一种简单、稳定、可靠的屏氧酶1基因多态性分析法

目的：建立一种简单、稳定、可靠的PON1基因多态性分析法。方法：取外周静脉血100μl，用ROSE法提取基因组DNA，用两对引物：P192F 5′-TAT TGT TGC TGT GGG ACC TGA G-3′,P192R 5′-CAC GCT AAA CCC AAA TAC ATC TC-3′；P55F 5′-GAA GAG TGA TGT ATA GCC CCA G-3′,P55R 5′-TTT AAT CCA GAG CTA ATG AAA GCC-3分别进行PCR扩增，用限制性内切酶AlwI,NlaⅢ对两种PCR产物分别进行酶切，3％琼脂糖凝胶电泳。结果：扩增的两个PCR产物在192、55多态性位点大小分别为99bp、170bp。Alw I酶切后，凝胶电脉得到完全酶切（66bp,33bp)，部分酶切（99bp、66bp、33bp)，未被酶切（99bp)三种类型DNA片段（RR，QR，QQ基因型）；NlaⅢ酶切后，凝胶电脉得到部分酶切（170bp,126bp,44bp)，未被酶切（170bp)两种类型DNA片段（LM，LL基因型）。经双盲重复检测，结果一致。应用此方法对胃癌患者血样标本检测，发现其PON1基因多态性在192位点频率较高。结论：采用一步PCR－RFLP技术可以建立简单、稳定、可靠的PON1基因多态性分析法。     

抗大肠癌噬菌体人单链抗体的初步鉴定及序列分析

目的 :对抗大肠癌细胞的单克隆噬菌体单链抗体进行初步鉴定和测序分析。方法 :采用细胞ELISA ,免疫组化 ,DNA序列测定和计算机分析方法 ,对 5个单克隆噬菌体抗体 (CH2 73,CH2 0 5 ,CH2 0 9,CHA12 ,CH72 3)进行初步鉴定和序列分析。结果 :5个抗体均对人大肠癌细胞、人胚肾上皮细胞和其它某些人肿瘤细胞反应 ,也与人正常肝细胞有弱阳性反应 ,但不与鼠源性的癌细胞和正常细胞反应。细胞免疫组化进一步证实了ELISA结果的正确性。大肠癌免疫组化对大肠癌组织有特异性的结合反应 ,而不与正常大肠组织反应。测序结果为CH2 73ScFv全长 732bp ;V ,D ,J分别属于VH3 30 D1 2 6 JH3 linker V1 13 JL2 ,GenBank序号为AY0 2 8777和AY0 2 8996 ;CH2 0 5全长 36 6bp ,V ,D ,J分别VH1 4 6 D6 13 JH3,GenBank序号为AF35 936 5 ;CH2 0 9,CHA12和CH72 3的ScFv基因完全相同 ,全长 72 3bp ,其VH DH JH与CH2 73ScFv基因中的VH DH JH 完全一致 ,V ,D ,J分别属于VH3 30 D1 2 6 JH3 linker L2 Jκ2 ,GenBank序号为AF36 3774。结论 :噬菌体抗体具有结合人大肠癌组织和细胞的活性 ,为进一步开发临床应用人源抗肿瘤抗体和小分子抗体片段奠定基础     

Increased occurrence of free immunoglobulin light chains in cerebrospinal fluid and serum in human immunodeficiency virus-1 infection

The presence of free immunoglobulin light chains (FLCs) in the cerebrospinal fluid (CSF) and sera of patients with human immunodeficiency virus-1 (HIV-1) infection, multiple sclerosis (MS), and neurologically healthy control individuals was investigated by paying special attention to ensure that only truly free light chains would be detected. The FLCs were extracted by specifically binding them to Sepharose-coupled anti-FLC monoclonal antibodies, and thereafter they were electrophoresed and immunoblotted with monoclonal antibodies to both light chain (LC) isotypes. A frequent occurrence of kappa and lambda FLCs was found in both CSF and sera of HIV-1 infected patients. In HIV-1 infection and in MS, the frequency of FLCs of the CSF was equal. In healthy controls, only occasional weak FLCs were observed in either CSF or serum. FLC bands of the CSF from patients with HIV-1 infection tended to be more intensive than those of the appropriately diluted sera. Both intrathecal synthesis of FLCs and their transudation from sera through the impaired blood-brain barrier (BBB) may contribute to this. Increasing severity of general HIV-1 infection was accompanied by an increase of FLC intensity in sera. A qualitative demonstration of FLC in the CSF may be meaningful only in the absence of altered BBB function.     

