral protein synthesis. Therefore, this gene was not regulated as strictly as an HSV-1 gene, but was influenced by the environment into which it was placed, presumably by factors that are present when the normal viral gene is on.     

Encoding of electrical, thermal, and mechanical noxious stimuli by subnucleus reticularis dorsalis neurons in the rat medulla

1. In anesthetized rats, recordings were made within the medullary subnucleus reticularis dorsalis (SRD) from neurons that exhibited convergence of nociceptive inputs from the entire body. Neurons with total nociceptive convergence (TNC) responded to suprathreshold percutaneous electrical stimuli (2-ms duration) with an early and a late peak due to activation of A delta- and C-fibers, respectively, no matter which part of the body was stimulated. Neurons with partial nociceptive convergence (PNC) responded to the same stimuli with an A delta-peak regardless of which part of the body was stimulated and with a C-peak of activation from some, mainly contralateral, parts of the body. The characteristics of the responses of these neurons to the application of graded intensities of electrical, thermal, and mechanical stimuli were analyzed. 2. All TNC neurons and 85% of PNC neurons responded to A delta- and C-fiber activation following percutaneous electrical stimulation of the contralateral hindpaw. With regard to A delta-fiber-evoked responses, a linear relationship between the logarithm of the applied current and the magnitude of the responses was found within the 0.25- to 6.0-mA and 0.5- to 24-mA ranges for TNC and PNC neurons, respectively; however, these curves were essentially similar. With regard to C-fiber-evoked responses, such a linear relationship was found within the 1.5- to 6.0-mA range for both types of SRD neurons, although the TNC neurons presented larger C-fiber-evoked responses than did the PNC neurons. 3. TNC and PNC neurons linearly increased their discharges during the application of noxious thermal stimuli to the contralateral hindpaw within the range 44-52 degrees C; the mean responses evoked by noxious heat from TNC neurons were of higher magnitude than those from PNC neurons. The majority of SRD neurons presented long-lasting afterdischarges, especially with the highest temperature employed (52 degrees C). 4. TNC neurons monotonically increased their discharges during graded mechanical or thermal stimulation of the tail. When mechanical stimuli were applied, a linear relationship was found between the logarithm of the strength of the mechanical stimulus and the neuronal discharges, in the 5.3- to 7.4-N/cm2 range. With thermal stimulation, TNC neurons linearly increased their discharges in the 44-52 degrees C range. When increasing amounts of the tail were immersed in a 50 degrees C waterbath, TNC neurons increased their discharges within a restricted range of tail surface areas (0.9-5.7 cm2); further increases in the stimulated surface size were not followed by increases in firing rate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal outcome.

Poor prognosis in nasopharyngeal carcinoma patients may result from resistance to the apoptosis-inducing effect of radio- and/or chemotherapy. Apoptosis depends on proper activation of caspase 3, resulting in cleavage of key proteins like PARP-1. To investigate whether disruption of the apoptosis pathway results in therapy-resistant tumour cells, we investigated whether absence of caspase 3 activation in tumour biopsies of nasopharyngeal carcinoma patients is related to poor clinical outcome. Moreover, we investigated whether absence of caspase 3 activation is related to loss of procaspase 3 expression or expression of the apoptosis regulators p53, bcl-2 and XIAP. We studied 36 Indonesian nasopharyngeal carcinoma patients without evidence of distant metastases who were treated with curative intent by radiotherapy only. Activation of caspase 3 and expression of the different markers were determined using specific antibodies. Levels of caspase 3 activation were determined by quantifying positively staining tumour cells. Nasopharyngeal carcinoma-derived C15 and C17 tumour cells were used as control. Absence of caspase 3 activation was strongly related to a poor clinical response to radiotherapy and to a higher T and N stage, resulting in a particularly poor clinical outcome with regard to progression-free (P<0.0001) and overall survival time (P<0.0001). Absence of caspase 3 activation was significantly correlated to loss of expression of procaspase 3 (P=0.04). In nasopharyngeal carcinoma patients treated with curative intent, absence of active caspase 3-positive neoplastic cells predicts rapid fatal outcome, and is associated with poor response to radiotherapy and high T and N stage at time of presentation.     

