转录激活因子3在糖皮质激素与邻苯二甲酸二丁酯联合作用致大鼠尿道下裂发生中的表达变化

目的 利用糖皮质激素与邻苯二甲酸二丁酯(DBP)联合作用致大鼠尿道下裂,观察转录激活因子3(ATF3)在正常大鼠生殖结节(GT)与不同严重程度尿道下裂大鼠生殖结节中的差异表达,并探讨ATF3在尿道下裂发生中的作用机制.方法 将SD孕鼠加只随机均分为4组,在妊娠14～18 d(GD14～18)分别给予:A组:大豆油灌胃2 ml/d;B组:地塞米松(DXM)0.1 mg/(kg·d)皮下注射;C组:DBP 700 mg/(kg·d)灌胃;D组:DXM 0.1 mg,/(kg·d)皮下注射加DBP 700 mg/(kg·d)灌胃.GD19将各组孕鼠处死剖腹统计雄性仔鼠出生体质量(BW)及肛门生殖器间距离(AGD),取雄性仔鼠生殖结节,运用免疫印迹法和免疫组织化学方法分析ATF3的表达.结果 DXM单独作用于孕鼠仅表现为仔鼠BW下降,但与DBP组合作用却能显著增强DBP的致畸作用,导致雄性仔鼠BW及AGD/BW明显减少.ATF3在A组的蛋白相对表达量为0.351±0.012,B组为0.336±0.015,C组为0.603±0.014,D组为0.851±0.016,C组或D组与A组,以及C组与D组之间的差异均有统计学意义(P＜0.05);免疫组织化学结果显示ATF3主要定位于生殖结节的尿道上皮和侧翼间质,C组和D组免疫组织化学染色强度明显强于A组,而D组染色强于C组.结论 DXM联合DBP进一步增强了DBP的致畸作用,提示尿道下裂的发生是环境中多因素协同作用的结果,ATF3在对照组与实验组生殖结节中的表达有明显变化,且随着尿道下裂程度的加重而表达增高,ATF3的高表达影响了生殖结节的发育及尿生殖褶的融合,这可能是尿道下裂发生的机制之一.

Abstract：

Objective To verify the differential expression of ATF3 in the genital tubercle (GT) of rats with hypospadias induced by maternal exposure to Di-n-butyl phthalate (DBP) or DBP + dexamethasone (DXM) in order to further explore the mechanism of hypospadias. Methods Forty pregnant SD rats were randomly divided into four groups which were given: group A, soybean oil oral gavage 2 ml/day;DBP (700 mg/kg gavage) + DXM (0. 1 mg/kg sc-injection) during gestational day (GD) 14-18. Pregnant rats were killed at GD 19, birth weight (BW) and anogenital distance (AGD) were recorded in the fetal rats, GT was harvested to verify the expression of ATF3 by Western blotting and immunohistochemistry. Results DXM alone had no effect except to reduce BW but could increase the teratogenic effect of DBP when combined with DBO. The BW and AGD/BW in group D were significantly lower than in group C. The expression of ATF3 had statisticall significant difference between groups C or D and group A, as well as between groups C and D (P ＜ 0. 05). ATF3 was mainly located in the urethral epithelium and mesenchyme. Conclusion These results suggests that exposure to common environmental chemicals in combination with environmental endocrine disrupters may increase the risk of male reproductive abnormalities,such as hypospadias. Up-regulation of ATF3 may affect urethral fold fusion, and then lead to the occurrence of hypospadias.     

转录激活因子3在糖皮质激素与邻苯二甲酸二丁酯联合作用致大鼠尿道下裂发生中的表达变化

Objective To verify the differential expression of ATF3 in the genital tubercle (GT) of rats with hypospadias induced by maternal exposure to Di-n-butyl phthalate (DBP) or DBP + dexamethasone (DXM) in order to further explore the mechanism of hypospadias. Methods Forty pregnant SD rats were randomly divided into four groups which were given: group A, soybean oil oral gavage 2 ml/day;DBP (700 mg/kg gavage) + DXM (0. 1 mg/kg sc-injection) during gestational day (GD) 14-18. Pregnant rats were killed at GD 19, birth weight (BW) and anogenital distance (AGD) were recorded in the fetal rats, GT was harvested to verify the expression of ATF3 by Western blotting and immunohistochemistry. Results DXM alone had no effect except to reduce BW but could increase the teratogenic effect of DBP when combined with DBO. The BW and AGD/BW in group D were significantly lower than in group C. The expression of ATF3 had statisticall significant difference between groups C or D and group A, as well as between groups C and D (P ＜ 0. 05). ATF3 was mainly located in the urethral epithelium and mesenchyme. Conclusion These results suggests that exposure to common environmental chemicals in combination with environmental endocrine disrupters may increase the risk of male reproductive abnormalities,such as hypospadias. Up-regulation of ATF3 may affect urethral fold fusion, and then lead to the occurrence of hypospadias.     

