利用组织芯片技术研究P73和mP53基因编码蛋白在胃癌中的表达及意义

目的:构建胃癌及其癌前病变组织芯片,采用免疫组化方法观察研究P73和mP53基因编码蛋白在胃癌中的表达并探讨其临床病理学意义.方法:收集2003-2004年辽宁省肿瘤医院和中国医大附属一院104例胃癌及癌前病变组织标本构建两个组织芯片蜡块,组织样品直径为1 mm.采用SABC免疫组化方法检测胃癌组织中P73和mP53蛋白的表达,观察分析其与胃癌病理生物学行为的关系.结果:P73基因编码蛋白在胃癌、肠上皮化生、不典型增生病变组织中的阳性表达率显著高于远癌正常胃黏膜(90.1%,44.0%,80.0%vs 17.9%,P＜0.01).Borrman Ⅲ/Ⅳ型胃癌P73蛋白阳性表达率(92.9%/100%)显著高于BorrmanⅡ型胃癌(57.1%)(P＜0.05).伴转移胃癌组P73蛋白阳性表达率(淋巴结转移组94.4%,肝转移组100%,卵巢转移100%)显著高于无转移组(76.2%)(P＜0.05).胃癌组织中P73蛋白表达与mP53蛋白表达密切相关(χ2=9.6736,P＜0.01).结论:P73蛋白表达与胃癌恶性病理生物学行为密切相关,其虽与抑癌基因P53同源,但与mP53蛋白表达呈正相关,提示其可能作为P53的一种模拟突变体在胃癌发生、发展中起作用.     

变异链球菌LuxS基因突变株生物被膜结构的电镜观察

目的：探讨变异链球菌Ingbritt C（血清C型）国际标准株与LuxS基因缺失的变异链球菌突变株之间生物被膜的形成差异。方法：分别将变异链球菌标准株和LuxS基因突变株接种于含有2％葡萄糖、2％蔗糖的乳酪消化胨酵母（TPY）液体培养基中,以牙釉质磨片为载体,于扫描电镜下观察形成24h的上述两菌株生物被膜。结果：（1）当以葡萄糖作为补充糖源时,变异链球菌标准株和LuxS基因突变株所形成的生物被膜表现型未见明显差异;（2）当以蔗糖作为补充糖源时。两菌株所形成的生物被膜有明显的不同,标准株生物被膜相对平滑均质,而LuxS基因突变株的生物被膜呈松散的蜂房状,基质间有较大的间隙,形成较大的团簇状菌落;（3）加入标准株的无菌体上清液可以使LarxS基因突变株的生物被膜表型部分恢复正常,提高LuxS基因突变株形成生物被膜的能力。结论：变异链球菌标准株和LuxS基因突变株均能形成生物被膜．但LuxS基因突变株的生物被膜结构发生改变;依赖于LuxS的群体感应系统会影响变异链球菌生物被膜的形成。     

Mutant ubiquitin and p62 immunoreactivity in cases of combined multiple system atrophy and Alzheimer’s disease

Recent studies have shown the co-existence of α-synuclein and phosphorylated tau (pTau) in several neurodegenerative diseases. Here, we report two autopsy cases of combined multiple system atrophy (MSA) and Alzheimer’s disease (AD). In both cases, abundant α-synuclein-positive glial and neuronal cytoplasmic inclusions were found in the brainstem, amygdala and hippocampal formation. pTau-positive neurofibrillary tangles (NFTs) were widely distributed in case 1 (Braak stage VI) and moderate in case 2 (Braak stage III). Although α-synuclein and pTau pathology co-occurred in the hippocampus and entorhinal cortex, only a few neurons showed co-existence of these two proteins. Immunoreactivity for p62, a ubiquitin proteasome system related protein, was found in the majority of NFTs, but in only a small proportion of neuronal α-synuclein inclusions. In addition, UBB+1, a mutant form of ubiquitin and a marker for proteasomal dysfunction, was present in the majority of NFTs, whereas co-existence of α-synuclein and UBB+1 was found in only a few neurons. These findings indicate that α-synuclein and phosphorylated tau co-occur in certain brain regions in cases of combined MSA and AD and that the proteasomal pathways differ between α-synuclein- and pTau-bearing neurons.     

