弓形虫ROP18重组腺病毒载体的构建及其对神经干细胞C17.2凋亡的影响

目的 构建弓形虫棒状体蛋白ROP18重组腺病毒载体,研究其对神经干细胞C17.2凋亡的影响.方法 将弓形虫ROP18从PEGFP-G2-ROP18重组质粒上亚克隆至腺病毒载体pHBAd-MCMV-GFP,重组腺病毒载体pHBAd-MCMV-GFP- ROP18经PCR,测序鉴定正确后,与骨架质粒pHBAd-BHG共转染HEK293细胞进行包装,收获,扩增重组腺病毒并对其滴度进行测定.以MOI=70的重组腺病毒感染神经干细胞C17.2,转染不同时间荧光显微镜观察基因的表达情况.转染48 h用CCK-8法检测C17.2的增殖情况;转染24 h采用Annexin V-APC/PI双染流式细胞术检测细胞凋亡情况;Western blot法检测半胱天冬氨酸蛋白酶3(caspase-3)的蛋白水平.结果 重组ROP18过表达腺病毒在C17.2神经干细胞内能有效表达.转染48 h,与空载体对照组相比,C17.2细胞增殖活性明显降低(P<0.05);转染24 h,流式细胞术检测结果显示,ROP18基因转染组C17.2细胞凋亡率明显增加(P<0.001);Western blot检测显示caspase-3活性片段的表达明显增加(P<0.05).结论 成功构建了弓形虫ROP18重组腺病毒并在神经干细胞C17.2中表达,ROP18重组腺病毒能诱导C17.2神经干细胞凋亡.     

BIM重组腺病毒转染对EGFR突变NSCLC细胞凋亡及BIM表达水平的影响

目的探讨BIM重组腺病毒转染对非小细胞肺癌（NSCLC）细胞株的凋亡诱导作用及机制。方法将NSCLC细胞株A549、H1650、H1975分别培养至对数生长期后随机分为三组,其中转染组以BIM重组腺病毒转染、阴性对照组（NC组）以不带BIM的空载体转染、空白对照组（BC组）不转染,其后继续培养48 h。采用Annexin PI单染法检测细胞凋亡率,Western blot法检测BIM蛋白表达。结果与NC组和BC组比较,转染组细胞凋亡率及BIM蛋白表达均显著增高,其中H1650、H1975细胞株均显著高于A549细胞株（P均〈0.01）。结论 BIM重组腺病毒转染对EGFR突变NSCLC细胞凋亡具有诱导作用,可能机制为上调细胞内BIM蛋白表达;此为NSCLC的基因治疗提供了依据。     

Smac基因过表达联合顺铂对SMMC-7721细胞增殖和凋亡的影响

目的 探讨Smac基因过表达联合顺铂对肝癌细胞增殖和凋亡的影响.方法 采用脂质体介导的转染方法 ,将重组质粒pcDNA3.1+hSmac导入人肝癌细胞株SMMC-7721中,采用Western blot和流式细胞术检测转染前后Smac蛋白表达情况.转染24 h后分别加入终浓度为5、15、25 μg/ml的顺铂诱导细胞凋亡.采用四甲基偶氮唑盐比色法检测癌细胞的增殖抑制作用,吖啶橙-溴化乙锭荧光染色法和膜联蛋白V/碘化丙啶双染标记流式细胞术检测细胞凋亡. 结果 Westernblot和流式细胞术检测结果证实转染后Smac蛋白表达明显增加(P＜0.01).与空白对照相比,Smac基因过表达可抑制癌细胞增殖,促进凋亡(P＜0.01).而且给予顺铂处理后,与空白对照组相比,细胞生长抑制率随剂量增加而显著上升,且转染Smac的细胞生长抑制率较相应未转染Smac的细胞明显升高(P＜0.01).吖啶橙-溴化乙锭荧光染色法和流式细胞术检测显示,转染Smac加顺铂处理组较单纯顺铂处理组细胞凋亡明显增加,差异具有统计学意义(P＜0.01).这表明Smac基因过表达可增强顺铂对肝癌细胞的增殖抑制和凋亡促进作用. 结论 促凋亡基因Smac可在肝癌细胞中过表达,抑制癌细胞的增殖和促进凋亡;而且过表达的Smac基因可增强癌细胞对化疗药物顺铂的敏感性,这为研究Smac在癌细胞凋亡过程中的调控作用以及肝癌化学治疗效果的改善提供了实验基础.     