转录激活因子3在糖皮质激素与邻苯二甲酸二丁酯联合作用致大鼠尿道下裂发生中的表达变化

Objective To verify the differential expression of ATF3 in the genital tubercle (GT) of rats with hypospadias induced by maternal exposure to Di-n-butyl phthalate (DBP) or DBP + dexamethasone (DXM) in order to further explore the mechanism of hypospadias. Methods Forty pregnant SD rats were randomly divided into four groups which were given: group A, soybean oil oral gavage 2 ml/day;DBP (700 mg/kg gavage) + DXM (0. 1 mg/kg sc-injection) during gestational day (GD) 14-18. Pregnant rats were killed at GD 19, birth weight (BW) and anogenital distance (AGD) were recorded in the fetal rats, GT was harvested to verify the expression of ATF3 by Western blotting and immunohistochemistry. Results DXM alone had no effect except to reduce BW but could increase the teratogenic effect of DBP when combined with DBO. The BW and AGD/BW in group D were significantly lower than in group C. The expression of ATF3 had statisticall significant difference between groups C or D and group A, as well as between groups C and D (P ＜ 0. 05). ATF3 was mainly located in the urethral epithelium and mesenchyme. Conclusion These results suggests that exposure to common environmental chemicals in combination with environmental endocrine disrupters may increase the risk of male reproductive abnormalities,such as hypospadias. Up-regulation of ATF3 may affect urethral fold fusion, and then lead to the occurrence of hypospadias.     

RNA干扰人膜联蛋白A3对胆囊癌细胞增殖的影响

目的 构建人膜联蛋白A3(annexinA3)基因RNA干扰(miRNA)表达载体,体外研究其对人胆囊癌细胞SGC-996的增殖及凋亡作用.方法 构建4组针对annexinA3的pcDNATM 6.2-GW/EmGFPmiR/miRNA表达载体和1组阴性对照序列.脂质体介导瞬时转染人胆囊癌细胞株SGC-996.用RT-PCR和Western印迹检测转染前后annexinA3基因的表达.挑选干扰效率最强的一组表达载体,以MTT法和流式细胞仪检测经miRNA干扰annexinA3对人胆囊癌细胞SGC-996的体外作用.结果 经测序鉴定,annexinA3-miRNA载体构建成功,转染人胆囊癌细胞SGC-996后荧光定量PCR和westem印迹检测显示:annexinA3基因表达及蛋白表达明显降低(P＜0.05);干扰后细胞体外增殖缓慢,增殖明显受抑(P＜0.05).annexinA3基因干扰对细胞周期无明显影响,但能明显增加胆囊癌细胞的凋亡(P＜0.05).结论 AnnexinA3-miRNA表达载体能有效抑制胆囊癌SGC-996细胞annexinA3的表达,抑制肿瘤细胞生长,引起胆囊癌细胞凋亡.AnnexinA3可能是胆囊癌治疗的一个分子靶点.     

胆囊良恶性组织中蛋白质组差异表达及膜联蛋白A3的功能

目的 分离并鉴定胆囊癌和胆囊良性组织的差异表达蛋白质,以发现可能用于早期诊断胆囊癌的肿瘤标志物.观察RNA干扰沉默膜联蛋白A3(AnnexinA3)基因对胆囊癌细胞增殖及周期的影响.方法 提取人胆囊癌和胆囊良性组织的总蛋白质,用双向电泳分离蛋白并进行比较.选择差异表达超过2倍的蛋白点进行MALDI-TOF/TOF质谱和生物学分析.miRNA干扰胆囊癌细胞株中AnnexinA3的表达后,通过噻唑蓝(MTT)实验及流式细胞仪观察癌细胞增殖能力及细胞周期的变化.结果 筛选出在胆囊癌组织中明显差异表达的46个蛋白点,共有17个蛋白质被成功鉴定,其中在胆囊癌组织中高表达的为9个,低表达的为8个,包括AnnexinA3、TTR蛋白等.RNA干扰胆囊癌细胞株AnnexinA3蛋白的表达后,MTT显示干扰后细胞增殖明显下降效率为44.14%(P<0.05).流式细胞仪观察显示干扰后G1期增加(P<0.05),S期减少(P<0.05).结论 胆囊癌组织相对于胆囊良性组织蛋白存在明显差异.干扰AnnexinA3蛋白的表达可以改善胆囊癌细胞的某些恶性生物学行为.     

