人精子有效抗原基因表达克隆的研究

我们从人睾丸组织中提取ＲＮＡ，并进一步纯化出ｍＲＮＡ；以此为模板，在反转录酶和ＤＮＡ多聚酶的作用下，合成ｃＤＮＡ；将ｃＤＮＡ与λｇＩＩ载体重组后转染大肠杆菌，构建人睾丸ｃＤＮＡ表达文库。运用遗传显色法和噬菌斑原位杂交法鉴定表达文库后，用兔抗人精子抗体筛选人精子抗原基因表达克隆（ＨＳＧ）。对ＨＳＧ表达抗原（ＨＳＧＡｇ）和特异抗体（ＨＳＧＡｂ）进行纯化和抗生育效应的测定。结果显示：（１）人睾丸ｃＤＮＡ表达文库容量为１．８２Ｘｌ０６ｐｆｕ，遗传显色法示重组率为６７％，噬菌斑原位杂交法示重组率为５１％。（２）经兔抗人精子抗体筛选２Ｘｌ０４ｐｆｕ，得８株人精子抗原基因表达克隆。（３）人血清、兔血清ＨＳＧ２Ａｂ和兔血清ＨＳＧ８Ａｂ对人精子具有补体依赖细胞毒作用。（４）兔血清ＨＳＧ３Ａｂ能阻断人精子在顶体反应时顶体后区ＣｏｎＡ受体的暴露。结果提示，ＨＳＧ２、ＨＳＧ３和ＨＳＧ８克隆的表达抗原是精子有效抗原，能作为精子免疫避孕疫苗的候选成分。     

不同海拔高度成年男性精子参数的比较

目的:了解不同海拔长期缺氧暴露对男性精子功能的影响。方法:以驻留海拔5340m1～3年的28例健康男性青年为高海拔组,以34例现驻平均海拔3800m2～5年的健康男性青年为中海拔组,对照组为31例常年生活在1300m海拔地区的成年男性。收集精液标本,采用计算机辅助精液分析系统(CASA)对精子参数进行分析。结果:高海拔组的精子密度、精子曲线运动速度(VCL)、直线运动速度(VSL)、平均路径速度(VAP)和直线性(LIN)分别为(51.12±14.61)×106/ml、(48.17±13.52)μm/s、(32.64±6.70)μm/s、(41.21±9.32)μm/s和(52.24±8.14)%,均明显低于对照组(P<0.01或P<0.05)。中海拔组的精子密度、精子活率、VSL、VCL、LIN、VAP和ALH等参数较对照组亦有不同程度的下降,但差异无显著性(P>0.05)。结论:随着海拔高度的增加,高原缺氧暴露对成年男性的精子参数的负面影响愈明显。     

人精子抗原基因表达产物HSG2Ag的纯化和鉴定

本研究运用离子交换层析法分离纯化人精子抗原基因表达克隆ＨＳＧ２的溶原蛋白，并用ＥＬＩＳＡ法鉴定其中的表达产物（ＨＳＧ２Ａｇ）。结果提示，用上述方法分离和纯化的ＨＳＧ２Ａｇ纯化度高，特异性高，适用应用于抗精子抗体检测试剂盒的制备，并为进一步研制精子抗原免疫避孕疫苗提供候选成分。     

人膀胱癌基因表达变化的基因芯片研究

目的 研究膀胱移行上皮细胞癌（TCC）和正常组织差异表达基因。方法 应用基因芯片技术对5例TCC癌组织和正常组织的mRNA进行检测。结果 在12800条目的基因中共发现差异表达基因384条。在癌组织中87条表达增加，297条表达降低；259条能在GeneBank中登录。结论 TCC的发生、发展涉及多基因改变，基因芯片技术可同时定量研究大量基因表达水平，是一种稳定、高效的方法。     

人精子抗原基因表达产物HSG_2Ag的纯化和鉴定

本研究运用离子交换层析法分离纯化人精子抗原基因表达克隆HSG2的溶原蛋白,并用ELISA法鉴定其中的表达产物(HSG_2Ag)。结果显示:(1)经离子交换层析法分离HSG_2溶原蛋白为12个主要蛋白峰;(2)用抗人精子抗体和纯化的人SIT阳性血清经ELISA法识别,在HSG_2溶原蛋白中,第3峰为人精子抗原基因表达克隆HSG_2的表达产物(HSG_2Ag)。结果提示,用上述方法分离和纯化的HSG_2Ag纯化度高,特异性高,适合应用于抗精子抗体检测试剂盒的制备,并为进一步研制精子抗原免疫避孕疫苗提供候选成分。     