吉非替尼在H3255突变细胞株凋亡中的作用及对BIM表达的影响

目的探讨吉非替尼在肺癌突变细胞株凋亡中的作用及可能机制。方法将H3255肺腺癌细胞株培养至对数生长期,将其随机分为5组,吉非替尼组加入1μmol/L吉非替尼培养48 h;DMSO对照组仅加入0.1%DMSO培养48 h;转染组以促凋亡蛋白(BIM)重组腺病毒转染,阴性对照组以不带BIM的空载体转染,空白对照组不转染,继续培养48 h。采用Annexin PI单染法检测各组细胞凋亡率;Western blot法检测各组BIM蛋白表达。结果吉非替尼组细胞凋亡率及BIM蛋白表达明显高于DMSO对照组(P均<0.01),与阴性对照组、空白对照组比较,转染组细胞凋亡率及BIM蛋白表达显著增高(P均<0.01),吉非替尼组与转染组细胞凋亡率无明显差异。结论吉非替尼对存在EGFR突变的H3255肺腺癌细胞株有明显促凋亡作用,且促凋亡作用与BIM重组腺病毒转染效果相当,其机制可能与增强BIM表达有关。     

两种关键信号转导通路活性状态与K562细胞对DNR、Ara-C敏感性的关系

目的观察两种关键信号转导通路的活性状态与白血病细胞株K562对柔红霉素(DNR)、阿糖胞苷(Ara-C)的敏感性的关系。方法将K562细胞分为5个组,经U0126、LY294002、雷帕霉素处理的细胞分别为抑制剂A、B、C组,经佛波酯(PMA)刺激的细胞为激活剂组,未作处理者为对照组。采用磷酸化流式细胞术检测细胞中的p-Akt T308、p-Akt S473及p-ERK1/2。将各组细胞分别与DNR、Ara-C作用48 h,采用AnnexinⅤ/PI标记法检测细胞凋亡率。结果抑制剂A组p-ERK1/2表达及抑制剂B组p-ERK1/2、p-Akt T308、p-Akt S473表达均低于对照组(P均<0.05);激活剂组p-ERK1/2、p-Akt S473表达高于对照组(P均<0.05);抑制剂C组p-ERK1/2、p-AktT308、p-Akt S473表达及激活剂组p-Akt T308与对照组相近(P均>0.05)。DNR和Ara-C作用后激活剂组细胞凋亡率均低于对照组(P均<0.05)。DNR作用后,抑制剂A、B、C组细胞凋亡率均高于对照组(P均<0.05);Ara-C作用后,抑制剂B、C组的细胞凋亡率高于对照组(P均<0.05),抑制剂A组与对照组凋亡率无显著差异(P>0.05)。结论在信号转导通路激活状态下,K562对DNR、Ara-C的敏感性降低,抑制Ras/PI3K/PTEN/Akt/mTOR通路能增强K562对DNR、Ara-C的敏感性,Ras/Raf/MEK/ERK通路激活可能与DNR耐药有关,但抑制该通路对Ara-C诱导的K562细胞凋亡无直接作用。     

NIRF基因通过PI3K/Akt信号通路诱导人肺癌细胞凋亡

目的探究NIRF基因对人肺癌细胞凋亡的影响以及机制研究。方法使用重组质粒siRNANIRF(siRNA-NIRF组)和空载体(siRNA-NC组)转染人肺癌细胞A549,以未转染细胞为对照(Control组),免疫印迹试验(Western blot)检测转染效果;噻唑蓝(MTT)检测转染细胞增殖情况,使用流式细胞术检测转染细胞凋亡状况,蛋白免疫印迹(Western blot)检测细胞中蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、磷脂酰肌醇-3激酶(PI3K)、磷酸化PI3K(p-PI3K)蛋白的表达量。结果转染重组质粒siRNA-NIRF后,细胞中NIRF蛋白的表达量显著低于对照组(P0.05);与对照组相比,siRNA-NIRF组细胞的存活率显著降低(P0.05),凋亡率显著增高(P0.05),siRNA-NC组无明显差异(P0.05);与对照组相比,siRNA-NIRF组和siRNA-NC细胞中Akt、PI3K蛋白的含量无明显变化(P0.05),但siRNA-NIRF组细胞中p-Akt和p-PI3K的含量显著低于对照组(P0.05)。结论干扰NIRF基因可抑制肺癌细胞A549增殖,促进其凋亡,其作用机制是影响PI3K/Akt信号通路的关键因子的表达量。     