半乳糖凝集素-3在邻苯二甲酸二丁酯致大鼠尿道下裂发生中的表达

目的 利用邻苯二甲酸二丁酯(DBP)胚胎期暴露导致SD大鼠尿道下裂发生的动物模型,观察半乳糖凝集素-3(galectin-3)在尿道下裂与正常大鼠生殖结节中的差异表达,探讨其在DBP致大鼠尿道下裂发生中的作用.方法 SD孕鼠20只,随机分为2组,妊娠14～18 d(GD14～18)实验组和对照组分别给予DBP 800 mg/(kg·d)和大豆油2 ml/d灌胃,GD19剖宫产后统计子鼠数、雄性子鼠出生体质量(BW)及肛门生殖器距离(AGD)、尿道下裂发生率,取两组雄性子鼠生殖结节,运用免疫印迹法和免疫组织化学方法分析galectin-3的表达.结果 尿道下裂仅实验组发生,发生率为40%,每窝子鼠数、雄性子鼠BW及AGD/BW在实验组分别为(8.90±1.25)只、(5.12±0.24)g、(0.57±0.03)mm/g,在对照组分别为(13.20±1.46)只、(6.72±0.42)g、(0.66±0.07)mm/g,差异有统计学意义(P＜0.05);galectin-3在尿道下裂组的相对表达量为0.603±0.014(n=10),正常对照组为0.851±0.015(n=10),差异有统计学意义(P＜0.05);galectin-3主要定位于生殖结节上皮细胞,尿道下裂组染色强度明显弱于对照组.结论 染毒期间DBP对雄性子鼠有明显毒性作用,改变了生殖结节中galectin-3的表达,影响了上皮细胞增殖、凋亡及尿生殖褶的融合.     

肝肺综合征大鼠肺组织膜联蛋白A2表达的变化

目的 评价肝肺综合征(HPS)大鼠肺组织膜联蛋白A2(ANXA2)表达的变化.方法 健康SD大鼠40只,雌雄不拘,3～4月龄,体重220～250 g.采用随机数字表法,将其分为3组:对照组(C组,n=10)、假手术组(S组,n=10)和肝肺综合征组(HPS组,n=20).HPS组采用胆总管结扎法建立肝肺综合征模型;C组不做任何处理;S组仅开腹暴露胆总管,但不结扎.术后5周时,处死大鼠,取肺组织,采用RT-PCR法检测ANXA2和平滑肌肌动蛋白α(SM-α-acdn)的mRNA表达,采用Western blot法检测ANXA2和SM-α-actin的蛋白表达.结果 与C组比较,S组肺组织ANXA2和SM-α-actin的mRNA及其蛋白表达差异无统计学意义(P＞0.05);与S组比较,HPS组肺组织ANXA2和SM-α-actin的mRNA及其蛋白表达上调(P＜0.05).结论 大鼠肝肺综合征时肺组织ANXA2表达上调.     

膜联蛋白A2和锌指转录因子Snail在人脑胶质瘤组织中的表达及意义

目的 探讨膜联蛋白A2(Annexin A2)和锌指转录因子Snail在胶质瘤组织的表达水平及两者与胶质瘤病理学特征的关系.方法 应用免疫组织化学法检测52例胶质瘤组织和22例正常脑组织中Annexin A2和Snail表达水平.结果 Annexin A2在胶质瘤组织中阳性表达率明显高于正常脑组织(80.77％比9.09％,P＜0.01),Snail在胶质瘤组织中阳性表达率明显高于正常脑组织(67.31％比13.64％,P＜0.01);Annexin A2及Snail表达水平与胶质瘤组织学分级有关(P＜0.05).结论 Annexin A2及Snail的过度表达与胶质瘤的发生发展和恶性程度有关,联合检测Annexin A2及Snail有助于判断胶质瘤患者预后.     