吸烟对重庆主城区成年男性精子凋亡及精液质量的影响

目的:探讨吸烟对重庆市主城区健康成年男性精子凋亡和精液质量的影响。方法:235例健康成年男性根据吸烟习惯分为吸烟组和不吸烟组。不吸烟组89例,吸烟组146例。采用计算机辅助精液分析系统检测精液常规参数;流式细胞术结合Annexin V-FITC/PI荧光染色检测精子凋亡(AN-/PI-、AN+/PI-、AN+/PI+、AN-/PI+精子比率),并对吸烟组和不吸烟组的各项参数进行比较研究。结果:与不吸烟组比较,吸烟组的早期凋亡精子(AN+/PI-)率高于不吸烟组[(8.1±5.1)%vs(6.8±3.8)%,P=0.039],而晚期凋亡精子(AN+/PI+)率两组间差异无显著性[(5.6±5.2)%vs(5.5±5.1)%,P=0.87];两组间精液量、精子密度、精子活动率、活力和正常形态精子率等精液常规指标的差异均无显著性(P=0.30;0.82;0.37;0.81;0.84)。结论:吸烟者早期精子凋亡率较不吸烟者高,提示吸烟可能诱导精子出现早期的细胞损害。精子凋亡可作为较精液常规指标更为敏感的生物标志物反映吸烟对男性精子造成的损伤。     

正常男性精子碱性核蛋白的研究

目的　观察正常人精子碱性核蛋白的组成及含量。　方法　8名正常人精子经解聚后，用聚丙烯酰胺凝胶电泳方法，于1、4、8、12、24、36、44周检测精子碱性核蛋白。　结果　正常人总组蛋白/总精核蛋白值为0.17±0.07，P1型/P2型精核蛋白值为1.05±0.21。　结论　正常男性精子碱性核蛋白指标可供临床评价精子受孕能力。     

正常生育男性成熟精子mRNA的种类和特征

目的:剖析具有正常生育能力男性成熟精子mRNA的种类和丰度。方法:健康并育有后代的成年男性10例,禁欲1周后取精液,上游法优化精子,Trizol试剂提取精子总RNA。混合所有总RNA进行基因表达系列分析(SAGE)。结果:所建SAGE文库共获877个克隆,测序得到21 052个标签,出现两次以上的独特性标签有2 712种。经与SAGEm ap数据库比对,19.7%的独特性标签没有基因匹配,代表新基因;其余能匹配的基因中,67%具有蛋白质结合或核酸结合能力,41%具有催化活性,13%与信号转导有关,与细胞运输、精子结构、转录调节相关者分别达到10%左右。结论:人成熟精子中存在数量庞大、种类繁多的mRNA。     

Interrogating Mouse Mammary Cancer Models: Insights from Gene Expression Profiling

Numerous mouse models for mammary cancer have been developed and characterized based upon their biological, molecular, and histopathological features. In an effort to dissect the molecular anatomy of such models and compare their gene expression profiles to those of human breast cancer, six models representing various oncogenic pathways have been investigated using cDNA microarray technology. Results of these analyses are presented and discussed in the context of technological challenges presented by analyzing data on such a large scale. Further expression profiling coupled with emerging proteomic technologies will more completely define and distinguish mouse models of mammary cancer from each other and provide a comprehensive basis for comparing such models with the human disease they are intended to represent.     

DNA Microarrays for Analysis of Gene Expression

Transformation of the prostatic epithelium is accompanied by changes in the expression of many genes, as reflected in altered steady-state levels of the mRNAs derived from them. The acquisition of androgen independence is likewise accompanied by changes in gene expression. Using DNA microarray technology, it is now possible to measure the levels of thousands of different mRNAs in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions; for example, comparing mRNA levels in malignant cells growing in the presence or absence of a hormone such as testosterone. Sufficient cDNA for hybridization to a microarray can be produced from as little as 1 mg of tissue. Coupled with DNA sequence information emerging from the Human Genome Project, this technique has the potential to rapidly identify genes that are differentially expressed in situations with direct relevance to prostate cancer. It is reasonably likely that diagnostic patterns of gene expression can be found for normal v benign proliferative malignant prostatic epithelium, androgen-dependent v androgen-independent tumors, and drug-sensitive v drug-resistant tumors. Once identified, informative cDNAs can be included in diagnostic microarrays with potential for clinical application.     