白藜芦醇对白血病K562细胞增殖、凋亡的影响及机制探讨

目的 探讨白藜芦醇对白血病K562细胞生长的影响及相关作用机制.方法 用不同浓度白藜芦醇作用于K562细胞,CCK-8法观察白藜芦醇对K562细胞生长增殖的影响,AnnexinV-FITC/PI双染法观察白藜芦醇对K562细胞凋亡的影响,PI染色法观察白藜芦醇对K562细胞周期的影响,Western blot法检测K562细胞中的Bcl-2、Bcl-xl、Bax、Cyclin D1蛋白.结果 白藜芦醇作用后的K562细胞的增殖被抑制并且凋亡增加,呈时间-剂量依赖性;同时,凋亡相关分子Bcl-2、Bcl-xl表达下调,Bax表达上调;白藜芦醇作用K562细胞后,G1期细胞增多,S期细胞减少,细胞周期调节蛋白Cyclin D1表达下降.结论 白藜芦醇可抑制K562细胞增殖并诱导其凋亡,其机制可能与下调细胞内Bcl-2、Bcl-xl、Cyclin D1表达并上调Bax的表达有关.     

海生素对K562/ADM细胞增殖和凋亡基因表达的影响

目的 探讨海生素(HSS)抗白血病作用及其机制.方法 分别以不同质量浓度(.1～1 000.0 μg/ml)HSS处理白血病耐药细胞K562/ADM,采用MTT法测定细胞增殖情况,免疫细胞化学法检测凋亡相关蛋白Bcl-2、Bax表达,流式细胞仪(FCM)测定凋亡率,Western blot法检测caspase-3表达.结果 HSS在较高质量浓度时可抑制K562/ADM细胞生长,且具有时效和量效性;经HSS处理的K562/ADM细胞呈现凋亡特有的亚G1峰,凋亡比率随HSS质量浓度和时间延长而增高;HSS处理后,K562/ADM细胞中Bcl-2呈阴性表达,Bax呈强阳性表达;caspase-3表达上调.结论 HSS在体外可明显抑制K562/ADM细胞增殖;可能通过下调Bcl-2表达、上调Bax及caspase-3表达诱导K562/ADM细胞凋亡.     

IGF-1重组腺病毒转染对缺氧诱导的神经干细胞凋亡的影响

将人胰岛素样生长因子-1(IGF-1)重组腺病毒(Ad IGF-1)转染C17.2神经干细胞;Western blot法检测IGF-1 蛋白的表达;建立神经干细胞缺氧模型,TUNEL 法检测细胞凋亡指数,流式细胞术测定细胞凋亡率的变化.发现转染Ad IGF-1的C17.2神经干细胞成功表达IGF-1 蛋白;缺氧诱导C17.2神经干细胞凋亡,转染Ad IGF-1的C17.2神经干细胞凋亡指数、凋亡率与对照组相比有显著差异.提示转染Ad IGF-1能减轻缺氧诱导的C17.2神经干细胞凋亡.     

