Ⅴ型腺病毒纤维蛋白基因真核表达质粒的构建及表达

目的：克隆VEGF受体sFET-1片段1-3区，并使之在原核中进行表达以探讨其对内皮细胞增殖和血管生成方面的抑制作用。方法：提取脐静脉血管内皮细胞总RNA，克隆sFLT-1基因1-3区，并使之在QIA表达系统进行表达，Ni^2 -Sepha-rose6B亲和层析纯化，复性，应用MTT和鸡胚绒毛尿囊膜血管生成模，探讨重组蛋白对血管生成的抑制作用。结果；该表达系统能表达sFLT-1基因片段，表达量低，纯化后，经复性，sFLT-1具有VEGF特异结合功能，1μg重组sFLT-1蛋白能抑制10ngVEGF诱导的脐静脉内皮细胞增殖和20ngVEGF诱导的鸡胚绒毛尿囊膜血管生成，结论：sFLT-1基因片段能在QIA表达系统中得到表达，复性后重组蛋白具有与VEGF结合和拮抗VEGF生物学活性的功能。     

从随机肽库中筛选血管内皮生长因子的抑制剂

目的：从随机噬菌体肽库中筛选血管内皮生长因子（VEGF165）的抑制剂,研究治疗实体瘤生长的小分子药物。方法：以VEGF165分子的受体KDR为靶分子,筛选随机噬菌体环7肽库,通过竞争性洗脱得到能特异结合KDR的阳性噬菌体,以ELISA、细胞免疫组化、细胞ELISA、鸡胚绒毛膜尿囊膜实验和MTT法,鉴定其结合活性和抑制活性。结果：得到5个能特异结合靶分子的阳性噬菌体克隆,它们均可以与表面有KDR表达的细胞结合,其中噬菌体克隆3和13还能抑制VEGF165诱导鸡胚绒毛尿囊膜新生血管形成的活性。但MTT法结果显示,这两种噬菌体小肽对高表达KDR的阳性细胞生长没有明显的抑制活性。结论：从随机噬菌体肽库筛选到了能抑制VEGF165生物活性的克隆,为进一步研究小分子抑瘤药物提供了一定基础。     

抗KDR胞外Ⅲ区单抗抑制血管内皮细胞增殖的研究

目的研制能够封闭血管内皮生长因子(VEGF)受体KDR的单克隆抗体,探讨其体外抑制VEGF165诱导的生物学活性。方法以原核表达的KDR胞外Ⅲ区融合蛋白免疫Balb／c小鼠,采用传统杂交瘤技术制备抗KDR胞外Ⅲ区单克隆抗体,采用ELISA和FACS方法鉴定其抗原结合特异性,采用免疫沉淀和[^3H]-TdR掺入的方法分析该单克隆抗体阻断VEGF165刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,抑制VEGF165岱诱导内皮细胞增殖的活性。结果抗KDR胞外Ⅲ区单抗Ycom1D3不仅能与可溶性KDR结合,亦可与细胞表面表达的KDR结合,并可竞争性抑制VEGF165与可溶性KDR的相互作用,阻断VEGF165刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,显著抑制由VEGF165诱导脐静脉内皮细胞的增殖。结论抗KDR胞外Ⅲ区单抗Ycom1D3可通过封闭KDR而抑制VEGF活性,在肿瘤及其他血管新生疾病治疗中具有潜在的应用前景。     

人血管抑制因子Vasostatin短片段(120-180 aa)的克隆、表达及功能研究

目的:利用基因工程技术,克隆并表达人血管抑制因子Vasostatin120-180aa功能区片段,探讨其对鸡胚绒毛尿囊膜新生血管抑制的作用.方法:采用PCR技术扩增人血管抑制因子Vasostatin120-180aa功能区基因,并利用pQE30原核表达系统诱导表达Vasostatin120-180aa,经镍金属螯合层析法纯化,通过鸡胚绒毛尿囊膜实验验证其抑制新生血管生成的作用.结果:PCR扩增出了长度为180 bp的Vasostatin120-180aa功能区基因,后经pQE30原核表达系统表达并纯化出Vasostatin120-180aa,SDS-PAGE显现一条约8 kD的阳性条带,Vasostatin120-180aa可显著抑制鸡胚绒毛尿囊膜新生血管的生成.结论:原核表达的Vasosta-tin120-180aa功能区片段具有生物学活性,可明显抑制鸡胚绒毛尿囊膜新生血管的生成,在一定范围内呈量效依赖性.     

