大鼠β细胞素蛋白的表达、纯化及生物学活性鉴定

目的:获得大量重组大鼠β细胞素并检测其活性。方法:用PCR法从大鼠肾组织中扩增534bp的β细胞素基因片段,并按读框克隆到原核表达载体pET28a( )中。以构建的重组质粒pET28a-rBTC转化大肠杆菌BL-21(DE3)菌株,在IPTG诱导下表达β细胞素蛋白,表达产物用SDS-PAGE和Westernblot检测。采用镍柱亲和层析法纯化目的蛋白。结果:经IPTG诱导后,在大肠杆菌中可表达相对分子质量Mr为20000的目的蛋白。目的蛋白主要以包涵体的形式表达;表达量约占菌体蛋白总量的20%～30%。表达的目的蛋白与抗His标签抗体具有良好的反应性。结论:大鼠β细胞素基因在PET表达系统中得到高效表达;蛋白复性后能有效地促进NIH3T3细胞的体外增殖。     

Differential modulation of proliferation, matrix metalloproteinase expression and invasion of human head and neck squamous carcinoma cells by c-erbB ligands

Evidence suggests that there is an association between the abnormal expression of members of the c-erbB receptor tyrosine kinase family and poor prognosis in head and neck squamous cell carcinomas (HNSCC). Until now, the relative contributions of different c-erbB ligands to HNSCC progression have not been clearly defined. In this paper we examined the effects of ligands with different c-erbB receptor specificities in terms of their stimulation of HNSCC proliferation, expression of matrix metalloproteinases (MMPs) and invasion. Heregulin-beta1 (HRG-β1; selective c-erbB3/B4 ligand) was found to stimulate proliferation in the majority of cell lines, whereas epidermal growth factor (EGF; EGFR ligand) and betacellulin (BTC; EGFR/B4 ligand) induced variable responses. All three ligands up-regulated multiple MMPs including collagenases, stromelysins, matrilysin and gelatinase B (MMP-9) but had minimal or no effects on gelatinase A (MMP-2), MT1-MMP and tissue inhibitors of MMPs (TIMPs). MMP-9 mRNA was induced to a higher level than other MMPs, although with slower kinetics. HRG-β1 was less active than EGF and BTC at the optimal concentration (relative potency of EGF:BTC:HRG = 3:4:1). In vitro invasion through Matrigel was also increased by all three ligands in proportion to their MMP up-regulation. A specific anti-EGFR monoclonal antibody (mAb ICR62) inhibited MMP up-regulation, migration and invasion induced by all three ligands, whereas an anti-c-erbB-2 mAb ICR12 inhibited mitogenic and motogenic responses following ligand stimulation but had no effect on MMP expression. These results suggest that c-erbB ligands may differentially potentiate the invasive phenotype of HNSCC via co-operative induction of cell proliferation, migration and proteolysis. The EGFR signalling pathway appears to be the dominant component controlling the proteolytic and invasive phenotype in HNSCC, whereas the c-erbB-2 signalling pathway is responsible, in part, for the mitogenic and motogenic effects of ligands. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interstitial Outburst of Angiogenic Factors During Skeletal Muscle Regeneration After Acute Mechanical Trauma

Angiogenesis is a key event during tissue regeneration, but the intimate mechanisms controlling this process are still largely unclear. Therefore, the cellular and molecular interplay along normal tissue regeneration should be carefully unveiled. To this matter, we investigated by xMAP assay the dynamics of some angiogenic factors known to be involved in tissue repair, such as follistatin (FST), Placental Growth Factor‐2 (PLGF‐2), epidermal growth factor (EGF), betacellulin (BTC), and amphiregulin (AREG) using an animal model that mimics acute muscle contusion injuries. In situ immunofluorescence was used for the evaluation and tissue distribution of their cellular sources. Tissue levels of explored factors increased significantly during degeneration and inflammatory stage of regeneration, peaking first week postinjury. However, except for PLGF‐2 and EGF, their levels remained significantly elevated after the inflammatory process started to fade. Serum levels were significantly increased only after 24 h for AREG and EGF. Though, for all factors except FST, the levels in injured samples did not correlate with serum or contralateral tissue levels, excluding the systemic influence. We found significant correlations between the levels of EGF and AREG, BTC, FST and FST and AREG in injured samples. Interstitial cells expressing these factors were highlighted by in situ immunolabeling and their number correlated with measured levels dynamics. Our study provides evidence of a dynamic level variation along the regeneration process and a potential interplay between selected angiogenic factors. They are synthesized, at least partially, by cell populations residing in skeletal muscle interstitium during regeneration after acute muscle trauma. Anat Rec, 298:1864–1879, 2015. © 2015 Wiley Periodicals, Inc.     

High Cortical Bone Mass Phenotype in Betacellulin Transgenic Mice Is EGFR Dependent

Signaling through the epidermal growth factor receptor (EGFR) by ligands such as epidermal growth factor (EGF), transforming growth factor α (TGFA), and amphiregulin (AREG) has been reported to have effects on skeletal growth. The role of betacellulin (BTC), another EGFR ligand, in skeletal development and bone metabolism is unknown. In previous experiments, transgenic mice overexpressing BTC ubiquitously under the control of the chicken β‐actin promoter (BTC‐tg) exhibited stunted growth and disproportionately sized long bones. In this study, we performed a detailed phenotypic analysis of BTC‐tg mice at 3, 6, and 9 wk of age. Osteoblastic cells from transgenic mice showed strong expression of BTC as determined by Western blots and by immunohistochemistry on bone sections. In femurs of male and female BTC‐tg mice, we found reduced longitudinal bone growth and a pronounced increase in total volumetric BMD. The increased femoral BMD was mainly caused by augmented endocortical bone apposition and subsequent cortical bone thickening. In contrast, vertebral BMD was reduced in BTC‐tg mice of both sexes. An overall similar phenotype was found in 6‐mo‐old BTC‐tg mice. The increase in cortical bone mass in the appendicular skeleton of BTC‐tg mice was largely blocked when they were crossed into the Egfr^Wa5 background characterized by a dominant negative EGFR. Our study showed that overexpression of BTC results in an EGFR‐dependent upregulation of cortical bone mass in the appendicular skeleton of mice, uncovering a potential novel anabolic pathway for cortical bone.     

