用实时荧光定量RT-PCR方法定量绵羊PrP基因的表达

为快速、准确定量绵羊PrP基因的mRNA,建立了绵羊PrP基因实时荧光定量聚合酶链反应检测方法。根据已报道的绵羊PrP基因序列,设计合成引物;采用RT-PCR方法扩增目的片段;构建标准重组质粒制备标准曲线,用于样品检测。结果发现,中枢神经系统组织PrP基因的表达量(copies/ng总RNA,39420)比外周组织(为7845)的高;在中枢神经系统中,脑干的PrP基因的表达量最高(为67020);外周器官中,淋巴结PrP基因的表达量最高(为29086),肾脏的表达量最低(为125)。建立绵羊PrP基因实时荧光定量PCR方法,对PCR扩增反应中每一个循环的产物进行定量分析,为进一步研究绵羊组织器官的PrP表达在传染性海绵状脑病发生中的作用提供基础数据。     

Antibodies to human c-myc oncogene product: evidence of an evolutionarily conserved protein induced during cell proliferation

Antisera to a synthetic c-myc peptide and to c-myc antigens synthesized from various portions of the human gene expressed in Escherichia coli were used in order to characterize the protein product of the human c-myc oncogene. Although the deduced molecular weight of the human c-myc protein is 49,000, these antisera precipitate a protein from human cells that migrates in sodium dodecyl sulfate-polyacrylamide gel as if its molecular weight were 65,000. In addition, the mouse c-myc protein, whether synthesized in cells or in a cell-free system directed by pure, synthetic messenger RNA, has analogous properties and is immunoprecipitated by the antiserum to the human c-myc protein. Similar proteins are immunoprecipitated from monkey, rat, hamster, and frog cells, suggesting evolutionary conservation of antigenic structure of the c-myc protein among vertebrates. In addition, and in a manner consistent with the behavior of its messenger RNA, the immunoprecipitable c-myc protein is sharply induced by the action of mitogens on resting human T cells.     

Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines

Alterations in proto-oncogene expression after stimulation of rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) have been investigated. A specific stimulation of c-fos messenger RNA and protein was detected 30 minutes after treatment. This induction was enhanced more than 100-fold in the presence of peripherally active benzodiazepines. The effect was specific as very little change was observed in the levels of c-rasHa, c-rasKi, c-myc, and N-myc messenger RNA's. Under the conditions used here, NGF treatment ultimately results in neurite outgrowth, with a reduction or cessation of cell division. Thus, stimulation of the c-fos gene in this system appeared to be associated with differentiation and not with cellular proliferation. The effect of benzodiazepines was stereospecific and represents a novel action of these compounds at the level of gene expression.     

cdc2 gene expression at the G1 to S transition in human T lymphocytes

The product of the cdc2 gene, designated p34cdc2, is a serine-threonine protein kinase that controls entry of eukaryotic cells into mitosis. Freshly isolated human T lymphocytes (G0 phase) were found to have very low amounts of p34cdc2 and cdc2 messenger RNA. Expression of cdc2 increased 18 to 24 hours after exposure of T cells to phytohemagglutinin, coincident with the G1 to S transition. Antisense oligodeoxynucleotides could reduce the increase in cdc2 expression and inhibited DNA synthesis, but had no effect on several early and mid-G1 events, including blastogenesis and expression of interleukin-2 receptors, transferrin receptors, c-myb, and c-myc. Induction of cdc2 required prior induction of c-myb and c-myc. These results suggest that cdc2 induction is part of an orderly sequence of events that occurs at the G1 to S transition in T cells.     

Pertussis toxin-sensitive pathway in the stimulation of c-myc expression and DNA synthesis by bombesin

The bombesin-like peptides are potent mitogens for Swiss 3T3 fibroblasts, human bronchial epithelial cells, and cells isolated from small cell carcinoma of the lung. The mechanism of signal transduction in the proliferative response to bombesin was investigated by studying the effect of Bordetella pertussis toxin on bombesin-stimulated mitogenesis. At nanomolar concentrations, bombesin increased levels of c-myc messenger RNA and stimulated DNA synthesis in Swiss 3T3 cells. Treatment of the cells with pertussis toxin (5 nanograms per milliliter) completely blocked bombesin-enhanced c-myc expression and eliminated bombesin-stimulated DNA synthesis. This treatment had essentially no effect on the mitogenic responses to either platelet-derived growth factor or phorbol 12,13-dibutyrate. These results suggest that the mitogenic actions of bombesin-like growth factors are mediated through a pertussis toxin-sensitive guanine nucleotide-binding protein. Furthermore they indicate that bombesin-like growth factors act through pathways that are different from those activated by platelet-derived growth factor.     

A point mutation in the c-myc locus of a Burkitt lymphoma abolishes binding of a nuclear protein

A 20-base pair region in the first intron of the human c-myc gene was identified as the binding site of a nuclear protein. This binding site is mutated in five out of seven Burkitt lymphomas sequenced to date. To investigate the protein-recognition region in greater detail, the abnormal c-myc allele from a Burkitt lymphoma line (PA682) that carries a t(8;22) chromosomal translocation was used. A point mutation in the binding region of the PA682 c-myc DNA abolished binding of this nuclear protein. This protein may be an important factor for control of c-myc expression, and mutations in its recognition sequence may be associated with c-myc activation in many cases of Burkitt lymphoma.     

G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.     

