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浙江大学学报（医学版） 2015年第3期44卷 285-292页

作者：辛廖冰江秀秀叶小磊吴瑞瑾徐开红马俊彦林俊浙江大学医学院附属妇产科医院妇科浙江杭州310006 浙江大学医学院附属邵逸夫医院妇产科浙江杭州310020

目的：探讨水通道蛋白（AQP）5基因对异位子宫内膜腺上皮细胞增殖及迁移的影响。方法：构建可靶向沉默AQP5基因的质粒，在293 T细胞中包装形成病毒颗粒后，感染原代培养的异位子宫内膜腺上皮细胞，分别通过逆转录PCR及蛋白质印迹法鉴定... 详细信息

目的：探讨水通道蛋白（AQP）5基因对异位子宫内膜腺上皮细胞增殖及迁移的影响。方法：构建可靶向沉默AQP5基因的质粒，在293 T细胞中包装形成病毒颗粒后，感染原代培养的异位子宫内膜腺上皮细胞，分别通过逆转录PCR及蛋白质印迹法鉴定异位子宫内膜腺上皮细胞中AQP5 mRNA和蛋白表达。 MTT法测定细胞增殖；Transwell技术检测细胞的体外迁移能力；蛋白质印迹法检测丝氨酸／苏氨酸蛋白酶（ AKT ）活化情况。构建裸鼠腹腔内子宫内膜异位症体内模型，考察阴性对照组和AQP5沉默组子宫内膜腺上皮细胞在裸鼠腹腔内瘤体生长情况及腹膜转移情况。结果：体外实验结果显示，AQP5沉默后异位子宫内膜腺上皮细胞的增殖能力在第7天时明显抑制（ P＜0．05）；AQP5沉默组异位子宫内膜腺上皮细胞磷酸化（ p－AKT）表达减少，而AKT表达无改变，p－AKT／AKT减少（ P＜0．05）；AQP5沉默组比阴性对照组迁移细胞数减少（ P＜0．05）。体内实验结果显示，与阴性对照组比较，AQP5沉默组瘤体结节数相对较少，体积较小，其中腹膜瘤体结节数少于阴性对照组（ P＜0．05）。结论：AQP5基因沉默可抑制异位子宫内膜腺上皮细胞的增殖及迁移能力，其机制可能与AKT活化有关。

关键词：子宫内膜异位症水通道蛋白质5 子宫内膜基因沉默上皮细胞/代谢细胞增殖细胞运动

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大鼠BMP-7重组腺病毒构建及在肾小管上皮细胞中表达

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浙江大学学报（医学版） 2010年第1期39卷 71-78页

作者：杨肃文王伟徐再春朱芸芸浙江大学医学院附属邵逸夫医院检验科浙江杭州310016 浙江省生物治疗重点实验室浙江杭州310016 浙江省立同德医院浙江杭州310012

目的:利用AdEasyTMsystem构建携带大鼠BMP-7基因的重组腺病毒,并鉴定其在原代肾小管上皮细胞中的表达。方法:将BMP-7全长序列分为46个片段分段合成,再采用PCR方法将合成的46个寡核苷酸片段拼接成BMP-7基因,并克隆到测序载体中测序鉴定。... 详细信息

