豚草花粉泛过敏原同源基因克隆与序列分析

目的　克隆豚草花粉泛过敏原同源基因。方法　采用生物信息学分析方法对众多的花粉过敏原基因进行序列同源性比较 ,以序列保守区域为依据设计合成简并引物 ,在特殊的RT PCR条件下 ,结合RACE技术对豚草花粉中的过敏原全长基因进行克隆 ;通过Northern杂交及序列分析初步确定基因产物是否为花粉过敏原。结果　获得了 3个新的全长基因。序列分析显示 :所得过敏原同源基因与数十种不同种属来源的过敏原肌动蛋白结合蛋白 (profilin)具有较高的同源性 ,初步认定其为泛过敏原 ,并暂命名为Amba 8(t)。Northern杂交证实该基因在花粉中表达。结论　采用本研究方法成功地在豚草花粉中克隆到 3个泛过敏原基因 ,为豚草花粉重组过敏原的蛋白质表达及标准化奠定了物质基础     

采用简并引物克隆葎草花粉过敏原全长同源cDNA

目的：建立一套稳定可靠的方法，对过敏原性物种中的过敏原同源基因进行快速克隆。方法：在分析生物信息数据库中积累的大量过敏原序列同源性的基础上，设计简并引物，基于高质量Lǘ草花粉RNA，逆转录合成cDNA。采用Touchdown方式在cDNA池中进行选择性PCR扩增。同时借助梯度PCR程序，对引物扩增的简并性作进一步强化，并结合RACE技术获取全长cDNA．进而对Lǘ草花粉中的过敏原同源基因进行克隆。结果：成功地获得3个全长cDNA克隆。序列分析显示．这些基因与已知过敏原的基因序列相似性高达79％-85％，初步认定其为泛过敏原肌球蛋白抑制蛋白(profilin)的同源基因。对比RACE技术获得的相应基因的全长序列发现，这些序列在引物结合处与简并引物序列之间存在4个碱基的差异，提示采用Touchdown方式的梯度PCR程序．可使引物的简并性得到进一步扩展。结论：简并引物与Touchdown梯度PCR相结合的方法．能够对以Lǘ草为代表的基因组未曾深入研究的物种中的过敏原同源基因进行有效克隆.     

刺苋花粉泛过敏原Profilin的抗原性评估与三级结构分析

目的 克隆刺苋花粉中Profilin蛋白基因,分析同源序列中不同性质氨基酸对抗原性及j级结构的贡献.方法 基于生物信息学分析已知泛过敏原Profilin的氨基酸序列并获得核心代表序列,以之为基础设计合成引物,采用Touchdown PCR技术从刺苋花粉的cDNA池中进行基因克隆,并经菌落PCR和双酶切验证;采用生物信息学软件MULTIPRED及SWISS-MODEL在线软件对所得基因编码蛋白进行抗原性评估及三级结构模拟.结果 从刺苋中获得两个序列不同的泛过敏原基因,分别命名为PRF7和PRF23.蛋白质三级结构显示:PRF7与已知的泛过敏原Profilin存在少数氨基酸的筹异,其空间结构与抗原性没有明显变化;而PRF23与北方豚草花粉泛过敏原Q64LH0之间相似程度较低,而空间结构也呈现明显的筹异;尽管Q64LH0与PRF23的伞序列抗原性均值差异无统计学意义,受一些区段上不同性质的氨基酸改变的影响,PRF23在这些区段上的抗原性显著低于Q64L-HO的抗原性.结论 基于Q64LHO和PRF23同源氨基酸序列的抗原性评估及三级结构分析,获得了不同性质氨基酸对抗原性及三级结构的贡献等信息,映射了南方花粉过敏症发牛率较低的原因所在,也为过敏原遗传改良过程中进行氨基酸置换指明了方向.     

金黄色葡萄球菌肠毒素A成熟肽基因序列的克隆及抗原性分析

目的 扩增金黄色葡萄球菌 (金葡菌)肠毒素A(SEA)基因并对其进行抗原性分析,为后续抗原性弱化及安全性免疫毒素的构建提供依据.方法 以金葡菌基因组DNA为模板,以SEA编码基因特异区段为引物,采用Touchdown PCR克隆SEA成熟肽编码基因,经菌落PCR筛选出阳性克隆后再进行DNA测序及BL2SEQ比对分析,并应用生物信息学软件对所得基因编码蛋白各区段的抗原性进行分析.结果PCR产物克隆后,菌落PCR挑选得到阳性克隆,DNA测序后进行的BL2SEQ比对显示,所得序列与GenBank中登录的序列之间一致性达100%.SEA成熟肽各区段上的抗原性均值较高,同时,有几个突出高值点分布在不同的区段上.结论 应用特定的生物信息学方法 从理论上确定了SEA成熟肽的高抗原性位点,为后续弱化SEA的抗原性研究提供了依据.     

