肝细胞癌肿瘤抑制基因的杂合性缺失和微卫星不稳定性研究

目的　了解肿瘤抑制基因(TSG)杂合性缺失(LOH)与微卫星不稳定性(MSI)在肝细胞癌发生机制中的作用,并探讨其临床病理学意义。方法　采用显微组织切割基础上的DNA直接测序法,从92例手术切除肝细胞癌中筛选出36例信息性肝细胞癌进行6种TSG(APC、DCC、MCC、OGG1、p53和RB1)的LOH检测,对其中15例肝细胞癌进行13个多态性微卫星位点的LOH和MSI检测,并与临床病理学参数的相关性进行统计学分析。结果　TSG的LOH总发生率为41 .7% (15 /36),仅MCC基因未出现LOH。15例肝细胞癌中有9例(60% )发生微卫星LOH,占检测微卫星的46 .2% (6 /13),但无1例肝细胞癌出现MSI。若将APC、OGG1和DCC基因LOH作为Ⅰ型(n=7 ),将p53和RB1基因LOH作为Ⅱ型(n=8)进行统计学处理,则两组肝细胞癌的平均瘤体直径分别为( 2. 9 ±1 .7)cm和(7 .2 ±3 .4)cm (P<0 .01),两组患者术后平均生存期分别为( 72. 0 ±38 6 )个月和(51 .0±30. 4)个月(P<0 .05 )。肝细胞癌基因变异型与患者的年龄、性别、血清甲胎蛋白水平、HBsAg阳性率、合并肝炎/肝硬化、肝细胞癌分化程度和组织学类型之间无明显相关性。结论　在肝细胞癌发生的多阶段演进与多基因变异过程中,LOH路径所起的作用要比MSI路径更大。Ⅰ型基因变异(APC、OGG1和DCC)主要在肝细胞癌早期阶段起作     

肝血管平滑肌脂肪瘤的形态学变异和免疫组织化学特征

目的探讨肝血管平滑肌脂肪瘤(AML)的临床病理学和免疫组织化学特征、诊断和鉴别诊断要点。方法对44例手术切除肝AML的临床病理学特征进行详细分析,并对10种免疫组织化学标志物的表达状况进行检测。结果肿瘤由平滑肌细胞、厚壁血管及脂肪3种成分混合组成,根据瘤组织成分的比例可分为经典型(13例)、肌细胞为主型(25例)、脂肪细胞为主型(4例)、血管瘤型(2例);肌细胞可呈多种形态变异,主要有上皮样细胞型、中间细胞型、梭形细胞型、嗜酸细胞型和多形细胞型5种;8例可见髓外造血。免疫组织化学染色显示,瘤细胞呈HMB45(44／44,100％)、SMA(38／38,100％)和CD117(30／38,78．9％)阳性。结论肝AML形态学变异较大,容易造成误诊,HMB45阳性瘤细胞具有重要的诊断意义,CD117可作为诊断AML的一个有用的辅助标记物。     

携带人livin α基因腺病毒载体的构建及其在树突状细胞的表达

目的:本研究旨在利用AdMax系统构建携带人抗凋亡基因livinα的重组腺病毒载体(rAd-livinα),并感染树突状细胞(Dendritic cells,DCs),制成DCs疫苗,为下一步用此疫苗抗肿瘤实验奠定基础。方法:以质粒pIRES2-EGFP-livinα为模板,通过PCR扩增出人livinα基因cDNA序列,再将livinα基因cDNA亚克隆于穿梭质粒pDC316-EGFP-cmv,获得重组质粒pDC316-EGFP-cmv-livinα。将鉴定后的重组穿梭质粒pDC316-EGFP-cmv-livinα与腺病毒辅助质粒pBHGlox(delta)E1,3Cre共转染HEK293细胞,同源重组获得重组腺病毒rAd-livinα。用重组腺病毒rAd-livinα感染人DCs后,Western blot方法分析livinα蛋白表达,流式细胞仪检测DCs的表型变化。结果:在HEK293细胞内能观察到重组腺病毒rAd-livinα绿色荧光蛋白的表达。rAd-livinα经PCR鉴定特异性地扩增出符合预期大小的片段。rAd-livinα感染的DCs中可检测到livinα蛋白的表达,并且感染的DCs与未感染DCs比较,可明显提高了细胞表面分子CD83、CD86和HLA-DR的表达(P<0.05)。结论:通过AdMax系统成功构建了重组腺病毒rAd-livinα,使livinα蛋白有效表达于DCs中,并且重组腺病毒感染的DCs的成熟度得到提高。     