HSPA2在牦牛不同组织器官中的表达差异

【目的】探索热休克蛋白70-2(heat shock 70kD protein-2, HSPA2)在牦牛不同组织器官中的表达差异。【方法】选取 3 头 1 岁龄的健康青海高原雄性牦牛为研究对象,在正常生理条件下,于 2013 年 9 月中旬采集不同组织器官（心脏、肝脏、脾脏、肺脏、肾脏、脑和睾丸）样本。（1）从牦牛不同组织器官中提取 RNA,将 RNA 反转录成第一链 cDNA,参照牦牛 HSPA2 和β-actin 基因序列（登录号为 KC790105.1和 DQ838049.1）设计特异性引物,首先采用 RT-PCR 来验证实时荧光定量 PCR（RT-qPCR）是否能应用于牦牛不同组织器官中 HSPA2表达差异的测定;然后采用 RT-qPCR 测定牦牛不同组织器官中 HSPA2相对表达量的差异。（2）将牦牛不同组织器官样本 4％多聚甲醛固定,制成石蜡切片,免疫组织化学法测定 HSPA2 在不同组织器官中的分布。采用 Image-Pro Plus 6.0 软件进行免疫组化图像分析,测定 HSPA2 阳性反应物的积分光密度,进行吸光度分析;通过 SPSS 19.0 统计软件,用单因素方差分析进行差异显著性测定。【结果】(1) RT-PCR 结果表明 RT-qPCR 法可用于牦牛不同组织器官中 HSPA2表达差异的测定。实时荧光定量 PCR 结果表明,HSPA2在睾丸中的相对表达量,分别是在脑、肾脏、心脏、脾脏、肺脏和肝脏中的 83.33、97.09、111.11、133.33、222.22和 285.71倍。(2)免疫组织化学结果显示,牦牛睾丸、肾脏、脑、心脏、肺脏、肝脏和脾脏中均有 HSPA2 的阳性表达。其中,在牦牛肾脏皮质肾小管、髓质肾小管,大脑皮质海马CA1区、小脑皮质,心肌细胞,肺泡上皮细胞,肝细胞,脾脏边缘区和红髓中有 HSPA2 阳性反应,着色深浅不等,大部分阳性反应位于细胞质,细胞核阳性反应极少;而在睾丸曲精小管中生精细胞细胞质和细胞核中均有 HSPA2 阳性反应,着色深浅不等;阴性对照组未观察到阳性反应。根据积分光密度值比较得出,HSPA2 蛋白的阳性表达量在睾丸中最高,脑、肾脏、心脏、肺脏和肝脏次之,脾脏最少。【结论】通过基因水平和蛋白水平两个层面的研究,发现 HSPA2 在牦牛各组织器官中存在表达差异;睾丸中 HSPA2 基因和蛋白表达量均高于脑、肾脏、心脏、脾脏、肺脏和肝脏,提示 HSPA2 可能与睾丸的生殖功能密切相关。     

无花果RNA的提取和半定量RT-PCR内参基因的优选

本试验以无花果的根、茎、叶、果作为材料,比较了CTAB法和Trizol法对无花果材料RNA的提取效果。以CTAB法提取的不同无花果材料的RNA为模板,反转成cDNA第一链,通过半定量RT-PCR,研究了植物常用内参基因18SrRNA、Actin和Tubulin的表达量变化。结果表明:CTAB法是适合不同无花果材料的RNA提取法;18SrRNA在无花果不同组织中的表达水平较高,且相对稳定,Tubulin在无花果不同组织中相对表达量较低,且相对稳定,是研究无花果不同组织基因表达水平较为适宜的内参基因。     

Autoimmunity and increased c-myb transcription

A single recessive gene, lpr, induces an autoimmune-lymphoproliferative syndrome in several strains of mice. The lymphoid organs of lpr/lpr mice contained cells with increased amounts of myb RNA, which codes for a protein found in the nucleus. A similar human lymphoproliferative disorder also had an increase in c-myb expression. Mouse T cells induced by mitogens to proliferate did not express large amounts of myb RNA, indicating that marked myb expression is not a general feature of lymphocyte activation and proliferation.     

丹龙醒脑方对脑缺血再灌注大鼠半暗带区神经细胞凋亡与即早基因表达的影响

目的 探讨丹龙醒脑方对局灶性脑缺血再灌注大鼠大脑皮质半暗带（ischemic penumbya,IP）区神经细胞凋亡与即早基因c-fos、c-jun、c-myc表达的关系。方法 将48只雄性SD大鼠随机均分为假手术组,脑缺血再灌注（模型）组,尼莫地平组及丹龙醒脑方小、中、大剂量组（丹小组、丹中组、丹大组）。后五组用线栓法制备大脑中动脉栓塞再灌注（MCAO/R）模型,再灌注24 h进行神经功能缺损评分后,取缺血侧大脑皮质,采用原位末端标记法（TUNEL）检测凋亡的神经细胞,免疫组化法分别检测c-fos、c-jun、c-myc蛋白。结果 与假手术组比较,其余各组的神经功能缺损评分、神经细胞凋亡指数（AI）及c-fos、c-jun、c-myc蛋白的积分光密度（IOD）值均升高,差异有统计学意义（P < 0.05,P < 0.01）;与模型组比较,尼莫地平组和丹龙醒脑方各剂量组的神经功能缺损评分、神经细胞AI及c-fos、c-jun、c-myc蛋白的IOD值均减小,差异有统计学意义（P < 0.05,P < 0.01）;尼莫地平组、丹龙醒脑方各剂量组组间比较,各指标差异均无统计学意义（P>0.05）。结论 丹龙醒脑方能显著降低脑缺血后神经功能缺损和减少神经细胞凋亡,其机制可能与下调即早基因c-fos、c-jun和c-myc的表达有关。