Cellular distribution of the new growth factor Pleiotrophin (HB-GAM) mRNA in developing and adult rat tissues

Summary Pleiotrophin (PTN), also known as HBGAM, belongs to an emerging cytokine family unrelated to other growth factors. We report here the first comprehensive study using in situ hybridization on the cellular distribution of this new heparin-binding growth factor mRNA in rat tissues. PTN mRNA was developmentally expressed in many — but not all — neuroectodermal and mesodermal lineages, whilst no PTN mRNA was detected in endoderm, ectoderm and trophoblast. PTN mRNA was found in the nervous system throughout development, with a post-natal peak of expression. In the adult nervous system, significant expression persisted in hippocampal CA1 pyramidal neurons and in cortical neurons, but also in different non-neuronal cells types in various locations (olfactory nerve, cerebellar astrocytes, pituicytes, Schwann cells surrounding the neurons in sensory ganglia). PTN mRNA was also found during development in the mesenchyme of lung, gut, kidney and reproductive tract, in bone and cartilage progenitors, in dental pulp, in myoblasts, and in several other sites. Expression was differently regulated in each location, but usually faded around birth. In the adult, PTN mRNA was still present in the meninges, the iris, the Leydig cells of the testis and in the uterus. PTN mRNA was also strongly expressed in the basal layers of the tongue epithelium, which is the only epithelium and ectodermal derivative to express PTN mRNA, and this only after birth. PTN is known to be a growth factor for perinatal brain neurons and a mitogen for fibroblasts in vitro. Recently, trophic effects on epithelial cells and a role as a tumour growth factor have been reported. The mechanisms of regulation and the functions of PTN are however still uncertain. Its expression pattern during development suggests important roles in growth and differentiation. Moreover, the presence of PTN mRNA in several adult tissues and the up-regulation of PTN mRNA expression in the gravid uterus indicate that PTN also has physiological functions during adulthood.     

性激素对心钠素基因表达影响的实验研究

为研究雄激素和雌激素对心钠素基因表达的影响,给完整的、去势的雄性大鼠皮下注射丙酸睾丸酮或雌二醇,持续2周。用冷酚法提取心房总RNA,用α-~(32)P标记ANF—cD-NA探针,经RNA电泳转移杂交和斑点吸印杂交。结果表明去势后ANF—mRNA含量下降,睾丸酮和雌二醇均使ANF—mRNA增加,然而小剂量的睾丸酮、雌二醇对去势大鼠ANF基因表达作用还需进一步研究。     

Galanin gene expression declines with adulthood in the cholinergic fields of the horizontal diagonal band of male rats

The neuropeptide galanin (GAL) influences leaming and memory processes, perhaps by inhibiting cholinergic function. We recently reported that, in the rat, the nucleus of the horizontal limb of the diagonal band (HDB) exhibits the highest level of GAL mRNA coexpression by basal forebrain (BF) cholinergic neurons and, in the HDB, virtually all GAL mRNA-expressing neurons correspond to the cholinergic cell type. Since GAL gene expression is induced across puberty in many brain regions, we used in situ hybridization histochemistry and quantitative autoradiography to assess GAL gene expression across the rostro-caudal extent of the HDB in prepubertal and adult male rats and to determine whether GAL gene expression is also regulated during maturation in this BF region. Our results show that the number of GAL mRNA-expressing cells per section is significantly reduced in the HDB with adulthood. Post-hoc analysis indicated that these age-associated differences in the number of GAL mRNA-expressing cells per section could be ascribed to the rostral and central subregions of the HDB. Age-related differences in the labeling intensity of GAL mRNA-expressing neurons were also detected in the rostral and central subregions of the HDB. No age-associated differences in GAL gene expression were found in the caudal HDB subregion. These results suggest that: (1) in contrast to other brain regions, GAL gene expression in the cholinergic BF may be negatively regulated by factors concomitant with puberty; and (2) the inhibition of cholinergic function by cosecreted GAL may be enhanced prior to puberty within cholinergic neurons of the rostral and central aspects of the HDB.     

白血病抑制因子反义寡核苷酸对髓母细胞体外生长的抑制作用

为进一步认识白血病抑制因子（LIF）对人髓母细胞瘤生长的生物学作用，我们使用LIF反义寡核苷酸，在髓母细胞内特异性阻断LIF基因表达并观察它对靶细胞的生物学效应。结果发现，被处理细胞的LIF表达降低至RT／PCR检出的阈值之下，抗LIF的免疫组织化学染色亦呈阴性，同时，这些细胞的生长速度明显减缓。相反，用与反义寡核苷酸互补的正义序列处理的细胞无论在基因表达／产生，还是在生长速度方面，均和正常培养细胞的细胞相似，本研究从而提示了一条以LIF为对象的髓母细胞瘤以及其它LIF生长依赖性肿瘤的基因和免疫治疗途径。     

p53 Protein expression in laryngeal squamous cell carcinomas bearing wild-type and mutated p53 gene

We performed an immunohistochemical analysis to investigate the expression of p53 protein in a panel of 18 laryngeal squamous cell carcinomas, 15 primary tumours and three in relapse, previously analysed by us for the presence of p53 gene mutations. Dysplastic and/or normal surrounding mucosa was evaluated in 15 different tumours. The results of our study are the following: (1) expression of p53 protein was observed in one out of five tumours positive for p53 gene mutations (20%) and in 10 out of 13 (80%) negative cases; (2), p53 protein over-expression was frequently observed in normal and/or dysplastic mucosa surrounding either wild-type (7/11) or mutated p53 tumours (2/4); (3), p53 immunoreactive cells showed a pattern of distribution in normal and mildly/moderately dysplastic mucosa (basal layers), different from that in severely dysplastic mucosa (whole thickness). These data further support the hypothesis that p53 protein over-expression may be a marker of the earliest phases of multistep tumorigenesis in laryngeal squamous cell carcinoma.     

