甲状腺过氧化物酶mRNA cRNA探针的构建及意义

目的 探讨甲状腺过氧化物酶(thyroperoxidase,TPO)mRNA cRNA探针的构建及原位表达的检测方法 和意义.方法 取甲状腺结节新鲜组织,在RT-PCR扩增TPO cDNA的基础上,构建pSPT19-TPO质粒,经Hind Ⅲ和BamHⅠ单酶切后成线性化模板,在SP6和T7 RNA聚合酶作用下,体外转录合成地高辛标记的TPO cRNA反义和正义探针,进行TPO mRNA的原位杂交实验.结果 TPO mRNA阳性杂交信号分布于甲状腺滤泡细胞的胞质,本组腺瘤2例、结节性甲状腺肿4例、瘤周甲状腺组织1例原位杂交阳性,乳头状癌2例、桥本病1例原位杂交阴性.核酸原位杂交和RT-PCR检测甲状腺组织TPO mRNA的表达,结果 一致.结论 成功构建甲状腺过氧化物酶mRNA 的cRNA探针;检测TPO mRNA是一种较准确反映甲状腺组织TPO状态的方法 ,TPO mRNA的原位检测可结合组织形态学了解甲状腺滤泡细胞的功能表达状况.     

地高辛标记的血小板T细胞活化抗原1cRNA探针的制备与鉴定

目的制备用地高辛 (digoxigenin ,Dig)标记的血小板T细胞活化抗原1(plateletandTcellactivationantigen1,PTA1)cRNA探针。方法构建重组质粒 pGEM 3ZF( ) PTA1 ,并做序列分析证实PTA1基因的插入方向正确后 ,用EcoRI消化得到线性DNA片段 ,再用SP6RNA聚合酶转录合成带有Dig标记的高比活度的单链cRNA探针。结果经斑点杂交证实 ,该探针敏感性高、特异性强。结论地高辛标记PTA1cRNA探针的制备 ,为进一步研究PTA1mR NA在组织、细胞的表达和分布提供了有效的工具。     

小鼠Brd2基因探针的制备及其在荧光原位杂交实验中的应用

目的:为观察溴结构域包含蛋白2(bromodomain containing protein 2,Brd2)基因在小鼠中枢神经系统的表达与分布,本研究制备了两条地高辛标记的Brd2 cRNA探针。方法:提取小鼠脑组织总RNA,设计两对Brd2引物,通过逆转录聚合酶链式反应(RT-PCR)方法,扩增得到两个Brd2的DNA片段,并将它们分别克隆到pCRⅡ-TOPO载体中。利用体外转录方法合成地高辛标记的cRNA探针,最后通过荧光原位杂交实验分析所标记探针的特异性及杂交效果。结果:本实验成功构建了两个Brd2/pCRⅡ-TOPO质粒,获得了特异性的地高辛标记的Brd2cRNA探针,在荧光原位杂交实验中应用两条探针得到了较好的杂交信号。结论:本实验制备的地高辛标记的cRNA探针可特异地检测Brd2 mRNA,为进一步观察Brd2 mRNA在中枢神经系统的分布及功能研究提供了工具。     

地高辛标记大鼠下丘脑生长激素释放因子cRNA探针的制备及应用

制备地高辛标记大鼠下丘脑释放因子ｃＲＮＡ探针；方法；大鼠下丘脑生长激素释放因子的重组质粒ｃＤＮＡ＝ＰＧＥＭ４经转化扩增后，用碱性裂妥法获取质粒ｃＤＮＲ并纯化。用限制性内切酶ＥＣＯＲＩ酶切，以线性ｃＤＮＡ为模板，在Ｔ７和Ｓｐ６ＲＮＡ聚合酶作用下，采用体外转录法分别合成地高辛素标记的大鼠下丘脑生激素释放因子ｃＲＮＡ和ＲＮＡ探针。     

