地高辛核酸探针的制备及研究进展

核酸分子杂交技术是分子生物学、临床医学研究的重要手段之一。目前非同位素核酸探针标记方法所用的配基及检测手段繁多。且各具优缺点,其中尤以地高辛配基标记的核酸探针技术突出,其检测灵敏度可达同位素标记探针。具有灵敏、安全、简便、实用等优点,具有广阔的应用前景。     

地高辛标记探针检测重组双功能水蛭素中的DNA残留量

为检测注射用重组双功能水蛭素半成品中的DNA残留量，应用地高辛标记重组双功能水蛭素工程菌DNA，并以此为DNA探针进行点杂交，同时做特异性及灵敏度实验，结果表明，该方法检测最低限值可达1pg,且重复性好，特异性强，操作较简便，可用于双功能水蛭素制备工艺的质控及半成品的检定。     

地高辛标记探针检测重组人肿瘤坏死因子α衍生物中残余DNA

本文用地高辛标记工程菌ＤＮＡ片段作探针，通过分子杂交和免疫显色检测ｒ－ｈＴＮＦαＤ半成品和制剂中的残余ＤＮＡ。该法对半成品和制剂分别采用蛋白酶Ｋ膜后处理和酚与氯仿抽提方法，消除了蛋白质或甘露醇对ＤＮＡ测定的干扰。本法特异、准确，灵敏度达１ｐｇ／点。用此法测定３批ｒ－ｈＴＮＦαＤ半成品和制剂（ｎ＝５），其残余ＤＮＡ均小于１００ｐｇ／剂量且重复性较好。     

检测流感疫苗中宿主细胞DNA残留量的地高辛标记探针杂交法的建立

目的  建立地高辛标记DNA探针杂交法,检测基于Madin-Darby犬肾（Madin-Darby canine kidney,MDCK）细胞生产的流感疫苗中宿主DNA的残留量。方法  提取MDCK细胞基因组DNA,制备地高辛标记探针。对地高辛标记DNA探针杂交法进行特异性、灵敏度及稳定性验证, 并用此法检测3批MDCK细胞流感疫苗中的DNA残留量。结果 MDCK细胞基因组DNA的质量浓度为33 ng/μl,260 nm与280 nm波长处的吸光度比值为1.9。制备的探针与非同源细胞DNA无杂交,最低检测限度为10 pg。探针在－70 ℃放置9个月后,检测灵敏度仍可达到10 pg。经检测,MDCK细胞流感疫苗成品中的DNA残留量＜10 ng。结论  建立的地高辛标记探针杂交法特异性强、灵敏度高、稳定好,并且操作简便,可用于MDCK细胞流感疫苗中DNA残留量的检测。     

服地高辛后红细胞受体数目及活性的变化

本前瞻性研究以外周红细胞作为心肌细胞的模型，动态观察（４０ｄ）８例心衰患者应用地高辛维持量疗法之红细胞洋地黄受体的数目，活性和泵单位活性的变化规律，并与８例未服用地高辛的心衰患者作平行对照。结果显示服用地高辛等３ｄ洋地黄受体的活性与数目明显降低（Ｐ＜０．０１），尔后同步上升，２０ｄ后达服药前水平；停药后受体的活性与数目一度下降，第１０ｄ恢复到原有水平；而泵单位活性不变；红细胞洋地黄受体的活性与数目     

神经生长因子高亲和力受体互补核糖核酸探针的研究

目的 制备用地高辛标记的神经生长因子高亲和力受体(Trk A)正、反义互补核糖核酸(cRNA)探针的研究。方法 设计Trk A引物，构建pGEM-T Easy-Trk A重组质粒，分别用ApaI和SacI进行酶切得到线性DNA片段，以SP6和T7RNA聚合酶转录合成带有地高辛标记的高比活度的正、反义单链cRNA探针。结果 经斑点杂交证实，该探针敏感性高、特异性强。Trk A-ApaⅠ探针稀释12500倍后的浓度为40ng／μl；Trk A-SacⅠ稀释1：2500倍后的浓度为8ng/μl。结论 成功制备了地高辛标记的Trk A cRNA探针，为毒理学中有关Trk A mRNA在组织、细胞的表达和分布的研究提供了有效工具。     

