嗜肝细胞腺相关病毒载体的构建

HBV具有天然嗜肝性,其中由HBVpreS1基因编码的前S1(PreS1)抗原位于病毒外壳表面,是HBV嗜肝性的主要分子机制[1].重组腺相关病毒(rAAV)是目前常用的基因治疗载体.我们以腺相关病毒载体系统为基础,用PreS1的编码基因HBVpreS1替代腺相关病毒载体系统的Cap基因,构建出以PreS1为衣壳的重组腺相关病毒载体(rA AVHBVpreS1),旨在探索构建肝病基因治疗特异性载体,为肝病靶向基因治疗提供实验研究基础.     

以乙型肝炎病毒为载体的基因治疗研究

目的 探讨乙型肝炎病毒(HBV)作为肝靶向性基因治疗载体的可能性。方法 用外源报告基因绿色荧光蛋白(GFP)取代HBV S基因读码框构建重组HBV载体,通过脂质体转染HepG2细胞,荧光显微镜下观察外源基因的表达,半巢式聚合酶链反应(PCR)检测细胞核内HBV 共价闭合环状DNA构型,常规PCR和Southern杂交检测上清液重组病毒DNA。结累 携带外源基因GFP的重组HBV载体能够在肝细胞内表达外源蛋白,此重组载体为复制缺损型,单独转染后不能在肝细胞包装与复制,在缺失包装信号ε的辅助HBV质粒下可被包装成携带外源基因的成熟重组HBV颗粒并分泌到胞外。结论 HBV可被改造成肝靶向性基因治疗载体。     

编码小鼠分泌型Klotho蛋白的cDNA克隆及其重组腺相关病毒载体构建

目的 克隆编码小鼠分泌型Klotho蛋白(secKlotho)的全长cDNA片段,构建secKlotho重组腺相关病毒载体.方法 以小鼠肾脏组织mRNA为模板,通过RT-PCR扩增获得小鼠Klotho基因大片段并克隆至pMD18-T载体中,再采用长臂引物进行二次PCR扩增获得编码secKlotho的全长cDNA片段,经EcoR I/Xho I酶切、连接、转化将目的 片段亚克隆至腺相关病毒载体pAAV-MCS中,酶切及DNA测序对阳性克隆进行鉴定.结果 RT-PCR成功扩增出1 600 bp的小鼠Klotho基因大片段,经二次PCR扩增得到编码secKlotho 的全长1 650 bp cDNA片段,并最终获得3个序列信息和读码框完全正确的pAAV/secKlotho克隆.结论 成功构建小鼠分泌型Klotho重组腺相关病毒载体,为Klotho基因转染治疗慢性肾功能衰竭及冠状动脉粥样硬化等疾病奠定了基础.     

HBcAg基因和PreS1（1～65）肽基因真核重组载体的构建和鉴定

目的:构建并鉴定包含人乙型肝炎病毒(HBV)HBcAg基因和PreS1(1～65)肽基因的真核重组载体.方法:采用PCR法扩增HBcAg基因,并通过基因突变在79～80位氨基酸处生成一个Nhe I酶切位点,然后将这段基因连接到真核表达载体pIRES2-EGFP启动子下游的EcoR I和BamH I之间.同时PCR扩增PreS1第1～65氨基酸基因,并在其两端分别引入Nhe I酶切位点,通过酶切、连接,将这段基因插入到HBcAg基因的Nhe I酶切位点上.将构建的重组载体pIREScS1转化大肠杆菌DH5α,分别用上述内切酶双酶切及DNA测序鉴定重组质粒.结果:酶切鉴定示,插入片段大小均与预计相符.测序结果与已知序列结果一致.结论:成功构建了HBcAg基因和PreS1(1～65)肽基因的真核重组载体.     

转基因表达干扰素-α1抑制乙型肝炎病毒的实验研究

目的 研究转基因表达干扰素-α1(IFN-α1)对乙型肝炎病毒(HBV)的抑制作用.方法 采用分子克隆技术,将HBV全基因插入真核细胞表达载体pBK-CMV中,用FuGENETM6,把pBK-HBV转染人肝癌细胞系SMMC-7721,建立HBV瞬时表达和稳定表达两种细胞模型;导入腺相关病毒载体介导的人IFN-α1重组体(pWP8A-IFN-α1),研究转基因表达IFN-α1对HBV的抑制作用.结果 pWP8A-IFN-α1转基因表达,在HBV瞬时表达的细胞培养系统中,对HBV有显著的抑制作用,HBeAg的产生被完全抑制;在HBV稳定表达系统中,对细胞培养液HBeAg的抑制率为70.6%,3 000 IU/ml外源重组IFN-α1b对HBeAg抑制率为53.6%.结论 转基因表达IFN-α1,能有效抑制HBV标志表达,其抑制作用与3 000 IU/ml的外源重组IFN-α1b的抑制作用相当.因而有可能应用转基因IFN-α1对HBV感染人群进行基因治疗.     

