四环素基因调控系统的研究进展

 大量临床前研究均证实了病毒作为治疗基因载体的有效性和可行性。但将病毒载体技术用于临床疾病的治疗还面临一些挑战。其中,如何对病毒携带基因的表达实施定时定量调控是必需解决的一个关键问题。目前常采用的方法是在病毒载体基因组中构建调控系统中,而四环素基因调控系统（Tet系统）是迄今为止基因治疗研究中使用最广泛的系统。该系统通过改变培养基、体内四环素或其衍生物（如强力霉素）的浓度,诱导或抑制目标基因的表达。本文简要综述了Tet系统的研究进程及其在不同疾病中的应用。     

严密型四环素调控系统转基因首建鼠的建立

目的制备ApoE-rtTA-tTS转基因小鼠1，为严密型四环素调控系统的体内研究提供调控部分的转基因小鼠，以便与反应部分小鼠交配得到双转基因小鼠。方法重组构建含目的基因的质粒pApoE-rtTA-tTS，应用显微注射法将其注入母鼠的受精卵，再植入代母输卵管，出生小鼠经PCR初步筛选出阳性，再经Southern杂交对阳性小鼠基因组DNA标本进一步鉴定。结果产生了2只整合ApoE-rtTA-tTS基因的首建鼠。结论成功制备了ApoE-rtTA-tTS转基因小鼠，为下一步建立严密型四环素调控系统的双转基因小鼠模型奠定了基础。     

四环素可调控基因表达系统的研究进展

近年来对人工合成可调控基因表达系统的研究较多,其中四环素调控表达系尤为突出,此系统已在基因功能研究、基因选择性表达分析方面得到了较好地应用。基因治疗是当前研究的热点之一,如何保证有效目的基因在体内适时、适量地表达是实现安全、有效的基因治疗的保障。因此,对此系统的改进及应用,将推动人类疾病基因治疗的进展     

乙型肝炎病毒启动子结构及调节机制的研究

乙型肝炎病毒(HBV)表达调控是近年来分子生物学研究的重点之一.各种肝细胞核因子和调节元件通过作用于HBV启动子而发挥转录调节作用.因此,研究HBV启动子调节机制对深入了解HBV的致病机理及寻找有效的抗病毒治疗方法具有重要意义.     

四环素调控的PTA1/CD226转基因小鼠的初步建立

为研究小鼠PTA1分子在体内的功能,建立四环素调控的小鼠PTA1/CD226转基因小鼠,我们构建了pBI-5-mPTA1载体,显微注射入B6D1F1受精卵,使用PCR检测新生小鼠基因组DNA中的PTA1与荧光素酶(luciferase)基因。将mPTA1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含有盐酸强力霉素(Dox)的培养基进行培养,检测细胞裂解液中荧光素酶的活性。将荧光素酶表达依赖Dox的小鼠与C57BL/6小鼠交配,采用PCR对子代鼠进行检测。最终共获得7只首建鼠,其目的基因表达高度依赖Dox,并得到了其中2只首建鼠的F1代小鼠。     

四环素调控的小鼠LAIR-1/CD305转基因小鼠的初步建立

目的：建立四环素调控的小鼠LAIR-1/CD305转基因小鼠,为进一步研究mLAIR-1分子的体内功能奠定基础.方法：构建pBI-5-mLAIR-1载体,显微注入B6D1F1受精卵,PCR检测新生小鼠基因组DNA中LAIR-1与荧光素酶（Luciferase）基因.将mLAIR-1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含盐酸强力霉素（Dox）的培养基进行培养,检测细胞裂解液中荧光素酶活性.将荧光素酶表达依赖Dox小鼠与C57BL/6交配,采用PCR对子代鼠进行检测.结果：共获得9只首建鼠,其目的基因表达高度依赖Dox,并得到其中5只首建鼠的F1代小鼠.结论：获得了四环素调控的小鼠LAIR-1转基因小鼠,可用于该分子体内功能的研究.     

