降钙素基因相关肽对血管平滑肌表型转化和增殖的影响

目的 探讨降钙素基因相关肽(CGRP)对大鼠血管平滑肌细胞(VSMCs)表型转化和增殖的影响以及它们之间的关系.方法 取大鼠主动脉先体外培养8d后再贴块培养(VSMCs);对照组直接用贴块法培养细胞.实验分为CGRP作用组和无CGRP作用组.用5-BrdU标记平滑肌细胞增殖变化;RT-PCR检测HRG-1和SM22α表达变化.结果 血管经体外培养8d后再贴壁培养的平滑肌细胞可见大量棕黄色标记增殖的细胞核,HRG-1和SM22α mRNA表达明显减少;而CGRP作用组标记的血管平滑肌增殖细胞明显减少,HRG-1和SM22α mRNA表达明显上调.结论 CGRP对VSMCs增殖有抑制作用并同时可使VSMCs从合成型向收缩型转化.     

冈比亚按蚊嗅觉结合蛋白基因agCP1588的克隆鉴定及其组织特异性表达谱分析

昆虫主要依靠嗅觉发现寄主 ,嗅觉在按蚊的寄主搜索行为中起关键作用。本文基于冈比亚按蚊的全基因组序列 ,设计特异引物 ,采用RT -PCR克隆了该按蚊嗅觉结合蛋白候选基因agCP15 88。测序分析结果表明该基因具有嗅觉蛋白的标志性结构域 ,通过半定量RT PCR技术研究了该基因的组织特异性表达谱 ,发现该基因只在雌蚊触角中表达。这一发现为进一步研究该嗅觉蛋白基因的功能奠定了基础     

不同胃癌细胞系中B7-H1分子的表达水平及定位

目的 通过检测胃癌细胞系SGC7901/VCR、SCC7901和BGC-823,以及永生化胃上皮细胞系GES中B7-H1的表达,探讨B7-H1与胃癌的发生及多药耐药(MDR)的关系.方法 上述细胞系培养于含10%灭活胎牛血清的RPMI-1640培养基中,利用RT-PCR技术、细胞免疫化学染色以及细胞免疫荧光染色技术与流式细胞术检测4种细胞系中B7-H1 mRNA与B7-H1蛋白的表达情况,并比较其在4种细胞系中的表达强度.结果 B7-H1 mRNA在4种细胞系中均有表达,表达强度按照GES-1、BGC-823、SGC-7901、SGC-7901/VCR的顺序递增;细胞免疫化学染色与免疫荧光染色表明,B7-H1蛋白主要表达在上述细胞的细胞膜和少量细胞浆中.结合流式细胞术检测进一步证实,B7-H1蛋白在4种细胞系中的表达结果与mRNA的表达一致.结论 B7-H1表达在胃上皮细胞上,可能通过抑制机体免疫反应,促进胃癌细胞生长及MDR基因表达.     

Expression of mucin genes in the human testis and its relationship to spermatogenesis

In this study we investigate the expression pattern of mucin genes in the human testis and evaluate the relationship between the expression of mucin genes and impaired spermatogenesis in the human testis. Thirty human testis tissues were collected from patients undergoing diagnostic testicular biopsy to investigate the cause of infertility. One part of the tissue underwent histological observation, and the other part of the tissue was subjected to semiquantitative RT-PCR of mucin genes, that is, mucin1, 2, 3, 4, and 9. The relative amount of mucin mRNAs was calculated by densitometry using glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control. The samples were histologically diagnosed as either obstructive azoospermia with normal spermatogenesis (n = 13) or non-obstructive azoospermia with impaired spermatogenesis (n = 17). In the human testis with normal spermatogenesis, mRNA expression of mucin1, 9, 13 and GAPDH were found, but RT-PCR products of mucin 2, 3 and 4 were not detected. In the testis with impaired spermatogenesis, however, RT-PCR product of mucin1 was not found. There was no difference in the other mucin mRNA expression patterns between the testis with either normal or impaired spermatogenesis. To our knowledge, this study is the first that has detected the mRNA of mucin9 and 13 in human testis. This study also shows that mucin1 expression might be closely related to spermatogenesis. Our findings should be substantiated by more direct evidence, such as mucin protein expression and localization.     

5′-Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma

 DNA methylation plays an important part in the regulation of gene expression. Alterations in DNA methylation in tumours have been reported and have been used to generate hypotheses about mutagenesis and silencing of tumour suppressor genes. However, the underlying mechanism is still poorly understood, and conflicting data on the levels of overexpression of 5′-cytosine DNA methyltransferase in sporadic colon carcinoma have been published. We used a competitive RT-PCR assay for quantification of mRNA of 5′-cytosine DNA methyltransferase in colon biopsies obtained from patients with hereditary colon carcinoma syndromes and compared the results with those obtained in a control group. No significant difference was found between the flat mucosa of FAP patients and the mucosa of the control group. In FAP and HNPCC patients, the 5′-cytosine DNA methyltransferase mRNA levels of adenomas were significantly higher (P<0.05) than of flat mucosa in the same group, but both showed great variability from patient to patient. Our findings suggest that the mRNA levels of methyltransferase cannot be used as predictive marker for screening in families affected by hereditary colon carcinoma. Received: 20 July 1998 / Accepted: 21 September 1998     

Large Envelope Glycoprotein and Nucleocapsid Protein of Equine Arteritis Virus (EAV) Induce an Immune Response in Balb/c Mice by DNA Vaccination; Strategy for Developing a DNA-Vaccine Against EAV-Infection