目的:利用AdEasyTMsystem构建携带大鼠BMP-7基因的重组腺病毒,并鉴定其在原代肾小管上皮细胞中的表达。方法:将BMP-7全长序列分为46个片段分段合成,再采用PCR方法将合成的46个寡核苷酸片段拼接成BMP-7基因,并克隆到测序载体中测序鉴定。经NotI和HindⅢ双酶切后接入pShuttle-CMV穿梭载体,构建重组腺病毒的穿梭质粒pShuttle-BMP-7。EcoRI酶切重组pShuttle-BMP-7,与pAdEasyTMDNA电穿孔共转化BJ5183感受态菌,抽提重组AdBMP-7质粒,PacI酶切线性化重组质粒AdBMP-7后,转染Ad293细胞进行病毒包装和扩增,腺病毒荧光检测试剂盒测定其滴度。用AdBMP-7感染大鼠肾小管上皮细胞,以ELISA法和W estern-blot法检测BMP-7在大鼠肾小管上皮细胞中的表达。RT-PCR方法检测-αSMA mRNA的表达水平,以鉴定重组腺病毒AdBMP-7的生物学活性。结果:构建了BMP-7基因的重组腺病毒载体,并制备出高滴度重组腺病毒保存液。该重组腺病毒在体外能有效转染大鼠肾小管上皮细胞并高表达BMP-7蛋白,所表达的BMP-7蛋白能有效逆转由TGF-β1诱导的α-SMA表达增高。结论:成功地构建了携带大鼠BMP-7基因的重组腺病毒,并初步证实其具有一定的抗肾纤维化功能。

关键词：骨形态发生蛋白质类/遗传学重组,遗传转染遗传载体腺病毒科/遗传学肾小管/细胞学上皮细胞/代谢肾硬化症/治疗

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工频磁场对人晶状体上皮细胞DNA双链断裂的影响

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浙江大学学报（医学版） 2008年第1期37卷 9-14页

作者：杜晓刚徐珊珊陈苘鲁德强许正平曾群力浙江大学医学院浙江省生物电磁学重点研究实验室浙江杭州310058 浙江省疾病预防控制中心浙江杭州310009

目的:采用γH2AX免疫荧光法观察工频磁场对人晶状体上皮细胞DNA双链断裂的影响。方法:将人晶状体上皮细胞暴露于0.4 mT、50 Hz工频磁场2 h、6 h、12 h、24 h、48 h,以0.1μmol/L的DNA损伤剂4-硝基喹啉-1-氧化物作用1 h作为阳性对照,结... 详细信息

目的:采用γH2AX免疫荧光法观察工频磁场对人晶状体上皮细胞DNA双链断裂的影响。方法:将人晶状体上皮细胞暴露于0.4 mT、50 Hz工频磁场2 h、6 h、12 h、24 h、48 h,以0.1μmol/L的DNA损伤剂4-硝基喹啉-1-氧化物作用1 h作为阳性对照,结束处理后进行γH2AX免疫荧光检测。计算细胞平均焦点数。将细胞平均焦点数及焦点阳性细胞率作为评价细胞DNA双链断裂程度的指标。结果:工频磁场暴露24 h的平均焦点数、γH2AX焦点阳性细胞率分别为(2.93±0.43)、(27.88±2.59)%,与假辐照组(1.77±0.37)、(19.38±2.70)%相比,差异具有显著性(P<0.05);工频磁场暴露48 h的平均焦点数、γH2AX焦点阳性细胞率分别为(3.14±0.35)、(31.00±3.44)%,与假辐照组相比,差异具有显著性(P<0.01);而工频磁场暴露2 h分别为(2.11±0.61)、(22.44±5.09)%,6 h分别为(2.18±0.47)、(22.59±3.08)%和12 h分别为(2.35±0.43)、(23.35±2.93)%,与假辐照组比较,差异没有显著性(P>0.05)。结论:体外实验表明,0.4 mT工频磁场长时间辐照可以使人晶状体上皮细胞DNA双链断裂增加。

关键词： DNA损伤辐射电离晶体/细胞学上皮细胞/代谢电磁场

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缝隙连接蛋白Cx43在大鼠经脉线上表达的实验研究

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中国针灸 2005年第9期25卷 629-632页

作者：郑翠红黄光英张明敏肖雁凌华中科技大学同济医学院附属同济医院中西医结合研究所武汉430030

目的:研究缝隙连接蛋白Cx43在正常健康大鼠经脉线上的表达及其意义。方法:应用二步法免疫酶技术和ASIAS2000自动扫描图像分析系统对大鼠多条经脉上的Cx43进行定位、定量分析。结果:缝隙连接蛋白Cx43广泛表达于表皮上皮细胞、真皮成纤维... 详细信息