地高辛精标记大鼠代谢型谷氨酸受体第5亚型RNA探针的制备研究

为制备用地高辛精标记的大鼠代谢型谷氨酸受体第 5亚型 c RNA探针 ,本实验用分子生物学技术重组质粒 p GEMm Glu R5 ,经限制性内切酶酶切分析 ,证实其确有 m Glu R5基因片段插入且方向正确。将此质粒再经限制性内切酶消化得到线性DNA片段 ,用 T7RNA聚合酶体外转录合成带有地高辛精标记的高比活性的单链 RNA探针 ;经斑点杂交实验证实该探针具有较高的敏感性和可靠性 ,可用于代谢型谷氨酸受体第 5亚型有关的研究     

正、反义β-1,4半乳糖基转移酶Ⅱ和Ⅴ地高辛标记RNA探针的制备和应用

目的　为了探讨 β1 ,4半乳糖基转移酶 Ⅱ和Ⅴ (β1 ,4 galactosyltransferaseI,β 1 ,4 GalT ⅡandⅤ )表达定位 ,本实验通过分子生物学手段 ,制备正、反义 β 1 ,4 GalT Ⅱ和Ⅴ地高辛标记的RNA原位杂交探针。 方法　设计引物 ,提取小鼠脑总RNA ,通过RT PCR方法 ,得到 β 1 ,4 GalT Ⅱ和Ⅴ基因序列 ,将其克隆到pGEM T载体。根据其多克隆酶切位点和Sp6及T7位置 ,分别酶切后作为转录模板 ,通过Sp6及T7RNA聚合酶 ,得到正、反义 β 1 ,4 GalT Ⅱ和Ⅴ地高辛标记的RNA原位杂交探针。检测标记探针的效价后 ,最后通过原位杂交分析标记探针的特异性和杂交效果。结果　本实验得到了高效价的正、反义 β 1 ,4 GalT Ⅱ和Ⅴ地高辛标记的RNA原位杂交探针 ,并表现出很好的杂交效果。结论　正、反义β 1 ,4 GalT Ⅱ和ⅤRNA原位杂交探针的制备 ,为进一步研究 β 1 ,4 GalT Ⅱ和Ⅴ在组织中的表达 ,尤其在神经组织的定位奠定基础     

第二军医大学举办第七期全国原位杂交组织化学技术学习班

1995年12月11日～17日在上海第二军医大学组织胚胎学教研室举办了第七期全国原位杂交组织化学技术学习班.来自北京、河北、山西、辽宁、吉林、山东、上海、江苏、浙江、河南、湖北、广东、广西、四川和贵州等15个省市高校的教研室与研究室共25人参加.他们中有正、副教授9名,博士生及博士后4名.有留学归国和等待出国的人员.该期学习班仍采用理论与实习(操作)相结合的方式进行.授课内容主要有:基因工程的基本原理及基本方法;核酸探针技术及其应用;真核细胞的原位杂交组织比学等.实习着重操作.质粒的扩增、抽提及纯化;质粒的酶切和回收(模板的制备);反意cRNA探针制备;组织切片制备、杂交、显色等技术.通过学习与研讨,大家一致认为,在国     

肝癌患者血清和癌组织中锌指蛋白216表达增加

目的 寻找肝癌相关抗原.方法 应用SEREX技术对肝癌组织cDNA文库进行血清学筛选,获得人类锌指蛋白ZNF216.利用原核表达和亲和层析技术获得ZNF216的6*His融合蛋白,建立间接ELISA方法,检测血清中产生的ZNF216抗体水平;利用RT-PCR技术检测肝癌患者肝癌组织及癌旁组织内ZNF216 mRNA的水平.结果 肝癌患者血清中产生的抗ZNF216抗体水平高于正常个体,ZNF216 mRNA在肝癌组织内的表达明显高于癌旁组织.结论 ZNF216在肝癌病人血清及癌组织内的过表达可能参与肝癌的发生,但还需要进一步研究证明.     

