缺血缺氧损伤对肠上皮细胞整合素极性及细胞凋亡的影响
陈前,秦环龙, 上海交通大学附属第六人民医院外科 上海市 200233
陈前,男, 1973-06-21, 上海市人, 汉族, 2004年硕士研究生毕业, 主治医师, 主要从事胃肠外科疾病研究.
国家自然科学基金资助项目, No. 30271286
上海市科委青年科技启明星跟踪计划资助项目, No. 02QMB1406
项目负责人:秦环龙, 200233, 上海市, 上海交通大学附属第六人民医院外科. chenqian2010@sohu.com
电话: 021-64942226 传真: 021-64368920
, http://www.100md.com
收稿日期: 2004-08-25 接受日期: 2004-12-09
Influence of ischemia and anoxia on distributionand polarity of integrin in intestine epithelial cells
Qian Chen, Huan-Long Qin
Qian Chen, Huan-LongQin, Department of Surgery, Shanghai Sixth People'sHospital, Shanghai JiaoTong University, Shanghai, 200233 China
Supported by National Science Foundation of China, No. 30271286;and the Fund from the Guiding Star Program of Youth Science and Technologyof Shanghai Science committee, No. 02QMB1406
, 百拇医药
Correspondence to: Huan-Long Qin, Department of Surgery, ShanghaiSixth People'sHospital, Shanghai Jiaotong University, 200233 Shanghai, China. chenqian2010@sohu.com
Received: 2004-08-25 Accepted: 2004-12-09
Abstract
AIM: To study the influence of ischemia and anoxia injury on the distribution and polarity of integrin in intestine epithelial cells (IEC).
, http://www.100md.com
METHODS: IEC ischemia and anoxia injury model was established. The cells were divided into four groups, namely, group A (control group), group B (anoxia group), group C (ischemia group), and group D (ischemia and anoxia group). Cell apoptosis and the polarity and distribution ofintegrins a3, a5, b1,b2, b5 were determined by flow cytometry (FCM) and laser scanning confocal microscope (LSCM), respectively.
RESULTS: Integrins a3,a5, b1,b5 were expressed exclusively on the basal and lateral surfaces of IECs in group A. After ischemia and anoxia injury, integrins a3,a5, b1,b5 distribution moved from the basolateral to the apical membrane. Cell apoptosis was increased in group B, C and D. Integrin a3 was not observed in the middle and apical membrane in group C. Integrin b2 distribution was not markedly changed in any group.
, 百拇医药
CONCLUSION: The change in the distribution of integrin is induced by ischemia and anoxia, which is correlated with IEC apoptosis. IEC apoptosis is more sensitive to the anoxia.
Key Words: Ischemia; Anoxia; Integrin; Polarity; Apoptosis; epithelial cells
Chen Q, Qin HL. Influence of ischemia and anoxia on distribution and polarity of integrin in intestine epithelial cells. Shijie Huaren Xiaohua Zazhi 2005;13(4):456-459
, http://www.100md.com
摘要
目的:研究缺氧缺血损伤对肠上皮细胞(IEC)整合素极性及分布的改变的凋亡机制.
方法:建立缺血缺氧与IEC凋亡的实验模型，实验分组:对照组(A组)，缺氧组(B组)，缺血组(C组)，缺氧缺血组(D组);采用流式细胞仪(FCM)检测IEC凋亡率，激光共聚焦显微镜(LSCM)检测IEC自基底侧至顶侧5个平面的整合素a3，a5，b1，b2，b5染色荧光强度并定量.
结果:A组细胞整合素a3，a5，b1，b5荧光标记主要分布于细胞基底侧，损伤后B、C、D组呈上移趋势，其分布和极性与细胞凋亡率相一致;整合素a3在C组的中间和顶层没有阳性表达.b2分布较弥散，无明显变化规律.
结论:缺氧缺血损伤对IEC整合素分布改变呈规律性变化，与IEC凋亡密切相关;对缺氧反应更敏感.