目的:研究缝隙连接蛋白Cx43在正常健康大鼠经脉线上的表达及其意义。方法:应用二步法免疫酶技术和ASIAS2000自动扫描图像分析系统对大鼠多条经脉上的Cx43进行定位、定量分析。结果:缝隙连接蛋白Cx43广泛表达于表皮上皮细胞、真皮成纤维细胞、皮下筋膜的肥大细胞以及肌肉肌膜细胞的胞浆胞膜中,经统计学分析,肾经足底段、膀胱经背部段Cx43的表达显著高于邻近对照线,两者相比差异有极显著性意义(P<0.01)。结论:缝隙连接蛋白及缝隙连接与经络高度相关,由缝隙连接介导的细胞间通讯可能在经络活动中起重要作用。

关键词：连接蛋白43/分析连接蛋白43/代谢上皮细胞/代谢肥大细胞/代谢经络实质免疫组织化学

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电磁噪声阻断1800MHz手机辐射所致的晶状体上皮细胞内活性氧增加及DNA损伤

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浙江大学学报（医学版） 2008年第1期37卷 34-38页

作者：吴炜姚克王凯军鲁德强何继亮徐立红孙文均浙江大学医学院附属第二医院眼科中心浙江杭州310009 浙江大学医学院浙江省生物电磁学重点研究实验室劳动卫生与环境卫生研究所生物化学与遗传学系浙江杭州310058

目的:探讨1800 MHz手机辐射对人晶状体上皮细胞内活性氧(ROS)和DNA损伤的影响,以及叠加电磁噪声后对该效应的阻断作用。方法:体外培养的人晶状体上皮细胞受比吸收率(specific absorption rate,SAR)为4 W/kg的1800 MHz手机频段间断辐射2... 详细信息

目的:探讨1800 MHz手机辐射对人晶状体上皮细胞内活性氧(ROS)和DNA损伤的影响,以及叠加电磁噪声后对该效应的阻断作用。方法:体外培养的人晶状体上皮细胞受比吸收率(specific absorption rate,SAR)为4 W/kg的1800 MHz手机频段间断辐射24 h或与2μT电磁噪声联合作用24 h后,利用荧光探针检测ROS水平及用彗星试验检测DNA损伤水平。结果:4 W/kg手机辐射可明显诱发人晶状体上皮细胞内ROS及DNA损伤(P<0.001);与2μT电磁噪声联合暴露后可明显阻断该效应,与手机单独辐射相比,差异有显著性(P<0.001),但与假辐照组比较差异无显著性(P>0.05)。结论:电磁噪声可阻断1800 MHz手机辐射所诱发的晶状体上皮细胞内ROS及DNA损伤。

关键词：噪声/副作用活性氧 DNA损伤电磁场彗星试验辐射晶体/细胞学上皮细胞/代谢

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Relationship between phosphatidic acid level and regulation of protein transit in colonic epithelial cell line HT29-cl19A

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第40期274卷 28652-28659页

作者： Auger, R Robin, P Camier, B Vial, G Rossignol, B Tenu, JP Raymond, MN Univ Paris 11 CNRS UMR 8619 Lab Biochim Transports Cellulaires F-91405 Orsay France

Colonic epithelial HT29-cl19A cells are polarized and secrete proteins among which alpha(1)-antitrypsin represents about 95%. Secretion occurs via a constitutive pathway, so that the rates of secretion directly reflect the rates of protein transit. In this paper we have demonstrated that: 1) in resting cells phospholipase D (PLD) is implicated in the control of apical protein transit;2) phorbol esters stimulate apical protein transit (stimulation factor 2.2), which is correlated with a PLD-catalyzed production of phosphatidic acid (PA) (2.45-fold increase);3) the stimulation of cholinergic receptors by carbachol results in an increase (stimulation factor 1.45) of apical protein transit which is independent of protein kinase C and PLD activities, but related to PA formation (1.7-fold increase) via phospholipase(s) C and diacylglycerol kinase activation;4) an elevation of the cAMP level enhances apical protein transit by a PA-independent mechanism;5) a trans-Golgis network or post-trans-Golgi network step of the transit is the target for the regulatory events. In conclusion, we have shown that PA can be produced by two independent signaling pathways;whatever the pathway followed, a close relationship between the amount of PA and the level of secretion was observed.