乙肝病毒核心区与"a"决定簇嵌合基因的体外表达及抗原性分析

目的评价乙肝病毒核心区与"a"决定簇嵌合基因表达产物的抗原性.方法应用基因工程技术将编码截短的乙型肝炎病毒核心抗原的主要免疫显性区替换为表面抗原"a"决定簇的基因片段,将嵌合基因装入原核表达载体pRESET-B内,在体外进行诱导表达,纯化目的蛋白并检测其抗原性.同时将嵌合基因亚克隆到真核表达载体pcDNA3内,瞬时转染 BHK细胞并采用间接免疫荧光法检测融合蛋白表达情况.结果诱导并纯化得到了分子量为28 ku的蛋白,经检测证实该蛋白具有一定的HBsAg抗原性和较弱的HBcAg抗原性.真核表达质粒可在 BHK细胞内表达,可检出良好的HBsAg抗原性及较弱的HBcAg抗原性.结论该嵌合质粒表达产物具有良好的HBsAg抗原性及较弱的HBcAg抗原性.     

铜绿假单胞菌外毒素A免疫毒素片段的抗原性评估及三级结构模拟

目的 克隆铜绿假单胞菌外毒素A(PEA)基因及分析不同克隆序列中不同性质氨基酸对抗原性及三级结构的贡献.方法 以铜绿假单胞菌基因组DNA为模板,以PEA编码基因特异区段为引物,采用Touchdown PCR克隆PEA编码基因.阳性克隆经菌落PCR鉴定后进行DNA测序及Clustal X(1.83)比对分析;采用生物信息学在线软件BIMAS及SWISS-Model对所得基因编码蛋白进行抗原性评估及三级结构模拟.人为置换关键部位的氨基酸残基,考察不同性质氨基酸对三级结构的影响.结果 从铜绿假单胞菌DNA中获得3个克隆.抗原性分析及三级结构模拟结果显示,3个克隆与公开发表的序列之间存在少数氨基酸的差异;个别区段上氨基酸的改变可引起相应位点抗原性的改变,但仅有个别位点的改变对空间结构产生影响.结论 基于抗原性评估及三级结构分析,获得了不同性质氨基酸对抗原性及三级结构的贡献等信息,为PEA编码基因用于弱化抗原性靶向性毒素的构建奠定了基础.     

诱发口腔鳞癌细胞凋亡嵌合基因表达载体的构建

目的：获取含凋亡基因fas表达调控元 件cdc25A片段和fas开放阅读框架的cdc25A -fas嵌合基因，构建和鉴定真核表达载体pAdTrack-CMV-cdc25A -fas和pAdTrack-cdc25A-fas，为将口腔鳞癌细胞固有的 促细胞增殖信号转变为促细胞凋亡信号的研究奠定基础。方法:根据fa s基因、cdc25A已知序列设计合成引物，采用PCR技术从pBLF58-1质 粒中扩增得到嵌合基因cdc25A-fas；然后定向克隆至真核表达载 体pAdTrack-CMV和pAdTrack，分别转化大肠杆菌E.coli DH5α感受态细胞；卡那霉素筛 选阳性克隆；进行PCR、酶切鉴定和序列分析。结果：PCR扩增得到特异 的 1 603 bp的cdc25A-fas片段；筛选并鉴定得到含pAdTrac k-CMV-cdc25A-fas和pAdTrack-cdc25A- fas的大肠杆菌E.coli DH5α阳性克隆。结论:成功构建了真核表 达载体pAdTrack-CMV-cdc25A-fas和pAdTrack-cdc25 A-fas,为下一步研究其在肿瘤基因治疗中的作用奠定基础。     

奶牛主要过敏原β-乳球蛋白基因的克隆及其原核表达载体的构建

目的克隆奶牛主要过敏原β-乳球蛋白（BLG）基因及其原核表达载体的构建。方法RT-PCR克隆奶牛主要过敏原BLG的全长基因,根据序列设计带有酶切位点的特异性引物,扩增奶牛BLG基因的完整开放阅读框,与pET-28a载体连接,构建原核表达载体。结果克隆了奶牛主要过敏原BLG基因,且构建了其原核表达载体。该基因含有长度为537bp的开放阅读框,编码178个氨基酸。该蛋白质的相对分子质量为19883,等电点为5.14（Gen Bank数据库中的登录号为EU883598）。序列同源性分析发现其与数据库中已知的BLG基因同源性很高。结论成功克隆了奶牛主要过敏原BLG基因及其原核表达载体的构建,为进一步奶牛主要过敏原BLG的重组表达和免疫活性鉴定等奠定基础。     