死亡受体介导的信号传导及调控

凋亡，又称程序性细胞死亡，是机体固有和基本的生物过程，对维持机体的正常发育和稳定起重要作用，一些细胞表面的被称为死亡受体（Death receptors,DR)的特异性感受器，该死亡受体识别外来死亡信号，并迅速启动细胞凋亡机制。由于凋亡失控可引起多中疾病，如肿瘤、神经退行性病变和获得性免疫缺陷综合征，故对死亡受体的理解有助于认识凋亡的生理和病理性意义。现就死亡受体的特性、介导的信号传导及调控的最新进展作一综述。     

地高辛联合β受体阻滞剂、ACEI类、醛固酮拮抗剂治疗慢性心力衰竭的临床疗效观察

目的观察地高辛联合β受体阻滞剂、ACEI类、醛固酮拮抗剂治疗慢性心力衰竭的临床疗效。方法选择2016年2月至2018年10月收治的56例慢性心力衰竭患者纳入研究范围,随机分为对照组和研究组,各28例。对照组患者选用美托洛尔、呋塞米、螺内酯片、卡托普利联合治疗,研究组患者在对照组的基础上增加地高辛治疗。比较两组患者治疗前后的N末端脑钠钛前体(NT-proBNP)水平。结果两组患者在治疗前的NT-proBNP水平检验结果差异无统计学意义(P>0.05)。治疗后,研究者患者的NT-proBNP水平明显低于对照组患者,下降幅度更为明显,差异显著,有统计学意义(P<0.05)。结论在治疗慢性心力衰竭患者上,使用美托洛尔、呋塞米、螺内酯片、卡托普利联合地高辛治疗,能有效提升患者的NT-proBNP水平,值得应用。     

地高辛标记探针检测重组人p43蛋白宿主DNA残留量的研究

目的:建立重组人p43蛋白原液中宿主DNA残留量的检测方法。方法:以重组人p43工程菌基因组DNA为模板,制备地高辛标记的探针,并以此探针与阳性对照品及供试品进行杂交及显色反应。结果:3批次重组人p43蛋白原液中每人份剂量的宿主DNA残留量均小于10 ng。结论:该方法灵敏度高,特异性强,重复性好,操作安全简便,可用于重组人p43制备过程中的原液检定及质量监控。     

细胞凋亡的死亡受体途径

死亡受体信号转导通路是诱导细胞凋亡的主要途径之一,目前已知的死亡受体有8种,其信号转导途径主要有3条,分别为TNFR、TRAIL和Fas/FasL信号途径.本文将就这三条通路的研究概况进行阐述.     

再生障碍性贫血患者死亡受体5的表达及意义

目的通过检测再生障碍性贫血(简称再障)患者骨髓单个核细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)的死亡受体5(deathreceptor5,DR5)mRNA的表达,探讨DR5诱导的凋亡在诱导再障造血干细胞凋亡中的作用。方法采用实时定量反转录聚合酶链反应(RT-PCR)对12例再障(再障组)、11例再障治疗后达缓解(缓解组)、12名健康人(对照组)骨髓单个核细胞DR5mRNA的表达进行检测。结果①再障组、缓解组、对照组DR5mRNA均有表达。②再障组DR5mRNA的表达高于缓解组、对照组,差异有统计学意义(P<0.05)。③缓解组与对照组间DR5mRNA的表达差异无统计学意义(P>0.05)。结论DR5mRNA在再障患者骨髓单个核细胞中表达增高,提示DR5介导的细胞凋亡可能在再障的发病机制中发挥一定的作用。     

Camptothecin sensitizes human hepatoma Hep3B cells to TRAIL-mediated apoptosis via ROS-dependent death receptor 5 upregulation with the involvement of MAPKs

