抑菌蛋白Reg3A的表达纯化与功能

利用原核表达系统表达人源抑菌蛋白Reg3A,经包涵体的复性和纯化获得有体外抑菌功能的活性抑菌蛋白,并对其体外抑菌功能进行初步研究。构建Reg3A原核表达载体PET-32a-Reg3A转化补充稀缺tRNA基因的表达菌株大肠杆菌BL21-Codonplus,阳性重组子采用诱导培养基诱导5h后,采用超声破碎的方法提取包涵体蛋白,经包涵体蛋白的纯化和透析复性后通过Ni-NTA亲和层析交换柱,获得纯度达95%的蛋白质。Western blot鉴定显示在15 kD处有特异性条带。使用纯化后的蛋白进一步进行抑菌圈实验和抑菌活性实验,对获得蛋白的体外抑菌活性进行评估,从而为进一步进行Reg3A蛋白功能的评估及应用奠定基础。     

噪声刺激后豚鼠耳蜗抗氧化能力的变化和α-硫辛酸对声损伤的保护作用

实验探讨了声刺激后豚鼠血清总抗氧化能力(total antioxidant capacity,TAC)和耳蜗组织一氧化氮(nitricoxide,NO)含量的变化及α-硫辛酸的抗氧化和对声损伤的保护作用。将豚鼠(350-400 g)随机分为无噪声对照组(n=20)、噪声+生理盐水组(n=20)和噪声+α-硫辛酸组(n=20)。噪声刺激(4.kHz倍频程,115 dB SPL 5 h)结束后立即测试脑于诱发电位(auditory brainstem responses,ABRs),取血清测TAC,制备耳蜗组织匀浆测NO的水平。所得结果如下:(1)无噪声对照组,动物听阈无明显变化;噪声刺激后生理盐水组,听阈上升的幅度明显高于α-硫辛酸组(P<0.05)。(2)噪声+生理盐水组,TAlC明显低于无噪声对照组(P<0.05);噪声+α-硫辛酸组同噪声+生理盐水组相比,TAC明显升高(P<0.05),同无噪声对照组相比,无显著性差异(P>0.05)。(3)噪声+生理盐水组,NO含量高于无噪声对照组(P<0.05);噪声+α-硫辛酸组同噪声+生理盐水组相比,NO含量明显减少(P<0.05),同无噪声对照组相比,差异无显著性(P>0.05)。上述结果提示,噪声刺激后血清TAC降低,耳蜗组织内NO含量增加;α-硫辛酸可通过抗氧化机制对噪声性听力损伤(noise induced hearing loss,NIHL)发挥保护作用。     

Caspase-3在老年豚鼠耳蜗螺旋神经节细胞的表达

目的检测caspase-3在老年豚鼠耳蜗的表达。方法实验分两组:实验组和对照组,实验组豚鼠年龄为33至35个月之间,对照组豚鼠年龄为2至3个月。用免疫组织化学方法检测caspase-3在两组豚鼠耳蜗的表达。结果Caspase-3在实验组耳蜗的表达呈阳性,阳性区域主要存在于耳蜗螺旋神经节细胞。在对照组耳蜗的表达呈阴性。结论Caspase-3在老年豚鼠耳蜗螺旋神经节细胞中呈阳性表达,提示caspase-3在豚鼠耳蜗老化过程中起重要作用。     

Caspase-3在豚鼠内淋巴积水耳蜗中的表达

目的观察Caspase-3在豚鼠内淋巴积水耳蜗中的表达。方法实验分正常对照组和实验组,每组10只豚鼠。用破坏并阻塞豚鼠内淋巴囊的方法造成豚鼠内淋巴积水模型。3周后处死豚鼠,取耳蜗分别用石蜡及火棉胶包埋、切片,免疫组织化学方法观察caspase-3在耳蜗的表达。结果caspase-3在豚鼠内淋巴积水耳蜗中表达呈阳性,阳性区域为耳蜗外侧壁和螺旋神经节细胞。结论caspase-3在豚鼠内淋巴积水耳蜗中呈阳性表达,提示在内淋巴积水病理过程中存在耳蜗细胞凋亡。     

