人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

CD40配体对结肠癌细胞株的体外抑制作用

研究免疫共刺激分子CD40在结肠癌细胞株的表达及重组可溶性人CD40配体(rshCD40L)对结肠癌细胞株的体外抑制作用。采用流式细胞仪分别检测4株结肠癌细胞(HCT116、Caco-2、SW48和COLO-320)CD40分子的表达,同时利用流式细胞仪检测γ干扰素(IFN-γ)对结肠癌细胞株CD40表达的影响。MTT法检测rshCD40L对结肠癌细胞的抑制作用,TUNEL法检测rshCD40L对结肠细胞的促凋亡作用,同时检测联合rshCD40L和IFN-γ抗结肠癌作用。结果流式细胞仪检测有3株结肠癌细胞(HCT116、Caco-2和SW48)表达CD40分子,IFN-γ能增强CD40+结肠癌细胞CD40分子的表达。rshCD40L能明显抑制CD40+结肠癌细胞的增殖作用(抑制率分别为HCT116:33.5%±5.5%;Caco-2:21.5%±3.6%;SW48:30.1%±4.2%),而对CD40-结肠癌细胞株(COLO-320)无明显抑制作用(P<0.05)。rshCD40L能诱导CD40+结肠癌细胞的凋亡作用(凋亡细胞百分比为HCT116:25.6%±4.52%;Caco-2:15.71%±2.27%;SW48:18.0%±3.7%),而对CD40-结肠癌细胞株(COLO-320)无诱导凋亡作用(P<0.05)。另外,IFN-γ能明显增强rshCD40L对结肠癌细胞的抑制增殖和促凋亡作用。重组人CD40配体与IFN-γ联合可显著诱导CD40阳性的结肠癌细胞凋亡,抑制其增殖,具有潜在的抗肿瘤作用。     

围产期母血与脐血乙肝标志物及DNA载量关联性研究

目的 研究围产期母血与新生儿脐血乙肝病毒标志物及核酸DNA载量的关联性,探索脐血乙肝五项模式与HBV-DNA载量之间关系,用于评估新生儿宫内感染乙肝病毒的风险性.方法 在知情同意的原则下选择住院分娩产妇612例作为乙肝研究对象,根据产妇乙肝五项不同模式共分为五组,均采用ELISA法检测产妇血清、新生儿脐血HBV标志物,同时用实时荧光定量PCR检测产妇血清和脐血新鲜血清HBV-DNA载量,探讨围产期母血与脐血间HBV-DNA载量的关联性.结果 年龄在18 ～ 44岁围产期妇女,A组传染性强的乙肝“大三阳”或1,3阳性者149例,产妇血清和脐血HBV-DNA阳性率分别为99.33％和32.21％;B组260例乙肝“小三阳”,产妇血清和脐血HBV-DNA阳性率分别为20.00％和3.08％;C组乙肝1、5阳性者58例,产妇血清和脐血HBV-DNA阳性率分别为65.52％和12.07％;D组乙肝病毒第5阳性者59例,产妇血清和脐血HBV-DNA阳性率分别为13.56％和1.69％;E组对照组:乙肝五项全阴性86例,产妇血清和脐血HBV-DNA阳性率分别为1.16％和0.A组与B组间脐血HBV-DNA阳性率,差别有统计学意义(x2=54.09,P＜0.01).乙肝产妇所生新生儿脐血HBeAg阳性率高达82.55％,脐血HBsAg的阳性率为32.89％,A组即HBsAg HBeAg阳性的产妇经胎盘传播乙肝病毒的概率(32.21％)明显高于B组“小三阳”产妇的脐血HBV-DNA感染率(3.02％),A组是B组的10.67倍;传染性强的A组母血是脐血HBV-DNA平均载量的6345倍.结论 ①新生儿宫内感染乙肝病毒阳性率与围产期母体内HBV-DNA载量呈正相关.②乙肝产妇生产的新生儿脐血HBeAg阳性率是HBsAg的2.51倍,要研制复合型乙型肝炎人免疫球蛋白:即增加高效价HBeAb成分可结合HBeAg,原有的HBsAb也可结合相应的HBsAg,增强阻断乙肝病毒垂直传播作用;③我国应加强乙肝知识的宣教工作,完善围产保健制度.     

