非同位素PCR方法检测脆性X 综合征FMR-1基因CGG重复序列数目

目的 建立一种可靠、简便的非同位素PCR方法检测脆性X综合征的突变基因。方法 采用生物素标记的CGG寡核苷酸探针，检测通过PCR扩增的FMR-1基因中CGG三核苷酸重复序列数目。从而判断所检样品中FMR—1基因是否正常。结果 该法可检测出正常人及携带者的CGG重复拷贝数。结论 该方法可以简便、安全、可靠地检测FMR—1基因中(CGG)n重复拷贝数，从而确定为正常人或携带者，可作为临床上筛查脆性X综合征的首选方法。     

Heterologous Expression of Rat Testis GABAA Receptor β3t Splicing Variant in CHO Cells

Objective To characterize a possible retention function of unique sequence in the 5'end of rat testis GABAA receptor β3t splicing variantMethods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined.Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively. The chimera product failed to be translocated into the cell surface when expressed in CHO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane.Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.     

A clinical and genetic study of 56 Saudi Wilson disease patients: identification of Saudi-specific mutations

Wilson disease (WD) is a hereditary disorder, with recessive transmission and genetic heterogeneity. Several mutations of ATP7B, the gene underlying WD, were reported in many ethnic groups. In this study, mutation screening in ATP7B of 56 Saudi Arabian WD patients was undertaken. The clinical data of all patients were recorded. The entire ATP7B coding sequence, including intron-exon boundaries were screened for mutation by the polymerase chain reaction (PCR)-based mutation detection technique and DNA sequencing. Thirty-nine patients were symptomatic at presentation and 17 subjects were pre-symptomatic siblings of affected patients. Fourteen patients had neurological, 11 patients had mixed (hepatic and neurological), and 14 patients had hepatic presentations. Family history suggestive of WD was present in 72% of cases and 68% had consanguineous parents. Genetic analysis showed disease-causing mutations in three exons (exons 8, 19 and 21) of the ATP7B gene in 28 patients (50%). Mutations in exons 21 (18 cases) and 19 (one case) were unique for Saudis. This large series of Saudi patients with WD has shown wide variability in the genomic substrate of WD. There is no correlation between genotype and clinical presentation.     

人野生型parkin基因真核表达载体的构建及其在PC12细胞中的表达

目的 克隆人野生型parkin基因并构建真核表达载体pCDNA3．1—parkin，将重组质粒转染PC12细胞获得高表达人野生型parkin基因的PC12细胞克隆。方法 从胎脑组织中提取总RNA，用RT—PCR方法获得人野生型parkin基因的全长cDNA，插入pCR2．1—TA克隆载体中进行序列测定，测序正确后将其亚克隆至表达载体pCD—NA3．1，利用脂质体将重组质粒转染PC12细胞，经G418筛选获得抗性细胞克隆，采用RT—PCR和Western Blot方法鉴定人野生型parkin基因在PC12细胞中的过表达。结果 经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒，RT—PCR和Western Blot证明经G418筛选得到的转基因PC12细胞克隆中存在人野生型parkin基因的表达。结论 成功构建了人野生型parkin基因的真核表达载体，获得了稳定表达人野生型parkin基因的PC12细胞克隆，为进一步研究parkin的生物学功能以及parkin在帕金森病发病机制中的作用奠定了良好的基础。     

烟酰胺对高糖介导胰岛细胞bax及bcl-2表达的影响

目的 探讨烟酰胺对高糖环境下胰岛细胞bax及bel-2蛋白表达的影响。方法 体外培养大鼠胰岛细胞，应用免疫组化方法检测培养液中葡萄糖终浓度为22．2mmol／L及加入烟酰胺后胰岛细胞bax及bcl-2蛋白表达。结果 培养液中葡萄糖终浓度为22．2mmol／L时，胰岛细胞bax表达阳性，bcl-2表达较对照组降低。加入烟酰胺后bax表达降低，bcl-2表达增强。结论 烟酰胺能够上调bcl-2表达，下调bax表达，在高糖介导的胰岛细胞损伤中起一定作用。     