, http://www.100md.com
关键词:缺血; 缺氧; 整合素; 极性; 凋亡; 上皮细胞
陈前, 秦环龙. 缺血缺氧损伤对肠上皮细胞整合素极性及细胞凋亡的影响. 世界华人消化杂志 2005;13(4):456-459
(PDF) 整合素分布趋势曲线. A: 整合素a3;B: 整合素a5; C: 整合素β1; D: 整合素b5.(以细胞底端平面为基准，以检测平面与细胞底端的相对距离为横坐标，相应检测平面的荧光强度为纵坐标，绘制整合素在IEC表面不同层面的分布曲线显示，正常IEC整合素主要分布于细胞基底部，损伤后出现向细胞侧面及顶侧分布的趋势.)
图2 A组整合素b1 LSCM扫描照片(A-B-C-D-E分别为底层到顶层)荧光强度逐渐由强变弱.
, http://www.100md.com
3 讨论整合素是由a和b亚单位组成的异二聚体跨膜糖蛋白，整合素因其a和b亚单位的不同组合而出现不同的亚型，a亚基质量在120-180ku之间，b亚基质量90-110ku之间.整合素主要功能是通过与相应的配体结合介导细胞与基质的黏附、信号传导、个体发育、分化、凋亡等病理生理过程.同大多数体细胞一样，在IEC表面也分布着多种整合素受体.与IEC表面有关的整合素有a2、a3、a5、b1、b4、b5[2].不同整合素在IEC的分布亦有不同，如a2b1分布于隐窝处，a3b1分布于绒毛底部，a5b1位于隐窝及绒毛的底部，a7b1则局限隐窝顶部及绒毛底部，而a6b4则均一地分布于整个IEC表面[3-4].近年来随着研究的深入，人们发现整合素还与IEC的凋亡密切相关[5]，整合素可能是IEC黏附于基底膜以维持细胞生存的主要方式，而且已引起了学者们的关注.有研究发现IEC经整合素a4封闭后，其分化、增生、Bcl-2、Bcl-X、Bax、Bak家族基因表达均明显受抑制[6].如犬经iv整合素a，b单抗1.0mg/kg持续2wk，可诱导出现消化道出血[2].还有学者发现，整合素调控着肠上皮的凋亡，a5b1似有抗凋亡作用[7].最近Levy et al[8]的一项研究发现丁酸(NaBT)可以使Caco-2细胞表面整合素b1的表达下降，导致细胞从基底膜脱落并发生凋亡.
, http://www.100md.com
有关整合素在细胞膜上的表达检测，国内外较为常用的办法是采用免疫组化、免疫荧光等技术，此二者不能较为精确地反应表达量，更无法检测极性分布改变、空间结构变化.近年来已有采用激光共聚焦显微镜(LSCM)研究整合素的报道.Gailitet al[9]利用LSCM观察体外培养的肾小管上皮细胞整合素a3呈极性分布，基底层和近基底层的侧层分布强，顶层和近顶层的侧层分布弱;在接受亚致死剂量的氧损伤后，细胞整合素a3的极性分布丧失，由基底层和近基底层向顶层和近顶层的侧层弥散;并且导致黏附于基质的细胞数量减少.Wuet al[6]在体研究发现，肾小管上皮细胞正常情况下整合素b1呈极性分布，缺血再灌注损伤后，也向顶侧弥散;Becket al[2]发现，在体的缺血再灌注损伤模型未发现整合素a3的顶层的侧层分布.以上实验结果提示，缺氧、氧损伤后整合素的极性分布丧失，是导致上皮细胞黏附性改变的基础;同时，也说明了LSCM能够很好地观察整合素的三维结构改变.
我们研究发现，IEC在实验因素刺激下整合素a3，a5，b1，b5发生了极性和分布的改变，并与细胞凋亡、脱落密切相关.其具体表现在，A组a3，a5，b1，b5整合素在细胞膜上呈极性分布，主要位于近基底膜侧;缺血缺氧后B，C，D组有向顶侧弥散的趋势，各层面的定量均存在差异，尤以第1层和第5层改变最为明显(P<0.01，P<0.05);整合素极性分布的明显改变趋势由大到小依次是缺血缺氧组、缺氧组、缺血组，且此三组之间也有差异(P<0.01，P<0.05);无论缺血缺氧还是单纯缺血或缺氧损伤与A组相比较，a3，a5，b1，b5在底层表达均明显下降，中间层和顶层均增加表达，说明有整体从基底向顶层移动的现象.缺血组a3在中间层及顶层没有出现阳性表达，说明其对缺血损伤可能不敏感.缺氧组、缺血缺氧组顶层各整合素亚型都发生变化，并都参与了细胞损伤作用，说明IEC对缺氧尤为敏感.b2整合素在细胞膜上的分布较均匀，缺血缺氧后改变不明显;这可能与整合素b2功能有关，或其本身没有直接参与细胞脱落、凋亡，只是其他某些亚型在IEC凋亡中起着重要调节作用.