噪声短时暴露所致的耳蜗电位改变

本实验观察115dB(SPL)白噪声暴露20min对豚鼠耳蜗直流电位(EP),复合听神经动作电位(CAP),微音器电位(CM)的影响。发现此种噪声暴露确可提高源于血管纹的正EP(P-EP),说明有血管纹功能的代偿性增强;而负EP(N-EP)变化不大。AP及CM输入-输出函数的变化说明噪声首先影响外毛细胞的主动运动功能。EP与耳蜗电图的对照分析表明,血管纹功能的改变确能影响噪声性听损伤的发展。     

VIP和SP在自发性高血压大鼠耳蜗中的表达与意义

目的通过研究两种神经肽VIP(血管活性肠肽)、SP(P物质)在自发性高血压大鼠耳蜗中的表达,探讨VIP、SP在高血压性内耳疾病中的作用.方法采用免疫组织化学SABC法,观察VIP、SP在自发性高血压大鼠耳蜗中的表达,并利用图象分析系统测量阳性表达区域平均光密度值,进行定量分析.结果基底转螺旋神经节细胞数目高血压组明显少于正常组(P<0.01).螺旋神经节细胞胞浆中和血管纹处均有VIP和SP表达.在螺旋神经节细胞胞浆中,VIP和SP的含量两组间差异无显著性意义(P>0.05);在血管纹中,VIP的表达高血压组高于正常组(P<0.05),而SP的含量差异无显著性意义(P>0.05).结论VIP和SP都是听觉传导通路的神经递质,而且VIP还参与耳蜗微循环的神经体液调节.     

n-3多不饱和脂肪酸调节饮食诱导肥胖大鼠miRNA表达变化

目的：研究n-3多不饱和脂肪酸（polyunsaturated fatty acids,PUFA）饮食对饮食诱导肥胖大鼠的miR NA表达影响。方法：将10只饮食诱导肥胖（diet induced obese,DIO）大鼠随机分成两组：n-3PUFA添加组和安慰剂添加组（对照组）;每周记录两组老鼠的体重、体长和进食量。对外周血miR NA的表达并进行分析和预测。结果：两组老鼠Lee指数有统计学差异（P〈0.05）;与对照组相比,在n-3组的外周血单核细胞中,29个miR NA上调,31个下调;其中rno-miR-200和rno-miR-211的表达量上调,rno-miR-29b和rno-miR-92b的表达量下调,其靶基因预测结果与神经营养因子,脂肪细胞因子,趋化因子和胰岛素信号通路有关。结论：n-3PUFA能够调节DIO大鼠的miR NA水平,其中有些与脂肪代谢相关。     

变压器噪声暴露对SD大鼠听力及应激状态的影响研究

目的:探究短时间内低声级强度低频的变压器噪声暴露对SD大鼠听力及应激状态方面的影响。方法:选取90只SPF级健康无听力障碍的(雌雄各半)SD大鼠作为实验对象,随机分为实验A、B组和对照C组,A、B组分别给予声级上限为65 dB SPL、60 dB SPL(频谱范围:100~800 Hz)的变压器噪声,噪声暴露时程为8周,每日噪声给予时间为22点至次日8点,C组在相同条件下饲养,不给予噪声暴露。噪声暴露结束后,通过DPOAE(畸变耳声发射)、ABR(听性脑干反应)检测、耳蜗铺片及毛细胞计数对SD大鼠听力学状况进行评估;通过血清中促肾上腺皮质激素(ACTH)、血清皮质醇(CORT)对SD大鼠的应激状态进行评估。结果:在变压器噪声暴露的8周内,各组大鼠生长状况良好,体重均呈正常生理性增长,组间无明显差异(P0.05);在变压器噪声暴露8周后,对A、B、C三组大鼠的听力学指标进行两两比较,组间均无明显差异(P0.05),对大鼠血清中促肾上腺皮质激素(ACTH)、血清皮质醇(CORT)的含量进行三组间比较,组间差异均无统计学意义(P0.05)。结论:连续暴露于声压级上限65/60 dB SPL,频谱范围为100~800 Hz的变压器噪声下8周(10小时/天)对SD大鼠听力未产生明显影响,未引发SD大鼠应激状态。     