重组腺相关病毒载体在脑缺血动物模型基因治疗中的应用

脑缺血的基因治疗是颇具前景的治疗手段,以病毒为载体的基因治疗的有效性和安全性关系到最终治疗的成败。重组腺相关病毒(recombinant adeno-associated virus,rAAV)载体作为目前安全性最好的病毒载体系统,在动物中枢神经系统(centralnervous systerm,CNS)中已进行了有效转导。     

转基因表达干扰素—a1抑制乙型肝炎病毒的实验研究

目的 研究转基因表达干扰素-a1(IFN-a1)对乙型肝炎病毒（HBV）的抑制作用。方法 采用分子克隆技术,将HBV全基因插入真核细胞表达载体pBK-CMV中,用FuGENE^TM6,把pBK-HBV传染入肝癌细胞系SMMC-77721,建立HBV瞬时表达和稳定表达两种细胞模型;导入腺相关病毒载体介导的人IFN-a1重组体（pWP8A-IFN-a1）,研究转基因表达IFN-a1对HBV的抑制作用。结果 pWP8A-IFN-a1转基因表达,在HBV瞬时表达的细胞培养系统中,对HBV有显著的抑制作用,HBeAg的产生被完全抑制;在HBV稳定表达系统中,对细胞培养液HBeAg的抑制率为70.6%,3000IU/ml外源组IFN-a1b对HBeAg抑制率为53.6%。结论 转基因表达IFN-a1,能有效抑制HBV标志表达,其抑制作用与3000IU/ml的外源重组IFN-a1b的抑制作用相当。因而有可能应用转基因IFN-a1对HBV感染人群进行基因治疗。     

小鼠全长膜型Klotho蛋白cDNA表达体系的构建与应用

目的克隆编码小鼠膜型Klotho(mKL)蛋白的cDNA片段,构建、包装Klotho重组腺相关病毒表达体系,并检测rAAV/mKL载体表达功能。方法选择RT-PCR扩增小鼠全长mKL蛋白的基因片段,将该片段亚克隆至腺相关病毒载体pAAV-IRES-hnGFP,采用酶切及DNA测序鉴定;利用AAV-293细胞包装rAAV/mKL,经转染7901细胞,检测其Klotho表达情况。结果本文成功克隆出序列信息和读码框完全正确的3 064 bp的小鼠Klotho基因片段,并构建pAAV/mKL克隆。在AAV-293细胞中包装出rAAV/mKL,病毒原液转染7901细胞后其Klotho mRNA表达上调,而细胞上清液Klotho蛋白也明显增加(P<0.01)。结论成功构建小鼠Klotho重组腺相关病毒载体(pAAV/mKL),获得了rAAV/mKL,并经转染7901细胞验证其基因表达功能正常,这就为进一步研究Klotho基因治疗衰老相关性疾病提供了技术基础。     

丁型肝炎病毒载体携带核酶在小鼠体内抑制乙型肝炎病毒复制

目的:研究用丁型肝炎病毒(HDV)作为载体携带乙型肝炎病毒(HBV)特异性的锤头状核酶所构建的重组体,在细胞体系及转染动物模型中对HBV基因表达和复制的影响.方法:将HDV-核酶重组体和HBV的共表达质粒转染Huh-7细胞以分析HDV-核酶重组体对HBV基因表达的影响;用小鼠尾静脉快速注射法将共表达质粒转染到小鼠体内,检测重组体在动物体内对HBV基因表达和复制的抑制作用.结果:转染细胞中,重组体对HBsAg的抑制与HDV重组位点和核酶靶位都有关;水压法注射的质粒在小鼠肝内得到表达,与对照相比重组HDV-核酶可有效抑制在肝和血清中HBV的基因表达以及复制,与细胞中的结果一致.结论:此项体内实验为进一步构建治疗性重组HDV病毒,发现靶向性抗病毒基因治疗手段奠定基础.     

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。     

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。     

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。     

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.