BSEP在高脂血症大鼠肝组织中表达的状况

目的:通过建立高脂血症大鼠模型,以胆盐输出泵(bile salt export pump,BSEP)为研究对象,探讨BSEP在高脂血症大鼠肝脏中的表达状况,初步思考其与高脂血症形成的相关性,为治疗高脂血症找到新的方向.方法:取♂Wistar大鼠60只,体质量145 g±7.5 g,随机分为2组,每组30只:普通饮食对照组(简称对照组)予以普通饮食、高脂饮食实验组(简称实验组)予以高脂饮食.喂食90 d,建立高脂血症模型,定期取血用自动生化仪检测胆固醇及胆汁酸含量,应用逆转录-聚合酶链反应(RT-polymerase chain reaction,RTP C R)技术检测两组模型肝脏组织的Bsep基因表达的强度,石蜡切片后用免疫组织化学SP(streptavidin-perosidase)法检测两组模型肝脏组织的BSEP表达的强度.结果:实验组胆固醇及胆汁酸含量明显高于对照组;电泳结果显示:实验中肝脏组织扩增产物都出现了内参β-actin基因的425 bp扩增带和Bsep基因的289 bp扩增带,实验组Bsep基因扩增带亮度强于对照组;免疫组织化学SP法结果显示,实验组Bsep表达阳性率为76.7%,对照组Bsep表达阳性率为12.5%,他们之间的差异有统计学意义(χ2=10.773,P<0.05).结论:实验组大鼠的Bsep基因的表达较对照组明显增加,提示我们可发展作用于Bsep的药物,为高脂血症及其相关疾病找到新的治疗方法.     

肝纤维化大鼠肝组织Smads基因表达状况及意义

目的:研究CCl4诱导的肝纤维化大鼠肝组织Smad基因表达的变化及其意义.方法:80只健康雄性SD大鼠分为2组:正常组(C组,n=40)和模型组(M组,n=40).以CCl4 sc 法诱导肝纤维化,HE和VG胶原染色观察肝脏胶原沉积情况,原位杂交法及免疫组化法检测肝组织Smads分子表达水平变化.结果:与C组比较,M组大鼠肝脏组织学积分显著增加(3.29±0.68 vs0,P<0.05),平均胶原面积显著增加(290.86±89.37 μm2 vs 56.12±21.45 μm2,P<0.01),肝组织Smad4蛋白表达率较C组明显增加(4.27%±0.43% vs 2.86%±0.86%,P<0.05),而Smad7蛋白表达虽然增加,但水平低下;Smad 3 mRNA表达A值明显增加(0.167±0.092 vs 0.010±0.002,P<0.05),Smad4 mRNA表达A值也明显增加(0.24 1±0.098 vs 0.021±0.004,P<0.05),Smad6、Smad7 mRNA虽然增加,但表达水平仍然低下.结论:实验性大鼠肝纤维化存在肝脏Smads分子表达水平的比例失调,TGF-Smad信号通路可能参与了肝纤维化的形成与发展.     

抵抗素基因转染诱导肝性胰岛素抵抗细胞的鉴定

目的 将编码人抵抗素基因的pcDNA 3.1(+)真核表达载体在HepG2肝癌细胞中进行稳定转染,以建立抵抗素诱导的肝性胰岛素抵抗细胞模型.方法 实验分3组:HepG2细胞组;空质粒组;抵抗素基因转染组,通过免疫细胞化学和RT-PCR方法 进行抵抗素基因和蛋白表达鉴定,用微量化的葡萄糖氧化酶法检测细胞对葡萄糖的摄取作用.结果 免疫细胞化学染色结果 表明:与对照组比较,抵抗素基因转染组抵抗素蛋白平均光密度值显著升高(P＜0.01);而空质粒组无显著差异(P＞0.05).RT-PCR扩增产物电泳表明:抵抗素基因转染组有目的 条带出现,而对照组和空质粒组无目的 条带出现,细胞葡萄糖摄取实验表明:抵抗素基因转染细胞对葡萄糖摄取作用下降.结论 过度表达人抵抗素基因的胰岛素抵抗的肝细胞建模成功.     