腰大肌作用力与脊柱伸展应力关系的生物力学实验研究

目的:探讨腰大肌作用力带动脊柱伸展应力的生物力学关系。方法:取家兔12只,分3组,每组4只,解剖后保留枢椎以下完整之脊柱及骨盆、髋关节、上段股骨,不损伤脊柱前、后纵韧带、椎间盘及所附着之腰大肌,保留脊柱背侧的竖脊肌、棘上韧带,置于生物力学拉伸测试仪(日本岛津制作所产AGS-J系列)。上端十字头分别夹枢椎(颈胸腰段)、第1胸椎(胸腰段)和第12胸椎(腰段),下端十字头夹股骨上部;分别作有腰大肌状态下和切断腰大肌状态下,股髋自屈曲位到过伸带动脊柱自屈曲位到过伸位拉伸试验,测定两种不同状态下脊柱各节段的伸展应力(N/mm2)。结果:有腰大肌状态和切断腰大肌状态下,股-髋-脊柱拉伸后脊柱伸展应力分别为:颈胸腰全段平均为306.6675N/mm2:78.7167N/mm2;胸腰段为680.8417N/mm2:373.0375N/mm2;腰段为1990.7944N/mm2:523.0608N/mm2;经统计学分析,具显著性差异,P<0.01。结论:腰大肌作用力对脊柱伸展应力影响显著,颈胸腰段占74.33%、胸腰段占45.21%,腰段占73.73%的伸展应力源自腰大肌。脊柱在腰大肌作用下产生腰椎向腹部的弯曲。     

应用噬菌体随机肽库技术筛选幽门螺杆菌中性粒细胞激活蛋白的抗原表位

目的 筛选和鉴定幽门螺杆菌(Helicobacter pylori,Hp)中性粒细胞激活蛋白(neutrophil-activating protein,NAP)的有效抗原表位,为Hp疫苗的研制提供基础.方法 以抗NAP的单克隆抗体作为固相筛选分子,经3轮吸附-洗脱-扩增免疫,筛选噬菌体随机7肽库,随机挑选噬菌体克隆,经噬菌体酶联免疫吸附试验(ELISA)、交叉反应试验及竞争抑制试验鉴定阳性克隆,测定阳性克隆所携带DNA序列并进行计算机辅助分析.以制备的阳性噬菌体克隆短肽液免疫小鼠,免疫血清与NAP经Western blot分析,以验证NAP的模拟表位.结果 经3轮免疫筛选后挑选到45个阳性克隆,经ELISA鉴定有12个阳性克隆,测序结果显示5种表位,其中P17噬菌体展示肽FAHLATQ与NAP氨基酸序列(137～143)高度同源,位于NAP高抗原区域(118～140),免疫血清可识别NAP.结论 用噬菌体随机7肽库成功筛选到了NAP的模拟表位,为基于NAP的诊断和疫苗的研制提供了基础.     

颈椎钩突邻近结构薄层断面与MRI对照研究

目的阐明颈椎钩突在薄层断面和MRI断面图像上与周围结构的毗邻关系,为颈椎退行性疾病提供影像学诊断依据。方法选取5例中国数字化可视人体（CVH）数据集中C3-C7椎体上缘清楚显示钩突的薄层断面图像,观察并测量钩突与邻近的椎动脉和颈神经的位置关系,选择对应平面的MRI断面图像对照分析。结果CVH数据集的薄层断面清晰显示颈椎钩突及其周围结构,在断面上测量并得出钩突与颈神经、钩突与椎动脉的距离的平均值,MRI清楚显示颈椎钩突及其邻近结构。结论将CVH数据集中的颈椎钩突平面的薄层断面与对应的MRI图像进行对照研究,可为颈椎病的影像学诊断和治疗提供形态学参考。     

三氧化二锑诱导急性早幼粒细胞白血病细胞凋亡的研究

目的 研究锑剂三氧化二锑（Sb2O3)对早幼粒细胞白血病细胞株NB4凋亡的诱导作用，以寻求早幼粒细胞白血病治疗的新方法。方法 采用细胞生长曲线，形态学及硝基四氮唑蓝（NBT）还原试验，判定NB4细胞的生长，分化及功能。采用细胞周期分析和DNA电泳研究细胞凋亡。结果 Sb2O3能诱导早幼粒白血病细胞凋亡，且具有时间，剂量依赖性。结论 Sb2O3能有效地诱导早幼粒白血病细胞凋亡，提示锑剂诱导细胞半亡的疗法，有望成为临床治疗早幼粒细胞白血病的新方法。     

国外乳腺癌病理诊断报告标准化概述及对国内标准化报告的建议

随着乳腺癌治疗方案的不断改进和发展,临床医师对乳腺癌病理诊断报告的内容和标准有了更多和更高的要求.