邻苯二甲酸二丁酯诱导大鼠尿道下裂发生的阴茎组织差异蛋白质组分析

我们通过比较邻苯二甲酸二丁酯（DBP）诱导尿道下裂与正常大鼠阴茎组织蛋白表达的差异，为阐明DBp导致尿道下裂的作用机制提供依据。[第一段]     

膜联蛋白A2和葡萄糖调节蛋白78表达与前列腺癌的关系

目的 分析膜联蛋白A2(Annexin A2)和葡萄糖调节蛋白78(GRP78)分别在人前列腺增生细胞系(BPH-1)、雄激素非依赖性前列腺癌细胞系(LNCaP)和雄激素依赖性前列腺癌细胞系(PC-3)中的差异表达. 方法 利用双向凝胶电泳结合质谱的蛋白质组学技术,分析Annexin A2和GRP78在三种细胞系的表达;分别采用免疫印迹法和免疫荧光染色法对其进行半定量和定位验证. 结果 Annexin A2在BPH-1和LNCaP中低表达,在PC-3细胞中高表达;GRP78在PC-3、BPH-1及LNCaP中表达量逐渐升高(P＜0.05). 结论 Annexin A2和GRP78的差异表达可能与前列腺癌发展的不同阶段激素依赖性有关,有望作为前列腺癌诊断和治疗的重要指标.     

恐惧应激对大鼠睾丸中Annexin5表达的影响

目的观察大鼠应激条件下睾丸组织中Annexin5表达的变化。方法建立SD大鼠的恐惧应激模型，分别取急性应激、急性对照、慢性应激、慢性对照SD大鼠睾丸，免疫组织化学和Western blotting分析Annexin5免疫定位和蛋白表达。结果急性应激组与对照组相比，免疫组化显示Annexin5都定位在间质细胞、支持细胞和成熟的精子头部，Western blotting分析发现Annexin5的表达降低了10．8％；慢性应激组与对照组相比，免疫组化结果显示Annexin5在精子头部的定位逐渐减少，应激1w和2w组，未见到Annexin5定位于精子头部，到了应激的3w和4w，发现Annexin5重新定位于精子的头部，而在间质细胞和支持细胞上的定位模式没有显著变化。Western blotting分析发现Annexin5的表达降低了17．4％。结论大鼠睾丸Annexin5参与了应激过程，急性应激条件下Annexin5表达降低。随着应激时间的延长，在慢性应激条件下，Annexin5的表达由减少之后而开始有缓慢增加。     

Annexin A1 involved in the regulation of inflammation and cell signaling pathways

With the deepening of research,proteomics has developed into a science covering the study of all the structural and functional characteristics of proteins and the dynamic change rules.The essence of various biological activities is revealed from the perspectives of the biological structure,functional activity and corresponding regulatory mechanism of proteins by proteomics.Among them,phospholipid-binding protein is one of the hotspots of proteomics,especially annexin A1,which is widely present in various tissues and cells of the body.It has the capability of binding to phospholipid membranes reversibly in a calcium ion dependent manner.In order to provide possible research ideas for researchers,who are interested in this protein,the biological effects of annexin A1,such as inflammatory regulation,cell signal transduction,cell proliferation,differentiation and apoptosis are described in this paper.     

Combined expression of S100A4 and Annexin A2 predicts disease progression and overall survival in patients with urothelial carcinoma

ObjectivesTo determine the expression patterns and prognostic value of S100A4 and Annexin A2 for urothelial carcinoma of the urinary bladder.Methods and materialsImmunohistochemical staining for S100A4 and Annexin A2 was performed in 315 archived radical cystectomies and 63 normal specimens. The immunoreactivity of these proteins was correlated to evaluate their clinical significance as prognostic factors.ResultsProtein levels of S100A4 and Annexin A2 were up-regulated in urothelial carcinoma compared with adjacent nontumor tissues. The increased expressions of S100A4 and Annexin A2 were associated with invasion depth, lymph node metastasis, and distant metastasis (P<0.05). High expression of S100A4 correlated with expression of Annexin A2. These alterations in expression were also associated with greater risk of disease progression and decreased chance of carcinoma-specific survival. Further multivariate analysis suggested that expressions of S100A4 and Annexin A2 were independent prognostic indicators for overall survival in urothelial carcinoma. The patients with S100A4-positive/Annexin A2-positive carcinomas presented the lowest 5-year survival rate compared with the other 3 groups.ConclusionsS100A4 and Annexin A2 proteins could be useful prognostic markers to predict tumor progression and prognosis in urothelial carcinoma. The expression patterns of S100A4/Annexin A2 interaction correlated well with the pathologic stage, disease progression, and carcinoma-specific survival. This finding could aid in identifying more biologically aggressive carcinomas and thus patients who might benefit from more intensive adjuvant therapy.     