K562/A02细胞核糖体蛋白L6的表达与多药耐药性的实验研究

目的观察核糖体蛋白L6(RPL6)基因表达的改变对白血病多药耐药性的作用。方法通过RT-PCR方法获得RPL6cDNA序列,用真核表达载体pcDNA3.1( )分别构建正向插入和反向插入的RPL6cDNA重组质粒,以脂质体将正义RPL6cDNA真核表达质粒转染K562细胞,将反义RPL6cDNA真核表达质粒转染K562/A02细胞,以MTT法检测细胞对阿霉素(ADM)、长春新碱(VCR)、米托蒽醌(MIT)和依托泊苷(VP16)耐药性的作用。结果RPL6在K562/A02细胞中的表达较K562增高(P<0.001),转染正义RPL6cDNA真核表达质粒后,K562细胞对ADM、VCR、MIT、VP16的耐药性比未转染的K562细胞分别增强3.25、1.95、2.00和3.48倍(P<0.05或0.01),转染反义RPL6cDNA真核表达质粒后,K562/A02细胞对ADM、VCR、MIT、VP16的耐药性比未转染的K562/A02细胞分别降低62%、50%、56%和47%(P<0.05或0.01)。结论RPL6基因过表达在K562/A02细胞耐药性的形成中起重要作用。     

胰岛素瘤的解剖分布特点分析

目的胰岛素瘤是最常见的胰腺神经内分泌肿瘤，因其临床表现多样，导致诊断困难。影像学诊断尤其是超声内镜(EUS)在胰岛素瘤的诊断中起着重要作用，拥有较高的敏感性和特异性。本研究拟通过明确胰岛素瘤的解剖分布特点，以期有助于提高影像学的诊断准确率和降低漏诊率，尤其是在教育和培训实践中对于EUS的学习者更具有指导价值。 方法回顾性分析解放军总医院第一医学中心病案资料数据库1993年1月至2019年11月经外科手术、病理确诊为胰岛素瘤的患者的临床资料，检索方法采取搜索术后病理诊断为"胰岛素瘤"的病例，通过查阅病例的方法，提取出胰岛素瘤的大小和解剖分布等数据，进一步分析其特点。 结果共检索到确诊为胰岛素瘤的患者116例，其中，男45例、女71例，年龄13～76岁，平均年龄(44.4±14.85)岁。胰岛素瘤单发110例(94.8%)、多发6例(5.2%)。位置分布：头颈部46例(39.7%)，单发45例、多发1例；体尾部68例(58.6%)，单发65例、多发3例；全胰腺多发2例(1.7%)。病变大小特点：最大径0.4～3.4 cm，平均大小(1.53±0.58)cm。≤1 cm 29例、>1 cm而≤1.5 cm41例、>1.5 cm而≤2.0 cm28例，≤3 cm 15例，>3 cm 3例。年龄与肿瘤的大小相关，≤44岁患者肿瘤平均大小为(1.36±0.51)cm、>44岁患者肿瘤平均大小为(1.70±0.60)cm，P<0.05。头颈部的肿瘤大于体尾部的肿瘤，头颈部肿瘤平均大小(1.66±0.63)cm，体尾部(1.42±0.52)cm，P<0.05。 结论胰岛素瘤在胰腺体尾部较头颈部更好发；绝大多数单发，但可以全胰腺多发；多数小于1.5 cm，肿瘤的大小与患者年龄和肿瘤的解剖分布相关。     

Pathogenesis of carcinoma of the papilla of Vater

Most adenomas and carcinomas of the small intestine and extrahepatic bile ducts arise in the region of the papilla of Vater. In familial adenomatous polyposis (FAP) it is the main location for carcinomas after proctocolectomy. In many cases symptoms due to stenosis lead to diagnosis at an early tumor stage. In about 80%, curative intended resection is possible. Operability is the most relevant prognostic factor. Most ampullary carcinomas resp. carcinomas of the papilla of Vater develop from adenomatous or flat dysplastic precursor lesions. They can be sited in the ampulloduodenal part of the papilla of Vater, which is lined by intestinal mucosa. They also can develop in deeper parts of the ampulla, which are lined by pancreaticobiliary duct mucosa. Intestinal-type adenocarcinoma and pancreaticobiliary-type adenocarcinoma represent the main histological types of ampullary carcinoma. Furthermore, there exist unusual types and undifferentiated carcinomas. Many carcinomas of intestinal type express the immunohistochemical marker profile of intestinal mucosa (keratin 7?, keratin 20+, MUC2+). Carcinomas of pancreaticobiliary type usually show the immunohistochemical profile of pancreaticobiliary duct mucosa (keratin 7+, keratin 20?, MUC2?). Even poorly differentiated carcinomas, as well as unusual histological types, may conserve the marker profile of the mucosa they developed from. These findings underline the concept of histogenetically different carcinomas of the papilla of Vater which develop either from intestinal- or from pancreaticobiliary-type mucosa of the papilla of Vater. Molecular alterations in ampullary carcinomas are similar to those of colorectal as well as pancreatic carcinomas, although they appear at different frequencies. In future studies, molecular alterations in ampullary carcinomas should be correlated closely with the different histologic tumor types. Consequently, the histologic classification should reflect the histogenesis of ampullary tumors from the two different types of papillary mucosa.     

