硒和维生素E对心肌成纤维细胞Ⅰ型胶原合成代谢的影响
摘要
心肌中纤维型胶原成份不成比例地增多称作心肌纤维化{cardiac
fibrosis)。心肌纤维化临床表现多无特征性，主要造成难治性心衰及严重 的心律失常。
心脏间质中各种纤维及无定型物质，统称为心脏的细胞外基质 (ECM)。胶原是ECM中最丰富的结构成分。心肌间质胶原成分主要为I型及Ⅲ型胶原，I型胶原约占80％，Ⅲ型约12％。心肌成纤维细胞(CFbs)是合成心肌胶原的主要细胞。研究表明，CFbs能感受、结合机械性刺激并产生功能性应答，还可以合成I、III型胶原、蛋白胶原酶及其他细胞外基质成分。I型胶原以前胶原(procollagen)的形式释放到培养基质中。
儿茶酚胺(CA)对CFbs有很强的促增殖作用。去甲肾上腺素(NE)通过
激活肾上腺素能受体，导致心肌细胞肥大和CFbs增殖。NE可促进大鼠I型前胶原 1[I]链及转化生长因子- (TGF- 1)的mRNA表达增加。临床上给予心肌梗死病人硒(Se)和维生素E(VE)作为辅助治疗，能够促进心功能指标的改善。李保玉等发现，心肌损伤后心肌内胶原蛋白含量增加，并与低Se、低VE相关，补Se、补VE后，心肌胶原蛋白含量下降，说明体内低Se、低VE状态易促进心肌胶原过度蓄积。低Se、低VE造成体内脂质过氧化物增多，不但加重细胞损伤，同时促进前胶原羟化和胶原蛋白的交联，致使心肌内胶原过度蓄积。说明二者与心肌纤维化具有很重要的关系。
本研究通过体外培养CFbs，移入6孔和24孔培养板，加入0．4％小
牛血清DMEM培养液、0．2g．μg／ml NE、不同浓度的Na2Seo3(0．05～0．2
μg／ml)和VE(1～100μg／m1)，培养48小时后，收集细胞及上清。测定上清液中的HYP和I型胶原含量、CFbs中的醄I]型胶原mRNA的表
达，以确定Se和VE对胶原合成的作用情况。实验内容如下：
一、CFbs的培养与鉴定．
(一)酶消化法体外培养CFbs，倒置显微镜下见贴壁的CFbs呈梭形，
境界清楚，核透亮，随着细胞的增殖，2～3天细胞渐至亚融合及融合状
态，此时行传代培养。实验用细胞为2～5代细胞。
(二)免疫组化法鉴定CFbs，S-ABC法对培养的CFbs进行波形蛋白单
克隆抗体(virnentin)染色。染色结果：光镜下见胞浆围绕核周围呈现棕
黄色反应，呈颗粒状，胞核不着色，阳性率达95％以上。
(三)透射电镜下见CFbs大小不等，细胞表面有较多的微绒毛，细胞
质基质深染，线粒体小，胞质空泡增多，可见髓样小体以及粗面内质网扩张。
二、NE对I型CFbs胶原合成的影响：
(一)NE对CFbs培养液I型胶原含量的影响
羟脯氨酸检测试剂盒测定培养液的羟脯氨酸(HYP)含量，间接EI,ISA．
法测定I型胶原蛋白的含量。结果如下，0．2μg／ml NE作用48小时后上清中：HYP和I型胶原含量增加，与对照组比较有显著性差异(P<0．01)。
(二)NE对CFbs醄αl [I]型胶原mRNA的表达的影响
用RT-PCR法对体外培养的CFbsαl [I]型胶原mRNA进行半定量分
析，记录醄I]型胶原和内参β-actin电泳条带强度×面积的比值。可见
NE上调Cfbsalαl [I]型胶原mRNA的表达，与对照组比较有显著性差异
(P<0．01)。表明NE可通过上调CFbsαl[I]型胶原mRNA的表达，使
CFbs I 型胶原合成与分泌明显增加。
三、Se和VE对细胞培养上清中胶原含量的影响
(一)收集0．2μg／ml NE与不同浓度Se和’VE作用48小时后的细胞和
培养上清液，各组培养上清中HYP含量随着Se浓度的增加(0．05、0．1、
0．2μg /ml)而逐渐降低，呈剂量依赖性，表明补Se可抑制胶原蛋白的合
成与分泌。NE和1~100lμg／ml VE作用48小时后，上清中的HYP含量与对照组、NE组比较，有显著性降低(p<0．01)，但无量效关系。联合补充0．05μg／ml Se与10μg／ml VE可降低培养上清中的HYP含量，较对照组、NE组、O．05μg /ml Se和10μg／ml VE组显著降低(p (二)间接ELISA法测定NE(0．21μg／m1)、NE+Se(0．05μg／m1)、NE
+VE(10μg／m1)和NE+Se、VE作用48小时后CFbs上清液中I型胶原含量(A值)，结果为：联用组较对照组、NE组、0．05μg／ml Se组A值显著性降低(p<0．01)，较10μg／ml VE组降低，说明o．05ugμg／ml Se和10ugμg／ml VE联用对于I型胶原合成有协同抑制作用。
(三)Se和VE对NE所致CFbs I型胶原mRNA表达的影响：记录 [I]型胶原和内参13-actin电泳条带强度×面积的比值，可见：0．05μg／ml Se和
10μg／mlVE组与对照组和NE组比较mRNA表达降低(P<0．01)，0．05μg／ml Se和10μg／ml VE联用组αl [I]型胶原mRNA表达与NE组、0．05μg／ml Se和10μg／mlVE组比较显著性降低(p<0．01)，二者具有协同效应。
根据研究结果得出结论：
1．一定浓度NE(0．2μg／m1)可促进培养上清中的HYP和I型胶原含
量的增加，并可促进αl [I]型胶原mRNA的表达。说明NE有促进胶原合
成的作用。
2．Se和VE可以抑制胶原蛋白的合成，Se在0．05,---,0．2μg／ml浓度范围内有剂量依赖性效应，一定浓度Se(0．05μg／ml)和VE(10μg／m1)联合应用对抑制I型胶原产生具有协同效应。