膜联蛋白7在转移性激素敏感前列腺癌原发灶组织中的表达及其意义

目的检测膜联蛋白7(ANXA7)在转移性激素敏感前列腺癌(HSPC)原发灶组织中的表达,探讨对其预后意义。方法取转移性HSPC原发灶组织106例,免疫组化检测标本ANXA7蛋白表达,分析其表达与常见临床病理指标和疾病无进展生存时间(PFS)及总生存时间(OS)的相关性。对照组为前列腺增生20例,去势抵抗性前列腺癌(CRPC)22例。结果转移性HSPC原发灶组织ANXA7阳性表达率为50/106(47.2%),前列腺增生组织ANXA7全部阳性表达,CRPC组织为3/22(13.6%),差异比较均有显著统计学意义(P0.05)。ANXA7表达减少与T分期、N分期级别高及Gleason评分高相关,生存分析显示ANXA7表达减少与较短的PFS和OS相关,ANXA7阳性和阴性表达者PFS中位值分别为24.4月和15.5月,OS中位值分别为56.4月和36.1月。结论 ANXA7在转移性HSPC原发灶组织中表达减少,其参与疾病进展过程,表达异常往往与患者不良预后相关。     

X染色体开放读码框架6在邻苯二甲酸二丁酯致雄性大鼠尿道下裂发生中的表达

目的 观察X染色体开放读码框架6(CXorf6)在邻苯二甲酸二丁酯(DBP)致大鼠尿道下裂发生中的作用.方法 于妊娠14～18 d(GD14～18)实验和对照组各10只孕鼠分别给予DBP 800 mg/(kg·d)和豆油2 ml/d灌胃,GD19剖宫产统计尿道下裂发生率,免疫印迹法和免疫组织化学法分析两组子鼠睾丸中CXorf6的表达.结果 尿道下裂仅实验组发生,发生率为40.7％;CXorf6在尿道下裂组的相对表达量为0.323±0.014(n=10),对照组为0.706±0.016(n=10),差异有统计学意义(P＜0.05);CXorf6主要定位于睾丸间质和支持细胞,尿道下裂组CXorf6着色细胞数量减少,染色强度弱于对照组.结论 DBP通过干预CXorf6的表达以影响睾丸的发育及雄激素的产生而导致尿道下裂的发生.     

邻苯二甲酸二丁酯致尿道下裂大鼠发育异常和阴茎病理学改变研究

目的邻苯二甲酸二丁酯（DBP）孕晚期染毒诱导子代雄鼠尿道下裂发生和探讨DBP致尿道下裂雄鼠发育异常和阴茎病理学改变。方法雌鼠怀孕14～18d，每天分别灌胃给予大豆油（A组），DBP500（B组）、800（C组）、1200（D组）mg／kg体重，分娩后统计仔鼠数和雄仔鼠体重。出生后（PND）7d测量雄仔鼠肛门生殖器距离（AGD），观察尿道下裂发生率和尿道下裂阴茎病理学改变。PND70,对尿道下裂雄仔鼠称重后解剖，评价发育情况。结果C组仔鼠数和雄仔鼠体重明显减少；D组出现孕鼠死亡和零产仔；尿道下裂仅C组发生。发生率为36．5％。PND7，B、C组雄仔鼠AGD和A组相比明显减小。阴茎连续性病理切片显示典型尿道下裂改变。解剖得尿道下裂雄仔鼠肝、肾、前列腺、睾丸、附睾脏器系数与A组相比明显减小，脑垂体脏器系数明显增大。结论800mg／kg体重DBP孕晚期染毒能高诱导雄仔鼠尿道下裂发生，DBP不仅损害雄仔鼠生殖系统，还引起发育异常。