关键词：卡巴胆碱/药理学结肠/细胞学结肠/代谢二酰甘油激酶/代谢酶激活酶激动剂类/药理学酶联免疫吸附测定上皮细胞/代谢高尔基体/代谢 HT29细胞动力学磷脂酸类/代谢磷脂酶D/代谢 C型磷脂酶类/代谢 α1抗胰蛋白酶/代谢人类

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PKN binds and phosphorylates human papillomavirus E6 oncoprotein

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第20期275卷 14824-14830页

作者： Gao, QS Kumar, A Srinivasan, S Singh, L Mukai, H Ono, Y Wazer, DE Band, V Tufts Univ New England Med Ctr Dept Radiat Oncol Boston MA 02111 USA Tufts Univ Sch Med Dept Biochem Boston MA 02111 USA Kobe Univ Grad Sch Sci & Technol Kobe Hyogo 6578501 Japan Kobe Univ Dept Biol Fac Sci Kobe Hyogo 6578501 Japan

The high risk human papillomaviruses (HPVs) are associated with carcinomas of cervix and other genital tumors. Previous studies have identified two viral oncoproteins E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high risk HPV E6 protein to immortalize human mammary epithelial cells has provided a single gene model to study the mechanisms of EG-induced oncogenic transformation. In recent years, it has become clear that in addition to EG-induced degradation of p53 tumor suppressor protein, other targets of E6 are required for mammary epithelial cells immortalization, Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid- and Rho small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HPVs were able to bind PKN, Furthermore, all the immortalization-competent and many immortalization-non-competent E6 mutants bind PKN. These data suggest that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells, Finally, we show that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a phosphoprotein, Our finding suggests a novel link between HPV E6 mediated oncogenesis and regulation of a well known phosphorylation cascade.

关键词：氨基酸取代乳房/细胞学细胞系细胞系转化无细胞系统细胞培养的克隆分子上皮细胞/细胞学上皮细胞/代谢基因文库诱变定点癌基因蛋白质类病毒性/代谢乳头状瘤病毒科/遗传学乳头状瘤病毒科/代谢磷酰化蛋白质结合蛋白激酶C 蛋白质丝氨酸苏氨酸激酶/代谢蛋白酪氨酸激酶类/代谢重组蛋白质类/代谢阻遏蛋白质类酿酒酵母菌转染女(雌)性人类

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Conditional expression of fibroblast growth factor-7 in the developing and mature lung

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第16期275卷 11858-11864页

作者： Tichelaar, JW Lu, W Whitsett, JK Childrens Hosp Med Ctr Div Pulm Biol Cincinnati OH 45229 USA

Effects of fibroblast growth factor-7 (FGF-7) on lung morphogenesis, respiratory epithelial cell differentiation, and proliferation mere assessed in transgenic mice in which the human FGF-7 cDNA was controlled by a conditional promoter under the direction of regulatory elements from either the human surfactant protein-C (SP-C) or rat Clara cell secretory protein (ccsp) genes. Expression of FGF-7 was induced in respiratory epithelial cells of the fetal lung by administration of doxycycline to the dam. Prenatally, doxycycline induced FGF-7 mRNA in respiratory epithelial cells in both Sp-c and Ccsp transgenic lines, increasing lung size and causing cystadenomatoid malformation. Postnatally, mice bearing both Ccsp-rtta and (Teto)(7)-cmv-fgf-7 transgenes survived, and lung morphology was normal, Induction of FGF-7 expression by doxycycline in the Ccsp-rtta x (Teto)(7)-cmv-fgf-7 mice caused marked epithelial cell proliferation, adenomatous hyperplasia, and pulmonary infiltration with mononuclear cells. Epithelial cell hyperplasia caused by FGF-7 was largely resolved after removal of doxycycline. Surfactant proteins, TTF-1, and aquaporin 5 expression were conditionally induced by doxycycline. The Sp-c-rtta and Ccsp-rtta activator mice provide models in which expression is conditionally controlled in respiratory epithelial cells in the developing and mature lung, altering lung morphogenesis, differentiation, and proliferation.