Combination of receptor-binding assays and designed mutant receptors for discerning agonists and antagonists

Competitive receptor-binding assays are convenient for analyzing interactions between receptors and their ligands and for screening pharmaceutical drugs and potential endocrine-disrupting chemicals. Although these assays can be used for high-throughput screening, they cannot discern antagonists and agonists. Based on three-dimensional structures of complexes between ligand-binding domain of human estrogen receptor-alpha and its ligands, we designed mutant receptors with modified mode of ligand-binding. In the current studies we examined the binding of endogenous ligands, artificial ligands, and potent endocrine-disrupting chemicals to wild-type and Asp351 mutants of the human estrogen receptor-alpha ligand-binding domain. The new combination assay showed the decrease of relative biding affinity (RBA) values for antagonists. For example, RBA for tamoxifen was changed from 4.8 (using the Asp351 receptor) to less than 1.5 (using the Glu351 receptor). On the other hand, the agonists showed increase of RBA values. For example, RBA for bisphenol A was changed from 0.011 (using the Asp351 receptor) to less than 0.030 (using the Glu351 receptor). The variation of RBA was dependant on the type of mutant receptors. The change of RBA from wild-type to mutant-type can be an index for discerning agonists and antagonists. Comparison of RBA values obtained by assays using wild-type and mutant receptors is a simple way of discerning agonists and antagonists, and this approach could be extended to other types of receptors, if information of the receptors was enough to construct a designed mutant receptor.     

含精氨酸-甘氨酸-天冬氨酸肽白细胞介素-24突变体蛋白的表达及其生物活性

目的 获得含精氨酸-甘氨酸-天冬氨酸(RGD)肽的白细胞介素(IL)-24突变体(RGD-IL-24)蛋白,研究其生物活性.方法 用重叠聚合酶链反应(PCR)技术在IL-24 cDNA序列中引入甘氨酸密码子GGC,构建RGD-IL-24 cDNA,将其克隆人质粒pET28a(+)获得pET28a/RGD-IL-24.pET28a/RGD-IL-24转化菌株BL21(DE3),采用亲合层析纯化蛋白,透析复性.SDS-PAGE电泳和Western blot鉴定RGD-IL-24蛋白.酶联免疫吸附试验(ELISA)法检测RGD-IL-24刺激外周血单核细胞(PBMC)产生IL-6、肿瘤坏死因子(TNF)-α和干扰素(IFN)-γ的能力来测定RGD-IL-24免疫活性.细胞黏附实验检测RGD-IL-24与多种肿瘤细胞结合特性.结果 成功获得RGD-IL-24基因,并构建表达质粒pET28a/RGD-IL-24.表达的RGD-IL-24蛋白约占菌体总蛋白的30%,纯化后蛋白纯度超过90%.RGD-IL-24处理的人PBMC培养上清IL-6、TNF-α和IFN-γ浓度(ng/L)分别为(780.5 ±98.3、2399.1 ±113.2、2130.1 ±98.6),显著高于IL-24处理组(551.0±52.1、1982.5 ±138.2、1762.9 ±119.8).黏附实验证实RGD-IL-24能增强与肿瘤A549、HeLa和ACHN细胞靶向结合作用.结论 含RGD肽的RGD-IL-24蛋白免疫生物活性及与肿瘤细胞靶向结合能力较IL-24明显增强.     

Oral MucoRice expressing double-mutant cholera toxin A and B subunits induces toxin-specific neutralising immunity

Rice-expressed cholera toxin B (CTB) subunit is a cold-chain-free oral vaccine that effectively induces enterotoxin-neutralising immunity. We created another rice-based vaccine, MucoRice, expressing nontoxic double-mutant cholera toxin (dmCT) with CTA and CTB subunits. Western-blot analysis suggested that MucoRice-dmCT had the shape of a multicomponent vaccine. Oral administration of MucoRice-dmCT induced CTB- but not CTA-specific serum IgG and mucosal IgA antibodies, generating protective immunity against cholera toxin without inducing rice-protein-specific antibody responses. The potency of MucoRice-dmCT was equal to that of MucoRice-CTB vaccine. MucoRice has the potential to be used as a safe multicomponent vaccine expression system.     

