昆明小鼠angiostatin基因的克隆与真核表达载体的构建

目的:从昆明小鼠肝组织中克隆血管抑素(angiostatin)基因,并构建真核表达载体pcDNA3.1( )-angiostatin.方法:从昆明小鼠肝组织中提取总RNA,经RT-PCR扩增出angiostatin基因,连入pGEMT载体后测序,并采用定向克隆构建出真核表达载体pcDNA3.1( )-angiostatin.结果:测序表明angiostatin基因与Gene Bank上的基因序列相比有2个碱基变异,其中之一编码氨基酸由组氨酸变成精氨酸.该基因序列已被Gene Bank收录(Accession Number:AY424276).结论:成功构建了昆明小鼠真核表达载体pcDNA3.1( )-angiostatin,在肿瘤抗血管生成治疗中有潜在应用价值.     

8型腺相关病毒介导的TRAIL基因促结肠癌细胞凋亡的实验研究

目的观察8型腺相关病毒介导的TRAIL基因对结肠癌HCT116细胞凋亡的诱导作用并探讨其作用机制.方法采用三质粒磷酸钙共沉淀方法转染HEK293细胞,制备出携带TRAIL基因的8型重组腺相关病毒;重组病毒感染结肠癌细胞及正常人肝细胞,利用台盼蓝染色计数法测定细胞生长活性;光镜下观察病毒感染后细胞大体形态改变;Hoechst染色荧光显微镜下观察细胞核形态的改变;DNA凝胶电泳观察细胞凋亡的发生;蛋白印迹检测凋亡相关基因caspase-3,caspase-8蛋白表达的变化.结果携带TRAIL基因的8型重组腺相关病毒能够激活caspase通路,促进结肠癌细胞凋亡,有效抑制结肠癌细胞的生长,而对正常人肝细胞的生长没有影响.结论8型腺相关病毒介导的TRAIL基因能够抑制结肠癌细胞HCT116的体外生长,对正常细胞没有毒性.     

三氧化二砷诱导肝癌Bel-7402细胞凋亡与线粒体跨膜电位关系的研究

目的研究三氧化二砷（As2O3）诱导人肝癌细胞系Bel-7402细胞凋亡及与线粒体跨膜电位的关系。方法应用不同浓度的As2O3作用于肝癌细胞后，观察As2O3对肝癌细胞生长状态的影响；通过噻唑蓝（MTT）比色法观察其对Bel-7402细胞增殖的影响；利用共聚焦显微镜及流式细胞术观察经Annexin V-FITC／PI双染后的细胞早期凋亡；通过共聚焦显微镜及流式细胞仪检测线粒体跨膜电位（MMP）的改变情况；并通过底物染色法反映Caspase-3的活性。结果不同浓度的As2O3作用于肝癌细胞后有明显的时间和剂量依赖性；经Annexin V-FITC／PI双染后，可观察到Bel-7402细胞的早期凋亡现象，2μmol／L As2O3作用24h细胞凋亡率为9．89％，作用48h细胞凋亡率为48．53％，而4μmol／L As2O3作用24h细胞凋亡率为18．27％，作用48h细胞凋亡率为67．52％；经As2O3药物作用后，细胞线粒体膜电位水平下降，且与药物作用时间和药物浓度有关（P〈0．05）；同时Caspase-3活性被激活。结论As2O3可明显抑制肝癌细胞Bel-7402的生长，并使细胞线粒体膜电位下降，诱导肝癌细胞凋亡。     

二氢青蒿素对胰腺癌生长的抑制作用

Objective To investigate the anti-tumor activity of dihydroartemisinin in pancreatic cancer in vitro and in vivo. Methods For cultured cells,cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis stained with Annexin V-FITG/PI. The protein expression in BxPC-3 cells was analyzed by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to dihydroartemisinin. Ki-67 staining and TUNEL assay were used to assess tumor cell proliferation and apoptosis in tumor tissue. Results After treatment by dihydroartemisinin in vitro, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and AsPC-1 reached up to (76.2 ± 3.5) % and (79.5 ± 2.9) %, and the apoptosis rates were up to (55.5 ± 3.2)% and (40.0 ± 3.5)%, the differences were significantly (P ＜ 0.01) compared with control [(2.0 ± 1.3) % and (0.9 ± 0.4) %]. Dihydroartemisinin inhibited the growth of pancreatic xenograft tumors in nude mice. The proliferation index and apoptusis index were (49.1 ± 3.9)% and (50.2 ± 4.4)% respectively in dihydroarternisinin 50 mg/kg treatment group, compared to those of (72.1 ± 3.3) % and (9.4 ± 2.9) % in control, the differences were significantly (P ＜0.01). Western blot assay indicated that dihydroartemisinin up-regulates expression of proliferation-associated protein p21WAF1 and down-regulates expression of PCNA, increases expression of apoptosis-associated protein Bax and decreases expression of Bcl-2 and activates caspase-9 in BxPC-3 cells. Conclusions Dihydroartemisinin exerts anti-tumor activity in pancreatic cancer both in vitro and in vivo by proliferation inhibition and apoptusis induction. Dihydroartemisinin can be used as a potential anti-tumor drug in pancreatic cancer.     