根除幽门螺杆菌疗效与细胞色素氧化酶P450 2C19基因多态性的关系

目的 比较雷贝拉唑与奥美拉唑三联疗法根除幽门螺杆菌（Hp）的疗效与细胞色素氧化酶P4502 C19（CYP 2C19）基因多态性的关系。方法 采用随机、对照研究方法，将169例因消化不良症状接受常规胃镜检查确诊为慢性胃炎且Hp阳性的连续患者分人两组：雷贝拉唑三联疗法组（RAC组85例）和奥美拉唑三联疗法组（OAC组84例）。Hp诊断依靠组织病理学检查并参考快速尿素酶试验、血清Hp抗体检测结果。RAC组、OAC组均给予三联治疗：RAC组：雷贝拉唑10mg，OAC组：奥美拉唑20mg，两组均联用羟氨苄青霉素1000mg和克拉霉素500mg，全部药物每日2次，疗程7d。采用聚合酶链式反应结合限制性内切酶技术（PCR-RFLP），进行CYP 2C19基因型分析，治疗结束后第28天用^14C尿素呼气实验检测Hp根除疗效。结果 160例完成治疗方案，RAC组及OAC组的Hp根除率按PP分析及ITT分析均无统计学差异（P〉0．05）。根据CYP 2C19基因型分析，160例中，弱代谢型（PM）、中间代谢型（IM）及强代谢型（EM）的Hp根除率分别为95．5%（21／22）、85．9％（73／85）和67．9％（36／53）。PM型及IM型的Hp根除率均显著高于EM型（P〈0．05），而PM型与IM型间差异无统计学意义（P〉0．05）。RAC组中，各基因型的Hp根除率差异无统计学意义（P〉0．05）。OAC组中。IM型与EM型间（P〈0．01）及EM型与PM型间（P〈0．05）差异均有统计学意义。结论 雷贝拉唑与奥美拉唑两种三联疗法均能有效根除Hp。总疗效差异无统计学意义。雷贝拉唑三联疗法疗效较稳定，个体间差异小。PM型及IM型的Hp根除率均较EM型为高。     

丙型肝炎病毒E1E2蛋白原核表达载体的构建及表达产物的鉴定

目的 :构建丙型肝炎病毒 (HCV)包膜糖蛋白 (E1E2 )基因的原核表达载体 ,为研究HCVE1E2蛋白属性及基因免疫奠定基础。方法 :设计HCVE1E2区基因上、下游引物 ,分别引入BglⅡ及EcoRI酶切位点 ,以含有HCVH株基因序列的质粒pBRTM/HCV1- 30 11为模板 ,通过PCR扩增获得HCVE1E2区基因片段 ,将其克隆入原核表达载体pRSETA ,然后通过酶切和序列测定对其进行鉴定 ;将此表达载体转化入表达菌BL2 1(DE3)pLySs诱导表达 ,并采用SDS -PAGE对表达产物进行鉴定。结果 :经酶切及序列测定分析表明 ,成功地构建了重组原核表达载体pRSETA -HCVE1E2 ;SDS -PAGE显示目的蛋白大量表达 ,且呈非溶状态。结论 :HCVE1E2融合蛋白可以在大肠杆菌中大量表达 ,为HCVE1E2蛋白的制备及基因疫苗的研制奠定了基础。     

大肠重复癌MMR、p53、Bax、PCNA表达及微卫星不稳定性研究

目的探讨微卫星不稳定性 (MSI)在大肠重复癌与单发癌中的变化规律 ,及MMR、p5 3、Bax、PCNA表达与MSI的关系。 方法采用免疫组化、PCR SSLP法对 38例大肠重复癌患者的 5 1处癌灶及 35例单发大肠癌分别进行MMR、p5 3、Bax、PCNA表达的检测和 5个微卫星位点MSI检测。结果重复癌组复制错误阳性率为 5 3% (2 7/ 5 1) ,单发癌组为 17% (6 / 35 ) ,差异有显著性意义 (χ2 =11 2 5 ,P <0 0 1)。重复癌组中 ,RER 与MMR表达缺失有密切的相关性 ;RER 与 p5 3表达呈负相关 ;RER 组PCNA标记指数显著低于RER-组 ;RER 与低分化、近端大肠癌密切相关。结论MSI在大肠重复癌的发生上起着重要作用 ,MSI可作为预测大肠重复癌发生的重要标志。     

短发夹状RNA对PC-3M细胞端粒酶逆转录酶基因表达和端粒酶活性的影响

目的:研究短发夹状RNA(shRNA)对PC 3M细胞中端粒酶逆转录酶(hTRT)基因表达及端粒酶活 性的影响。方法:构建针对hTRT基因的shRNA表达质粒psilencer TRT,在质脂体介导下转染人前列腺癌PC 3M细胞,应用RT PCR及免疫组织化学检测hTRTmRNA及蛋白表达水平,应用SYBR Green染色法检测端粒 酶活性的变化。结果:重组体psilencer TRT转染细胞24h和48h后显著下调hTRTmRNA及蛋白水平,作用 48h后,其抑制率分别为85.39%和79.17%,较空白对照组差异有统计学意义(P<0.01)。同时,随着时间的延 长,端粒酶活性亦有明显下降(P<0.01)。结论:利用短发夹状RNA干扰技术能有效抑制靶基因的表达,为前 列腺癌的基因治疗提供新思路。