关键词：水通道蛋白质5 水孔蛋白质类/代谢多西环素/药理学酶抑制剂/代谢上皮细胞/代谢成纤维细胞生长因子10 成纤维细胞生长因子7 成纤维细胞生长因子基因表达调控发育期生长物质/生物合成生长物质/遗传学原位杂交肺/生长和发育膜蛋白质类小鼠转基因核蛋白质类/代谢磷脂酶A/拮抗剂和抑制剂蛋白质类/遗传学蛋白质类/代谢蛋白脂质类/遗传学蛋白脂质类/代谢肺表面活性剂/遗传学肺表面活性剂/代谢转录因子/代谢转基因子宫珠蛋白/代谢动物人类小鼠大鼠

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Phorbol ester-induced expression of airway squamous cell differentiation marker, SPRR1B, is regulated by protein kinase Cδ/Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第41期275卷 32250-32259页

作者： Vuong, H Patterson, T Shapiro, P Kalvakolanu, DV Wu, R Ma, WY Dong, ZG Kleeberger, SR Reddy, SPM Johns Hopkins Univ Dept Environm Hlth Sci Div Physiol Sch Publ Hlth Baltimore MD 21205 USA Univ Maryland Sch Pharm Baltimore MD 21201 USA Univ Maryland Sch Med Greenbaum Canc Ctr Baltimore MD 21201 USA Univ Calif Davis Dept Internal Med Sch Med Davis CA 95616 USA Univ Minnesota Hormel Inst Austin MN 55912 USA

The transcriptional induction of SPRR1B by phorbol 12-myristate 13-acetate (PMA) is mainly mediated by the first -152-base pair 5'-flanking region containing two functional AP-1 sites. In this study, we have analyzed the signaling pathways that mediate the induction in tracheobronchial epithelial cells. PKC inhibitor ablated PMA-stimulated expression of endogenous SPRR1B and reporter gene expression driven by SPRR1B promoter. PKC activator promoted the transcription. The dominant negative protein kinase C delta (dn-PKC delta) and rottlerin (PKC delta inhibitor) completely suppressed PMA-stimulated promoter activity, dn-Ras or dn-MEKK1 inhibited PMA-stimulated promoter activity, while their corresponding constitutively active mutants augmented it. dn-c-Raf-1 did not have any effect on reporter gene expression. Since MEKK1 activates multiple parallel pathways, we examined involvement of JNK/SAPK, p38, and MKK1 in promoter regulation. Go-expression of the dominant negative forms of MKK4, MKK7, JNK/SAPK, MKK3, MKK6, or p38 alpha did not suppress PMA-stimulated reporter gene expression. However, MKK1 inhibitors UO126 and PD98095 suppressed gene expression. Consistent with this, expression of dn-MKK1 strongly suppressed PMA-stimulated promoter activity, while the constitutively active MKK1 augmented it. However, MKK1-mediated induction of SPRR1B probably does not depend on extracellular signal-regulated kinases 1 and 2, suggesting the requirement of another kinase(s). dnc-Jun mutants abolished PMA-stimulated expression supporting an important role for AP-l proteins in SPRR1B expression. Together, these results suggest that a PKC delta/Ras/MEKK1/MKK1-dependent/AP-1 pathway regulates the PMA-inducible expression of the SPRR1B in tracheobronchial epithelial cells.