血管内皮生长因子受体KDR的靶向制剂的制备

目的：研究从噬菌体肽库中筛选到的与血管内皮生长因子受体Ⅱ(KDR)有特异结合活性的小肽，做为KDR靶向药物的先导物质的应用。方法：从噬菌体肽库中筛选能特异结合KDR的噬菌体克隆，挑选结合力最强的克隆测序并化学合成小肽P5，梯度ELISA、阻断实验和竞争结合实验测定小肽在体外与KDR的结合活性。将P5与生物素(NHS-d-Biotin)、BSA化学偶联，ELISA法和细胞免疫组化实验检测偶联物与KDR的结合活性。结果：化学合成的小肽P5在体外能特异结合KDR，Kd=168．6nmol／L，约是KDR与配体血管内皮生长因子(VEGF165)亲和力的1／30，P5能阻断VEGF165与KDR的结合活性，但不能竞争VEGF165与KDR的结合活性。将P5作为导向物质化学合成的P5-BSA-Biotin，在体外同样具有与KDR和细胞表面KDR分子结合的特性。结论：化学合成的小肽P5有望作为先导分子，在以KDR为靶点的肿瘤靶向治疗中得到应用。     

肿瘤中血管内皮生长因子受体的表达

血管内皮生长因子(vascular endothelial cell growth factor,VEGF)是肿瘤血管生长的最主要调节者.VEGF通过与VEGF受体的结合,介导其生物学功能.flt-1与flk-1/KDR是VEGF的两个高亲和的受体,不同的受体可介导VEGF不同的生物学功能.本文对VEGF受体的结构与特性,VEGF受体在肿瘤标本、肿瘤细胞系、内皮细胞与裸鼠皮下接种肿瘤动物模型中的表达以及VEGF受体表达的调控等方面作一综述.     

重组可溶性KDR及其抗体对内皮细胞的增殖抑制作用

目的：分析可溶性KDR（sKDR)及其抗体对内皮细胞增殖的抑制作用。方法：通过ELISA分析大肠杆菌表达的sKDR纯化产物与VEGF165结合的能力；sKDR免疫家兔制备KDR262抗血清，Wenstern blot分析该抗血清与KDR262蛋白结合的特异性；用^3H-TdR掺入法，MTT法和细胞计数3种方法分析重组sKDR及其抗体对内皮细胞的增殖抑制作用。结果：sK-DR蛋白可与VEGF165特异性结合，肝素可增强其结合能力。KDR262抗血清具有特异性识别KDR蛋白的能力，其滴度为1：2000，用^3H-TdR掺入法和MTT2种方法分析sKDR蛋白及其抗体对内皮细胞的增殖抑制作用结果一致，sKDR蛋白浓度在10μg/ml,2μg/ml,0.4μg/ml时对内皮细胞增殖抑制率平均为56%，44%和32%；抗KDR262抗体在稀释度为50，200和800倍时对内皮细胞增殖的抑制率平均为70%，56%和43%，细胞计数法测定结果，sKDR及抗KDR262抗体组与VEGF165单独刺激组，GST和PBS对照组相比，内皮细胞增殖被明显抑制，随浓度增加抑制活性明显增强，但抗体的抑制活性比sKDR蛋白高。结论：大肠杆菌表达的sKDR及其抗体对内皮细胞均具有明显的增殖抑制作用。     

血管内皮生长因子部分多肽抗血管生成的研究

目的：观察血管内皮生长因子（VEGF）部分多肽（3－4外显子）抗血管生成的作用。方法：抽提LoVo细胞总RNA,进行RT－PCR,克隆VEGF部分多肽cDNA,构建VEGF部分多肽原核表达载体,用限制性酶切和DNA测序进行鉴定,通过聚丙烯酰胺凝胶电泳（SDS－PAGE）分析表达产物,表达产物经亲和层析纯化后,以人脐静脉内皮细胞（HUVEC）和鸡胚尿囊膜（CAM）血管测定其生物学活性。结果：表达产物以可溶性分子形成存在于菌体中,具有良好的抗原性和特异性,并具有抑制HUVEC增殖及CAM血管形成的活性。结论：VEGF部分多肽具有竞争抑制血管生成的功能,在肿瘤生物靶向治疗中具有一定潜在的价值。     

血管内皮生长因子受体导向毒素的制备

目的 以噬菌体肽库中筛选的与血管内皮生长因子受体(KDR)有特异结合活性的小肽为先导分子,制备KDR的导向毒素。方法 KDR为靶蛋白,以亲和筛选和竞争性洗脱法,从噬菌体环7肽库中,筛选到一段能特异结合KDR的小肽P5,将P5与大肠杆菌分泌的志贺氏毒素的毒性亚基StxA部分,以融合蛋白的形式原核表达,制备导向毒素。结果 获得了融合表达毒素P5-StxA。ELISA和Western blotting检测结果表明,融合毒素P5-StxA在体外能特异结合KDR。细胞毒性实验显示,P5-StxA具有与单独StxA分子同样的毒性。鸡胚绒毛尿囊膜实验结果表明,P5-StxA对鸡胚绒毛尿囊膜新生血管的形成有抑制活性。结论 导向毒素P5-StxA对KDR有特异结合活性,小肽P5有希望成为导向药物的先导小分子。     

