以白细胞介素-2为免疫佐剂的幽门螺杆菌粘附素核酸疫苗制备及其免疫预防作用

目的构建含幽门螺杆菌（Hp）粘附素（HpaA）基因和白细胞介素（IL）-2的核酸疫苗，体外转染COS-7细胞，鉴定其表达蛋白的免疫原性和免疫保护作用。方法应用聚合酶链反应（PCR）技术从Hp标准菌株CCUG17874基因组DNA扩增HpaA基因；从重组质粒pCIneo—IL-2扩增小鼠IL-2基因，并通过TA克隆分别克隆人pUCmT载体。检测HpaA及IL-2的核苷酸序列，酶切、连接反应将HpaA和IL-2同时克隆人真核表达载体pIRES，再经PCR法和酶切反应进行鉴定；通过脂质体法将重组载体pIRES-HpaA—IL-2转染COS-7细胞，SDS-PAGE及Western印迹法检测表达蛋白的免疫原性。重组载体转化减毒鼠伤寒沙门菌LB5000，抽提质粒，转化人SL7207，反复传代，鉴定重组核酸疫苗菌的稳定性。以该疫苗菌经口接种小鼠，4周后再用Hp攻击，鉴定感染状况。结果测序结果证实扩增的HpaA基因与HpHpaA序列一致，IL-2序列和小鼠IL-2序列一致。PCR和酶切鉴定结果证实，HpaA和IL-2基因克隆人载体pIRES，成功构建含HpaA和IL-2基因的核酸疫苗质粒pIRES-HpaA—IL-2，Western印迹法检测到相对分子质量分别为30000和14000的HpaA和IL-2蛋白条带。小鼠体内实验显示HpaA—IL-2及HpaA组分别有75．0％、58．4％获免疫保护，与PBS组差异有统计学意义（P〈0．01）。结论成功构建了HpaA和IL-2的Hp减毒沙门核酸疫苗菌，其免疫原性和保护性均得到证实，免疫佐剂IL-2可提高免疫保护率。     

幽门螺杆菌热休克蛋白60核酸疫苗的构建和免疫原性鉴定

背景:核酸疫苗是新兴的第三代疫苗。目前幽门螺杆菌(H.pylori)疫苗的研究主要集中在减毒、灭活和亚单位疫苗方面,有关H.pylori核酸疫苗的研究罕见报道。目的:构建含H.pylori热休克蛋白60(hsp60)基因的核酸疫苗,并鉴定其表达蛋白的免疫原性。方法:抽提H.pylori标准菌株CCUG17874基因组DNA为模板,应用聚合酶链反应(PCR)扩增hsp60基因并插入测序载体pUCmT,测定插入的hsp60基因的核苷酸序列。通过一系列酶切、连接反应将hsp60基因克隆入真核表达载体pIRES,然后转化入感受态大肠杆菌DH5α,筛选阳性克隆,行酶切和PCR鉴定。采用脂质体法将所构建的重组载体pIRES鄄hsp60转染真核细胞COS鄄7,蛋白质印迹法(Western blotting)检测pIRES鄄hsp60表达蛋白的免疫原性。结果:以H.pylori基因组DNA为模板,PCR扩增出约1 640 bp的hsp60基因片断,测序结果表明其与GenBank中H.pylorihsp60原序列的同源性达98%。酶切和PCR鉴定结果证实hsp60基因已克隆入真核表达载体pIRES,成功构建了含hsp60基因的H.pylori核酸疫苗pIRES鄄hsp60。蛋白质印迹法检测显示经pIRES鄄hsp60转染的COS鄄7细胞在约60 kDa(1 Da=0.992 1 u)处出现特异性蛋白条带。结论:成功构建了具有免疫原性的含hsp60基因的H.pylori核酸疫苗,为进一步探索其免疫作用奠定了     

溶藻弧菌热休克蛋白70基因真核表达载体的构建及鉴定

目的构建溶藻弧菌热休克蛋白70(HSP70)真核表达载体pcDNA3.1。方法提取溶藻弧菌基因组DNA,PCR扩增HSP70基因片段,克隆至TA载体,通过PCR、酶切及测序鉴定后,将HSP70基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.1,构建pcDNA3.1重组质粒,通过PCR、酶切及序列分析对HSP70基因pcDNA3.1重组质粒进行鉴定。结果扩增出1 914bp的溶藻弧菌HSP70基因片段,并成功构建溶藻弧菌HSP70真核表达载体pcDNA3.1。结论成功构建了溶藻弧菌HSP70真核表达载体pcDNA3.1,为HSP70基因疫苗研制奠定了基础。     

