人源性抗结核分枝杆菌噬菌体展示单链抗体文库的构建

目的 构建人源抗结核分枝杆菌噬菌体展示单链抗体文库,为结核特异性单链抗体的筛选奠定基础. 方法 从抗结核分枝杆菌抗体阳性患者淋巴细胞中提取总RNA,逆转录成cDNA,PCR扩增获得抗体的重、轻链可变区基因,然后拼接装配成单链抗体基因,重组于噬菌粒载体pCANTAB5S,转化大肠埃希菌E.coli TG1,以辅助噬菌体拯救后,构建人源性抗结核分枝杆菌噬菌体展示单链抗体库. 结果 成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体文库,库容量达107. 结论 以结核患者淋巴细胞免疫球蛋白可变区基因片段和噬菌粒展示载体pCANTAB5S为基础,成功构建人源性抗结核分枝杆菌噬菌体展示单链抗体库,并达到建库标准,可进一步从中筛选获得结核特异性单链抗体.     

胃肠道肿瘤靶向抗癌胚抗原单链抗体与T细胞表面受体基因的融合分子构建

目的 将胃肠道癌胚抗原(CEA)特异性单链抗体基因与人T细胞CD3ξ胞内区、跨膜区基因克隆入真核表达载体，表达相应的融合分子，以期制备消化道肿瘤靶向性杀伤T细胞。方法 应用PCR法扩增抗CEA特异性单链抗体(scFv)基因；用RT-PCR法从正常人外周血T细胞扩增出CD3ξ胞内区、跨膜区基因；将单链抗体插入CD3ξ基因序列的上游，构建重组真核表达载体scFv-CD3ξ-pcDNA3．0并测序。结果 抗CEA scFv cDNA片段为810bp，与已知的序列相符；CD3ξ胞内区、跨膜区的cD-NA片段为438bp，与基因库公布的序列一致。重组的表达载体经酶切琼脂糖电泳及测序加以证实。结论 成功制备了具有抗肿瘤相关抗原CEA靶向性及CD3ξ胞内区、跨膜区信号转导功能的重组分子，为制备消化道肿瘤靶向性嵌合锚定T细胞奠定了实验基础。     

特异性抗肝癌单链抗体二聚体在毕赤酵母中的表达、纯化及生物学活性鉴定

目的:实现特异性抗肝癌单链抗体二聚体在毕赤酵母中高效表达、纯化并鉴定其生物学活性.方法:构建酵母表达载体pGAPZαA-BDM,转化感受态大肠杆菌DH5α,选择测序正确的阳性克隆进行扩增后,电转化酵母细胞,表达抗体二聚体,对表达抗体进行纯化、浓度检测,并鉴定其对肝癌细胞的结合活性,免疫组织化学检测二聚体对肝癌组织抗原的特异性.结果:测序显示成功构建酵母表达载体pGAPZαA-BDM,表达96 h抗体收获量最大,二聚体表达量为30 mg/L菌液,为大肠杆菌的300倍.抗肝癌单链抗体二聚体BDM与三种肝癌细胞结合,而与正常肝细胞不结合,结合效价为1:128.免疫组织化学显示二聚体与肝癌组织结合的阳性率比肝硬化、胃癌、正常肝组织高,差异有统计学意义.结论:成功制备了高表达量、高特异性、较好的活性及稳定性的抗肝癌单链抗体二聚体BDM,为制备免疫纳米颗粒及开展肝癌的放射免疫诊断和靶向治疗奠定了基础.     

抗甲状腺刺激性免疫球蛋白单链抗体噬菌体抗体库的初建

目的 建立抗甲状腺刺激性免疫球蛋白单链抗体噬体抗体库,为进一步获得功能性抗体可变区基因,制备单链抗体和单链抗体－辣根过氧化物酶融合蛋白,用于甲状腺自身免疫性疾病的治疗和检测打下基础,方法 利用ＲＴ－ＰＣＲ技术从分泌抗甲状腺刺激性免疫球蛋白的杂交瘤细胞中扩增出轻重链可变区基因,与噬粒表达载体ｐ３ＳＣＭＨ连接,转化大肠杆菌,以甲状腺刺激性免疫球蛋白阳性患者血清ＩｇＧ为抗原对表达的噬菌体抗体进行筛选,结     

抗HBV PreS2单链噬菌体抗体基因在噬菌体载体中的克隆及初步表达

本文利用基因工程技术,在成功构建抗HBV PreS2 3B9mAb单链可变区抗体(ScFV)基因的基础上,在389 ScFv基因中引入限制性内切酶位点,克隆的噬菌体表达载体pCANTAB5噬菌粒中。经转染TG1     

核糖体蛋白L5在胃癌中的表达及功能

目的:探讨核糖体蛋白L5(ribosomal protein L5,RPL5) 在胃癌细胞中的表达及对胃癌细胞生长的影响．方法:Western blot检测RPL5在胃癌细胞系中的表达, 构建RPL5特异性siRNA载体,转染细胞,Western blot进行鉴定,MTT方法和流式细胞术检测转染细胞的生长变化．结果:RPL5在胃癌细胞系AGS、MKN45、SGC7901、 MGC803中的表达均明显强于在GES-1和正常胃黏膜上皮中的表达．成功构建RPL5特异siRNA载体U6- RPL5A和U6-RPL5B,转染AGS细胞,进行稳定筛选,发现U6-RPL5A能显著抑制RPL5的表达,其相应的细胞系AGS-U6-RPL5A的生长速度减慢．细胞周期检测结果显示AGS-U6-RPL5A细胞中处于增殖期的细胞减少了约5％．结论:对RPL5功能的进一步深入研究可能会有助于胃癌的诊断和治疗．     

