小鼠乳腺中瘦素的信号转导通路

为研究小鼠乳腺发育、泌乳和退化过程中瘦素的信号转导通路,分别取妊娠12d、泌乳12d以及退化12d小鼠乳腺组织进行体外培养,对照组培养基为无血清培养基,处理组培养基中则只添加瘦素。培养48 h后取出分别提取乳腺上皮细胞总蛋白,并用免疫印迹的方法检测对照组和处理组乳腺上皮细胞中信号分子MAPK、STAT3和STAT5的磷酸化情况。结果表明,虽然在妊娠期、泌乳期和退化期乳腺上皮细胞中均有MAPK、STAT3和STAT5信号分子存在,但是在妊娠期瘦素只能专一性的诱导MAPK磷酸化,泌乳期瘦素可以激活STAT3和STAT5,而在退化期瘦素则特异性地激活STAT3,启动乳腺上皮细胞的凋亡过程。     

Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility

EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates sphingosine kinase and increases intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1, as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.     

Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation

Closely related signals often lead to very different cellular outcomes. We found that the differentiation of human mesenchymal stem cells into bone-forming cells is stimulated by epidermal growth factor (EGF) but not platelet-derived growth factor (PDGF). We used mass spectrometry-based proteomics to comprehensively compare proteins that were tyrosine phosphorylated in response to EGF and PDGF and their associated partners. More than 90% of these signaling proteins were used by both ligands, whereas the phosphatidylinositol 3-kinase (PI3K) pathway was exclusively activated by PDGF, implicating it as a possible control point. Indeed, chemical inhibition of PI3K in PDGF-stimulated cells removed the differential effect of the two growth factors, bestowing full differentiation effect onto PDGF. Thus, quantitative proteomics can directly compare entire signaling networks and discover critical differences capable of changing cell fate.     

信号转导和转录激活子5b(STAT5b)及其STAT5b基因的研究进展

信号转导和转录激活子5b(STAT5b)是STATs家族的重要成员,在细胞内信号转导和转录激活中发挥关键的作用。STAT5b具有广泛的生物学效应,参与动物生长、繁殖、泌乳和代谢。为了对STAT5b及其STAT5b基因有一全面、深入的了解,综述了STAT5b基因的定位、STAT5b基因的结构、STAT5b的生物学功能以及STAT5b的表达、多态及遗传效应研究,该基因在动物生产性能方面,具有广阔的研究前景。     

水牛 STAT5a 和 STAT5b 基因启动子克隆及其活性分析

为了解转录激活与信号转导因子5(STAT5)在水牛乳腺发育及泌乳生理中的功能,对水牛STAT5a和STAT5b基因的5′调控区启动子序列进行了克隆,并比较其活性.根据GenBank已公布的牛STAT5a和STAT5b基因序列设计特异性引物,以广西本地水牛乳腺组织基因组DNA为模板,通过PCR法分别扩增不同长度STAT5a和STAT5b基因启动子片段并进行生物信息学分析.结果表明:克隆得到的水牛STAT5a基因启动子片段(P1和P2)大小是500bp,700bp,STAT5b基因启动子片段(P3,P4和P5)大小是500bp,800bp,1 500bp.在线分析结果显示,仅P2片段中存在高甲基化位点,且富含SP1,AP2等转录因子结合位点.将构建的不同长度启动子片段分别连入pGL3-Basic载体,分别转染水牛乳腺上皮细胞,检测荧光素酶表达水平.与未转染组相比,P1~P5质粒转染组的荧光素酶比值均显著提高(p0.05);添加5mg/mL质量浓度催乳素(PRL)处理乳腺上皮细胞,P3质粒转染组的荧光素酶比值显著高于其他各组(p0.05).以上结果表明,水牛STAT5a和STAT5b基因启动子活性均受到PRL调节,两者表达水平在水牛乳腺上皮细胞中不同,且STAT5b基因表达受PRL调控更为明显.     

葛根素对脑缺血再灌注大鼠海马组织P-STAT3、P53表达的影响

目的 探讨葛根素对大鼠脑缺血再灌注损伤后海马组织磷酸化的转录激活蛋白3（STAT3）、凋亡因子P53表达的影响及其保护机制。方法 采用线栓法制备大鼠左侧大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）模型,利用葛根素干预,分别于缺血2 h再灌注6 h、24 h、72 h 3个不同时间点采用Longa评分法进行神经功能缺损评分、TUNEL法检测凋亡细胞数,免疫组织化学法检测海马组织磷酸化的STAT3（P-STAT3）及细胞凋亡因子P53的表达。结果 与假手术组比较,葛根素组与缺血再灌注组的神经功能缺损加重,凋亡细胞数增多,P-STAT3和P53表达增高,差异均具有统计学意义（P<0.05）;与缺血再灌注组比较,葛根素组神经功能缺损评分明显下降,凋亡阳性细胞数均见明显减少,P-STAT3和P53表达显著降低,差异均具有统计学意义（P<0.05）。结论 脑缺血再灌注损伤后STAT3被异常激活,其磷酸化水平显著增高;葛根素可能是通过抑制STAT3的磷酸化水平,降低P53的表达起到抗神经细胞凋亡作用,从而改善大鼠脑组织的缺血性损伤。     