幽门螺杆菌基因hpaA克隆、融合表达和鉴定

目的克隆幽门螺杆菌粘附素基因hpaA,构建幽门螺杆菌hpaA基因与麦芽糖结合蛋白基因融合表达载体,并进行诱导表达,鉴定融合蛋白免疫原性,为幽门螺杆菌疫苗研究提供依据。方法利用PCR技术从H.pylori郑州分离株MEL-HP27染色体DNA上扩增出hpaA基因,序列分析后,将其克隆到表达载体pMAL-c2X中,转化大肠杆菌(E.coliTB1),用IPTG诱导目的基因表达,SDS-PAGE方法对表达产物进行分析,Western blot鉴定其免疫原性。结果用PCR方法扩增的hpaA基因长度为783bp,编码260个氨基酸,经酶切鉴定和测序,插入到克隆载体的基因片段与预期目的DNA片段相一致;SDS-PAGE结果显示表达产物相对分子质量约为29kDa,融合蛋白的表达量约占全菌总蛋白的26%。结论本研究成功构建了hpaA基因与麦芽糖结合蛋白基因融合原核表达系统,为幽门螺杆菌基因工程组分疫苗的研究奠定基础。     

弓形虫GRA8基因真核表达重组质粒的构建、鉴定及测序

目的 构建弓形虫RH株pcDNA3，1( )-GRA8真核表达重组质粒，为进一步DNA免疫做准备。方法 用聚合酶链反应(PCR)从弓形虫RH株的基因组DNA中扩增编码致密颗粒蛋白(GRA8)的基因片段，纯化后重组入pUC-19克隆载体，再经含IPTG，XGal氨苄培养基蓝白筛选，挑选白色克隆酶切，低溶点琼脂糖纯化，回收目的基因亚克隆入pcDNA3．1( )，经氨苄培养基过夜培养挑选6个克隆提纯酶切，PCR鉴定和测序。结果 从RH株基因组DNA中扩增出特异的GRA8基因片段，克隆成功pcDNA3．1( )-GRA8重组质粒。测序表明GRA8这部分基因与GENEBANK相应基因序列完全一致。结论 本结果为研究抗弓形虫核酸疫苗打下基础。     

HIV-1B亚型核心蛋白gag基因真核表达载体的构建及其在HepG2细胞中的表达

目的克隆人类免疫缺陷病毒Ⅰ型B亚型核心蛋白gag基因,构建真核表达载体,并在真核细胞中表达,为进一步制备自行设计的以λ噬菌体作为载体的HIV核酸疫苗奠定基础。方法以克隆好的HIV1B亚型U26942全基因质粒DNA作为模板,根据Genbank中gag基因的核苷酸序列设计引物,并在引物的5’端分别引入BamHⅠ及XhoⅠ酶切位点,特异性的扩增gag基因。TA克隆后经双酶切、测序等鉴定重组质粒,再经双酶切、连接构建含gag编码基因的真核表达载体,并进行酶切鉴定分析pcDNA3.1(+)/gag。在脂质体介导下转染HepG2细胞,经G418压力筛选建立稳定转染gag基因的细胞系,用RT PCR及Western印迹检测其在HepG2细胞中的表达。结果重组质粒经BamHⅠ、XhoⅠ双酶切成5.4kb与1.5kb的片断,表明表达载体pcDNA3.1(+)中插入了gag基因片断,测序结果表明编码框正确。RT PCR及Western印迹证实稳定转染gag基因的HepG2细胞系中有该基因的表达。结论成功构建了HIV1B亚型核心蛋白gag基因的真核表达载体pcDNA3.1(+)/gag,并在HepG2细胞中获得稳定表达。     

幽门螺杆菌中性粒细胞激活蛋白核酸疫苗实验研究

目的 构建幽门螺杆菌(Hp)中性粒细胞激活蛋白(Hp-NAP)口服DNA疫苗，初步评价其免疫治疗作用，为Hp疫苗研制奠定基础。方法 PCR扩增全长Hp-NAP基因(napA)，测序并同源性分析后，亚克隆入真核表达载体pIRES，双酶切并PCR鉴定。将重组质粒plRES-napA转化减毒鼠伤寒沙门菌SL7207，口服接种长期感染Hp之BALB／c小鼠，观察疗效。结果PCR扩增出-435bp产物，序列分析表明，所克隆序列与CenBank中SSl-napA核苷酸及蛋白质同源性均＞98％。PCR及双酶切证实，成功构建了携带napA的重组减毒鼠伤寒沙门菌DNA疫苗。疫苗接种后4周，治疗组3／4小鼠快速尿素酶检测阴性，对照组均阳性(P=0．0476)；治疗组血清抗Hp-NAP抗体效价明显升高。结论 成功构建了具有较好免疫治疗作用的Hp—NAP口服重组DNA疫苗，为进一步研制多价抗HpDNA疫苗奠定了基础。     