大鼠GLUT3启动子的克隆及其缺糖调控活性的测定

目的: 构建葡萄糖转运体3(GLUT3)启动子的报告基因,并在正常和缺糖情况下检测其转录活性。方法: 使用生物信息学软件预测了GLUT3 的启动子序列,长度1 292 bp, 包含第1个外显子, 通过PCR及双酶切方法,从大鼠全血基因组DNA中获得GLUT3 基因编码序列, 包含GLUT3 启动子序列,然后克隆到报告基因pGL3-Basic载体上,构建GLUT3 启动子的报告基因;用脂质体转染法将pGL3-GLUT3与胸腺嘧啶脱氧核苷激酶(pRL-TK)共转染入PC12细胞中,以pRL-TK载体作内参照,分别给予正常和缺糖培养,采用双萤光素酶报告系统评估GLUT3 启动子的活性。结果: 经PCR方法扩增出GLUT3 启动子序列,测序与GenBank序列一致;转染后检测显示,该pGL3-GLUT3明显具有转录活性,而且在缺糖24 h情况下其双萤光素酶活性有明显升高。结论: 成功构建出GLUT3 启动子报告基因,pGL3-GLUT3表现出很好的缺糖诱导活性,缺糖24 h是研究pGL3-GLUT3在缺糖情况下转录调控很有意义的时间点。     

不同原料制备乙型肝炎病毒特异性转移因子的研究

目的　为临床治疗乙肝提供一种安全、有效的免疫调节剂。方法　用组织匀浆、反复冻融破碎细胞、透析灭活和过滤除菌等技术 ,获得乙型肝炎病毒特异性转移因子 (HBV STF) ;用Lowry法测多肽含量 ;用苔黑酚显色法测定核糖核酸 (RNA)含量 ,用氨基酸分析仪分析测定HBV STF的水解氨基酸总含量。结果　来源于HbsAg阳性胎盘和HbsAg阳性血液的多肽含量分别为 0 4 9± 0 0 14和 0 2 9± 0 0 13(P <0 0 1) ,RNA含量分别为 0 34± 0 0 10和 0 2 2± 0 0 0 8(P <0 0 1)氨基酸总含量为 198 6± 1 5 6 2和 12 3 4± 1 5 2 1(P <0 0 1)。结论　用HbsAg阳性胎盘制备的乙型肝炎病毒特异性转移因子质量优于HbsAg阳性血液。     

鼻咽癌细胞中ezrin基因启动子上游序列转录调控特性的研究

目的:研究鼻咽癌细胞中ezrin基因启动子上游序列的转录调控特性。方法:构建一系列携带ezrin基因-1541/-706序列的报告基因表达载体,ezrin基因-1541/-706序列以正向和反向分别连接至不含启动子的报告基因上游、ezrin启动子上游、SV40启动子上游、ezrin启动子或SV40启动子控制的报告基因下游,将质粒转染CNE2细胞,检测荧光素酶活性。结果:CNE2细胞中,当-1541/-706序列正向位于报告基因上游时表现出启动子活性,其转录激活作用约为ezrin启动子的50%;反向连接时无启动子活性。而且,当-1541/-706序列正向位于ezrin启动子或SV40启动子上游时,显著提高荧光素酶表达;当反向位于启动子下游、正向或反向位于启动子控制的报告基因下游时,转录增强作用消失。结论:CNE2细胞中ezrin基因启动子上游序列具有转录激活和转录增强作用,这种作用具有DNA序列位置和方向依赖性。     

Cyclin H基因启动子区-1 SNP对转录活性的影响

目的 探讨CCNH基因启动子区-1SNP位点碱基变异对CCNH基因转录活性的影响.方法 通过PCR和定点突变技术,构建了含CCNH基因基础启动子及-1G突变启动子,用双荧光素酶报告系统观察此SNP对CCNH基因启动子转录活性的影响.结果 在AD293细胞中,-1G突变启动子活性比CCNH基础启动子活性低26%(P<0.05).结论 -1T→G碱基变异可显著降低CCNH基因启动子转录活性.     