可溶性血管内皮生长因子受体2片段的克隆、表达及其在肿瘤血管形成中的作用

目的 对可溶性血管内皮生长因子受体2(VFGFR2)片段阻断血管内皮细胞生长因子(VEGF)与相应受体结合抑制血管形成的作用进行体内外实验研究。方法 应用RT-PCR技术,从胎鼠肝脏扩增Flk-1／KDR片段,重组于逆转录病毒载体PLXSN和表达载体pFT-28b( ),并行表达、纯化和鉴定。以原代培养的小鼠内皮细胞,观察可溶性受体蛋白对内皮细胞生长的影响。以脂质体法转染肿瘤细胞系S180和B16,观察基因转染后的体内生物学特点。结果 在受精后第9,11天的胎鼠肝组织中分离出1000bp大小的可溶性VEGFR2片段,连接TA克隆载体,经测序此片段为VEGFR2胞外段部分序列。将可溶性VEGFR2片段克隆入表达载体pET-28b( ),体外实验显示,可溶性受体蛋白能有效抑制内皮细胞的生长和增殖。将可溶性VEGFR2片段克隆入逆转录病毒载体PLXSN并成功转染肿瘤细胞系S180和B16,体内实验显示,转基因细胞系的瘤重减轻,体积明显缩小,且其血管密度明显降低,而Flkl蛋白表达明显增高。结论 可溶性VEGFR2片段是一种有效的抑制血管形成的生物工程产品,有望作为抗血管形成基因治疗的靶点。     

Schwannomas of Head and Neck and Review of Literature

Benign nerve cell tumours have been given various names like schwannoma, neurilemmoma, neurinoma, neurofibroma, spindle cell tumours etc. Extra cranial head and neck schwannomas usually present as solitary and well-demarcated lesions. The lesion can cause secondary symptoms, such as nasal obstruction, dysphasia, and hoarseness, depending upon the location of the lesion. Fine needle aspiration cytology, CT scans, and MRI may be of limited help in the diagnosis of schwannomas. The treatment is complete surgical excision of the benign tumour and postoperative histopathological examination establishes the final diagnosis.     

A comparative study of knowledge and awareness of colorectal and breast cancerA comparative study of knowledge and awareness of colorectal and breast cancer

Aims: To assess and compare knowledge and awareness of colorectal cancer and breast cancer in a sample of the general population. Methods: Eleven hundred visitors to six different outpatient clinics, in a University Hospital, were given a study-specific questionnaire, based on educational material from the British Association of Cancer United Patients (CancerBACUP). The questionnaire consisted of 12 statements on the incidence, presentation, detection, treatment and prognosis of colorectal and breast cancer. Results: One thousand and sixty-eight individuals returned the questionnaire. One thousand and four completed questionnaires were analysed. The mean age (SD) of respondents was 50.1 (17.2) years, and the male to female ratio was 2:3. Respondents had read more about breast than about colorectal cancer (60.3%vs 32.4%,P <0.0001, McNemar's test). The proportion of correct answers for each statement on breast cancer was higher than for answers to corresponding items on colorectal cancer. Mean overall scores (95% CI) for breast and colorectal cancer were 88.1 (86.9, 89.2) and 64.4 (62.5, 66.3) respectively, the mean difference (95% CI) being 23.7 (22.0, 25.5). Scores were higher for breast cancer irrespective of age or gender. Conclusion: There is a low level of understanding of colorectal cancer in the general population when compared to breast cancer. This highlights the importance of public education in this common cancer.     

Quality of life and care in leukemia,myeloma and non-malignant disease. Opinions of patients and relatives,and effects of geography and time

In a questionnaire study 140 subjects answered 4200 questions in 1980 and 1986. They consisted of patients with myeloma, acute leukemia, lung carcinoma, and non-malignant disease and their relatives. In 22 additional cases the questionnaire was not answered. The results show that myeloma patients are less content with the general care than leukemia patients (P < 0.05). Similarly, relatives of deceased myeloma patients are less satisfied with the information given to them than relatives of deceased leukemia patients (P < 0.001). The information has improved with time, however, since the patients were more satisfied in 1986 than in 1980 (P < 0.001) and relatives of myeloma patients still alive were more satisfied than relatives of patients who had died earlier (P < 0.001).     

miRNA与肿瘤侵袭转移

目前,microRNA (miRNA)已成为肿瘤研究中最基本的参与者,主要通过与靶标基因3 'UTR(非翻译区)的完全或不完全配对,降解靶标基因mRNA或抑制其翻译,从而参与调控个体发育、细胞凋亡、增殖及分化等生命活动.miRNA作为调控基因表达的重要分子在肿瘤侵袭转移中的作用越来越受到重视,表明miRNA在肿瘤侵袭和转移中的作用机制具有重要的理论意义,同时也可为肿瘤的诊断和治疗提供新方法.本文就miRNA通过调控上皮间质转化及肿瘤干细胞导致肿瘤侵袭转移的最新研究进展作一综述.     