同种大鼠颈部脾脏异位移植模型的建立

目前 ,虽然我国临床脾脏移植在国际相关领域处于领先地位 ,但是在实验动物模型方面却缺乏同种大鼠带血管异位脾移植模型建立成功的报道。这里我们借鉴Bitter Suermann〔1〕 和Li等〔2〕建立的同种大鼠异位脾脏移植模型的经验 ,建立了同种大鼠颈部脾脏异位移植模型。此模型操作简单、创伤小、观察取材方便、可以再次手术、便于推广和应用 ,现报道如下。材料与方法1.材料 :(1)仪器、设备 :SXP 1B手术显微镜 (上海医用仪器厂 ) ,显微外科器械 (上海精密仪器厂 ) ,双极电凝 (北京电子仪器厂 ) ,8 0医用无损伤缝合线(强生中国医疗器材公司 ,上…     

二氢青蒿素对胰腺癌生长的抑制作用

Objective To investigate the anti-tumor activity of dihydroartemisinin in pancreatic cancer in vitro and in vivo. Methods For cultured cells,cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis stained with Annexin V-FITG/PI. The protein expression in BxPC-3 cells was analyzed by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to dihydroartemisinin. Ki-67 staining and TUNEL assay were used to assess tumor cell proliferation and apoptosis in tumor tissue. Results After treatment by dihydroartemisinin in vitro, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and AsPC-1 reached up to (76.2 ± 3.5) % and (79.5 ± 2.9) %, and the apoptosis rates were up to (55.5 ± 3.2)% and (40.0 ± 3.5)%, the differences were significantly (P ＜ 0.01) compared with control [(2.0 ± 1.3) % and (0.9 ± 0.4) %]. Dihydroartemisinin inhibited the growth of pancreatic xenograft tumors in nude mice. The proliferation index and apoptusis index were (49.1 ± 3.9)% and (50.2 ± 4.4)% respectively in dihydroarternisinin 50 mg/kg treatment group, compared to those of (72.1 ± 3.3) % and (9.4 ± 2.9) % in control, the differences were significantly (P ＜0.01). Western blot assay indicated that dihydroartemisinin up-regulates expression of proliferation-associated protein p21WAF1 and down-regulates expression of PCNA, increases expression of apoptosis-associated protein Bax and decreases expression of Bcl-2 and activates caspase-9 in BxPC-3 cells. Conclusions Dihydroartemisinin exerts anti-tumor activity in pancreatic cancer both in vitro and in vivo by proliferation inhibition and apoptusis induction. Dihydroartemisinin can be used as a potential anti-tumor drug in pancreatic cancer.     

吻合器直肠黏膜环形切除联合经直肠闭式修补术在直肠前突中的应用

直肠前突 (rectocele ,RC)又称直肠前膨出 ,为常见的出口阻塞综合征。我们自 2 0 0 0～ 2 0 0 2年采用吻合器直肠黏膜环形切除联合经直肠闭式修补术治疗RC 8例 ,效果满意 ,现报告如下。资料与方法1.一般资料 :本组 8例均为女性 ,年龄 2 7～ 6 9岁 ,平均( 4 2± 11)岁 ;分娩史 1次者 3例 ,2次者 2例 ,3次者 2例 ,5次者 1例 ;病程 2～ 35年 ,平均 ( 12± 8)年 ;排便时间 2 7～ 6 9min ,平均 ( 34± 6 )min。临床主要表现为排便困难、肛门阻塞感 ,常需口服泻剂或手助排便 ;排便时间延长 ,肛门及会阴坠胀、有排空不全感 ,部分病人出现下腹及肛…     

叶酸、VitB12及血浆同型半胱氨酸水平对Alzheimer病的影响

目的:探讨叶酸、VitB12及血浆同型半胱氨酸(Hcy)水平对Alzheimer病(AD)的影响。方法:运用多聚酶链反应-限制性内切酶片段长度多态性技术(PCR-RFLP)检测42例AD及40例正常老年人MTHFR基因多态性,用高效液相色谱仪和电化学检测法测定42例AD及40例正常老年人血浆总Hcy水平,同时测定血浆叶酸及VitB12水平。结果:MTHFR基因型有3种,即纯合子(T/T)型,杂合子(T/C)型,纯合子(C/C)型。患者组基因型和等位基因频率与正常对照组相比,差异均无显著性(P>0.05)。AD患者血浆总Hcy水平显著高于正常组(t=16.64,P<0.001)。AD组血浆叶酸及VitB12水平明显低于对照组(t=7.24,9.11,P<0.001)。结论:MTHFR基因突变与AD发生无关;高同型半胱氨酸血症是AD发病的一个新的危险因素。     

大鼠肝硬化半定量计分系统的建立

大鼠肝纤维化、肝硬化模型制作方法多种多样。本研究采用肝毒性药物———硫代乙酰胺 ( thioacetamide,TAA)诱导肝纤维化、肝硬化。该方法诱导肝纤维化、肝硬变模型稳定可靠。1.材料和方法:(1)动物:健康成年雄性Wistar大鼠,清洁级,6 月龄,体重180~200 g, 由哈尔滨医科大学第一临床医学院动物实验中心提供。(2)诱导方法:Wistar大鼠 8 只一笼,自由饮水,全价颗粒饲料喂饲。模型组大鼠共 48只,以200 mg/kg体重给予 TAA(美国Sigma公司)。电子天平称量 TAA,以蒸馏水无菌操作配成 2% (w/v)溶液,每次使用前新鲜配制。每周 3 次腹腔内注射,…     

呼吸系统医院感染菌株对悉复欢药物敏感性研究

１９９３年１～１０月间，从我院临床呼吸系统医院感染病人痰标本中共分离细菌１２种２１８株，并分别对近几年新问世的抗生素做药物敏感试验，初步认为悉复欢（Ｃｉｆｒａｎ又称环丙氟哌酸）效果较为理想，其结果表明：２１８株细菌中革兰氏阳性菌株６７株，６１株敏感，６株中度敏感；革兰氏阴性杆菌１５１株，１３８株敏感，１３株中度敏感，无一株耐药。悉复欢在临床医院感染治疗中具有广泛应用前途，其机理有待进一步研究。