Differentiation of Helicobacter pylori isolates by polymerase chain reaction—restriction fragment length polymorphism

Objective: To investigate the association between the diversity of urease gene and urease activity of clinical isolates of Helicobacter pylori (H. pylori). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of urease gene and rapid urease activity test were used to study the urease activity of different clinical isolates of H. pylori. Results: H. pylori clinical isolates were divided into 4 types according to their PCR-RFLP results of urease gene and urease activity. Type Ⅰ , possessing strong urease activity (0. 11) and presented 1 fragment of 1. 7 kb by PCR-RFLP, had close relations with gastric ulcer; type Ⅱ , with the weakest urease activity (0. 07) and 2 fragments (1. 3 and 0. 4 kb respectively) , was associated with duodenal bulb ulcer; type Ⅲ , with the strongest urease activity (0. 12) and 2 fragments (0. 4 and 0. 17 kb) with or without 1 fragment (0. 23 or 0. 37 kb) , was responsible for gastritis; type Ⅳ , with weak urease activity (0. 09) and 2 fragments     

微卫星改变与肺癌发生发展的关系

目的 :研究肺癌组织中第 3号染色体短臂 (3P)和第 9号染色体短臂 (9P)上微卫星标志物的改变与肺癌发生发展的关系。方法 :应用聚合酶链反应 (PCR)结合微卫星银染法检测 5 6例肺癌和 2 3例良性肺部病变 3P和 9P上的微卫星标志物的改变。结果 :5 6例肺癌组织标本中 3P上有微卫星改变 2 6例 ,检出率 46% ,9P上有微卫星改变的 2 3例 ,检出率为 41% ,5 6例肺癌组织中 ,3P和 9P上有微卫星改变的 3 9例 ,检出率 69 6% ,2 3例肺部良性疾病组织中均未出现微卫星标志物改变。肺癌组织标本中 3P上的微卫星改变与肺癌的分化程度有关 ,低分化肺癌的微卫星改变较高、中分化肺癌的微卫星改变明显增多 (P <0 .0 1)。而 9P上的微卫星改变与淋巴结转移有关 ,有淋巴结转移的肺癌组织标本上微卫星改变较不伴淋巴结转移的微卫星改变检出率明显增多 ,(P <0 .0 5 )。结论 :3P和 9P上的微卫星改变与肺癌发生、发展和转移密切相关 ,检测肺癌组织 3P和 9P上的微卫星改变对肺癌的早期诊断和判断有无淋巴结转移有一定的价值     

Presence of beta human papillomaviruses in nonmelanoma skin cancer from organ transplant recipients and immunocompetent patients in the West of Scotland

Background  Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of the genus beta (betaPV).
Objectives  We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.
Methods  One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.
Results  In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples ( P  =   0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) ( P  <   0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.
Conclusions  A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis.     

聚合酶链反应检测患儿尿标本中的巨细胞病毒DNA

本文建立了聚合酶链反应(PCR)检测人巨细胞病毒(HCMV)方法,探讨了该方法的某些实验条件。用PCR检测24例临床患儿尿标本。证明PCR方法特异、敏感、简便而快速。同科不同属病毒如HSV、EBV等以及人源正常细胞均无假阳性结果:最小检出量可达0.1fg DNA,相当于6个基因拷贝;用水浴箱手控时间即可进行PCR循环,30次循环仅需97min30s;PCR和DNA杂交检测尿标本中HCMV阳性例数分别为20和14例。该结果证明HCMV感染儿童的广泛性和严重性,初步表明儿童肾病综合症与HCMV有关。     

环氧合酶-2反义核酸对膀胱癌细胞恶性表型的抑制作用

目的探讨环氧合酶-2(COX-2)反义核酸对膀胱癌细胞恶性表型的抑制作用。方法采用脂质体介导方法,分别构建转染COX-2反义真核表达载体和空载体的膀胱癌细胞系5637-AS细胞和5637-P细胞,RT-PCR及W estern b lot分析转染细胞COX-2 mRNA及蛋白表达水平。MTT比色实验、Boyden侵袭小室法和裸鼠成瘤实验分别检测转染细胞体外增殖速度、体外侵袭力和体内成瘤性。结果RT-PCR结果显示,5637-AS细胞COX-2/磷酸甘油醛脱氢酶(GAPDH)mRNA比值(0.65)明显低于5637(0.92)和5637-P细胞(0.90);W estern b lot提示5637-AS细胞COX-2/β-actin比值(0.29)明显低于5637细胞(0.46)和5637-P细胞(0.44)。MTT比色实验显示5637-AS细胞较5637-P、5637细胞生长速度减慢,三者倍增时间分别为3.6 d、2.9 d和2.9 d。体外侵袭实验结果表明,5637-AS侵袭细胞数显著低于5637细胞及5637-P细胞(18.20±5.97比45.40±8.12,18.20±5.97比44.10±6.47,P<0.01)。裸鼠成瘤实验中,5637-P、5637细胞接种裸鼠后第5天肿瘤长出,而5637-AS细胞第7天肿瘤长出;30 d后5637-AS细胞接种组瘤体重量(392.15 mg±33.58 mg)明显小于5637细胞(738.26 mg±66.32 mg),和5637-P细胞接种组(698.53 mg±88.76 mg),P<0.01。结论膀胱癌细胞中COX-2过表达与细胞的恶性表型相关,反义技术抑制COX-2表达可以逆转膀胱癌细胞的恶性表型。