免疫球蛋白可变区基因的体外扩增、序列分析及人-鼠嵌合重链抗体的表达

本文用聚合酶链反应法(PCR)扩增了抗人小细胞肺癌单克隆抗体的轻、重链可变区基因(V_k、V_H基因)。逐步克隆将V_H基因与表达质粒重组,最终与载有人恒定区基因的载体拼接,得嵌合质粒的在哺乳类细胞中表达了人-鼠嵌合重链抗体。 1 免疫球蛋白可变区基因的体外扩增用酸性异硫氰酸胍-苯酚-氯仿抽提抗人小细胞肺癌单克隆抗体的细胞,得较高质量的总RNA,用oligo(dT)为引物合成第一链     

纳米水平分子成像与诊疗一体化研究进展与挑战

分子成像能以非侵入性的方式重现活体细胞的生理功能和生物学过程,提高疾病的早期和特异性诊断水平。纳米颗粒/材料具有物理性质可控性高、易于表面修饰、血液循环时间长和可功能化等优点,在疾病诊断与治疗中显示出巨大潜力。但如何阐明纳米材料多功能间的内在联系、解决其代谢及安全性等关键机制难题、实现纳米颗粒/材料多功能性到临床多功能...     

Association between functional alterations of senescence and senility and disorders of gait and balance

OBJECTIVES:

Declines in cognition and mobility are frequently observed in the elderly, and it has been suggested that the appearance of gait disorders in older individuals may constitute a marker of cognitive decline that precedes significant findings in functional performance screening tests. This study sought to evaluate the relationship between functional capacities and gait and balance in an elderly community monitored by the Preventive and Integrated Care Unit of the Hospital Adventista Silvestre in Rio de Janeiro, RJ, Brazil.

METHODS:

Elderly individuals (193 females and 90 males) were submitted to a broad geriatric evaluation, which included the following tests: 1) a performance-oriented mobility assessment (POMA) to evaluate gait; 2) a mini-mental state examination (MMSE); 3) the use of Katz and Lawton scales to assess functional capacity; 4) the application of the geriatric depression scale (GDS); and 5) a mini-nutritional assessment (MNA) scale.

RESULTS:

Reductions in MMSE, Katz and Lawton scores were associated with reductions in POMA scores, and we also observed that significant reductions in POMA scores were present in persons for whom the MMSE and Katz scores did not clearly indicate cognitive dysfunction. We also demonstrated that a decline in the scores obtained with the GDS and MNA scales was associated with a decline in the POMA scores.

CONCLUSIONS:

Considering that significant alterations in the POMA scores were observed prior to the identification of significant alterations in cognitive capacity using either the MMSE or the Katz systems, a prospective study seems warranted to assess the predictive capacity of POMA scores regarding the associated decline in functional capacity.     

Alterations of regular and mature monocytes are distinct,and dependent of intensity and duration of exercise

Circulating monocytes comprise functionally distinct regular (CD14^bright+) and mature (CD141^low+) cells. Cell surface receptors were determined by three colour flow cytometry in 8 healthy control subjects. Compared to regular monocytes, mature monocytes had lower levels of the high affinity Fcy receptor 1 (CD64), complement receptor 3 (CDllb), CD45RO and higher levels for HLA-DR, LFA-1 (CD11a/CD18), interleukin-2 receptor (CD25), CD45RA and the Fc receptor 3 (CD16). Both regular and mature monocytes were measured before and up to three hours after four different types of exercise (Ex) in endurance trained athletes (n=9-16). Immediately after anaerobic exercise of I min with a maximal lactate concentration (la_max) of I2.3 (SD I.4) mmol · l^–1 and exhaustive exercise of 24 (SD 8) min with a maximal lactate concentration (la_max) of 7.4 (SD 2.6) mmol· l^–1 mature monocytes increased more than regular monocytes. Exhaustive endurance exercise of 87 (SD 21) min [la_max 3.7 (SD I.0)] led to a similar increase of regular and mature monocytes. 15–33 min after a 100km run regular monocytes increased significantly, whereas mature monocytes decreased. Up to three hours after the end of all exercises mature monocytes fell below pre-exercise values. In conclusion, duration and intensity of exercise alter distinct maturation stages of monocytes differently. It is probable that the avidity of adhesion molecules like LFA-1 to their endothelial ligands is increased to enable the firm attachment to the endothelium.     

Triptorelin and cetrorelix induce immune responses and affect uterine development and expressions of genes and proteins of ESR1, LHR,and FSHR of mice

Context: GnRH immunity can reduce the expression of pituitary GnRH levels, and cause the changes in reproductive behaviors. It is unclear whether triptorelin (TRI) and cetrorelix (CET) immunity influences uterine development and expression of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and estradiol receptor 1 (ERS1) in the uterus.

