摘要：目的研制离体肾脏灌流装置。方法自行设计、制造一种离体肾脏灌流装置,并进行6例新西兰白兔离体肾脏常温下灌流实验,以评估此灌流装置的工作性能。结果灌流装置能与尿量偶联自动补充灌流液,机器能持续工作,性能稳定。最长灌流保存时间达80.65h。灌流24、48h,肾脏静脉流出液乳酸脱氢酶(LDH)活性、钾钠离子浓度和尿N-乙酰βD-氨基葡萄糖苷酶(NAG)活性无显著变化,肾组织形态学基本正常,当灌流65h,肾静脉流出液LDH活性和钾离子浓度显著增高,钠离子浓度显著降低,尿NAG活性显著增高,肾小管上皮细胞变性肿胀。结论该肾脏体外灌流装置操作简单,使用方便,能满足医学科研的要求。

摘要：目的 :筛选出能与HBV DNAC启动子形成三链结构的高亲和力寡核苷酸。方法 :以体外合成的3 2 P标记的HBV DNAC启动子区序列 ( 173 4～ 175 4)为靶序列 ,合成 5种有可能与该靶序列形成三链结构的寡核苷酸链 (TFO) ,同时设计对照靶序列和对照TFO ,用凝胶滞留试验、酶足迹试验等方法对比分析这些TFO与靶序列是否形成三链结构及其结合的亲和性、特异性。结果 :在 3 7°C、pH7 4条件下 ,CT TFO和GT TFOp与3 2 P 靶序列DNA结合的亲和力十分微弱 ,其Kd均 >10 -6mol/L ;GT TFOap和AG TFOs与靶序列DNA结合的亲和力较高 ,Kd分别为 5× 10 -7mol/L和 2 5× 10 -8mol/L ;AG TFO1与靶序列结合的亲和力最高 ,Kd为 3× 10 -9mol/L ,而且两者的结合具有序列特异性。结论 :含AG或GT的TFO可与HBVC启动子区类同聚嘌呤链逆平行方向结合成三链DNA ,其中AG TFO1与靶序列结合的亲和力最高 ,反应完全 ,可使靶双链DNA均转变成三链DNA ,经一定修饰后 ,可试用于HBV基因转录抑制实验。

摘要：Objective: To screen a triple helix-forming oligodeoxyribonucleotide (TFO) that can bind HBV core promoter at target site with high affinity and specificity, and to observe the ability of manganese porphyrin modified TFO to combine and cleave HBV DNA. Methods: Similar homopurine domain (1 734 - 1 754) in HBV core promoter was selected as target sequence. Several corresponding TFOs were synthesized. The affinities and specificities of TFOs binding target sequence were tested with electrophoretic mobility shift and DNase I footprinting assays. The selected best TFO was modified with manganese porphyrin and acridine. The ability of the TFO derivative to cleave HBV DNA was observed with cleavage experiment. Results: Under the condition of 371 and pH 7. 4, the TFO consisting of cytidylate and thymidylate (CT-TFO) and the parallel TFO consisting of guanylate and thymidylate (GT-TFOp) bound the target sequence weakly with Kd values much more than 10 -6 mol/L. The affinities of anti-parallel GT-TFO ( GT-TFOap) and short TFO consisting of adenine nucleotide and guanylate (AG-TFOsh) binding the target sequence were higher than those of the formers, with Kd values of 5 μ 10-7 mol/L and 2. 5 μ 10-8 mol/L respectively. Long AG-TFO (AG-TF01) had the highest binding affinity with a Kd value of 3 μ 10 -9 mol/L among all the TFOs studied for sequence specificity. In the presence of potassium monopersulfate, KHSO5, TFO modified with manganese porphyrin and acridine cleaved the target sequence where the triplex DNA formed. Conclusion: TFO containing AG or GT binds homopurine in HBV core promoter in adverse parallel direction to form triple helix. AG-TFO1 has the highest binding affinity among all the TFOs studied. After modified with manganese porphyrin, AG-TFO1 completely binds and cleaves the target HBV DNA sequence where triplex DNA is formed.