, 百拇医药
4 参考文献
1 StupackDG, Cheresh DA. Get a ligand, get a life:integrins, signaling and cellsurvival. J Cell Sci 2002;115:3729-3738
2 Beck R, Nebe B,Guthoff R, Rychly J. Inhibition of lens epithelial cell adhesion by thecalcium antagonist Mibefradil
correlates with impair integrindistribution and organization of the cytoskeleton. Graefes Arch ClinExp Ophthalmol
, http://www.100md.com
2001;239:452-458
3 Goncalves I,Hughan SC, Schoenwaelder SM, Yap CL, Yuan Y, Kachson SP. Integrin alphaIIb beta 3-dependent
calcium signals regulateplatelet-fibrinogen interactions under flow:Involvement of phospholipase Cgamma2.
J Biol Chem 2003;278:34812-34822
4 Nebe B,Holzhausen C, Rychly J, Urbaszek W. Impaired mechanisms of leukocyteadhesion in vitro by the calcium
, http://www.100md.com
channel antagonist mibefradil. CardiovascDrugs Ther 2002;16:183-193
5 Nebe B, Kunz F,Peters A, Rychly J, Noack T, Beck R. Induction of apoptosis by the calciumantagonist mibefradil
correlates with depolarization of themembrane potential and decreased integrin expression in human lensepithelial
cells. Graefes Arch Clin Exp Ophthalmol 2004;242:597-604
6 Wu X, Davis GE,Meininger GA, Wilson E, Davis MJ. Regulation of the L-type calcium channelby alpha 5b1 integrin
, 百拇医药
requires signaling between focal adhesionproteins. J Biol Chem 2001;276:30285-30292
7 Matlin KS, HausB, Zuk A. Integrins in epithelial cell polarity:using antibody to analyzeadhesive function and
morphogenesis. Methods 2003;30:235-245
8 Levy P, RobinH, Bertrand F, Kornprobst M, Capeau J. Butyrate-treated colonic Caco-2cells exhibit defect
integrin-mediated signaling together withincreased apoptosis and differentiation. J Cell Physiol 2003;197:336-347
9 Gailit J,Clarke C, Newman D, Tonnesen MG, Mosesson MW, Clark RA. Human fibroblastsbind directly to fibrinogen at
RGD sites through integrin alpha(v)beta3. ExpCell Res 1997;232:118-126
编辑 潘伯荣 审读 张海宁, 百拇医药( 陈 前,秦环龙)
陈前,男, 1973-06-21, 上海市人, 汉族, 2004年硕士研究生毕业, 主治医师, 主要从事胃肠外科疾病研究.
国家自然科学基金资助项目, No. 30271286
上海市科委青年科技启明星跟踪计划资助项目, No. 02QMB1406
项目负责人:秦环龙, 200233, 上海市, 上海交通大学附属第六人民医院外科. chenqian2010@sohu.com
电话: 021-64942226 传真: 021-64368920
, http://www.100md.com
收稿日期: 2004-08-25 接受日期: 2004-12-09
Influence of ischemia and anoxia on distributionand polarity of integrin in intestine epithelial cells
Qian Chen, Huan-Long Qin
Qian Chen, Huan-LongQin, Department of Surgery, Shanghai Sixth People'sHospital, Shanghai JiaoTong University, Shanghai, 200233 China
Supported by National Science Foundation of China, No. 30271286;and the Fund from the Guiding Star Program of Youth Science and Technologyof Shanghai Science committee, No. 02QMB1406
, 百拇医药
Correspondence to: Huan-Long Qin, Department of Surgery, ShanghaiSixth People'sHospital, Shanghai Jiaotong University, 200233 Shanghai, China. chenqian2010@sohu.com
Received: 2004-08-25 Accepted: 2004-12-09
Abstract
AIM: To study the influence of ischemia and anoxia injury on the distribution and polarity of integrin in intestine epithelial cells (IEC).