芳香化酶在生后早期小鼠脑内基因表达的原位杂交研究

芳香化酶细胞色素P450能催化特定脑区雄激素转化为雌激素。在中枢神经系统发育中,局部雌激素的形成可以影响神经结构的性差异,调节神经内分泌和性生殖功能和参与学习记忆。而脑内雌激素的有效浓度依赖于芳香化酶的基因表达及其活性。芳香化酶活性仅在脑发育中特定的脑区如下丘脑和边缘系统检测到。关于其基因表达的研究结果不尽一致。本研究采用人胎盘芳香化酶cDNA为模板,体外转录地高辛标记的cRNA为探针,应用原位杂交技术研究了芳香化酶mRNA在小鼠生后早期(P2～P15)脑内的分布。结果显示,阳性信号主要分布于大脑皮质,海马、丘脑及下丘…     

地高辛标记的大鼠p75 RNA探针的制备和应用

目的制备用地高辛标记的神经生长因子低亲和力受体(p75)的RNA探针.研究p75在海马组织中的表达.方法设计p75引物,构建p75/pGEM-T重组质粒,分别用ApaⅠ和Sac Ⅰ进行酶切得到线性化DNA片段,以Sp6和T7聚合酶转录合成酶合成地高辛标记的(dig-)正反义RNA探针.运用点膜杂交的方法检验探针的敏感度,运用该探针,通过原位杂交分析p75在海马中的表达.结果构建了p75/pGEM-T质粒,获得高效价的正、反义dig-p75 RNA探针,应用该探针发现p75 mRNA在海马中的表达.结论成功制备了地高辛标记的p75RNA探针,为进一步研究p75在海马中发育和损伤过程中的表达打下基础.     

聚(丙基，3-羟丙基)DL-天冬酰胺的体内外降解及键合阿司匹林复合物的释放行为的研究

选用两种不同亲疏水性的聚天冬酰胺修饰材料进行体内外降解实验,研究结果表明,聚天冬酰胺材料的降解过程系酶解,材料的亲疏水性不同,则在体内的降解速度不同,同种材料在埋植部位、肾、肝中的代谢速度也不同。对两种材料的键合阿司匹林复合物的体内外释放试验表明,酶解有利于药物的释放,材料的亲脂性增加有利于药物的释放。     

牛卵母细胞体外成熟和体外培养的研究进展

本文主要对牛卵母细胞体外成熟时超微结构和分子水平的变化、受精卵体外培养的条件及其影响因素作初步探讨.     

聚乙交酯丙交酯体内外生物降解性能的相关研究

通过对聚乙交酯丙交酯 (PGL A)进行体内和体外生物降解性的实验研究 ,探讨两种降解过程之间的关联性。体外实验是将 PGL A材料 (1cm× 1cm)分别浸泡在人工唾液和 PBS溶液中 ,体内 (动物 )试验是将材料直接植入 Wistar大白鼠的皮下组织 ,浸泡后或植入后 1～ 10周 ,每周计算材料的质量损耗率 ,每 2周进行分子量测定。实验结果显示 :PGL A材料在人工唾液中的降解要快于在 PBS液中的降解 ;材料质量损耗的发生慢于分子量的变化 ;体外的降解周期大约为 9～ 10周 ,体内降解周期为 8周左右 ,体内组的降解速率是体外组的 1.33倍。结论为PGL A体外降解主要是化学降解过程 ,通过酯键的水解来进行。体内降解过程中 ,应力环境和生物因素都会对材料的降解动力学产生影响 ,使降解过程明显加快 ,但体内和外降解都符合脂肪族聚酯降解的动力学模型 ,PGL A的体内外生物降解性之间存在一定的相关性。     

Wireless and inductively powered implant for measuring electrocardiogram

The development of an active implantable device for measuring electrocardiogram (ECG) is presented. The study is a part of a project which aims at developing implantable ECG instrumentation with wireless data and power transfer (). The developed implant presented here has all the measurement electronics as well as power and data communication instrumentation included. The implant itself contains no battery, while power for the implant is transferred electromagnetically from an external reader device. The results of testing the implant attached on the body surface and in vitro in a water container are also presented. The developed system was also successfully tested in in vivo measurements, which were conducted on four cows with an implantation time of 24 h. The in vivo testing of implant in cows was conducted by a veterinarian in supervised conditions under approved animal experiment licence.     