Equine arteritis virus (EAV) is a member of the Arteriviridae family, that includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian haemorrhagic fever virus (SHFV). Equine arteritis is a contagious disease of horses and is spread via respiratory or reproductive tract. The objective of the present study is to evaluate the possibility for developing a model system for prevention horses against an EAV infection by DNA vaccination. A cDNA bank from the RNA of EAV was established. This gene library contains the translation unit of the EAV open reading frames (ORF) 1 to 7. The identity of the cDNA was confirmed by nucleotide sequence analysis. Using this defined EAV cDNA gene library the cDNA sequence of the viral ORFs were molecularly cloned into the corresponding sites of well characterized and powerful expression vectors (pCR3.1, pDisplay, and/or pcDNA3.1/HisC).The capability of these recombinant plasmids expressing the gene products of the individual viral ORFs 3 to 5, and 7 in induction of an immune response in mouse system was investigated. The Balb/c mice (ten mice per assay) were inoculated with the DNA of the constructed expression vectors harboring and expressing the EAV cDNA of the viral ORFs. The Balb/c mice were injected with about 100 g DNA diluted in 100 l PBS. The DNA was injected subcutaneously and into the tibialis cranialis muscle (Musculus gastrocnemius). The mice were boosted 3 to 5 times with the same quantities of DNA and under the same conditions at about two week intervals. Control mice received the same amount of parental expression vectors via an identical route and frequency.The pre- and post-vaccinated sera of the individual animals were screened by neutralization tests (NT). Neutralizing antibodies against EAV were detected when the animals were inoculated with the DNA of the expression vectors harboring cDNA of the EAV ORFs 5 and 7. Highest NT-titers were observed when the animals were administered with the cDNA of ORF 5 and/or with the cDNA of the neutralization determinants of EAV that is located on the N-terminal ectodomain of the gene product of ORF 5 between the amino acid positions 1–121. These results obtained from these studies justified proofing the capability of the EAV cDNA sequences of the viral genes including ORFs 5 and 7 in the autologous animal system horse.     

酪氨酸激酶受体RON在上皮性卵巢癌中的表达及其意义

目的:研究酪氨酸激酶受体 RON 在上皮性卵巢癌组织中的表达及其与临床病理学参数的关系.方法:采用逆转录聚合酶链反应和免疫组织化学方法分别检测 42 例上皮性卵巢癌新鲜组织 RON mRNA 及其对应 32 例石蜡组织中 RON蛋白的表达.结果:上皮性卵巢癌新鲜组织中 RON mRNA 阳性表达率为57.14%,对应的上皮性卵巢癌石蜡组织中 RON蛋白平均面密度值为(0.060 1±0.028 4),RON mRNA 和蛋白表达均与上皮性卵巢癌组织的临床分期、组织学分级及淋巴结转移有关.结论:RON 的过度表达与上皮性卵巢癌的进展、转移密切相关,检测 RON 的异常对判断肿瘤的临床进展及转移有一定的参考价值,RON 可能成为诊断治疗的新靶点.     

SHP-1和JAK1基因在初治急性白血病患者中的表达

目的：探讨SHP-1和JAK1基因在初治急性白血病（AL）细胞的转录表达及其与初治AL患者化疗效果的关系。方法：采用半定量逆转录-聚合酶链反应（RT-PCR）方法检测60例初治AL患者骨髓单个核细胞及20例健康人外周血单个核细胞中SHP-1和JAK1 mRNA的表达。结果：60例初治AL患者SHP-1和JAK1的mRNA表达阳性率分别为30%，100%；初治AL患者SHP-1 mRNA表达水平明显低于正常对照组（P<0.001），JAK1 mRNA表达水平较正常对照组略增高，但差异无统计学意义（P=0.180）,初治AL患者SHP-1 mRNA阳性组的诱导化疗完全缓解（CR）率为88.9%，阴性患者组CR率为54.8%，差异有统计学意义（P=0.005）。SHP-1与JAK1 mRNA表达呈负相关（P=0.046）。结论：SHP-1可能是白血病潜在的抑制基因，可望作为判断初治急性白血病患者疗效和预后的指标。     

Bronchiolar expression of aquaporin-3 (AQP3) in rat lung and its dynamics in pulmonary oedema

Aquaporins (AQPs) are water channel proteins that permit osmotically driven water movement. To determine their dynamics in pulmonary oedema, we examined the expression of mRNA and protein for AQP1, AQP3, AQP4, and AQP5 in the lungs of normal and thiourea-treated rats. In the thiourea group, lung water content increased significantly (vs. controls) with the peak at around 4 h. Semi-quantitative RT-PCR showed that AQP3 mRNA in the thiourea group rose significantly, peaking at around 4–8 h. The expression of AQP1, AQP4, AQP5, ENaC and CFTR mRNA each decreased significantly some time after the peak in lung water content. Immunoblot analysis showed that glycosylated AQP3 protein was increased 4–10 h after treatment. Expression of the other AQP proteins was not significantly altered, except for that of AQP4. Immunohistochemical examination revealed that AQP1 was expressed in endothelia, AQP3 in the basal cells of the large airways and in cuboidal cells in the bronchioles, AQP4 in the basolateral membrane of airway cells and AQP5 in type-I pneumocytes. Our results suggest that AQP3 is expressed not only in large airways, but also in bronchioles, and is related to water movement in pulmonary oedema.