Activation of the lama2 gene in muscle regeneration: abortive regeneration in laminin alpha2-deficiency

Mutations in laminin alpha2, a subunit of the basement membrane protein laminin-2/merosin, cause merosin-deficient congenital muscular dystrophy. To gain insight into the molecular mechanism of disease, we generated and used a mutant mouse, dyW, in which the lacZ gene was inserted into the lama2 gene so that beta-galactosidase would be expressed in place of laminin alpha2. Heterozygous and homozygous mutant mice are normal at birth, but homozygous mice develop muscular dystrophy at 2 to 3 weeks of age. The lama2/lacZ gene was highly expressed in muscle in the early stages of embryonic myogenesis, but was down-regulated at later stages in both heterozygous and homozygous mice. No beta-galactosidase activity was detected in skeletal muscle after birth in adult heterozygous mice. In contrast, high beta-galactosidase activity was detected in postnatal homozygous mice. Induction of injury in heterozygous mice resulted in intense reexpression of beta-galactosidase in the injured muscle early in regeneration, with a decline in enzyme activity as repair of the tissue progressed. Although the initial response to injury was similar in heterozygous and homozygous mice with abundant beta-galactosidase-positive, mononucleated cells in the injured area, repair was rarely completed in the homozygous mice, evidently caused by excessive death of cells associated with immature myofibers. The defect in muscle repair was very efficiently corrected in homozygous dyW mice expressing a human LAMA2 transgene in skeletal muscle. The data show the importance of laminin alpha2 in muscle regeneration and suggest that a major contributor to disease in muscular dystrophy is abortive regeneration.     

深圳地区肠道病毒71型VP1基因变异趋势研究

目的 研究深圳地区肠道病毒71型VP1基因变异趋势.方法 用肠道病毒71型特异性引物进行RT-PCR,并对EV71的VP1进行扩增,所得的序列与肠道病毒71型A、B、C基因型代表株的核苷酸序列比较,用DNA Star、BioEdit和Mega 3.1软件进行系统进化分析.结果 35株病毒间VP1核苷酸同源性为92.1%-100%,它们与A、B基因型代表株VP1区核苷酸同源性差异较大,为81.4%-91.1%,与C4基因型代表株接近,其核苷酸同源性在93%-97.4%之间.1998-2004年深圳地区EV71主要流行株为C4b亚型,从2003年开始逐步向C4a亚型过渡,至2006年已全部变迁为C4a亚型,且从2003年至2008年,深圳地区EV71 C4a株与安徽阜阳株FY23的核苷酸的同源性分别为：94.5%-94.7%,95.7%-95.8%,96.2%,95.4%-97.5%,96.3%-99.2%,有逐年增高的趋势.2006年两株EV71和2008年EV71代表株核苷酸序列在VP1区的第66位点,均由A→C,从而导致VP1区氨基酸的第22位点由谷酰胺变为组氨酸（Q→H）.结论 深圳地区EV71流行株逐渐由C4b亚型向C4a亚型演变,2006年部分EV71及2008年EV71 VP1第66核苷酸位点发生有义突变.     

Chlorotoxin-modified macromolecular contrast agent for MRI tumor diagnosis

Clinical diagnosis of cancers using magnetic resonance imaging (MRI) is highly dependent on contrast agents, especially for brain tumors which contain blood-brain barrier (BBB) at the early stage. However, currently mostly used low molecular weight contrast agents such as Gd-DTPA suffer from rapid renal clearance, non-specificity, and low contrast efficiency. The aim of this paper is to investigate the potential of a macromolecular MRI contrast agent based on dendrigraft poly-l-lysines (DGLs), using chlorotoxin (CTX) as a tumor-specific ligand. The contrast agent using CTX-modified conjugate as the main scaffold and Gd-DTPA as the payload was successfully synthesized. The results of fluorescent microscopy showed that the modification of CTX could markedly enhance the cellular uptake in C6 glioma and liver tumor cell lines, but not in normal cell line. Significantly increased accumulation of CTX-modified conjugate within glioma and liver tumor was further demonstrated in tumor-bearing nude mice using in vivo imaging system. The MRI results showed that the signal enhancement of mice treated with CTX-modified contrast reached peak level at 5 min for both glioma and liver tumor, 144.97% ± 19.54% and 158.69% ± 12.41%, respectively, significantly higher than that of unmodified counterpart and commercial control. And most importantly, the signal enhancement of CTX-modified contrast agent maintained much longer compared to that of controls, which might be useful for more exact diagnosis for tumors. CTX-modified dendrimer-based conjugate might be applied as an efficient MRI contrast agent for targeted and accurate tumor diagnosis. This finding is especially important for tumors such as brain glioma which is known hard to be diagnosed due to the presence of BBB.     