血管活性物质在大鼠肝肺综合征发病机制中的作用

目的:探讨内皮素-1(endothelin-1,ET-1)、一氧化氮(nitric oxide,NO)、降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在大鼠肝肺综合征(hepatopulmonary syndrome,HPS) 发病机制中的作用.方法:健康Wistar大鼠32只随机平均分为假手术组、CBDL术后3 wk组、4 wk组、5 wk组.采用胆总管结扎(common bile duct ligation, CBDL)术制备大鼠HPS模型成功后,行肝功能及肝肺病理检查.放射免疫分析法检测大鼠血浆和肝组织、肺组织匀浆中ET-1和CGRP的水平,利用硝酸还原酶法检测大鼠血清和肝、肺组织匀浆中NO的水平.结果:在大鼠HPS形成过程中,肝细胞变性、坏死,大量纤维组织增生,形成假小叶.肺泡毛细血管增生、扩张.伴肺泡间隔增宽、容量减小.术后3-5 wk,CBDL组大鼠血浆和肝、肺组织匀浆中ET-1、NO、CGRP水平显著升高,且与ALT水平呈正相关(血浆,ET-1:r=0.9889,P=0.0111:NO:r-=0.9935,P=0.0065;CGRP:r=0.9714.P=0.0286;肝组织:r=0.9969,P=0.0035;r=0.9993,P=0.0070;r=0.9507,P=0.0493;肺组织:r=0.9939,P=0.0061;r=0.9991,P=0.0009;r=0.9557,P=0.0443).结论:在大鼠HPS形成过程中,血浆和肝、肺组织匀浆中ET-1,NO和CGRP水平持续升高,提示血管活性物质ET-1,NO和CGRP在其发病机制中可能起着一定作用.     

shRNA干扰SMYD3对肝癌细胞c-Myc表达及凋亡的影响

目的:观察SMYD3(SET and MYND-domain containing 3)基因沉默后c-Myc的表达及对HepG2细胞凋亡的影响.方法:构建针对SMYD3的shRNA干扰质粒Pgenesil-1-s1、Pgenesil-1-s2和阴性对照质粒Pgenesil-1-hk,同时设空白对照组,采用Lipofectamine2000脂质体介导转染法转染质粒.转染后24、48、72 h,RT-PCR检测HepG2细胞SMYD3和c-Myc的表达情况.流式细胞术法检测各组细胞的凋亡.结果:SMYD3、c-Myc基因在HepG2细胞中强表达.RT-PCR显示Pgenesil-1-s1、Pgenesil-1-s2转染组与阴性对照质粒转染组Pgenesil-1-hk转染24、48、72 h后相比,SMYD3基因表达均明显受到抑制 (F=67.46,P<0.01:F=176.79,P<0.01;F=175.28,P<0.01),同时c-Myc表达下调(三组之间:F=11.58,P=0.009; F=126.41,P<0.01;F=261.25,P<0.01).Pgenesil-1-s1、Pgenesil-1-s2转染组细胞早期凋亡率与Pgenesil-1-hk转染组 (LSD-t=-13.58,-12.62,均P<0.01)、空白组(LSD-t=-18.62,-17.67,均P<0.01)相比有显著性差异.结论:RNA干扰技术特异性沉默HepG2细胞SMYD3基因后,抑制了c-Myc的表达,促进了HepG2细胞的凋亡.     

肝细胞生长因子与肝再生增强因子重组表达质粒对大鼠肝纤维化的影响

Objective To evaluate the therapeutic effects of recombinant expression plasmid containing hepatocyte growth factor (HGF) and augmenter of liver regeneration (ALR) on rats with hepatic fibrosis. Methods Ninety Sprague-Dawley rats, which had been established into hepatic fibrosis models, were equally divided into 6 groups: blank group, pcDNA3.1 therapy group,pcDNA3.1-HGF therapy group, pcDNA3. 1-ALR therapy group, pcDNA3.1-HGF and pcDNA3. 1-ALR combined therapy group, and pcDNA3. 1-HGF-ALR therapy group. Zero point one μmol of blank or plasmid was injected into model rats in each group by tail vein once a day for 3 days. Model rats in blank group didn't receive any treatment. Additional 10 rats were chosen as control group, which were not given any interference during the experiment. All rats were sacrificed 4 days after end of treatment. Liver tissues were reserved for observing pathologic changes after HE staining and detecting proliferating cell nuclear antigen (PCNA) and c-jun by immunohistochemistry. Measurement data were compared by single-factor analysis of variance. Comparison between groups was done by SNK test. Enumeration data were analyzed by Fisher's exact test. Results In blank group and pcDNA3.1 therapy group, hyperplasia of fibrous connective tissue was very obvious, false lobules were formed. There was no significant difference between these two groups (x2 =0. 317,P= 1. 000).In the 4 remaining groups, hepatic fibrosis all achieved different degree of amelioration, and the therapeutic effect of pcDNA3.1-HGF-ALR was optimal. In control group, the expressions of PCNA and c-jun in liver tissues were low, with absorbance value of 8.6±1.9 and 3.2 ± 1.2, respectively. In blank group and pcDNA3. 1 therapy group, the expressions of PCNA and c-jun were obviously increased, with absorbance value of 24. 1±3.0, 24.5±4.3 and 23.8±3.1, 24.9±4.2, respectively,which were significant different from control group (all P＜0.01). In the 4 remaining groups, the expressions of PCNA were all obviously increased, and expressions of c-jun were all obviously decreased. The maximum change scope was observed in pcDNA3. 1-HGF-ALR therapy group.Conclusions The recombinant expression plasmid pcDNA3. 1-HGF-ALR can effectively ameliorate experimental hepatic fibrosis of rats. The anti-fibrosis effects are achieved probably by up-regulating PCNA expression and down-regulating c-jun expression.     