Demonstration of the mechanism of action of biguanides

Summary Palmitic acid oxidation in rat diaphragm homogenate is depressed by biguanide concentrations that are still incapable of inhibiting oxidative phosphorylation. Glucose oxidation is not directly effected by the same biguanide concentrations: however, the inhibitory effect of palmitic acid on glucose oxidation is partly removed by biguanides. Inhibition of fatty acid oxidation, which accounts for most of the metabolic effects caused by these drugs, can be regarded as the fundamental mechanism of action of biguanides. There is some evidence suggesting that these drugs might interact with carnitine, thus preventing long-chain fatty acids from being transported across the mitochondrial membrane to the site of oxidation. Traduzione a cura degli AA.     

Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease

BACKGROUND AND AIM: Both the clinical presentation and the degree of mucosal damage in coeliac disease vary greatly. In view of conflicting information as to whether the mode of presentation correlates with the degree of villous atrophy, we reviewed a large cohort of patients with coeliac disease. PATIENTS AND METHODS: We correlated mode of presentation (classical, diarrhoea predominant or atypical/silent) with histology of duodenal biopsies and examined their trends over time. RESULTS: The cohort consisted of 499 adults, mean age 44.1 years, 68% females. The majority had silent coeliac disease (56%) and total villous atrophy (65%). There was no correlation of mode of presentation with the degree of villous atrophy (p=0.25). Sixty-eight percent of females and 58% of males had a severe villous atrophy (p=0.052). There was a significant trend over time for a greater proportion of patients presenting as atypical/silent coeliac disease and having partial villous atrophy, though the majority still had total villous atrophy. CONCLUSIONS: Among our patients the degree of villous atrophy in duodenal biopsies did not correlate with the mode of presentation, indicating that factors other than the degree of villous atrophy must account for diarrhoea in coeliac disease.     

血吸虫童虫生长发育及其机制的研究进展

血吸虫童虫是宿主免疫系统攻击的重要靶标,包括皮肤型、肺型和肝门型童虫。宿主分子对童虫生长发育具有重要作用。童虫生长发育机制包括免疫调节、信号转导、性别发育及凋亡等。肌动蛋白、组织蛋白酶、烯醇化酶和葡萄糖基转移酶等分子为血吸虫童虫生长发育的重要分子。本文对血吸虫童虫生长发育及其机制的研究进展做一综述。     

124例耐多药结核分枝杆菌基因突变特征分析

目的对临床分离的耐多药结核分枝杆菌相关基因的突变特征进行分析。方法对124例耐多药结核分枝杆菌以及50株敏感株的耐药相关基因(包括异烟肼inh A、kat G、oxyR-ahp C间隔区以及利福平rpo B)进行序列测定,分析其基因突变情况。结果异烟肼耐药inh A基因突变率为14.5%;kat G基因突变率为70.2%(87/124),主要位于315位;oxyR-ahp C间隔区突变率为15.3%;inh A、kat G两种基因同时突变率75.0%,三种基因同时突变率为89.5%。利福平rpo B基因突变的检出率高达95.2%,突变主要发生在531、526、516位点。结论我省耐多药菌异烟肼耐药相关基因最常见突变为kat G 315、inh A C-T(-15)、axyR-ahp C间隔区(-10)C-T,利福平为rpo B531、526、516。结合MDR-TB耐药相关基因的特征分析,可以建立一种快速、准确、特异的适合于我省的检测结核菌耐多药性的新方法。     