中国男性原发不育的遗传高风险因子:Y染色体DAZ基因拷贝缺失

目的 探讨Y染色体无精症因子C区(azoospermia factor C，AZFe)无精症缺失基因(deleted-in-azoospermia，DAZ)家族基因拷贝缺失与中国男性原发不育之间的关系。方法 运用多重PCR与PCR-RFLP检测技术，对210例已生育男性、216例原发无精症与189例严重少精症患者Y染色体AzFc区域DAZ基因家族的基因拷贝数进行分析。结果 在所有已生育男性中未检出DAZ基因拷贝的完全或部分缺失，而在原发无精症与严重少精症患者中DAZ基因拷贝完全缺失率分别为8．8％和12．2％，DAZ1／DAZ2共缺失率分别为8．3％和5．3％。结论 在中国男性原发无精症与严重少精症患者中存在较高频率的DAZ基因拷贝缺失现象，提示Y染色体AZFc区域DAZ基因家族基因拷贝的完全与部分缺失是中国男性原发不育的遗传高风险因子。     

重症肌无力抗乙酰胆碱受体单链抗体基因的构建

[目的 ]构建重症肌无力抗乙酰胆碱受体主要免疫原区单链抗体A 7基因 .[方法 ]应用PCR技术及基因克隆技术分两步完成重链和轻链可变区基因的克隆 ,再对构建的单链抗体A 7基因进行序列测定检测其核苷酸序列 .[结果 ]构建的重组质粒经序列分析 ,重链可变区基因长度为 36 9bp ,轻链可变区基因长度为 32 1bp ,单链抗体基因长度为 735bp ;单链抗体A 7基因正确地插入在载体质粒pHEN 2开放读码框架内 .[结论 ]成功地构建重症肌无力抗乙酰胆碱受体主要免疫原区单链抗体A 7基因 ,为进一步制备基因工程抗体奠定了基础 .     

枳鳖胶囊抗大鼠肝纤维化形态学及肝组织Ⅲ型胶原mRNA表达的实验研究

目的:观察枳鳖胶囊抗肝纤维化形态学和Ⅲ型胶原mRNA表达的变化。方法:选择66只健康清洁级SD大鼠,雌雄各半,随机分为:正常对照组,模型组,秋水仙碱组,枳鳖胶囊大、中、小剂量组。以40%四氯化碳花生油皮下注射结合高脂饲料、酒水饮料复合因素诱导大鼠肝纤维化模型。造模结束后,除正常组不予处理外,其他组分别以蒸馏水,秋水仙碱混悬液,高、中、低浓度枳鳖胶囊溶液灌胃,疗程36d。疗程结束后,观察大鼠新鲜肝脏的表面情况、色泽、质地等大体形态,测定肝组织Ⅲ型胶原mRNA的表达,光镜观察肝组织HE染色和网状纤维染色的病理形态学改变。结果:模型组大鼠肝细胞损害、肝脏脂肪变性和胶原纤维增生的程度最显著,Ⅲ型胶原mRNA的表达最强。枳鳖胶囊组上述改变明显减轻,且可抑制Ⅲ型胶原mRNA的表达,疗效优于秋水仙碱组。结论:枳鳖胶囊可较好地保护肝细胞,并能阻止肝纤维化进程甚或逆转肝纤维化病理改变,抑制胶原基因表达,抑制胶原基因表达可能是其抗肝纤维化的作用机理之一。     

阴道毛滴虫Rab11鸟苷三磷酸酶eDNA克隆和序列分析

目的 近年研究表明Rab11鸟苷三磷酸酶在调节各种真核细胞的膜转运通道中发挥着重要作用。但是，对于原生动物毛滴虫膜转运系统的研究仍甚少。本研究旨在克隆和分析阴道毛滴虫Rabll鸟苷三磷酸酶基因，以便进一步探讨其功能。方法 使用SMART cDNA文库构建试剂盒构建阴道毛滴虫cDNA表达文库，用生物信息学进行TvRab11同源分析，鉴定TvRab11基因，用PCR方法扩增基因组DNA，TA克隆TvRab11 cDNA、测序及序列分析。结果 在分离出的cDNA克隆中发现一个Rab11鸟苷三磷酸酶同源基因。该cDNA序列长710bp，读码框含636bp，推测蛋白质序列具211个氨基酸。序列分析表明该基因系Rab11亚家族的同源基因，其推测肽链与拟南芥Rab11c的同源性晟高（一致性60％，相似性79％），与人类Rab11b次之（一致性58％，相似性78％）。该氨基酸序列拥有Rab鸟苷三磷酸酶家族的所有保守结构域和特异性Rab结构域（RabF1-5）。进化树分析也表明该基因系Rab11的同源基因。序列分析还显示该基因的基因组DNA序列与cDNA序列完全一致．提示该基因无内含子。结论分析表明该基因是阴道毛滴虫Rab11鸟苷三磷酸酶同源基因，其功能有待进一步研究。