Abnormal development of pacinian corpuscles in double trkB;trkC knockout mice

Pacinian corpuscles depend on either A or Aβ nerve fibers of the large- and intermediate-sized sensory neurons for the development and maintenance of the structural integrity. These neurons express TrkB and TrkC, two members of the family of signal transducing neurotrophin receptors, and mice lacking TrkB and TrkC lost specific neurons and the sensory corpuscles connected to them. The impact of single or double targeted mutations in trkB and trkC genes in the development of Pacinian corpuscles was investigated in 25-day-old mice using immunohistochemistry and ultrastructural techniques. Single mutations on trkB or trkC genes were without effect on the structure and S100 protein expression, and caused a slight reduction in the number of corpuscles. In mice carrying a double mutation on trkB;trkC genes most of the corpuscles were normal with a reduction of 17% in trkB−/−;trkC+/− mice, and 8% in trkB +/−;trkC −/− mice. Furthermore, a subset of the remaining Pacinian corpuscles (23% in trkB−/−;trkC+/− mice; 3% in trkB+/−;trkC−/− mice) were hypoplasic or atrophic. Present results strongly suggest that the development of a subset of murine Pacinian corpuscles is regulated by the Trk-neurotrophin system, especially TrkB, acting both at neuronal and/or peripheral level. The precise function of each member of this complex in the corpuscular morphogenesis remains to be elucidated, though.     

Adaptation of HIV-1 to cells expressing rhesus monkey TRIM5α

The cross-species transmission of retroviruses is limited by host restriction factors that exhibit inter-species diversity. For example, the TRIM5α proteins of Old World monkeys block the early, post-entry steps in human immunodeficiency virus (HIV-1) infection. We adapted an HIV-1 isolate to replicate in cells expressing TRIM5α_rh from rhesus monkeys, an Old World species. A single amino acid change in the cyclophilin-binding loop of the HIV-1 capsid protein allowed virus replication in cells expressing TRIM5α_rh. The capsid of the escape virus exhibited a reduced affinity for TRIM5α_rh, but retained the ability to bind cyclophilin A efficiently. Thus, a preferred HIV-1 escape pathway involves decreased binding to TRIM5α, a capsid-destabilizing factor, and retention of binding to cyclophilin A, a capsid-stabilizing factor.     

rmhTNF对人胃癌细胞株裸鼠移植瘤模型的治疗作用

背景:重组改构人肿瘤坏死因子(rmhTNF)是原型TNF-α的改构体,前期实验显示其对体外培养的人胃癌细胞株具有抑制增殖和诱导凋亡作用。目的:初步研究rmhTNF对人胃癌细胞株裸鼠移植瘤模型的治疗作用。方法:雄性BALB/c裸鼠皮下接种人胃癌细胞株BGC-823构建移植瘤模型,随机予rmhTNF、TNF-α,5-氟尿嘧啶(阳性对照)和0.9%NaCl溶液(阴性对照)进行干预,观察各组裸鼠一般情况、移植瘤生长情况及其组织病理学改变。结果:建模裸鼠成瘤率为100%。各药物干预组移植瘤生长均明显减慢,其中rmhTNF组生长抑制最为明显,生长曲线较阴性对照组明显下移,抑瘤率显著高于TNF-α组和阳性对照组(83.1%对59.8%和50.1%,P0.01),一般情况与接种前相比无明显改变,移植瘤组织中可见较多凋亡和坏死细胞。结论:BGC-823细胞在BALB/c裸鼠皮下有良好的成瘤性。rmhTNF在体内能直接诱导胃癌细胞凋亡和坏死,对人胃癌细胞株裸鼠移植瘤模型具有治疗作用。     

Decline in Daily Running Distance Presages Disease Onset in a Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of lower motor neurons resulting in paralysis and death. Epidemiological and clinical findings suggest that a decline in athletic performance may presage the clinical onset of ALS, but this possibility has not been tested in an animal model. By placing running wheels in each mouse’s cage to measure their exercise activity, we show that presymptomatic G93A SOD1 ALS mice are more active runners (15–20 km/day) than control mice (7–9 km/day). The ALS mice then exhibit a sharp decline in daily running distance 10–20 days prior to the onset of clinical disease. Within the group of ALS mice, there were no significant correlations between cumulative lifetime running distance and age at clinical disease onset or age at death, suggesting that amount of exercise did not affect the course of the disease process. Our data show that presymptomatic ALS mice have a propensity for running long distances, and then dramatically reduce the amount they run prior to the appearance of clinical symptoms. The monitoring of voluntary running distance may provide a valuable biomarker to evaluate the efficacy of potential therapeutic interventions for ALS in preclinical studies. Daniel A. Bruestle and Roy G. Cutler contributed equally to this work.