肝细胞生长因子与肝再生增强因子重组表达质粒对大鼠肝纤维化的影响

Objective To evaluate the therapeutic effects of recombinant expression plasmid containing hepatocyte growth factor (HGF) and augmenter of liver regeneration (ALR) on rats with hepatic fibrosis. Methods Ninety Sprague-Dawley rats, which had been established into hepatic fibrosis models, were equally divided into 6 groups: blank group, pcDNA3.1 therapy group,pcDNA3.1-HGF therapy group, pcDNA3. 1-ALR therapy group, pcDNA3.1-HGF and pcDNA3. 1-ALR combined therapy group, and pcDNA3. 1-HGF-ALR therapy group. Zero point one μmol of blank or plasmid was injected into model rats in each group by tail vein once a day for 3 days. Model rats in blank group didn't receive any treatment. Additional 10 rats were chosen as control group, which were not given any interference during the experiment. All rats were sacrificed 4 days after end of treatment. Liver tissues were reserved for observing pathologic changes after HE staining and detecting proliferating cell nuclear antigen (PCNA) and c-jun by immunohistochemistry. Measurement data were compared by single-factor analysis of variance. Comparison between groups was done by SNK test. Enumeration data were analyzed by Fisher's exact test. Results In blank group and pcDNA3.1 therapy group, hyperplasia of fibrous connective tissue was very obvious, false lobules were formed. There was no significant difference between these two groups (x2 =0. 317,P= 1. 000).In the 4 remaining groups, hepatic fibrosis all achieved different degree of amelioration, and the therapeutic effect of pcDNA3.1-HGF-ALR was optimal. In control group, the expressions of PCNA and c-jun in liver tissues were low, with absorbance value of 8.6±1.9 and 3.2 ± 1.2, respectively. In blank group and pcDNA3. 1 therapy group, the expressions of PCNA and c-jun were obviously increased, with absorbance value of 24. 1±3.0, 24.5±4.3 and 23.8±3.1, 24.9±4.2, respectively,which were significant different from control group (all P＜0.01). In the 4 remaining groups, the expressions of PCNA were all obviously increased, and expressions of c-jun were all obviously decreased. The maximum change scope was observed in pcDNA3. 1-HGF-ALR therapy group.Conclusions The recombinant expression plasmid pcDNA3. 1-HGF-ALR can effectively ameliorate experimental hepatic fibrosis of rats. The anti-fibrosis effects are achieved probably by up-regulating PCNA expression and down-regulating c-jun expression.     

福州汉族献血者RHD和RHCE基因的多态性分析

目的：探讨福州市汉族献血者RHD和RHCE基因的多态性。方法：应用PCR-SSP等技术分析206名福州市汉族献血者的RHD和RHCE基因。用Arlequin3.5软件计算RHD、RHCE基因频率,检验Hardy-Weinberg平衡,计算最大可能性RH单倍型频率。结果：206名福州市汉族献血者都携带RhD抗原,都存在所检测的RHD基因第1、3、4、5、6、7、9和10外显子。206名献血者中,RHD、RHd、RHDel、RHCe、RHcE、RHce和RHCE基因频率分别为0.9660、0.0267、0.0073、0.6788、0.1667、0.0857和0.0688;DCe、DcE、Dce、DCE、dCe、dce、DelCe和DelcE单倍型频率分别为0.6580、0.1661、0.0733、0.0685、0.0145、0.0122、0.0065和0.0008。福州市汉族献血者RHD、RHEC各基因座基因频率均符合Hardy-Weinberg平衡定律（P〉0.05）。结论：福州市汉族人群RHD、RHd、RHCe、RHcE、RHce和RHCE等基因和DCe、DcE、Dce、DCE、dCe、dce等单倍型都达到多态水平。在RhD血型阳性中国人中,可能有相当数量的人携带诸如RHd、RHD1227A、RHD207A、RHD710delC等基因。     