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various types of malignant cancer cells, but several cancers have acquired potent resistance to TRAIL-induced cell death by unknown mechanisms. Camptothecin (CPT) is a quinolone alkaloid that induces cytotoxicity in a variety of cancer cell lines. However, it is not known whether CPT triggers TRAIL-induced cell death. In this study, we found that combined treatment with subtoxic doses of CPT and TRAIL (CPT-TRAIL) potentially enhanced apoptosis in a caspase-dependent manner. CPT-TRAIL effectively induced the expression of death receptor (DR) 5, which is a specific receptor of TRAIL, and treatment with a chimeric blocking antibody for DR5 reduced CPT-TRAIL-induced cell death, indicating that CPT functionally triggers DR5-mediated cell death in response to TRAIL. CPT-induced generation of reactive oxygen species (ROS) also preceded the upregulation of DR5 in response to TRAIL. The involvement of ROS in DR5 upregulation confirmed that pretreatment with antioxidants, including N-acetyl-L-cysteine and glutathione, significantly inhibits CPT-TRAIL-induced cell death by suppressing DR5 expression. The specific inhibitors of ERK and p38 also decreased CPT-TRAIL-induced cell death by blocking DR5 expression. In conclusion, our results suggest that CPT sensitizes cancer cells to TRAIL-mediated apoptosis via ROS and ERK/p38-dependent DR5 upregulation.     

110例心力衰竭患者地高辛血药浓度监测及影响因素分析

目的:监测心力衰竭患者地高辛的血药浓度,并对其影响因素进行分析。方法:用荧光偏振免疫法测定110例患者地高辛血药浓度,对地高辛血药浓度与疗效的关系及影响地高辛血药浓度的因素进行分析。结果:以0.8ngmL～2.0ngmL为地高辛有效血药浓度,16例患者<0.8ngmL,65例在有效血药浓度范围之内,>2.0ngmL的29例患者中毒反应发生率较高(65.5%)。结论:地高辛血药浓度受多种因素的影响,包括肾功能、电解质、合并用药和地高辛样免疫活性物质等。     

地高辛血药浓度监测在ICU的临床应用与研究

目的 总结并分析我院重症监护室患者进行地高辛血药浓度监测的情况,为临床合理用药提供参考.方法 采用荧光偏振免疫法测定地高辛血药浓度,利用有关统计学方法对所得数据进行处理,并作回顾性分析.结果 74份病历共进行TDM 104次,其中测定地高辛46例次,达有效血药浓度34例,占73.91%;未达有效血药浓度5次,占10.87%;达中毒血药浓度7例,占15.22%.结论 ICU患者基础疾病复杂,内环境紊乱,联合使用多种药物.所以使用地高辛时,剂量不能完全决定血药浓度,血药浓度也不能完全决定临床疗效,因此必须在参考TDM结果的同时,结合患者具体生理、病理及临床表现等各种情况来调整给药方案.     

Characterization of 5-ht6 receptor and expression of 5-ht6 mRNA in the rat brain during ontogenetic development

We have determined the pharmacological characteristics of the rat 5-ht₆ receptor stably expressed in CHO cells. Moreover, using RT-PCR experiments the in vivo expression of the gene encoding this receptor was studied in rat at various embryonic days (ED) starting from ED₁₀ to birth (PN₀) and at post-natal days (PN) up to PN₃₆. The pharmacological analysis of the [³H]5-HT binding in stably transfected CHO cells expressing rat 5-ht₆ receptors revealed the presence of a single class of high affinity saturable binding sites for 5-HT corresponding to an affinity constant: Kd = 27.2±3.4 nM. This receptor also exhibited a high affinity for a number of typical and atypical antipsychotics, tricyclic antidepressant drugs and ergot alkaloïds. In stably transfected CHO cells, serotonin elicited a potent stimulation of adenylyl cyclase activity which was blocked by antipsychotic and antidepressant drugs. These results confirm the hypothesis that 5-ht₆ receptors may correspond to an important target for atypical antipsychotics and reveal an original pharmacological profile for this receptor. The study of the ontogeny of the 5-ht₆ mRNA in rat developing brain showed that 5-ht₆ mRNA were first detectable with a high level on ED₁₂, slighly decreased up to ED₁₇ and then remained stable at high level until the adult age. The ontogenetic pattern of 5-ht₆ mRNA expression appeared to correlate with the occurence of the first cell bodies of serotonergic neurons; the early expression of 5-ht₆ mRNA and the fact that this receptor is positively coupled to the production of cAMP may suggest a role for 5-ht₆ receptor in the early growth process involving the serotonergic system.     