人源性抗-HBc单链噬菌体抗体库的构建

目的 构建人源性单链噬菌体抗体库，为筛选人源性抗—HBc单链抗体奠定基础。方法 利用逆转录—聚合酶链反应(RT—PCR)和噬菌体表面展示技术，直接从乙肝病毒核心抗体(抗—HBc)阳性患者淋巴细胞中提取总RNA，逆转录成cDNA；合成全套人抗体可变区引物扩增抗体可变区基因，并将重、轻链可变区基因进行拼接装配成单链抗体(ScFv)基因，重组于噬菌粒载体叶pHEN1，转化抑制型大肠埃希菌E.coliTG1，以辅助噬菌体援救后，构建成人源性单链噬菌体库。结果 成功地构建了人源性抗—HBc单链噬菌体库，库容量达10^6。结论 利用RT—PCR和噬菌体表面展示技术可以成功构建人源性单链抗体库，并达到建库标准，可进一步从中筛选人源性单链抗体。     

一种新型胃肠道癌特异性单链抗体的表达及其在胃癌中的检测

目的通过原核可溶性表达将我们制备的抗胃肠道肿瘤特异性单链抗体（single chain antibody variable fragment,scFv）基因表达出单链抗体蛋白,初步鉴定并检测其在几种胃癌细胞中的表达。方法通过基因原核表达、Western blot、ELISA等方法检测单链抗体的表达;通过免疫细胞化学方法检测其对几种胃癌细胞的肿瘤相关抗原的识别。结果 获得的单链抗体分子量31 KD,与理论分子量基本一致,并通过Western blot、ELISA加以证实;免疫细胞化学方法证明该单链抗体可识别胃癌细胞KATOⅢ、HGC-27、肝癌细胞SMMC7721等细胞,不能识别SGC7901。结论通过对单链抗体的原核表达,明确了单链抗体对胃癌细胞的识别能力,为进一步临床应用提供理论基础。     

四株人结肠腺癌细胞CEA表达水平的检测及其意义

目的:检测四株结肠癌细胞株中癌胚抗原(carcinoembryonic antigen,CEA)的表达,并探讨其意义．方法:四株人结肠腺癌细胞(LS174T,LoVo, SW480及HCT-8)分别应用ELISA法测定细胞培养上清中CEA含量;免疫组化检测细胞CEA蛋白表达;半定量RT-PCR检测细胞CEA mRNA表达丰度．结果:LS174T和SW480细胞在蛋白和mRNA水平均可检测到CEA的表达且前者表达量高于后者(细胞培养上清中CEA含量:1050±25．0 ng／107 cells vs 66±5．6 ng／107 cells,P<0．0001;半定量检测CEA mRNA表达丰度:1．137±0．155 vs 0．399±0．135,P=0．003);而LoVo细胞在蛋白和和mRNA水平均未检测到CEA的表达:HCT-8细胞免疫组化有弱阳性染色．结论:四株结肠癌细胞株的实际CEA表达情况与既往文献报道不尽相同．这种细胞株CEA表达特性的改变有可能影响其体外生物学行为从而导致实验结果的不确定性．     

抗HBV Pre S2单链噬菌体抗体基因在噬菌体载体中的克隆及初步表达

目的 利用基因重组技术构建ＨＢＶ前Ｓ２单链抗体基因并使其在大肠杆菌中永生化和表达活性的抗ＨＢＶ前Ｓ２单链抗体（ＳｃＦｖ），方法和结果 利用基因重组技术，在构建抗ＨＢＶ前Ｓ２３Ｂ９ＳｃＦｖ基因的基础上，在此基因中引入限制性同切酶位点，克隆到噬菌体表达载体－ｐＣＡＮＴＡＢ５噬菌粒中，经亲和筛选得到２株阳性重组克隆，诱导表达产物在特异性阻断ＥＬＩＳＡ实验中具有一定阻断亲本３Ｂ９单抗与ＨＢＶ前Ｓ２抗原的结     

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.     

Study of constancy of hydrogen-consuming flora of human colon

The constancy of the hydrogen consuming flora of the human colon was studied in 15 healthy subjects via two measurements obtained 18 to 36 months apart. Hydrogen disappearance rate and the major products of H₂-consuming bacteria, methane and sulfide, were measured during incubation of fecal homogenates with excess hydrogen and sulfate. In 11/15, the hydrogen consumption rate and the predominant hydrogen-consuming pathway (methanogenesis, sulfate reduction, or neither) remained constant. However, major shifts in these pathways were observed in four subjects, with two losing and two gaining the ability to produce methane. Methanogenesis was associated with the highest hydrogen consumption rate. This study demonstrates that clinically unrecognizable, major alterations of the colonic flora occur in healthy subjects. Understanding of the factors responsible for these alterations might allow for therapeutic manipulation of the colonic flora.Supported in part by the Department of Veterans Affairs and NIDDKD RO1 DK 13309-25.