外源性组织型纤溶酶原激活物信号肽突变体增强蛋白质在哺乳细胞中的表达及分泌

目的 比较不同tPA信号肽突变体对目的蛋白表达和分泌水平的影响,优化并筛选通用性外源信号肽序列.方法 通过定点突变技术将人类组织型纤溶酶原激活物（tPA）的信号肽第22位氨基酸由脯氨酸（P）突变为丙氨酸（tPA22P/A）或甘氨酸（tPA22P/G）,并将突变前后的3种信号肽序列分别插入HIV-1 p24基因的N末端,构建不同的p24蛋白表达载体,这些重组表达载体体外瞬时转染人胚肾HEK293T细胞,转染72 h后,通过SDS-PAGE与western blot检测并比较各组培养上清和细胞中p24蛋白的表达水平.结果 成功构建了3个p24蛋白的真核表达载体P24T.tPA、P24T.tPA22P/A和P24T.tPA22P/G,经序列测定证实基因序列和方向与预期相符;重组载体转染293T细胞72 h后均检测到分泌性p24的表达.与P24T.tPA组相比,P24T.tPA22P/A转染组培养上清中和细胞内p24蛋白表达水平分别提高62%和29%,P24T.tPA22P/G转染组分别提高8%和6%.结论 tPA信号肽22位由脯氨酸突变为丙氨酸或甘氨酸,这就提高了目的蛋白的表达和分泌水平,为优化外源蛋白在哺乳细胞中的表达和分泌提供了实验基础.     

投射物钝挫伤的研究进展

钝挫伤是生活中尤其是交通事故中常见的伤类之一。非致命性武器击中人体或子弹击中防弹衣可造成钝挫伤,其造成的损伤效应与通常交通事故钝挫伤有所不同,对投射物钝挫伤的研究可以了解人体组织对投射物撞击的力学响应,确定人体损伤与投射物载荷之间的量效关系。本文就当前投射物造成钝挫伤的致伤机制、研究现状和评估标准进行综述。     

鼻咽癌侵犯周围结构与肿瘤分期关系的MRI研究

目的 应用MRI探讨鼻咽癌侵犯周围结构的规律及其与肿瘤T分期的关系.方法 回顾性分析1573例经病理证实的鼻咽癌初诊患者,根据2008年鼻咽癌T分期,观察鼻咽癌向周围结构侵犯的MRI表现和规律.对鼻咽癌周围不同方向结构的侵犯率均采用Z检验分析.结果 鼻咽癌对周围结构的侵犯:咽颅底筋膜1299例(82.58%)、咽旁间隙1090例(69.29%)、鼻腔304例(19.33%)、口咽49例(3.12%)、颈动脉间隙514例(32.68%)、翼内肌661例(42.02%)、翼外肌210例(13.35%)、颅底骨质943例(59.95%)、颅神经630例(40.05%)、鼻窦242例(15.38%).T分期分布:T1期为242例(15.38%),T2期为288例(18.31%),T3期为410例(26.06%),T4期为633例(40.24%).鼻腔受累的病例中90.46%(275/304)合并T3以上的结构受累;口咽受累均伴有T3以上结构受累;翼内肌受累的病例中69.14%(457/661)伴有T4结构侵犯;鼻窦受累的病例中92.15%(223/242)伴有T4结构侵犯.鼻咽癌侵犯周围结构的模式为向侧方(1299例)多于向上(943例)侵犯(Z=14.025,P＜0.01);向侧方侵犯多于向下(49例)(Z=45.032,P＜0.01);向上侵犯多于向下(Z=34.301,P＜0.01);向前侵犯(304例)多于向下(Z=14.404,P＜0.01).结论 鼻咽癌侵犯周围结构的模式为向侧方侵犯多于向上;向侧方侵犯多于向下;向上侵犯多于向下;向前侵犯多于向下.     

