Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

HPLC法同时测定鸡骨香中6个萜类成分

目的建立同时测定鸡骨香Croton crassifolius中6个萜类成分(chettaphanin I、山藿香定、crassifolin B、cyperenoic acid、crassifolin A、cyperenol)的HPLC方法。方法采用Kromasil 100-5 C18色谱柱(250 mm×4.6 mm,5μm);以乙腈(A)-0.02%三氟乙酸水(B)梯度洗脱:0~35 min,35%A;35~55 min,35%~60%A;55~80 min,60%A。体积流量为1.0 m L/min;检测波长为210 nm;柱温为25℃。结果 6个萜类化合物均有良好的分离度,线性关系良好(r0.999 7),chettaphanin I、山藿香定、crassifolin B、cyperenoic acid、crassifolin A、cyperenol的加样回收率分别为100.2%、99.13%、98.48%、99.22%、101.1%、102.5%,RSD分别为0.48%、0.48%、0.96%、1.10%、1.35%、0.95%。结论该方法简单准确,具有良好的重复性和稳定性,可为鸡骨香质量控制提供科学依据。     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

胆管囊状扩张症:新的临床分型与治疗策略

目的 提出胆管囊状扩张症的临床分型及针对不同分型的治疗策略和手术方法.方法 回顾性分析1993年6月至2010年6月解放军总医院收治的434例胆管囊状扩张症患者的临床资料.收集和重新分析患者腹部CT、MRI、MRCP和胆道造影检查结果,根据胆管囊状扩张病变累及胆管树的部位及范围,结合其临床病理特征、发病因素及适用的手术方式提出一种新的胆管囊状扩张症的临床分型;分析新分型中不同胆管囊状扩张症的临床表现、手术方式、围手术期结果、随访结果等资料,针对不同分型胆管囊状扩张症制订治疗策略和手术方法.率的比较采用x2检验,理论频数＜5或总观测频数＜ 30时,采用Fisher确切概率法.结果 根据囊状扩张病变累及胆管树的部位及病理特征将其分为5种类型:(1)A型:周围肝管型肝内胆管囊状扩张.A1型:囊状扩张病变局限分布于部分肝段;A2型:囊状扩张病变弥漫分布于全肝.(2)B型:中央肝管型肝内胆管囊状扩张.B1型:单侧肝叶中央肝管囊状扩张;B2型:囊状扩张病变同时累及双侧肝叶主肝管及左、右肝管汇合部.(3)C型:肝外胆管型胆管囊状扩张.C1型:囊状扩张病变未累及胰腺段胆管;C2型:囊状扩张病变累及胰腺段胆管.(4)D型:肝内外胆管型胆管囊状扩张.D1型:囊状扩张病变累及单叶中央肝管和肝外胆管;D2型:囊状扩张病变累及双侧肝叶中央肝管和肝外胆管.(5)E型:壶腹胆管型胆管囊状扩张.本组434例胆管囊状扩张症患者中,A型24例(A1型17例、A2型7例),B型13例(B1型10例、B2型3例),C型300例(C1型56例、C2型244例),D型96例(D1型17例、D2型79例),E型1例.24例A型患者中,14例伴有先天性肝纤维化,16例合并多囊肾病,区别于其他各型患者.手术方式:24例A型患者中,17例A1型患者行部分肝切除术,3例A2型患者行肝移植,1例A2型患者行囊状扩张病变穿刺引流术,3例A2型患者采用非手术治疗;13例B型患者中,12例患者行肝切除术,1例患者合并胆管癌,采用非手术治疗;300例C型患者中,286例患者行肝外囊状扩张病变切除+胆管空肠吻合术,14例患者因囊状扩张胆管恶变行胆管癌根治性切除术;96例D型患者中,35例患者行肝外胆管囊状扩张病变切除+肝切除(肝内胆管囊状扩张病变累及的部分肝组织)+胆管空肠吻合术,59例患者仅行肝外囊状扩张病变切除术,1例D1型和1例D2型患者伴有胆管癌,行根治性切除术;1例E型患者行EST治疗.399例患者获得随访,随访时间l～15年,平均随访57个月.33例患者出现胆管空肠吻合口狭窄和(或)结石.24例患者并发胆管癌,其中15例患者于随访期内死亡.46例患者因复发性胆管炎伴有吻合口狭窄或结石、肿瘤实施再次手术治疗(胆管空肠再吻合术、内镜下胆管取石术、胆管癌根治性切除术等).其余患者无相关临床症状或偶发轻度的胆管炎,经对症治疗缓解.D型患者中联合肝切除者与仅行肝外胆管囊状扩张病变切除者的症状缓解率、狭窄和(或)结石复发率、再手术率分别为88.2％(30/34)、8.8％ (93/34)、11.8％ (4/34)和64.4％(38/59)、28.8％(17/59)、35.6％ (21/59),两者比较,差异有统计学意义(P＜0.05).结论 新的胆管囊状扩张症分型基于囊状扩张病变累及胆管树的部位及其病理特征,对于不同分型的胆管囊状扩张症选择不同的治疗策略和手术方法具有明确的指导作用.     