关键词：细胞分化细胞系上皮细胞/细胞学上皮细胞/药物作用上皮细胞/酶学上皮细胞/代谢基因表达调控/药物作用基因报告基因 jun/遗传学同工酶类/拮抗剂和抑制剂同工酶类/代谢 MAP激酶激酶1 MAP激酶激酶激酶1 MAP激酶信号系统/药物作用膜蛋白质类丝裂原激活蛋白激酶激酶类/拮抗剂和抑制剂丝裂原激活蛋白激酶激酶类/遗传学丝裂原激活蛋白激酶激酶类/代谢丝裂原激活蛋白激酶类/拮抗剂和抑制剂丝裂原激活蛋白激酶类/遗传学丝裂原激活蛋白激酶类/代谢模型生物学突变/遗传学蛋白激酶C/拮抗剂和抑制剂蛋白激酶C/代谢蛋白激酶Cδ 蛋白质丝氨酸苏氨酸激酶/拮抗剂和抑制剂蛋白质丝氨酸苏氨酸激酶/遗传学蛋白质丝氨酸苏氨酸激酶/代谢蛋白质类/遗传学蛋白质类/代谢 RNA 信使/遗传学 RNA 信使/代谢呼吸系统/细胞学呼吸系统/药物作用呼吸系统/酶学呼吸系统/代谢十四酰佛波乙酯/药理学转录因子AP-1/代谢 ras蛋白质类/代谢人类

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Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第26期275卷 20157-20167页

作者： Ariizumi, K Shen, GL Shikano, S Xu, S Ritter, R Kumamoto, T Edelbaum, D Morita, A Bergstresser, PR Takashima, A Univ Texas SW Med Ctr Dept Dermatol Dallas TX 75235 USA

Dendritic cells (DC) are special subsets of antigen presenting cells characterized by their potent capacity to activate immunologically naive T cells. By subtracting the mRNAs expressed by the mouse epidermus-derived DC line XS52 with the mRNAs expressed by the J774 macrophage line, we identified five novel genes that were expressed selectively by this DC line. One of these genes encoded a type II membrane-integrated polypeptide of 244 amino acids containing a putative carbohydrate recognition domain motif at the COOH-terminal end. This molecule, termed "dectin-1," was expressed abundantly at both mRNA and protein levels by the XS52 DC line, but not by non-DC lines (including the J774 macrophage line). Dectin-1 mRNA was detected predominantly in spleen and thymus (by Northern blotting) and in skin-resident DC, i.e. Langerhans cells (by reverse transcription-polymerase chain reaction). Affinity-purified antibody against dectin-1 identified a 43-kDa glycoprotein in membrane fractions isolated from the XS52 DC line and from the dectin-1 cDNA-transfected COS-1 cells. His-tagged recombinant proteins containing the extracellular domains of dectin-1 showed marked and specific binding to the surface of T cells and promoted their proliferation in the presence of anti-CD3 monoclonal antibody at suboptimal concentrations. These in vitro results suggest that dectin-1 on DC may bind to as yet undefined ligand(s) on T cells, thereby delivering T cell co-stimulatory signals. Not only do these results document the efficacy of subtractive cDNA cloning for the identification of unique genes expressed by DC, they also provide a framework for studying the physiological function of dectin-1.

关键词：氨基酸序列印迹法 RNA COS细胞细胞培养技术/方法细胞系细胞膜/代谢克隆分子 DNA 互补/代谢树突细胞/代谢上皮细胞/代谢流式细胞术免疫印迹法外源凝集素类/代谢膜蛋白质类/化学膜蛋白质类/遗传学小鼠近交BALBC 分子序列数据神经组织蛋白质类/化学神经组织蛋白质类/遗传学表型重组融合蛋白质类/代谢逆转录聚合酶链反应序列同源性氨基酸 T淋巴细胞/代谢组织分布动物女(雌)性小鼠兔

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