重组腺病毒mIκBα基因联合γ射线治疗肝细胞癌的实验观察

目的 探讨重组腺病毒mIkBα基因（AdmIkBα）联合γ射线对人高转移性肝细胞癌HCC9204细胞和裸鼠HCC9204移植瘤生长的影响。方法利用有限稀释法测定病毒滴度，以不同感染复数（MOI）的AdIxBαm（10、20、30、50）转染HCC9204细胞，Westemblotting法检测转染前后细胞1：）65表达和mIxBa表达。采用4Gyγ射线对各组细胞进行治疗。MTT法检测转染组、转染组＋放射组、对照组细胞生长抑制率。建立裸鼠HCC9204肝癌动物模型，肿瘤局部注射AdmIkBα30MOI并联合6Gyy射线治疗，在不同时间测量肿瘤体积。结果病毒滴度1．252×10^9pfu／ml。mIkBa蛋白随AdmlteBa剂量增加而进行性增加，P65蛋白随AdmIkBα剂量的增加呈进行性递减。转染组体外细胞生长随AdmIkBα剂量的增加逐渐受抑制，经7射线照射后生长抑制更加明显。AdmIkBα转染和放射联合治疗后7～28d，裸鼠肿瘤生长速率明显低于单纯照射组、单纯转染治疗组。结论AdmIkBα转染联合Y射线治疗可增强对肝痛细胞杀灭作用．联合治疗明品优于单纯放疗组和单纯基因治疗组。     

NEP1-40和甲基强的松龙联合治疗对脊髓修复的影响

目的：探讨甲基强的松龙（MP）和NEP1-40联合治疗对大鼠脊髓损伤后脊髓的功能恢复的影响。方法：选用SD大鼠60只,随机分为联合治疗组、MP治疗组、NEP1-40治疗组和对照组4组。建立大鼠中段胸部脊髓后半部横切模型。MP的给药方法为术后30 min内经鼠尾静脉给予MP 30 mg/kg,后给予剂量5.4 mg/（kg.h）,持续23 h;NEP1-40的给药方法为术后3 d于硬膜下管给予NEP1-40 12.5μg/（20μl PBS.d）,连续7 d。对照组给予等量生理盐水。不同时间点进行运动诱发电位（MEP）测定和行为学评价。结果：联合治疗组大鼠潜伏期延迟时间较MP、NEP1-40治疗组和对照组短,5 w时接近正常,其P1波的潜伏时间为5.42 ms,N1波的潜伏时间为5.55 ms（P〈0.01）,其BBB运动评分5 w时为17分（P〈0.01）;联合治疗组的空洞面积百分比不到对照组的一半（P〈0.01）;NF200阳性染色的神经元数目为20.19个/1000μm^2。结论：MP和NEP1-40联合治疗可减少病变范围,减轻白质纤维脱髓鞘病变,增多NF200阳性神经元数目,促进神经再生,改善由于脊髓后半横切所引发的脊髓电生理的障碍,促进了脊髓功能恢复。在治疗脊髓损伤时,MP与NEP1-40有协同作用,促进脊髓功能恢复。     