Replication of HIV-1 in vivo and in vitro

A complex relationship exists between HIV and its cellular targets. The lethal effect of HIV on circulating CD4⁺ helper T lymphocytes parallels the degree of the infected individual's immunodeficiency and ultimately the transition to AIDS and death. However, as with other members of the Lentivirus family of retroviruses, the ubiquitous, mobile macrophage is also a prime target for HIV infection, and apparently, in most instances, is the initial infected cell, since most people are infected with a CCR5 chemokine-tropic virus. Unlike the lymphocyte, the macrophage is apparently a more stable viral host, capable of a long infected life as an HIV reservoir and a chronic source of infectious virus. Published in vitro studies have indicated that whereas lymphocytes replicate HIV solely on their plasma membrane, macrophages have been envisaged to predominantly replicate HIV within cytoplasmic vacuoles, and thus have been likened to a “Trojan horse,” when it comes to the immune system. Recent studies have revealed an ingenious way by which the cultured monocyte-derived macrophage (MDM) replicates HIV and releases it into the medium. The key macrophage organelle appears to be what is alternatively referred to as the “late endosome” (LE) or the “multivesicular body” (MVB), which have a short and a long history, respectively. Proof of the association is that chemically, LE/MVB and their vesicles possess several pathopneumonic membrane markers (e.g., CD63) that are found on released HIV particles. The hypothesis is that HIV usurps this vesicle-forming mechanism and employs it for its own replication. Release of the intravacuolar virus from the cell is hypothesized to occur by a process referred to as exocytosis, resulting from the fusion of virus-laden LE/MVB with the plasma membrane of the macrophage. Interestingly, LE/MVB are also involved in the infection stage of MDM by HIV. Close review of the literature reveals that along with the Golgi, which contributes to the formation of LE/MVB, the MVB was first identified as a site of HIV replication by macrophages many years ago, but the full implication of this observation was not appreciated at the time. As in many other areas of HIV research, what has been totally lacking is an in vivo confirmation of the in vitro phenomenon. Herein, the ultrastructure of HIV interaction with cells in vitro and in vivo is explored. It is shown that while HIV is regularly found in LE/MVB in vitro, it is infrequently the case in vivo. Therefore, the results challenge the “Trojan horse” concept.     

Replication of HIV-1 in Vivo and in Vitro

A complex relationship exists between HIV and its cellular targets. The lethal effect of HIV on circulating CD4⁺ helper T lymphocytes parallels the degree of the infected individual's immunodeficiency and ultimately the transition to AIDS and death. However, as with other members of the Lentivirus family of retroviruses, the ubiquitous, mobile macrophage is also a prime target for HIV infection, and apparently, in most instances, is the initial infected cell, since most people are infected with a CCR5 chemokine-tropic virus. Unlike the lymphocyte, the macrophage is apparently a more stable viral host, capable of a long infected life as an HIV reservoir and a chronic source of infectious virus. Published in vitro studies have indicated that whereas lymphocytes replicate HIV solely on their plasma membrane, macrophages have been envisaged to predominantly replicate HIV within cytoplasmic vacuoles, and thus have been likened to a “Trojan horse,” when it comes to the immune system. Recent studies have revealed an ingenious way by which the cultured monocyte-derived macrophage (MDM) replicates HIV and releases it into the medium. The key macrophage organelle appears to be what is alternatively referred to as the “late endosome” (LE) or the “multivesicular body” (MVB), which have a short and a long history, respectively. Proof of the association is that chemically, LE/MVB and their vesicles possess several pathopneumonic membrane markers (e.g., CD63) that are found on released HIV particles. The hypothesis is that HIV usurps this vesicle-forming mechanism and employs it for its own replication. Release of the intravacuolar virus from the cell is hypothesized to occur by a process referred to as exocytosis, resulting from the fusion of virus-laden LE/MVB with the plasma membrane of the macrophage. Interestingly, LE/MVB are also involved in the infection stage of MDM by HIV. Close review of the literature reveals that along with the Golgi, which contributes to the formation of LE/MVB, the MVB was first identified as a site of HIV replication by macrophages many years ago, but the full implication of this observation was not appreciated at the time. As in many other areas of HIV research, what has been totally lacking is an in vivo confirmation of the in vitro phenomenon. Herein, the ultrastructure of HIV interaction with cells in vitro and in vivo is explored. It is shown that while HIV is regularly found in LE/MVB in vitro, it is infrequently the case in vivo. Therefore, the results challenge the “Trojan horse” concept.     

