EGCG对大鼠放射性肺损伤的防治作用及机制探索

目的:研究表没食子儿茶素-3-没食子酸酯(EGCG)对大鼠放射性肺损伤的防治作用及相关机制。方法:用60Co源γ射线单次照射SD大鼠全肺,构建放射性肺损伤模型,激素治疗组和EGCG治疗组大鼠分别给予地塞米松注射液和EGCG治疗;以肺系数、HE染色、Masson染色和肺组织羟脯氨酸(HYP)含量观察及评价EGCG对放射性肺炎及纤维化的改善情况;检测血清总超氧化物歧化酶(T-SOD)活力和丙二醛(MDA)含量;用Western印迹检测肺组织Nrf-2、HO-1、NQO-1的表达。结果:EGCG可明显减轻肺脏充血水肿,减低肺系数、肺组织HYP含量、肺泡炎及肺纤维化评分;EGCG可明显降低血清MDA水平,提高血清T-SOD活力,上调大鼠肺组织细胞Nrf-2、HO-1、NQO-1的表达水平。结论:EGCG能明显改善放射性肺损伤及纤维化病变,可能是通过Nrf2-ARE信号途径增加抗氧化酶表达,提高机体抗氧化能力而发挥治疗作用的。     

自噬相关基因LC3-I的基因表达及单克隆抗体的制备

目的：通过克隆LC3．I基因,体外原核表达LC3-I蛋白后制备抗LC3单克隆抗体,作为自噬研究中的标记分子检测自噬的发生和发展过程。方法：RT．PCR方法从RAW264．7细胞基因组中克隆LC3基因,亚克隆至pQE80L原核表达载体后转化E．cobDH5a进行诱导表达,SDS—PAGE电泳及Westemblot鉴定表达蛋白。蛋白纯化后免疫BALB／c小鼠。采用淋巴细胞杂交瘤技术,制备分泌抗LC3．I杂交瘤细胞株,体内诱生腹水制备mAb,间接ELISA法测定其效价,辛酸一硫酸铵沉淀法及亲和层析法纯化mAb。结果：成功克隆了LC3一I基因,并对其在E．coilDH5a进行诱导表达,SDS-PAGE分析表明在相对分子量Mr为20×10^3有特异条带,Westernblot验证表达产物具有一定的生物学活性。建立了3株稳定分泌特异性抗LC3-ImAb的杂交瘤细胞株,诱导产生的腹水获得的抗体效价在10^5-10^7之间,结论：在E．coli中对LC3-I进行表达,并制备特异性较强抗LC3-I蛋白的单克隆抗体。为自噬研究提供了良好的标记分子,可对自噬形成和发展进行有效的检测。     

类风湿性关节炎伴间质性肺病中肿瘤标志物对肺功能的影响分析

目的：探究类风湿性关节炎伴间质性肺病中肿瘤标志物对肺功能的影响。方法：选取2011年3月至2013年3月我院收治的类风湿性关节炎患者88例,根据其是否伴有ILD,分为RA组53例和RA-ILD组35例。检测两组肺功能指标用力肺活量（FVC）、1s用力呼气容积（FEV1）、最大通气量百分比（MVV）、一氧化碳弥散量（DLCO）,肿瘤标志物癌胚抗原CEA、癌抗原125（CA125）、癌抗原199（CA199）及抗环胍氨酸肽抗体（抗CCP抗体）的值,并利用统计学方法分析肿瘤标记物与肺功能指标、抗CCP抗体的相关性。结果：RA．ILD组FVC、FEV1、MVV、DLCO均比RA组低,CEA、CA125、CA199均比RA组高,结果比较差异显著具有统计学意义（P〈0．05）。但两组抗CCP抗体含量相比却无明显差异,不具有统计学意义（P〉0．05）。相关性分析显示,CEA与FVC、FEV1、MVV、DLCO呈负相关（P〈0．05）,而CA125、CA199却与肺功能指标无相关性,CEA、CA125、CA199与抗CCP抗体均无相关性（P〉0．05）。结论：RA-ILD的肺功能指标均有明显下降,而肿瘤标志物表达水平却明显增高,CEA对肺功能损害影响较大,却与关节损害的关系不大,这为临床对类风湿性关节炎伴间质性肺病早诊断、早治疗提供了依据。     

Vascular endothelial growth factor regulates primate choroid-retinal endothelial cell proliferation and tube formation through PI3K/Akt and MEK/ERK dependent signaling

Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic protein that exhibits a broad range of biological and pathological effects in wet age-related macular degeneration and proliferative diabetic retinopathy. However, its specific mechanism is still not fully understood. Here, we examined the effects of VEGF on choroid-retinal endothelial cells (RF/6A) proliferation and tube formation, and the underlying signal pathways responsible in this process. RF/6A cells were pretreated with MEK inhibitor or PI3K inhibitor, and then incubated in a hypoxia chamber. Real-time PCR and Western blot analysis were carried out to explore VEGF expression on mRNA and protein levels. Hypoxia inducible factor-1α (HIF-1α) and VEGFR2 expression levels were also investigated in the presence and absence of hypoxic conditions. CCK-8 analysis and tube formation assay were tested under hypoxia, exogenous recombinant VEGF, and different signal pathway inhibitors, respectively. Mean while, the PI3K/Akt and MEK/ERK pathways in this process were also investigated. Our results showed that VEGF, HIF-1α, VEGFR2, p-ERK, and p-Akt were up-regulated in RF/6A cells under hypoxic conditions. MEK inhibitor (PD98059) and PI3K inhibitor (LY294002) decreased ERK and Akt activity, respectively, and reduced VEGF expression. VEGF-induced RF/6A proliferation and tube formation requires MEK/ERK and PI3K/Akt signaling, and both of the two pathways were needed in regulating VEGF expression. These suggest that VEGF plays an important role in RF/6A proliferation and tube formation, and MEK/ERK and PI3K/Akt pathway may be responsible for this process.     