Immunogenicity and protective efficacy of a fusion protein vaccine consisting of antigen Ag85B and HspX against Mycobacterium tuberculosis infection in mice

Subunit vaccines have the potential advantage to boost Mycobacterium bovis Bacillus Calmette‐Guérin (BCG)‐primed immunity in adults. However, most candidates are antigens highly expressed in replicating bacilli but not in dormant or persisting bacilli, which exist during Mycobacterium tuberculosis infection. We constructed M. tuberculosis fusion protein Ag85B‐Mpt64_190–198‐HspX (AMH) and Ag85B‐Mpt64_190–198‐Mtb8.4 (AMM), which consist of Ag85B, the 190–198 peptide of Mpt64, HspX (Rv2031c) and Mtb8.4 (Rv1174c), respectively. AMH and/or AMM were mixed with adjuvants composed of dimethyl‐dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA‐BCG PSN) to construct subunit vaccines. Mice were immunized thrice with Ag85B, AMH and AMM vaccines and the immunogenicity of the fusion protein vaccines was determined. Then, mice were primed with BCG and boosted twice with Ag85B, AMH, AMM and AMM + AMH vaccines, respectively, followed by challenging with M. tuberculosis virulent strain H37Rv, and the immune responses and protective effects were measured. It was found that mice immunized with AMH vaccine generated high levels of antigen‐specific cell‐mediated responses. Compared with the group injected only with BCG, the mice boosted with AMM, AMH and AMM + AMH produced higher levels of Ag85B‐specific IgG1 and IgG2a and IFN‐γ‐secreting T cells upon Ag85B and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation. It is interesting that only mice boosted with AMM + AMH had significantly lower bacterial count in the lungs than those receiving BCG, whereas mice boosted with AMH or AMM did not. The results suggest that AMH consisting of HspX, the antigen highly expressed in dormant bacilli, could be combined with antigens from replicating bacilli to enhance BCG primed immunity so as to provide better protection against both growing and non‐growing bacteria that occur during the infection process.     

Intranasal immunization with autolysin (LytA) in mice model induced protection against five prevalent <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> serotypes in China

In order to evaluate immunogenicity and protective efficacy of LytA from Streptococcus pneumoniae, we subcloned the full-length lytA-encoded autolysin (LytA) from 5 major pathogenic serotype isolates in China and obtained purified rLytA. Bioinformatics analysis showed that sequences of LytA were highly conserved in all strains we used in this work, and western blot analysis demonstrated that rLytAs from heterogeneous serotypes were cross-recognized by serum of mice infected with 23F strain SH137. Mice were intranasally immunized with purified rLytA, and serum anti-rLytA IgG, IgA and secretory IgA were elicited. More importantly, rLytA intranasal-immunized mice showed a significantly higher survival rate and lower bacterial carriage in response to infection by Streptococcus pneumoniae. The fact that mice immunized with rLytA from strain SH137 also had a higher survival rate after intraperitoneal injection of other four serotype strains of living S. pneumoniae suggested that it possessed cross-protection effect. Our study revealed that intranasal immunization with rLytA may protect mice against mucosal and systemic pneumococcal infection; hence, it was an attractive vaccine candidate.     

甲状腺球蛋白抗体对电化学发光免疫分析法测定甲状腺球蛋白的影响

采用电化学发光免疫分析法(ECLIA)同时测定甲状腺球蛋白(Tg)、自身甲状腺球蛋白抗体(TgAb)值,并用回收实验研究TgAb对Tg测定值的影响。甲状腺疾病患者血样84例,测定Tg、TgAb。回收实验分为三组。第一组分别加入50、100和200 ng/ml的标准Tg;第二组TgAb≥4 000 IU/ml的血样对倍稀释5次,分别测定Tg、TgAb值,并加入100 ng/ml标准Tg;第三组TgAb浓度梯度组,分别加入100 ng/ml标准Tg。计算回收率(R)。结果显示血清对倍稀释后,TgAb实测值逐渐下降;Tg实测值、Tg计算值逐渐上升,两者的差值随稀释倍数的增加而增大。当R<80%时,有22人(91.7%)TgAb>115 IU/ml。ECLIA测定Tg时,TgAb的干扰导致Tg测定值低于真实值,并呈浓度依赖性。TgAb的存在是导致回收率降低的主要原因。