嗜肺军团菌htpA基因的克隆及其原核表达

目的克隆并检测嗜肺军团菌htpA基因在原核系统中表达情况,为进一步研究HtpA蛋白的免疫性能作必要的准备。方法采用聚合酶链反应(PCR)从嗜肺军团菌基因组DNA中扩得军团菌热休克蛋白A基因htpA,并将其定向克隆至原核表达载体pGEX-4T-1,构建原核表达重组质粒pGhtpA,重组子经限制性内切酶分析、聚合酶链式反应及测序鉴定后,转化宿主菌大肠杆菌JM109,IPTG诱导表达,产物进行SDS-PAGE电泳、免疫印迹分析鉴定。结果扩增出了291bp完整的htpA基因,构建了原核表达重组质粒pGhtpA,并检测到约36kDa的GST-htpA融合蛋白质表达条带。结论成功克隆了嗜肺军团菌htpA基因并使HtpA蛋白在原核表达系统中得到了有效的表达,为进一步研究其免疫学特性奠定了基础。     

恶性疟原虫氯喹抗性的研究进展

恶性疟原虫氯喹抗药性的产生是一个多基因、多因素作用的复杂过程。该文通过对恶性疟原虫氯喹抗性产生机制以及与抗性相关的pfmdr1基因、pfcrt基因和cg2基因等的研究进展进行综述。探讨氯喹抗性产生的机制，为恶性疟原虫氯喹抗性机制的研究及新药设计提供参考。     

胃癌组织中Survivin、PTEN、P53基因的表达及意义

目的　探讨凋亡抑制基因 Survivin和抑癌基因 PTEN、P53在胃癌组织中的表达及其与胃癌发生发展、临床病理特征之间的关系。方法　应用免疫组织化学链霉亲和素方法 (SABC) ,检测 Survivin、PTEN和 P53基因在 6 5例胃癌、2 6例不典型增生胃黏膜、10例浅表性胃炎和 30例正常胃黏膜组织中的表达。结果　胃癌组织中Survivin、PTEN、突变型 P53基因的阳性表达率分别为 70 %、4 8%、6 5 % ,不典型增生胃黏膜中分别为 4 2 %、85 %、31% ,Survivin和突变型 P53基因在正常胃黏膜和浅表性胃炎组织中不表达 ,PTEN基因在正常胃黏膜和浅表性胃炎组织中全部呈阳性表达。胃癌组织中 PTEN基因的表达与正常胃黏膜组织中的表达差异有显著性 (P<0 .0 5 ) ,胃癌组织、不典型增生胃黏膜以及正常胃黏膜中 Survivin和突变型 P53基因的表达差异均有显著性 (P<0 .0 5 )。随临床分期增加、胃癌分化程度降低、浸润深度加深、淋巴结的转移 ,突变型 P53基因的阳性表达率逐渐上升 (P<0 .0 5 ) ,PTEN基因的阳性表达率逐渐降低 (P<0 .0 5 )。在胃癌组织中 Survivin与突变型 P53基因的表达呈正相关 (P<0 .0 5 ) ,与 PTEN基因的表达呈负相关 (P<0 .0 5 ) ,PTEN基因与突变型 P53基因的表达呈负相关 (P<0 .0 5 )。结论　 Survivin、PTEN和 P53     

Loss of heterozygosity at adenomatous polyposis coli,mutation in colorectal cancer and deleted in colorectal cancer genetic loci in colorectal cancers

AIM: To evaluate the role and analyze the loss of heterozygosity (LOH) of adenomatous polyposis coli (APC), mutation in colorectal cancer (MCC) and deleted in colorectal cancer (DCC) genes in the development and progression of colorectal cancers. METHODS: LOH at APC, MCC and DCC genes was examined in 41 surgically resected specimens of colorectal carcinomas by polymerase chain reaction and restriction fragment length polymorphism analysis technique. RESULTS: LOH of APC and MCC were observed in 7 of 25 (28.0%) and 8 of 22 (36.4%) of informative cases, respectively. When considered as one locus, the LOH frequency for APC/MCC was 14 of 36 (38.9%). LOH at DCC gene locus was detected in 21 of 38 (55.3%) of informative cases. No correlation was found between the LOH at APC or MCC gene and tumor histological types, size, invasion, lymph node metastasis and Dukes’ stages (P > 0.05). However, LOH rates at DCC locus in the group with lymph-node metastasis (80.0%) and in Dukes’ stages III and IV (71.4%) were significantly higher than those without lymph node metastasis (39.1%) and in Dukes’ stages I and II (35.3%) (P < 0.05). CONCLUSION: LOH at APC and/or MCC may occur more frequently in the early stages and plays a role in the initiation of colorectal cancer while LOH at DCC is frequent at late event and associated with the progression and metastasis of colorectal cancer.     