芦荟大黄素及芦荟提取物的诱变性和抗诱变性

目的:用L5178Y小鼠淋巴瘤细胞体外微核试验评价芦荟大黄素和芦荟提取物的诱变和抗诱变作用,为其安全性评价提供依据。方法:设溶剂对照、阳性对照和抗诱变对照,芦荟大黄素和芦荟提取物诱变和抗诱变试验各设4个剂量组,处理L5178Y细胞12 h后按常规方法进行体外微核试验分析。结果:较高浓度(6.67μg/ml)的芦荟大黄素可致微核细胞率增加,与对照组比较,差异有统计学意义(P0.05);而芦荟提取物未见此效应。在一定剂量范围内,芦荟大黄素(0.22～6μg/ml)和芦荟提取物(20～180μg/ml)对甲磺酸甲酯(MMS)所致微核细胞率均有一定程度的拮抗作用,与对照组比较,差异有统计学意义(P0.01)。结论:芦荟大黄素具有一定的诱变作用,而在本实验剂量范围内的芦荟提取物未见遗传毒性。两种受试物在一定范围内均能较好地拮抗MMS所致的染色体损伤。     

甲状腺手术的技巧及副损伤的预防和处置

鉴于甲状腺手术是普外科的常见手术，为求其日渐完美，以有益于病人，现根据作者的体会，并结合阅读相关文献，就其手术操作、喉返神经处理、甲状旁腺处理进行扼要阐述。     

甲胎蛋白与自噬在肝癌发生发展中的作用及其相互关系

甲胎蛋白（AFP）作为临床诊断肝癌最常用的肿瘤标志物,有抑制免疫、促进细胞生长、抑制癌细胞凋亡的作用。自噬是一种维持细胞生存的重要途径之一,其与肝癌的发生发展及治疗有着密切联系,对肝癌既有抑制又有促进作用。PI3K/AKT作为两者共有的信号通路,它们是否有着相互关系来促进肝癌的发展尚需进一步研究。     

Erythrocyte levels of cadmium and lead and risk of B-cell non-Hodgkin lymphoma and multiple myeloma

Cadmium and lead are persistent environmental toxins that are known or probable carcinogens, based on evidence for causality for nonhematologic cancers. Associations of these metals with risk of non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) are unknown but biologically plausible. To examine the associations of circulating levels of lead and cadmium exposure with risk of B-cell NHL (B-NHL) and multiple myeloma, we conducted a nested case-control study among 299 incident B-cell NHLs and 76 MM cases within the Cancer Prevention Study-II Nutrition Cohort (CPS-II NC). Each case was incidence-density matched to two eligible controls on age, race, sex and blood draw date. Conditional logistic regression was used to estimate relative risks (RR) and 95% confidence intervals (CI) for lymphoid malignancies overall and stratified by subtype. We observed a significant positive association between high erythrocyte lead concentration and risk of lymphoid malignancies overall (RR = 1.16, 95% CI: 1.02-1.33 per 17.6 μg/L (1 standard deviation [SD])) and follicular lymphoma in particular (RR = 1.80, 95% CI: 1.15-2.80 per SD). In contrast, there was no association between erythrocyte cadmium and risk of B-NHL (RR = 0.89, 95% CI: 0.75-1.06 per 0.37 μg/L [1 SD]), or any B-NHL subtypes; but a strong inverse association with MM risk (RR = 0.59, 95% CI: 0.38-0.89, per SD). Results from our study suggest a positive association between erythrocyte lead level and risk of lymphoid malignancies and a possible inverse association between cadmium and myeloma. Additional research is needed to confirm and further explore these findings.     

Comparison of the Efficacy and Safety of Ceftibuten and Cefaclor in the Treatment of Pneumonia and Bronchiectasis

Summary

In a multicentre, international study of 187 adult patients with bacterial pneumonia or bronchiectasis, the safety and efficacy of a regimen of 200 mg ceftibuten administered twice-daily was compared with cefaclor given in a dosage of 500 mg three times a day. Of the 94 evaluable patients, 66 received ceftibuten and 28 received cefaclor. The overall bacteriological response was similar in the two treatment groups with elimination of the original pathogen in 91% and 89% of the patients receiving ceftibuten and cefaclor, respectively. The overall clinical response mirrored the bacteriological results with a successful clinical outcome in 92% of ceftibuten-treated patients compared with 93% in patients receiving cefaclor. Adverse experiences were, in general, few and mild, being reported in 8% and 17% of patients receiving ceftibuten and cefaclor, respectively.