水菠菜叶乙醇提取物对MG-63骨质疏松相关基因表达的影响

为探讨水菠菜叶乙醇提取物与抗骨质疏松作用的关系及其作用机制,用水菠菜叶乙醇提取物处理人成骨样细胞MG-63 48 h后,提取水菠菜叶乙醇提取处理组和对照组细胞的总RNA,将两组RNA纯化为mRNA,逆转录成cDNA,用Cy3和Cy5两种不同的荧光染料进行线性扩增标记,然后与骨质疏松相关基因表达谱芯片杂交,扫描后对获得的数据用LuxScan 3.0软件分析.研究发现经水菠菜叶乙醇提取物诱导后MG-63细胞的244个骨质疏松相关基因中,有c-Fos、JNK、ODF、c-src、OSCAR、RANKL、Samd4、Samd6、Samd7 9个基因表达明显上调,LRP5、Dkk1、TGFB、OPG 4个基因明显下调.结果表明水菠菜叶乙醇提取物能改变骨质疏松相关基因的表达,抗骨质疏松作用机制可能与Wnt/β-catenin蛋白信号传导途径和骨保护素/核因子受体激活信号传导途径有关,与雌激素内分泌途径无关.     

PDEA-coated magnetic nanoparticles for gene delivery to Hep G2 cells

Poly(2-(diethylamino)ethyl methacrylate) coated magnetic nanoparticles (PDEA-MNPs) were synthesized as a new gene nanocarrier to delivery plasmids (pEGFPN1 and pRL-TK) into human hepatoma (Hep G2) cells. The PDEA-MNPs shows the pH-sensitive property. These nanoparticles are positively charged at acidic pH and negatively charged at neutral or alkaline pH. The PDEAMNPs exhibited a low cytotoxicity in Hep G2 cells. PDEA-MNPs could bind and protect DNA from DNase I degradation. The transfection study demonstrated that the PDEA-MNPs could carry plasmid into Hep G2 cells and exhibited a high gene transfection efficiency. These results indicated that the novel magnetic nanoparticles could enhance gene transfection in vitro and hold the potential to be a promising non-viral nanodevice.     

水稻Rop基因OsRac5表达特性

Rop在植物生长、发育、免疫及环境信号应答等多种生物学过程中具有重要作用。已有研究显示水稻Rop基因OsRac5可能与育性控制有关,但是该基因的表达特性,以及非生物胁迫和植物生长物质对其表达的影响尚不清楚。本文采用qRT-PCR技术检测了OsRac5在水稻生长发育过程中、非生物胁迫以及植物生长物质处理条件下的表达特性,结果显示OsRac5在水稻生长发育过程中在多种组织广泛表达,尤其在根和雌雄蕊形成期的幼穗中高表达;干旱、高盐和低温等非生物胁迫均能诱导OsRac5表达;ABA、GAs、6-BA等植物生长物质能上调OsRac5基因表达,提示该基因与水稻幼穗发育、抗逆性及细胞生长等过程相关。     

中药渣蚓粪对玉米生长及土壤肥力特性的影响

采用盆栽试验,研究了中药渣蚓粪对玉米生长及土壤肥力的影响.结果表明: 随着蚓粪施用量的增加,玉米的株高、茎粗、叶面积、叶绿素含量均显著增加;生长60 d收获时,多数蚓粪处理的土壤容重显著降低;蚓粪处理的土壤pH显著高于对照和相应的化肥处理.蚓粪处理的土壤全氮、有机质含量也明显高于化肥处理,且随蚓粪施用量的增加,效果越趋显著.中药渣蚓粪可作为一种高效有机肥,其合理施用有助于改善土壤物理结构,缓解土壤的酸化进程,提高土壤有机质和氮素含量,有效促进作物生长.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Human umbilical cord-derived MSC culture: the replacement of animal sera with human cord blood plasma

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great potential for their therapeutic use in various clinical diseases. Many publications have reported on human blood-derived alternatives to animal serum for culturing mesenchymal stem cells, such as human serum, allogenic umbilical cord blood serum, and human platelet derivatives. However, it is not clear whether human umbilical cord blood plasma (UCBP), as the surplusage of umbilical cord blood mesenchymal stem cell extraction, could be used. In this study, in order to make the best of umbilical cord blood, the human UCBP was dialyzed to replace fetal bovine serum (FBS) in the culture medium. hUC-MSCs were cultured in the new medium. Cell growth rate, specific biomarkers, and differentiation properties were detected to characterize the cell proliferation and MSC-specific properties. The hUC-MSCs cultured in such derived medium were verified with proliferation rate, cluster differentiation markers, cell cycle, as well as differentiation capabilities. Such dialyzed human UCBP is fully comparable with, if not superior to, FBS in deriving and culturing hUC-MSCs.