不同体质量指数的糖尿病患者使用预混胰岛素治疗后疗效及体重变化的观察

目的:探讨不同体重指数的2型糖尿病患者在口服降糖药继发失效后,加用预混胰岛素治疗3个月后的疗效及体重变化。方法:选择口服降糖药失效的2型糖尿病患者共50例,按体重指数分为A组(BMI<25 kg/m2)和B组(BMI≥25 kg/m2)各25例,停用口服药物,三餐前加用门冬胰岛素30注射液皮下注射(商品名:诺和锐30)治疗3个月后,分别比较两组治疗前后空腹血糖、餐后2 h血糖、糖化血红蛋白、体重、血脂变化。结果:两组的空腹血糖、餐后2 h血糖、糖化血红蛋白均较治疗前明显下降,差异有统计学意义(P<0.01),A组BMI有轻度升高(P>0.05),而B组BMI未见明显改变。结论:对于不同体质量指数的2型糖尿病患者在口服降糖药继发失效后,加用新型预混胰岛素强化治疗,均有明确的疗效,且无明显体重增加的不良反应。     

蛋白激酶C在调节血管内皮细胞通透性中的作用

蛋白激酶C(proteinkinaseC,PKC)是一种钙或/和磷脂依赖性蛋白磷酸化酶,广泛存在于人体的各种组织细胞中.它能被细胞外生物活性因素(生长因素、神经递质、细胞因子等)激活,完成靶细胞蛋白的磷酸化,通过蛋白磷酸化后生物活性的改变而完成细胞外源性信号的应答,构成细胞内重要的信号转导系统.许多实验证明蛋白激酶C的激活在血管内皮细胞屏障功能和血管通透性的变化调节中起着重要的作用.本实验从组织、细胞和分子水平对PKC在血管通透性调节中的作用进行了系统的研究.目的和方法1.采用游离灌注的猪冠状微静脉模型,在严格控制生理环境的条件下,通过荧光倒置显微镜,利用荧光比率测定技术,测定了游离猪冠状微静脉的通透性,并观察蛋白激酶C的激活与抑制对微静脉通透性的影响.2.培养单层脐静脉内皮细胞株ECV-304,用同位素标记和液体闪烁计数法检测细胞浆和细胞膜的PKC活性,观察PKC在热损伤刺激后激活和移位的情况.3.应用免疫荧光技术,观察PKC激活和热损伤刺激对人脐静脉内皮细胞株ECV304的F-actin的细胞定位和结构变化的影响,从形态学的角度证明PKC对内皮细胞的作用.结果1.利用PKC的特异性激动剂佛波酯醇(PMA)激活PKC可显著增加血管通透性约至300%.PKC的特异选择性抑制剂bisindolylmaleimide(BIM)阻断了PMA增加通透性的作用;在单纯PMA刺激下,Pa值是对照组的290.94%±78.13%(n=7,血管平均口径D=63.86±3.14μm),而在BIM的影响下,PMA只使Pa值升高到对照的139.97%±20.82%(n=7,D=43.25±3.08μm).2.用PKC特异性激动剂PMA和热损伤刺激都可以导致ECV-304细胞浆中的PKC的激活和向胞膜移位.未受刺激的ECV-304细胞,胞浆中的PKC活性是43.47fmol·mg     

烧伤刺激介导内皮细胞蛋白激酶C的激活转位

目的：探讨烧伤刺激对血管内皮细胞蛋白激酶C（PKC）活性的影响。方法：用10％烧伤血清分别刺激细胞0、5、10、30和60分钟后,通过细胞降解和离心获得细胞浆和细胞膜蛋白粗提液。用同位素标记和液体闪烁计数法检测细胞浆和细胞膜的PKC活性。PKC的激动剂佛波酯醇（PMA）100nmol／L刺激细胞作为阳性对照组。选用PKC特异性抑制剂PKC（19－36）预处理细胞后,分别观察烧伤血清和PMA介导的内皮细胞PKC活性的变化。结果：烧伤血清和PKC的激动剂PMA都可以激活内皮细胞的PKC,并使其发生由胞浆至胞膜的转位;用PKC的特异性抑制剂预处理细胞可以分别抑制这种变化。结论：烧伤血清可以介导内皮细胞PKC的激活和转位;烧伤可以通过激活内皮细胞的PKC而导致内皮细胞功能如通透性的变化。     

无痛肠镜结合复方聚乙二醇电解质散在老年结肠息肉中的治疗效果

目的:探究无痛肠镜结合复方聚乙二醇电解质散在老年结肠息肉中的治疗效果.方法:选取100例老年结肠息肉患者为研究对象,随机分组,各50例,对照组采用无痛肠镜联合甘露醇,观察组采用无痛肠镜结合复方聚乙二醇电解质散.比较两组治疗效果.结果:观察组息肉共63枚,对照组息肉65枚,对照组息肉切除率(90.77％)低于观察组息肉切除率(100.0％),但数据无统计学差异(P>0.05).观察组肠道总清洁度(96.0％)高于对照组(82.0％),有统计学差异(P<0.05).结论:针对老年结肠息肉患者采用无痛肠镜联合复方聚乙二醇电解质散可实现良好肠道准备,实现更佳的肠道清洁.     

影响原发性肝癌肝移植治疗的预后因素分析

目的分析影响肝癌肝移植术后生存率和无瘤生存率的危险因素,探讨国内肝移植治疗肝癌的选择标准。方法对67例接受同种异位原位肝移植治疗的原发性肝癌病人的基本资料和肿瘤相关资料包括术前病情分级、血清AFP水平、术前辅助治疗以及肝癌大小、数目、pTNM分期、肿瘤恶性程度分级等因素进行单因素和多因素分析。结果术后1年、2年累积生存率为77%、67%,6个月和12个月无瘤生存率为66%和58%。单因素分析显示对肝癌肝移植术后累积生存率影响有统计学意义的因素为CHILD分级(MELD积分)和肝外大血管侵犯;多因素分析影响肝癌肝移植术后无瘤生存率有统计学义的因素是肿瘤大小、大血管侵犯和肿瘤分化程度。结论影响肝癌肝移植术后生存率的因素仍是术前患者肝功能状态。对存在大血管侵犯的肝癌患者需严格控制肝移植术适应证,而无血管侵犯的患者在选择肝移植治疗时肿瘤大小指标可较米兰标准适当放宽。