编码弓形虫表面抗原P30、P22复合基因真核表达载体的构建

目的　构建编码弓形虫RH株表面抗原P30、P22复合基因的真核表达重组质粒, 为进一步表达融合蛋白及研制核酸疫苗做准备。　方法　用弓形虫RH株腹腔接种小鼠,收集腹水,酚/氯仿法抽提弓形虫基因组 DNA;用 PCR技术从基因组DNA中扩增编码表面抗原 P30、P22 的基因片段,分别重组入 pMD18 T载体中。将 pMD18 T载体中的P30、P22基因片段分别酶切,定向克隆入 pUC18克隆载体中, pUC18 P30 P22 中的 P30 P22 片段经酶切、纯化后,亚克隆入 pcDNA3.1( )真核表达载体,用酶切、PCR及测序的方法对重组子进行鉴定。　结果　从弓形虫 RH株基因组DNA中扩增出特异的P30及P22片段;大小均与预测值相符;克隆 pUC18 P30 P22 重组质粒的酶切片段分别与 P30、P22基因大小一致;经亚克隆、筛选鉴定获得了 pcDNA3.1 P30 P22重组质粒,所测P30、P22基因序列与文献报道一致。结论　成功构建弓形虫 pUC18 P30 P22重组质粒和 pcDNA3.1 P30 P22 重组质粒,为研制弓形虫 DNA疫苗奠定了基础。     

编码弓形虫表面抗原P30、P22复合基因真核表达载体的构建

目的 构建编码弓形虫RH株表面抗原P30、P22复合基因的真核表达重组质粒，为进一步表达融合蛋白及研制核酸疫苗做准备。方法 用弓形虫RH株腹腔接种小鼠，收集腹水，酚／氯仿法抽提弓形虫基因组DNA；用PCR技术从基因组DNA中扩增编码表而抗原P30、P22的基因片段，分别重组入pMD18T载体中。将pMD18-T载体中的P30、P22基因片段分别酶切，定向克隆入pUC18克隆载体中，pUC18-P30-P22中的P30P22片段经酶切、纯化后，亚克隆入pcDNA3．1(-)真核表达载体，用酶切、PCR及测序的方法对重组子进行鉴定。结果 从弓形虫RH株基因组DNA中扩增出特异的P30及P22片段；大小均与预测值相符；克隆pUC18-P30P22重组质粒的酶切片段分别与P30、P22基冈大小一敛：经亚克隆、筛选鉴定获得了pcDNA3．1-P30-P22重组质粒，所测P30、P22基因序列与文献报道一致。结论 成功构建弓形虫pUC8-P30P21重组质粒和pcDNA3．1-P30-P22重组质粒，为研制弓形虫DNA疫苗奠定了基础。     

弓形虫ROP2基因的克隆与鉴定

目的 体外扩增弓形虫棒状体分泌抗原2（ROP2）靶基因，构建真核表达载体pc-DNA3-ROP2。 方法 收集、纯化RH株弓形虫速殖子，提取基因组DNA；根据基因库ROP2基因序列设计合成1对引物，应用PCR扩增ROP2基因片段，回收纯化后克隆入TA载体质粒pUCm-T；用限制性内切酶EcoRⅠ、HindⅢ双酶切该重组子，将切下的ROP2基因在T4DNA连接酶作用下插入真核细胞表达载体质粒pc-DNA3，并进一步作双酶切、PCR及测序鉴定。 结果 以弓形虫基因组DNA为模板，PCR扩增出1.7 kb ROP2基因片段，克隆于pUCm-T载体中，再将ROP2基因亚克隆于真核表达载体质粒pc-DNA3，经筛选鉴定，构建pc-DNA3-ROP2重组质粒；测序结果显示，重组质粒包含了ROP2蛋白基因读码框内的完整序列，能完整表达ROP2的抗原蛋白。 结论 弓形虫ROP2基因片段，经TA克隆及亚克隆，构建弓形虫pc-DNA3-ROP2重组质粒。     

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.     

治疗高血压药物的经济学评价

重视高血压治疗中的经济学评价,对利用我国有限的卫生资源来遏制高血压对人民群众的危害有着重要的现实意义。药物经济学对于药物治疗的成本和治疗的结果给予同样的关注。因为治疗高血压的费用,不仅涉及药物价格,还包括患者的危险水平,降压疗效和对临床终点事件的影响,以及治疗的依从性和安全性。因此药物经济学更强调整体成本和价-效比。低危病人,若非药价低廉,治疗的价-效比不够理想。而在高危的患者,价-效比越小越经济而不是药费越便宜越好。