多能性转录因子mRNA转染山羊胎儿成纤维细胞条件优化

【目的】针对转染试剂脂质体2000和转染方法进行研究,用以优化外源多能性转录因子oct4、sox2、klf4、c-myc的mRNA转入山羊胎儿成纤维细胞（goat embryonic fibroblast,GEF）的条件。【方法】采用胰酶消化法,从山羊胎儿腿部肌肉组织中分离并获得了纯化的GEF细胞。根据mMESSAGE mMACHINE® T7 Ultra Kit试剂盒说明书,体外转录步骤主要有：①前期的质粒准备：首先用质粒小提试剂盒对摇菌获得的各体外转录载体质粒进行提取,随后对各体外转录载体目的基因下游进行单酶切,获得线性化质粒,最后用DNA纯化试剂盒对线性化的质粒进行纯化;②mRNA的“加帽”：以线性化DNA为模板,合成带有5′端帽子结构的mRNA,添加TURBO DNase以除去模板DNA;③mRNA的“加尾”：以ATP为原料,利用试剂盒自带的Poly(A)聚合酶,对已经“加帽”的mRNA进行“加尾”,以合成结构完整的mRNA;④完整mRNA的纯化：按照MEGAclear^TM Kit试剂盒对mRNA进行纯化。随后,将体外转录获得各转录因子的mRNA进行浓度和OD值测定。分别比较总mRNA（EGFP和4种多能性转录因子的mRNA的质量分别为0.2μg）和脂质体2000 的比例为1:0.5、1:1.0、1:1.5、1:2.0以及在最佳总mRNA与脂质体2000比例体系下第2、4、6代GEF细胞的mRNA转染效率。对最佳转染体系下转染多能性转录因子mRNA的GEF细胞进行免疫荧光检测,并对转染多能性转录因子mRNA后GEF细胞的形态和数目进行观察。【结果】mRNA和脂质体2000 的比例为1:1.0以及在最佳总mRNA与脂质体2000比例体系下第2代GEF细胞的mRNA转染效率均显著高于其他组（P＜0.05）,且本组细胞在形态和生长方面相对较好;免疫荧光检测表明,4种多能性转录因子的mRNA在GEF细胞中定位于细胞核,并获得稳定表达,在细胞质中不见多能性转录因子oct4、sox2、klf4、c-myc的表达;GEF细胞在转染4种mRNA 24h后,细胞形态由梭状慢慢向圆形靠拢;细胞活力低于对照组（山羊成纤维细胞正常生长组）。【结论】GEF细胞在总mRNA和脂质体2000 的比例为1:1.0以及在最佳总mRNA与脂质体2000比例体系下第2代GEF的条件下更适合mRNA的转染。研究工作为mRNA转染体细胞或干细胞的研究及诱导性多能干细胞（induced pluripotent stem cells,iPS）的获得提供参考资料,并对mRNA在成纤维细胞中的去向提供间接证据,有利于更加深入地了解多能性转录因子oct4、sox2、klf4、c-myc的mRNA相关功能。     

Imaging sites of receptor dephosphorylation by PTP1B on the surface of the endoplasmic reticulum

When bound by extracellular ligands, receptor tyrosine kinases (RTKs) on the cell surface transmit critical signals to the cell interior. Although signal termination is less well understood, protein tyrosine phosphatase-1B (PTP1B) is implicated in the dephosphorylation and inactivation of several RTKs. However, PTP1B resides on the cytoplasmic surface of the endoplasmic reticulum (ER), so how and when it accesses RTKs has been unclear. Using fluorescence resonance energy transfer (FRET) methods, we monitored interactions between the epidermal- and platelet-derived growth factor receptors and PTP1B. PTP1B-catalyzed dephosphorylation required endocytosis of the receptors and occurred at specific sites on the surface of the ER. Most of the RTKs activated at the cell surface showed interaction with PTP1B after internalization, establishing that RTK activation and inactivation are spatially and temporally partitioned within cells.     

Localized Rac activation dynamics visualized in living cells

Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) was developed to quantify the spatio-temporal dynamics of the Rac1 nucleotide state in living cells. FLAIR revealed precise spatial control of growth factor-induced Rac activation, in membrane ruffles and in a gradient of activation at the leading edge of motile cells. FLAIR exemplifies a generally applicable approach for examining spatio-temporal control of protein activity.     

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.