Slug在胰腺癌中的表达及干扰Slug对胰腺癌转移的影响

目的 探讨Slug和E-CADHERIN(E-cadherin)表达与胰腺癌转移的关系以及干扰Slug转录因子对胰腺癌转移的影响.方法 采用免疫组化和RT-PCR技术分别检测36例胰腺癌组织中Slug、E-cadherin和mRNA表达.将肝脏高转移潜能的胰腺癌细胞株SW1990H4,分为3组:对照组、空载体质粒(shRNA-1)和转染干扰S1ug质粒(Slug-shRNA-1),观察Slug对E-cadherin mRNA表达的逆转作用,及对SW1990H4体外侵袭、运动的抑制作用.结果 胰腺癌组织中Slug高表达,而E-cadherin低表达.转移组胰腺癌组织中Slug蛋白和mRNA表达明显高于非转移组,差异分别有统计学意义(P＜0.05),而E-cadherin在转移组胰腺癌中无表达.PCR产物凝胶电泳结果显示,Slug mRNA在空白对照组SW1990H4中表达为0.985±0.016,E-cadherin mRNA表达为0.120±0.001.shRNA-1时转染48 h时,Slug mRNA的表达水平为0.973±0.014,E-cadherin mRNA表达水平分别0.160±0.001,与对照组比差异分别无统计学意义(P＞0.05).转染Slug-shRNA-1组瞬时转染48 h时,Slug mRNA的表达水平为0.554±0.011,E-cadherin mRNA表达水平为0.36±0.002,与对照组和shRNA-1组比差异分别有统计学意义(P＜0.05).SW1990H4稳定转染后,shRNA-1和Slug-shRNA-1组Slug mRNA表达水平分别为0.968±0.015,0.206±0.017,两组比较,差异有统计学意义(P＜0.05),而E-cadherin mRNA表达水平分别为0.18±0.002,0.727±0.006,两组比较,差异有统计学意义(P＜0.05).体外细胞运动实验表明,对照组、转染shRNA-1和SlugshRNA-1组跨膜细胞数393±28、352±24、96±13,差异有统计学意义(P＜0.01).重组细胞基底膜(Matrige1)侵袭实验显示,3组穿透基底膜细胞数分别为223±69、202±64、65±19,差异有统计学意义(P＜0.05).结论 Slug高表达,E-cadherin低表达与胰腺癌的转移相关.胰腺癌细胞中存在Slug mRNA与E-cadherin mRNA表达的逆转关系,抑制Slug mRNA的表达对胰腺癌细胞的侵袭和运动有抑制作用,可能为胰腺癌转移的基因治疗提供新的靶点.

Abstract：

Objective To investigate expression of slug and E-cadherin in pancreatic cancer tissues and determine the inhibitory effects of anti-Slug, an anti-sense plasmid, on the invasion of pancreatic cancer cell lines in vitro. Methods Slug and E-cadherin protein and mRNA was analyzed by IHP and RT-PCR in 36 cases of pancreatic cancer. Then anti-Slug plasmid was transfected into herin and Slug expression. The inhibitory effects of anti-sense Slug were also detected by Transwell motility assay and Matrigel invasion assay. Results The expression of Slug and mRNA in metastatic pancreatic cancer tissue was higher than that in non-metastatic tissue. E-cadherin and mRNA was lower in metastasis tissues(P＜0.05). The inverse relationships were further observed by transient transfection of anti-Slug into SW1990H4 cells. The downregulated expression of Slug and re-expression of E-cadherin were found. The Slug mRNA levels were 0.985±0.016,0.973±0.014, 0. 554±0. 011 after 0, 48 h of transfection of anti-sense Slug, and that of E-cadherin were 0.120±0.001, 0.360±0.002, 0. 727±0. 006, respectively. The diference was significant between different time points (P＜0.05). The Slug mRNA levels were 0. 206±0.017, 0.968±0.015, and that of E-cadherin were 0. 18±0.002,0.727±0.006 after stable transfection of anti-sense Slug, and control plasmid, respectively. The diference was significant (P＜0.05). The motility activity(393±28, 352±24, 96 ±13 )and the invasion activity (223 ± 69, 202 ± 64, 65 ±19) of1 antisense Slug transfectant cells were significantly decreased as compared with those of control cells (P＜0.05). Conclusions Higher expression of slug and lower expression of E-cadherin is related to the invasion and metastasis in pancreatic cancer. A reverse corelation of E-cadherin and Slug expression exists in pancreatic cancer. Slug is possibly a potential target for cancer gene therapy blocking invasion and metastasis in human pancreatic cancer.     

X线下经鼻-空肠置管小肠造影的临床应用

目的:介绍一种简便易行、图像清晰的小肠造影检查方法。方法:选择临床需要小肠造影的87例患者实施经鼻-空肠置管小肠造影检查,造影图像经放射科两位以上副主任医师分别阅片。结果:87例患者插管造影成功率达100%,检查过程中患者能够耐受,配合良好,所得造影图像清晰,有助于诊断。结论:X线下经鼻-空肠置管小肠造影,插管技术较易掌握,图像显示清晰,值得推广应用。