大鼠脊髓挤压伤后NT-3、NT-4在腹角运动神经元表达的变化

我们前面的研究已证实,神经生长因子和脑源性神经营养因子与成年大鼠挤压性脊髓损伤修复有关。在本研究中,通过免疫组织化学ABC法,我们探讨了挤压伤后不同时间脊髓腹角神经元NT-3和NT-4的表达。结果显示,在对照组,NT-3和NT-4的阳性反应主要分布在脊髓腹角神经元,挤压性脊髓损伤后7天和21天,NT-3阳性神经元的数量较对照组和24小时组明显增加,比较之,损伤后24小时和7天,NT-4阳性神经元的数量已较正常者增多,且NT-T的反应强度21天者较24小时和7天者有增多。结果表明NT-3和NT-4的表达在挤压性损伤后的脊髓腹角神经元被不同程度地上调,提示NT-3和NT-4可能与挤压性脊髓损伤修复有关。     

肝纤维化进程中miRNA-19b表达变化的研究

目的:观察微小RNA(miRNA)-19b在肝纤维化大鼠肝脏组织的表达,探讨miRNA-19b在肝纤维化进程中的作用.方法:将20只SD大鼠随机分成正常对照组和肝纤维化模型组.采用改良式复合因素法复制大鼠肝纤维化模型,分别在6、7周后,取各组大鼠肝脏组织,用荧光实时定量PCR方法检测miRNA-19b的表达.结果:6周后肝纤维化模型组大鼠肝脏组织miRNA-19b的表达低于正常对照大鼠组(P＜0.05),7周模型组大鼠肝脏组织miRNA-19b的表达低于6周模型组(P＜0.05).结论:miRNA-19b在肝纤维化大鼠肝脏组织中表达降低,可能与纤维化进程相关,且可能成为肝纤维的诊断以及预后的一个指标.     

再生基因家族的研究

再生基因家族自1988年被发现以来,其在糖尿病、炎症创伤与肿瘤尤其在消化系统肿瘤中的作用日渐被重视。越来越多的该家族成员被发现,并已开始考虑在临床治疗中应用。这些研究开始显示再生基因家族的潜在应用价值。 Abstract:Since the first member of Reg gene was discovered in 1988,it has been verified that Reg genes play important roles in diabetes,inflammation and injury,and tumors.More members were cloned and their application in treatment was studied.With the development of related research,there is a great potential of Reg family in biomedical field.     

Expression of Reg/PAP family members during motor nerve regeneration in rat

In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-associated protein (PAP) family members following hypoglossal nerve injury in rats. In addition to four rat family members (RegI, Reg-2/PAP I, PAP II, and PAP III) that had been identified, we newly cloned and sequenced a type-IV Reg gene in rats. Among these five family members, the expression of Reg-2/PAP I mRNA was predominantly enhanced in injured motor neurons after axotomy. Furthermore, a marked induction of PAP III mRNA was observed in the distal part of the injured nerve. A polyclonal antibody was raised against PAP III, and a Western blotting analysis using this antibody confirmed an increased level of PAP III protein in the injured nerve. These results suggest that Reg family members would be new mediators among injured neurons and glial cells, and may play pivotal roles during nerve regeneration.     

The gut peptide Reg3g links the small intestine microbiome to the regulation of energy balance,glucose levels,and gut function

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Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization

Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the MutS strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies.     

Central and peripheral actions of α-MSH in the thermoregulation of rats

The effect of α-MSH on thermoregulation in rats at room temperature was examined. α-MSH (1 μg ICV or 30 μg IP) alone did not alter temperature. However, this peptide was a potent antipyretic when administered centrally or peripherally in rats treated with pyrogen derived from Salmonella typhi.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.