, http://www.100md.com
METHODS: IEC ischemia and anoxia injury model was established. The cells were divided into four groups, namely, group A (control group), group B (anoxia group), group C (ischemia group), and group D (ischemia and anoxia group). Cell apoptosis and the polarity and distribution ofintegrins a3, a5, b1,b2, b5 were determined by flow cytometry (FCM) and laser scanning confocal microscope (LSCM), respectively.
RESULTS: Integrins a3,a5, b1,b5 were expressed exclusively on the basal and lateral surfaces of IECs in group A. After ischemia and anoxia injury, integrins a3,a5, b1,b5 distribution moved from the basolateral to the apical membrane. Cell apoptosis was increased in group B, C and D. Integrin a3 was not observed in the middle and apical membrane in group C. Integrin b2 distribution was not markedly changed in any group.
, 百拇医药
CONCLUSION: The change in the distribution of integrin is induced by ischemia and anoxia, which is correlated with IEC apoptosis. IEC apoptosis is more sensitive to the anoxia.
Key Words: Ischemia; Anoxia; Integrin; Polarity; Apoptosis; epithelial cells
Chen Q, Qin HL. Influence of ischemia and anoxia on distribution and polarity of integrin in intestine epithelial cells. Shijie Huaren Xiaohua Zazhi 2005;13(4):456-459
, http://www.100md.com
摘要
目的:研究缺氧缺血损伤对肠上皮细胞(IEC)整合素极性及分布的改变的凋亡机制.
方法:建立缺血缺氧与IEC凋亡的实验模型，实验分组:对照组(A组)，缺氧组(B组)，缺血组(C组)，缺氧缺血组(D组);采用流式细胞仪(FCM)检测IEC凋亡率，激光共聚焦显微镜(LSCM)检测IEC自基底侧至顶侧5个平面的整合素a3，a5，b1，b2，b5染色荧光强度并定量.
结果:A组细胞整合素a3，a5，b1，b5荧光标记主要分布于细胞基底侧，损伤后B、C、D组呈上移趋势，其分布和极性与细胞凋亡率相一致;整合素a3在C组的中间和顶层没有阳性表达.b2分布较弥散，无明显变化规律.
结论:缺氧缺血损伤对IEC整合素分布改变呈规律性变化，与IEC凋亡密切相关;对缺氧反应更敏感.
, http://www.100md.com
关键词:缺血; 缺氧; 整合素; 极性; 凋亡; 上皮细胞
陈前, 秦环龙. 缺血缺氧损伤对肠上皮细胞整合素极性及细胞凋亡的影响. 世界华人消化杂志 2005;13(4):456-459
(PDF) 整合素分布趋势曲线. A: 整合素a3;B: 整合素a5; C: 整合素β1; D: 整合素b5.(以细胞底端平面为基准，以检测平面与细胞底端的相对距离为横坐标，相应检测平面的荧光强度为纵坐标，绘制整合素在IEC表面不同层面的分布曲线显示，正常IEC整合素主要分布于细胞基底部，损伤后出现向细胞侧面及顶侧分布的趋势.)
图2 A组整合素b1 LSCM扫描照片(A-B-C-D-E分别为底层到顶层)荧光强度逐渐由强变弱.