HBV—LP检测HBVDNA复制的敏感性与特异性研究

目的 检测乙肝患者的HBV-LP及HBV DNA,探讨HBV-LP的敏感性和特异性及其临床应用价值.方法 HBV-LP检测采用ELISA法,HBV DNA检测采用RT-PCR法.结果 HBV-LP检测及HBV DNA检测的敏感性分别为64.89%、99.68%和60.63%、100%（P〉0.05）;两检测的＋LR和-LR分别为202.78、60.63和0.3522、0.3937,＋LR之间差异有统计学意义（P〈0.005）.结论 HBV-LP检测有与HBV DNA检测相近的敏感性及特异性,阳性似然比更高于HBV DNA检测,为较好的检测HBV DNA复制的血清学指标.     

Striatal influences on paravermal cerebellar activity

Summary Units were recorded extracellularly in paravermal cortex (lobule VI) of the cerebellum of chloralose anesthetized cats. Electrical stimulation of the striatum evoked excitation followed by inhibition in these neurons. In addition, the somatosensory properties of these cells were also affected by the striatum. A conditioning-test paradigm (C-T) was used in which conditioning stimulation was applied to the striatum. Test responses were evoked in cerebellar neurons by facial stimulation. As a function of the C-T interval, striatal stimulation could either enhance or suppress the test facial responses. In another procedure, a moveable electrode was used to map the thresholds for affecting the cerebellum from different points in the striatum. The lowest mean threshold was in the putamen followed respectively by the internal capsule and caudate nucleus. Control experiments suggested that striatal effects on the cerebellum were due neither to extra-striatal current spread nor antidromic activation of corticostriatal fibers. These data were discussed with regard to models of striatal motor functioning that indicate a role in postural control and sensory gating.Supported by NIH grant NS 21418     

The control of trunk Hox specificity and activity by Extradenticle

We characterize a 37-bp element (fkh[250]) derived from the fork head (fkh) gene, a natural target of the Hox gene Sex combs reduced (Scr). In vitro, Scr cooperatively binds to this DNA with the Hox cofactor Extradenticle (Exd), and the activation of this enhancer in vivo requires Scr and exd. Other Hox/Exd heterodimers do not activate this element in vivo and do not bind this element with high affinity in vitro. The amino-terminal arm of the Scr homeodomain is crucial for the specific activation of this element in vivo. By mutating two base pairs within this element, we can convert the Scr/Exd-binding site to a Hox/Exd consensus site that binds several different Hox/Exd heterodimers. This element, fkh[250(con)], is activated by Scr, Antennapedia (Antp), and Ultrabithorax (Ubx) but repressed by abdominal-A (abd-A). We also show that Scr and Exd are only able to activate the fkh[250] element during the early stages of embryogenesis because, by stage 11, Scr negatively regulates the gene homothorax (hth), which is required for the nuclear localization of Exd. These results suggest that Exd is a specificity cofactor for the trunk Hox genes, and that the control of Exd subcellular localization is a mechanism to regulate Hox activity during development.     

癌症患者血清中与Co^2＋—反应的白蛋白的观察

经Co~(2+)处理而产生的Cp蛋白,在癌症患者血清中的含量明显低于正常血清中的含量。可以认为是一种能反映癌症的相关指数。本文证明了Cp的来源是白蛋白,它们的表现分子量分别为Cp_1≈130000、Cp_2≈110000道尔顿,接近自蛋白的一倍。Cp生成的适宜条件是偏于碱性溶液。本文还通过一批病例的检测数据,评阶了Cp含置作为癌症相关指数的意义。     

Protein method influences on calculation of tissue receptor concentration

Many protein methods are used for estimation of tissue receptor concentration. The authors compared performance, analytic variability, and accuracy of six protein methods used in these calculations. They found the Lowry protein procedure standardized with bovine serum albumin (BSA), usually considered the reference method, to be the most imprecise and most time consuming method. When the BSA standards from the Lowry procedure were assayed with the other methods, results ranged from 74 to 141% of expected. For three other protein standards, reactivity among the six methods varied almost twofold. Comparison of Lowry protein concentrations in cytosols from 46 tumors biopsies with other methods indicated best agreement was with an automated turbidometric (TCA) or a Coomassie dyebinding procedure. Use of protein standardization for the two direct spectrophotometric procedures decreased overestimation of receptor protein concentrations. Because receptor concentration is the quotient of receptor quantity and protein concentrations, tissue receptor results are dependent in part on standardization and choice of protein method.