氯硝柳胺悬浮剂的毒性评价

目的评价氯硝柳胺悬浮剂的毒性，为现场大规模应用灭螺提供依据。方法按照中华人民共和国国家标准GB 15670-1995《农药登记毒理学试验方法》和鱼类毒性试验方法进行。结果经口、经皮肤的LDso雌、雄性大鼠均>5 000 mg/kg，经呼吸道的LCso雌、雄性大鼠均>5 000mg/m3，该药经口、经皮肤、经呼吸道毒性均属微毒类药物；兔眼用药后，观察期内无不良反应，对眼无刺激性；皮肤用药后对皮肤无刺激性。与氯硝柳胺原药、氯硝柳胺乙醇胺盐原药和氯硝柳胺乙醇胺盐可湿性粉剂相比，氯硝柳胺悬浮剂对鱼急性毒性最低。结论氯硝柳胺悬浮剂属微毒类药物，对鱼的毒性低于其乙醇胺盐可湿性粉剂，适合于现场应用。     

Assessment of quality of life of parents of children with juvenile chronic arthritis

The aim of the study was to assess the quality of life (QOL) and the psychological status of parents of children with juvenile chronic arthritis (JCA). The QOL, anxiety and depression of the parents of 28 children with JCA were evaluated and compared to those of the parents of 28 healthy children. Mothers of JCA children and mothers of healthy children reported similar QOL. The reported anxiety and depression levels were similar for mothers and fathers in both groups. The parents of children with pauciarticular-type JCA reported lower QOL and higher levels of anxiety and depression than the parents of children with other types, namely polyarticular and systemic JCA. These findings may be explained by the fact that the pauciarticular patients had shorter disease duration and were less frequently seen in the outpatient clinic. The QOL of mothers of children with JCA was found to be slightly impaired in the group of children with pauciarticular JCA. Future larger studies are needed to confirm these results, as the number of subjects in the three groups was rather low. Received: 26 September 2001 / Accepted: 8 February 2002     

Numerical Simulation of the Effect of Rate of Change of Glucose on Measurement Error of Continuous Glucose Monitors

Background

A 5-day in-patient study designed to assess the accuracy of the FreeStyle Navigator® Continuous Glucose Monitoring System revealed that the level of accuracy of the continuous sensor measurements was dependent on the rate of glucose change. When the absolute rate of change was less than 1 mg•dl⁻¹•min⁻¹ (75% of the time), the median absolute relative difference (ARD) was 8.5%, with 85% of all points falling within the A zone of the Clarke error grid. When the absolute rate of change was greater than 2 mg•dl⁻¹•min⁻¹ (8% of the time), the median ARD was 17.5%, with 59% of all points falling within the Clarke A zone.

Method

Numerical simulations were performed to investigate effects of the rate of change of glucose on sensor measurement error. This approach enabled physiologically relevant distributions of glucose values to be reordered to explore the effect of different glucose rate-of-change distributions on apparent sensor accuracy.

Results

The physiological lag between blood and interstitial fluid glucose levels is sufficient to account for the observed difference in sensor accuracy between periods of stable glucose and periods of rapidly changing glucose.

Conclusions

The role of physiological lag on the apparent decrease in sensor accuracy at high glucose rates of change has implications for clinical study design, regulatory review of continuous glucose sensors, and development of performance standards for this new technology. This work demonstrates the difficulty in comparing accuracy measures between different clinical studies and highlights the need for studies to include both relevant glucose distributions and relevant glucose rate-of-change distributions.     

Angiographic diagnosis of the degree of serosal invasion of carcinoma of the colon

Angiography using Prostaglandin El^® was performed on 38 patients with carcinoma of the colon in order to diagnose the degree of serosal cancer invasion. The findings at angiography were classified into four groups:1) AG-S3, abnormal change (irregularity and/or encasement) up to marginal vessels; 2) AG-S2, abnormality up to vasa recta; 3) AG-S1, abnormality of penetrating branches of vasa recta within the wall of the colon; and 4) AG-S0, no distinct findings of abovementioned vessels. These angiographic findings were compared with both macroscopic and microscopic serosal cancer invasion. Angiographic diagnosis is in accord with the macroscopic findings in 84.2 percent of cases. Angiographic diagnosis is in accord with the microscopic findings in 32.4 percent of cases. Macroscopic findings confirm the angiographic diagnosis precisely but the conflict with microscopic findings should not be overlooked. This may be the result of inflammatory change, adhesion, and fibrosis around carcinoma of the colon.