黑色素瘤抗原基因MAGE-A1在直肠癌组织的表达

[目的]检测黑色素瘤抗原基因MAGE-A1在直肠癌组织中的表达,探索其与直肠癌临床病理的关系及其在直肠癌免疫治疗中的应用价值。[方法]采用RT-PCR法,对66例直肠癌患者的癌组织、癌旁"正常"黏膜组织和手术切缘组织(乙状结肠端)以及3例直肠息肉标本(无瘤)的MAGE-A1的表达情况进行检测,并对RT-PCR扩增产物中的目的基因片段进行DNA测序验证。[结果]66例直肠癌患者的直肠癌组织、癌旁"正常"黏膜组织、手术切缘组织MAGE-A1基因的表达阳性率分别为30.30%(20/66)、12.12%(8/66)、12.12%(8/66),3例直肠息肉标本未见MAGE-A1表达。肿瘤组织MAGE-A1基因表达阳性率均显著高于癌旁"正常"黏膜组织、手术切缘组织(P0.05);而与年龄、性别、组织学类型、Dukes分期及淋巴结转移无关(P0.05)。[结论]基于MAGE-A1基因在直肠癌中的高表达率,MAGE-A1表达蛋白可以作为一种有前途的靶点用于免疫治疗,同时有望成为一种筛查和随访指标。     

中国大陆人散发性先天性巨结肠症患者内皮素受体B基因的研究

目的 探讨中国大陆人散发性先天性巨结肠症（sHD）易患基因内皮素受体B（EDNRB）的突变与多态性特征。方法 以92例sHD及其中32例患儿双亲为研究对象，并以60例正常儿为对照。提取受检者外周静脉血DNA，采用聚合酶链反应-单链构象多态性技术（PCR-SSCP）对EDNRB基因外显子-2（exon-2）进行分析，并通过DNA测序检测阳性标本的核苷酸改变方式，与文献报道的其他种族sHD同一基因特征做比较。结果 全部标本EDNRB基因exon-2均未发现突变与多态性位点的存在。结论 中国大陆人sHD患者EDNRB基因的exon-2不存在突变与多态性位点。     

粪便基因甲基化分析对大肠癌的早期诊断价值

目的探讨人粪便中分泌型卷曲相关蛋白(SFRP2)及增生性息肉蛋白(HPP1)基因甲基化分析对患者大肠癌诊断的价值。方法从30例结直肠癌患者、30例结直肠腺瘤患者及30例正常对照者的粪便中分别提取DNA,采用甲基化特异性PCR(MSP)技术分析其SFRP2及HPP1基因甲基化状态。结果大肠癌、腺瘤和正常对照组的SFRP2基因甲基化阳性率分别为66.6%(20/30)、50.0%(15/30)和3.3%(1/30)。大肠癌、腺瘤和正常对照组的HPP1基因甲基化阳性率分别63.3%(19/30)、43.3%(13/30)和6.6%(2/30)。结论 SFRP2和HPP1基因甲基化是大肠癌进展过程中的早期事件。粪便SFRP2和HPP1基因甲基化分析可望成为大肠癌早期无创诊断的新途径。     

Whole-genome analysis reveals molecular innovations and evolutionary transitions in chromalveolate species

The chromalveolates form a highly diverse and fascinating assemblage of organisms, ranging from obligatory parasites such as Plasmodium to free-living ciliates and algae such as kelps, diatoms, and dinoflagellates. Many of the species in this monophyletic grouping are of major medical, ecological, and economical importance. Nevertheless, their genome evolution is much less well studied than that of higher plants, animals, or fungi. In the current study, we have analyzed and compared 12 chromalveolate species for which whole-sequence information is available and provide a detailed picture on gene loss and gene gain in the different lineages. As expected, many gene loss and gain events can be directly correlated with the lifestyle and specific adaptations of the organisms studied. For instance, in the obligate intracellular Apicomplexa we observed massive loss of genes that play a role in general basic processes such as amino acid, carbohydrate, and lipid metabolism, reflecting the transition of a free-living to an obligate intracellular lifestyle. In contrast, many gene families show species-specific expansions, such as those in the plant pathogen oomycete Phytophthora that are involved in degrading the plant cell wall polysaccharides to facilitate the pathogen invasion process. In general, chromalveolates show a tremendous difference in genome structure and evolution and in the number of genes they have lost or gained either through duplication or horizontal gene transfer.