β细胞素基因-226A＞G多态性与糖耐量减低人群的相关性研究

目的 探讨β细胞素基因(BTC)-226A＞G多态性与云南省昆明地区糖耐量减低人群的相关性.方法 根据口服75 g葡萄糖耐量试验(OGTT)将1 076例云南省昆明地区人群分为糖耐量减低组(IGT)、2型糖尿病组(T2DM)和健康对照组(NGT),用稳态模型法胰岛素抵抗指数(HOMA-IR)、稳态模型法β细胞功能指数(HOMA-β)、早期胰岛素分泌指数(△I30/△G30)等进行组间比较.同时采集血样后提取基因组DNA,应用高分辨率熔解曲线分析方法(HRM)检测BTC基因-226A＞G基因型,观察3组人群中基因型和等位基因的分布情况,同时分析糖耐量减低人群中不同基因型相关临床变量的差异性.结果 ①BTC基因-226A＞GGG基因型及A等位基因在T2DM组中的频率分别为0.133和0.657,在IGT组中为0.085和0.715,差异均无统计学意义(P＞0.05);G等位基因在T2DM组中为0.343,在IGT组中为0.285,两组之间差异具有统计学意义(P=0.041).②IGT组-226位点AA基因型和AG+GG基因型的表型间的空腹血糖(FBG)、空腹胰岛素(Fins)、OGTT 2 h血糖(PBG)、OGTT 2 h胰岛素(PINS)、HOMA-IR、HOMA-β和△I30/△G30比较差异无统计学意义(P＞0.05).结论 BTC基因-226A＞G多态性可能与云南省昆明地区人群糖耐量减低相关,推测含有G等位基因的糖耐量减低人群更易进展为T2DM.     

人BTC蛋白表达及生物学活性鉴定

目的获得大量具有生物活性的人成熟β细胞素(BTC)蛋白。方法以人胰岛细胞瘤cDNA为模板,PCR扩增BTC基因成熟蛋白编码区的全部序列,并按读框克隆入原核细胞表达载体pET32a( )中,构建重组质粒pET32a( )-hBTC。重组质粒转化大肠杆菌BL21,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导及亲和层析法纯化获得融合蛋白,运用SDS-PAGE和Western blot进行鉴定。以不同浓度BTC连续5 d作用于NIH3T3细胞,MTT比色法检测细胞增殖能力。结果融合蛋白以水溶性形式分泌于大肠杆菌BL-21胞浆中,其作用于NIH3T3细胞可明显促进细胞增殖。结论人成熟BTC蛋白在pET32a( )表达系统中得到高效表达,所纯化蛋白具有促进NIH3T3细胞体外增殖的作用。     

β细胞素基因多态性与2型糖尿病的相关性

目的在陕西人群中筛查β细胞素基因(BTC)外显子1 TGC19GGC、5’非翻译区A-226G的基因多态性,并探讨这两个位点基因多态性与2型糖尿病的相关关系。方法 2型糖尿病患者130例,健康对照96例;分别采用限制性片段长度多态性-PCR、等位基因特异性引物-PCR对两个位点进行基因多态性的筛查。结果在β细胞素基因外显子1 TGC19GGC中,G等位基因、TG基因型频率在2型糖尿病组与健康对照组之间的差异有统计学意义(P<0.05)。在β细胞素基因5’非翻译区A-226G中,G等位基因频率、GG基因型频率在2型糖尿病组与健康对照组之间的差异无统计学意义(P>0.05)。两个位点单体型总体分布在2型糖尿病组与健康对照组之间的差异有统计学意义(P<0.05);利用SHEsis软件分析,两个位点间不存在有意义的连锁不平衡(D’=0.386,r~2=0.009)。结论在陕西人群中,BTC基因外显子1 TGC19GGC、5’非翻译区A-226G存在基因多态性。BTC基因外显子1 TGC19GGC基因多态性与2型糖尿病的发病有相关性;BTC基因5’非翻译区A-226G基因多态性与2型糖尿病的发病无相关性。两个位点不存在有意义的连锁不平衡。     

Elevated specific peripheral cytokines found in major depressive disorder patients with childhood trauma exposure: A cytokine antibody array analysis

Taking into consideration the previous evidence of revealing the relationship of early life adversity, major depressive disorder (MDD), and stress-linked immunological changes, we recruited 22 MDD patients with childhood trauma exposures (CTE), 21 MDD patients without CTE, and 22 healthy controls without CTE, and then utilized a novel cytokine antibody array methodology to detect potential biomarkers underlying MDD in 120 peripheral cytokines and to evaluate the effect of CTE on cytokine changes in MDD patients. Although 13 cytokines were identified with highly significant differences in expressions between MDD patients and normal controls, this relationship was significantly attenuated and no longer significant after consideration of the effect of CTE in MDD patients. Depressed individuals with CTE (TD patients) were more likely to have higher peripheral levels of those cytokines. Severity of depression was associated with plasma levels of certain increased cytokines; meanwhile, the increased cytokines led to a proper separation of TD patients from normal controls during clustering analyses. Our research outcomes add great strength to the relationship between depression and cytokine changes and suggest that childhood trauma may play a vital role in the co-appearance of cytokine changes and depression.