Synthesis and cytotoxic activity of G3 PAMAM-NH2 dendrimer-modified digoxin and proscillaridin A conjugates in breast cancer cells

The objective of this study was to determine the cytotoxicity, antiproliferative activity, and apoptosis induction activity of two modified glycosides – digoxin and proscillaridin A – conjugated to a generation 3 polyamidoamine dendrimer (G3 PAMAM-NH₂) in hu-man breast cancer cells. The results suggest that conjugation with the G3 PAMAM-NH₂ dendrimer enhances the cytotoxicity of modified digoxin and proscillaridin A both in MCF-7 and in MDA-MB-231 breast cancer cells. Additionally, the conjugate-induced apoptosis was significantly greater than apoptosis evoked by free modified digoxin and proscillaridin A.     

Differential effect of potassium on the action of digoxin and digoxigenin in guinea-pig heart.

The effect of potassium on the binding of digoxin or digoxigenin to isolated Na+, K+-ATPase was compared with that of potassium on the positive inotropic action of the agents in guinea-pig hearts. The binding of digoxigenin to the enzyme in vitro was reduced to a greater extent by potassium than was the binding of digotoxin. The digoxigenin-induced increase in the force of contraction of left atrial preparations estimated at steady state was reduced at higher potassium concentrations. Potassium had a lesser effect when digoxin was used as the inotropic agent. In contrast, potassium concentrations. Potassium had a lesser effect when digoxin was used as the ininotropic agent. In contrast, potassium reduced the rate of development and also the rate of loss of the positive inotropic action of digoxin observed with left atrial and Langendorff preparations, respectively, to a greater extent than those of digoxigenin. The loss of the positive inotropic effect was more rapid with digoxigenin than with digoxin at each KCl concentration. These data support the contention that the extent of the interaction of digitalis with Na+,K+-ATPase determines the degree of the positive inotropic effect.     

A comparison of the effects of digoxin and digitoxin on systolic time intervals and colour vision

Summary Animal studies suggest that for the same inotropism hydrophilic cardiac glycosides produce greater depression of atrioventricular (AV) conduction than lipophilic ones. This has been explained on the basis of a greater vagomimetic effect with hydrophilic agents and a greater sympathomimetic effect with lipophilic agents.In this randomized, cross-over study we investigated the effects of placebo, digoxin (relatively hydrophilic), and digitoxin (relatively lipophilic) in twelve healthy volunteers. For both drugs steady-state serum concentrations in the mid-therapeutic range were achieved.Both drugs produced the same positive inotropic effect as measured by systolic time intervals (QS₂c). There was a trend for digoxin to have a greater effect on AV conduction than digitoxin.After atropine or propranolol there was no difference between the effect of the two cardiac glycosides on AV conduction. No significant effects on colour vision were seen. We conclude that, there do not seem to be pharmacodynamic differences between digoxin and digitoxin at mid-therapeutic serum concentrations.     

The uptake and subcellular distribution of radio-labeled metabolites of digoxin in the isolated perfused guinea-pig heart

Summary Cardiac glycosides like digitoxin and digoxin with three digitoxoside sugar residues have been reported to undergo step-wise degradation to yield the corresponding genins, and the importance of the digitoxoside side-chain for the pharmacodynamic property of cardiac glycosides has also been suggested. A systematic study was therefore undertaken on the cleavage products of digoxin. Digoxigenin-bis-digitoxoside (with two sugar residues) digoxigenin-monodigitoxoside (with one sugar residue) and digoxigenin (with no sugar) were compared with the parent compound, digoxin. The radio-labeled compounds were perfused through isolated guinea pig hearts using 10^–7 M concentration in the perfusion medium for a fixed period of 64 min followed by an 8 min period of wash-out with normal medium. The uptake and sub-cellular distribution of the drugs were thereafter measured by scintillation counting. All the compounds produced positive inotropic responses, the monodigitoxoside producing the greatest effect, digoxigenin next in order of inotropic response magnitude, the bis-digitoxoside produced the least effect, and digoxin was intermediate between the genin and bis-digitoxoside. The uptake of the monodigitoxoside was the highest, and in general, the quantitative uptake was related to the inotropic response. The greatest binding of each digitoxoside was found in the microsomal fraction. Both mechanical activity and uptake of all four drugs were uniformly reduced by an increase in potassium concentration in the perfusion medium.Supported by grant HE-07051 from the National Heart Institute