Objective: The study investigated the effects of active immunity of GnRH agonist and antagonist on uterine development, microstructures, expression of hormone receptors mRNAs, and proteins in uteri.

Materials and methods: One hundred and five mice were assigned into CET, TRI, and control groups (CG). Mice in CET-1, CET-2, and CET-3 (n?=?15) were subcutaneously injected with 10, 20, and 40?μg CET antigens for seven days, respectively. Mice in TRI-1, TRI-2, and TRI-3 were injected with 10, 20, and 40?μg TRI antigens for seven days, respectively. The qPCR and Western blot were implemented to determine expressions of ESR1, LHR and FSHR mRNAs, and proteins.

Results: Compared with CG, the uterine weights of CET-1, CET-2, and CET-3 increased by 42.86, 62.86, and 10.00% on day 35 (p?p?p?p?p?Conclusions: CET immunity promoted the uterine development, improved EET and UWT, and also promoted the expressions of ESR1 and FSHR protein levels. It lessened the LHR protein levels. TRI immunity blocked EET and UWT, inhibited uterine growth and development. The efficacy of CET immunity was more obvious than TRI.     

韧带和肌腱的生物力学和力学生物学研究

国际韧带和肌腱研讨会(The International Symposium on Ligaments and Tendons,ISI&T)于2000年在美国佛罗里达州奥兰多市首次召开。研讨会的宗旨是引起对韧带和肌腱研究的重视,并为生物工程师、生物学家、临床医师提供一个可以分享、评论、讨论韧带和肌腱最新研究成果的论坛。从2000年起,国际韧带和肌腱研讨会已经开展了15届;每届研讨会上涌现了大量令人振奋的关于当前韧带和肌腱研究热点和未来挑战的讨论。多年来,韧带和肌腱领域内的研究数量大幅增加,研究质量不断提升。为纪念《医用生物力学》杂志创刊30周年,本文总结过去30年里韧带和肌腱研究的主要进展,包括组织力学、力学生物学、损伤与治愈机制、组织修复和再生。     

男性乳腺发育症临床病理分析及雌,孕激素受体检测

对１１３例男性乳腺发育症进行临床病理分析。同时检测其中３０例乳腺组织中雌激素受体和孕激素受体分布情况，结果发现两者阳性率分别为８０．０％和８３．３３％。结合文献讨论了男性乳腺发育症的发生与高血清激素浓度及乳腺组织高受体水平的关系。     

Morphology and cytochemistry of leucocytes and thrombocytes of six species of fish

Cytochemical reactions of blood leucocytes and thrombocytes from six species of fish, rainbow trout (Onchorhynchus mykiss), coho salmon (Onchorhynchus kisutch), white sturgeon (Acipenser transmontanus), goldfish (Carassius auratus), striped bass (Morone saxatulis), and channel catfish (Ictalurus punctatus) were determined. Because the staining reactions were generally similar to the reactions found in mammalian leucocytes with similar morphological features, it is reasonable to classify fish leucocytes using the same terminology as is used for mammalian leucocytes. However, in some species leucocytes with features similar to mammalian eosinophils or basophils were not found. In goldfish leucocytes were found that had segmented nuclei and unstained, moderately refractile cytoplasmic granules. These cells were classified as segmented, granular leucocytes. Although these cells do not appear similar to any mammalian or avian leucocyte, the pattern of positive cytoplasmic alkaline phosphatase staining and negative granular staining is similar to that of equine eosinophils.     

Pathology and genetics of phaeochromocytoma and paraganglioma

Phaeochromocytoma and paraganglioma (PHEO/PGL) are rare tumours with an estimated annual incidence of 3 per million. Advances in molecular understanding have led to the recognition that at least 30–40% arise in the setting of hereditary disease. Germline mutations in the succinate dehydrogenase genes SDHA, SDHB, SDHC, SDHD and SDHAF2 are the most prevalent of the more than 19 hereditary genetic abnormalities which have been reported. It is therefore recommended that, depending on local resources and availability, at least some degree of genetic testing should be offered to all PHEO/PGL patients, including those with clinically sporadic disease. It is now accepted that that all PHEO/PGL have some metastatic potential; therefore, concepts of benign and malignant PHEO/PGL have no meaning and have been replaced by a risk stratification approach. Although there is broad acceptance that certain features, including high proliferative activity, invasive growth, increased cellularity, large tumour nests and comedonecrosis, are associated with an increased risk of metastasis, it remains difficult to predict the clinical behaviour of individual tumours and no single risk stratification scheme is endorsed or in widespread use. In this review, we provide an update on advances in the pathology and genetics of PHEO/PGL with an emphasis on the changes introduced in the WHO 2017 classification of endocrine neoplasia relevant to practising surgical pathologists.