The utility of in situ--based methodologies including in situ polymerase chain reaction for the diagnosis and study of viral infections

Molecular in situ-based assays are a useful adjunct to the diagnosis of viral infections by the surgical and cytopathologist. In some cases, the viral nucleic acids and/or proteins are abundant and easily detected by in situ hybridization or immunohistochemistry. In other cases, such as the one integrated provirus typical of latent retroviral localization, in situ polymerase chain reaction amplification is required to localize the virus in the intact cell. Direct correlation of viral localization and the histologic changes will demonstrate in many cases that routine histopathology often does not provide sufficient information to determine what specific cells are infected and/or the number of infected cells in a given biopsy. By combining this information with, for example, cytokine localization, one can elucidate much about the pathophysiology of the viral infection that cannot be afforded by polymerase chain reaction-based methods alone.     

乙型肝炎病毒携带者睾丸中HBV DNA的检测研究

目的检测乙型肝炎病毒携带者的睾丸活检组织是否携带乙肝病毒。方法11例乙型肝炎病毒携带者的睾丸活检组织制成石蜡切片,应用特异性乙肝病毒DNA（HBV DNA）的探针以原位杂交法检测这些患者的睾丸组织切片。结果11例患者中有5例检测到HBV DNA,分布于间质细胞、支持细胞及各级生精细胞的细胞核。结论男性乙肝病毒携带者存在经生殖细胞向子代垂直传播乙肝的可能。     

人未成熟卵母细胞的体外培养和体外受精

收集人次级卵泡中的卵母细胞（ｎ＝６９）进行体外培养和体外受精。２３个卵母细胞经体外培养后用组织学方法观察，１９个（成熟率８３％）排出第一极体。４６个经体外培养后再进行体外受精的卵母细胞中有２４个（受精率５３％）成为受精卵。其中有１３个（卵裂率５４％）在体外发育到２－细胞胚或４－细胞胚。本实验结果提示人次级卵泡中的初级卵母细胞经体外培养可达成熟并具有受精能力。     

伊氏锥虫分离株对安锥赛抗性的体内、外检测结果的相关性研究

为探讨筛选安锥赛治疗伊氏锥虫病有效剂量的快速方法 ,采用生长繁殖抑制法测定伊氏锥虫XJCA、HBM、ZJB1、JSB1、JSB2和ZJB2分离株对安锥赛的敏感性 ,结果它们的IC50 依次是 0 0 0 15 11、 0 0 0 8436、0 0 15 5 0、 0 0 16 2、 0 0 5 90 6和 0 0 82 6 0 μg mL。采用小白鼠体内试验 ,测定这 6株伊氏锥虫的CD10 0 ,每个分离株伊氏锥虫有安锥赛 3个剂量组和不治疗对照组 ,每个治疗组 10只小白鼠 ,对照组 5只小白鼠 ,每只小白鼠经腹腔接种 1 0× 10 6条锥虫 ,以给药剂量 ,将XJCA株分为 3 5mg kg组、 5 0mg kg组和 7 1mg kg组 ,HBM株分为 3 5mg kg组、 5 0mg kg组和 7 1mg kg组 ,ZJB1分为 5 0mg kg组、 7 0mg kg组和 9 0mg kg组 ,JSB1株分为 5 0mg kg组、 7 1mg kg组和 10 1mg kg组 ,JSB2株分为 7 0mg kg组、 9 0mg kg组和 11 0mg kg组 ,ZJB2株分为 9 0mg kg组、 11 0mg kg组和 13 0mg kg组 ,结果这 6个伊氏锥虫分离株的CD10 0 依次为3 5mg kg、 5 0mg kg、 7 0mg kg、 7 1mg kg、 11 0mg kg和 13 0mg kg。它们的IC50 与CD10 0 的相关系数经计算和检验分析 ,IC50 与CD10 0 之间相关系数为 0 2 2 2 ,这表明它们呈曲线性正相关 ,以IC50 与CD10 0 作出它们的相关“曲线”。