附睾特异性基因的表达与调控

哺乳动物的附睾是一条高度卷曲的管道系统,附睾上皮是执行附睾功能的重要组成部分。附睾上皮蛋白质的合成与分泌,为精子提供了一个特殊的、不断改变的管腔液体环境,使其在附睾内运行过程中获得运动能力并最终达到功能成熟。附睾特异性表达的基因具有高度区域化的特征,受雄激素和(或)睾丸因子的调控,在出生后发育中呈现时空特异性的表达模式,这些都提示它们在附睾中发挥着重要而独特的作用。     

Unique Early Gene Expression Patterns in Human Adult-to-Adult Living Donor Liver Grafts Compared to Deceased Donor Grafts

Because of inherent differences between deceased donor (DD) and living donor (LD) liver grafts, we hypothesize that the molecular signatures will be unique, correlating with specific biologic pathways and clinical patterns. Microarray profiles of 63 biopsies in 13 DD and 8 LD liver grafts done at serial time points (procurement, backbench and postreperfusion) were compared between groups using class comparisons, network and biological function analyses. Specific genes were validated by quantitative PCR and immunopathology. Clinical findings were also compared. Following reperfusion, 579 genes in DD grafts and 1324 genes in LDs were differentially expressed (p < 0.005). Many upregulated LD genes were related to regeneration, biosynthesis and cell cycle, and a large number of downregulated genes were linked to hepatic metabolism and energy pathways correlating with posttransplant clinical laboratory findings. There was significant upregulation of inflammatory/immune genes in both DD and LD, each with a distinct pattern. Gene expression patterns of select genes associated with inflammation and regeneration in LD and DD grafts correlated with protein expression. Unique patterns of early gene expression are seen in LD and DD liver grafts, correlating with protein expression and clinical results, demonstrating distinct inflammatory profiles and significant downregulation of metabolic pathways in LD grafts.     

Activation of Spermatozoal Forward Migration in vitro by Hydrocortisone

The effects of hydrocortisone and cortisone on spermatozoal motility in vitro were tested. Hydrocortisone at concentrations of 50, 100 and 1000 nmoles/ml was effective in activating in vitro the forward migration and the motility of boar spermatozoa recovered from the cauda epididymidis. Where boar epididymal spermatozoa were incubated with hydrocortisone at concentrations of 50, 100 and 1000 nmoles/ml for between 0 and 24 h at 25°C in vitro , the spermatozoal motility was significantly higher than where no hydrocortisone was added. With ejaculated boar spermatozoa, hydrocortisone at concentrations of 100 and 1000 nmoles/ml increased the spermatozoal motility for between 0 and 2 h and at a concentration of 50 nmoles/ml increased spermatozoal motility for between 2 and 24 h at 25°C in vitro. After 4 h incubation, the effect of hydrocortisone at a concentration of 100 nmoles/ml on boar ejaculated sperm motility was not significantly different from the control. But, hydrocortisone at a concentration of 1000 nmoles/ml inhibited the forward migration of boar ejaculated sperm after it had been incubated with the sperm for 6 h. Cortisone, although structurally similar to hydrocortisone, had no significant effect in improving the motility of boar spermatozoa.
Both hydrocortisone and cortisone had no demonstrable effect on the forward migration and the motility of human spermatozoa in vitro. ,     

Kidney Transplant Rejection and Tissue Injury by Gene Profiling of Biopsies and Peripheral Blood Lymphocytes

A major challenge for kidney transplantation is balancing the need for immunosuppression to prevent rejection, while minimizing drug-induced toxicities. We used DNA microarrays (HG-U95Av2 GeneChips, Affymetrix) to determine gene expression profiles for kidney biopsies and peripheral blood lymphocytes (PBLs) in transplant patients including normal donor kidneys, well-functioning transplants without rejection, kidneys undergoing acute rejection, and transplants with renal dysfunction without rejection. We developed a data analysis schema based on expression signal determination, class comparison and prediction, hierarchical clustering, statistical power analysis and real-time quantitative PCR validation. We identified distinct gene expression signatures for both biopsies and PBLs that correlated significantly with each of the different classes of transplant patients. This is the most complete report to date using commercial arrays to identify unique expression signatures in transplant biopsies distinguishing acute rejection, acute dysfunction without rejection and well-functioning transplants with no rejection history. We demonstrate for the first time the successful application of high density DNA chip analysis of PBL as a diagnostic tool for transplantation. The significance of these results, if validated in a multicenter prospective trial, would be the establishment of a metric based on gene expression signatures for monitoring the immune status and immunosuppression of transplanted patients.