, http://www.100md.com
3 讨论整合素是由a和b亚单位组成的异二聚体跨膜糖蛋白，整合素因其a和b亚单位的不同组合而出现不同的亚型，a亚基质量在120-180ku之间，b亚基质量90-110ku之间.整合素主要功能是通过与相应的配体结合介导细胞与基质的黏附、信号传导、个体发育、分化、凋亡等病理生理过程.同大多数体细胞一样，在IEC表面也分布着多种整合素受体.与IEC表面有关的整合素有a2、a3、a5、b1、b4、b5[2].不同整合素在IEC的分布亦有不同，如a2b1分布于隐窝处，a3b1分布于绒毛底部，a5b1位于隐窝及绒毛的底部，a7b1则局限隐窝顶部及绒毛底部，而a6b4则均一地分布于整个IEC表面[3-4].近年来随着研究的深入，人们发现整合素还与IEC的凋亡密切相关[5]，整合素可能是IEC黏附于基底膜以维持细胞生存的主要方式，而且已引起了学者们的关注.有研究发现IEC经整合素a4封闭后，其分化、增生、Bcl-2、Bcl-X、Bax、Bak家族基因表达均明显受抑制[6].如犬经iv整合素a，b单抗1.0mg/kg持续2wk，可诱导出现消化道出血[2].还有学者发现，整合素调控着肠上皮的凋亡，a5b1似有抗凋亡作用[7].最近Levy et al[8]的一项研究发现丁酸(NaBT)可以使Caco-2细胞表面整合素b1的表达下降，导致细胞从基底膜脱落并发生凋亡.
, http://www.100md.com
有关整合素在细胞膜上的表达检测，国内外较为常用的办法是采用免疫组化、免疫荧光等技术，此二者不能较为精确地反应表达量，更无法检测极性分布改变、空间结构变化.近年来已有采用激光共聚焦显微镜(LSCM)研究整合素的报道.Gailitet al[9]利用LSCM观察体外培养的肾小管上皮细胞整合素a3呈极性分布，基底层和近基底层的侧层分布强，顶层和近顶层的侧层分布弱;在接受亚致死剂量的氧损伤后，细胞整合素a3的极性分布丧失，由基底层和近基底层向顶层和近顶层的侧层弥散;并且导致黏附于基质的细胞数量减少.Wuet al[6]在体研究发现，肾小管上皮细胞正常情况下整合素b1呈极性分布，缺血再灌注损伤后，也向顶侧弥散;Becket al[2]发现，在体的缺血再灌注损伤模型未发现整合素a3的顶层的侧层分布.以上实验结果提示，缺氧、氧损伤后整合素的极性分布丧失，是导致上皮细胞黏附性改变的基础;同时，也说明了LSCM能够很好地观察整合素的三维结构改变.
我们研究发现，IEC在实验因素刺激下整合素a3，a5，b1，b5发生了极性和分布的改变，并与细胞凋亡、脱落密切相关.其具体表现在，A组a3，a5，b1，b5整合素在细胞膜上呈极性分布，主要位于近基底膜侧;缺血缺氧后B，C，D组有向顶侧弥散的趋势，各层面的定量均存在差异，尤以第1层和第5层改变最为明显(P<0.01，P<0.05);整合素极性分布的明显改变趋势由大到小依次是缺血缺氧组、缺氧组、缺血组，且此三组之间也有差异(P<0.01，P<0.05);无论缺血缺氧还是单纯缺血或缺氧损伤与A组相比较，a3，a5，b1，b5在底层表达均明显下降，中间层和顶层均增加表达，说明有整体从基底向顶层移动的现象.缺血组a3在中间层及顶层没有出现阳性表达，说明其对缺血损伤可能不敏感.缺氧组、缺血缺氧组顶层各整合素亚型都发生变化，并都参与了细胞损伤作用，说明IEC对缺氧尤为敏感.b2整合素在细胞膜上的分布较均匀，缺血缺氧后改变不明显;这可能与整合素b2功能有关，或其本身没有直接参与细胞脱落、凋亡，只是其他某些亚型在IEC凋亡中起着重要调节作用.
, 百拇医药
4 参考文献
1 StupackDG, Cheresh DA. Get a ligand, get a life:integrins, signaling and cellsurvival. J Cell Sci 2002;115:3729-3738
2 Beck R, Nebe B,Guthoff R, Rychly J. Inhibition of lens epithelial cell adhesion by thecalcium antagonist Mibefradil
correlates with impair integrindistribution and organization of the cytoskeleton. Graefes Arch ClinExp Ophthalmol
, http://www.100md.com
2001;239:452-458
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编辑 潘伯荣 审读 张海宁, 百拇医药( 陈 前,秦环龙)