3．Se和VE降低对αl [I]型胶原mRNA表达，一定浓度Se(0．05μg／m1)和VE(10μg／m1)联用对αl [
Cardiac fibrosis means excessive fibril collagen deposffs inter
myocardiocytes disproportionately．Usually patients with cardiac tibrosis have
no symptoms，but it can lead to incurable heart failure and serious arrhythmia．
All types of collagen and unformed material are called extmcellular
matrix(ECM)．Type I collagen is the most abundant material in ECM．The
Type I collagen is the main component of secreted ECM by CFbs，about 80％．
Cardiac fibroblasts(CFbs)are the primary cells，which can synthesize
collagen．Investigations suggest that CFbs are sensitive to mechanical
stimulation and Can produce functional responses．CFbs Can synthesize Type I
and III collagens，collagenase and other ECM．Type I collagen is released to
the out of cell in the form of procollagen．Catecholamine Can make CFbs
proliferate significantly．Norepinephrine(NE)Can make myocardiocytes
hypertrophia and CFbs proliferate by exciting receptor of NE，and it can
increase the expression of mRNA of collagen al[I]and TGF．p1．
Selenium(Se)and Vitamin E(VE)have been used in the myocardial
‘infarction patients as assistant treatment，which can improve the heart index．
Bao Yu Li et al，found that when the myocardiocytes are injured，collagen in
the matrix increased，which was associated、with low Se and low VE．After Se
and VE were added，the activity of GSH：．Px ascended and the content of
collagen protein in the heart increased，which suggests that low Se and Low
VE Can lead to collagen deposition．Low-level Se and VE Can increase lipid
peroxide in the body，which Can not only injure the cell seriously,but also
enhance the procollagen hydroxidation and collagen crosslinking and then
lead to the excessive accumulation of the collagen inter the myocardiocytes．It
could be concluded that Se and VE have important relationship with the
cardiac nbrosis．
The purpose of this study was to investigate the effect of Se and VE On
collagen I synthesizing metabolism of cultured CFbs treated with NE at
protein and gene level．Cell and culture medium were collected after 48h
culture．Then we examined the hydroxyproline(HYP)and Type I content as
well as the expression of 51[I]I collagen mRNA．The experiment suggested
that there is a close relationship between collagen production and Se and VE．
The major results are as follows：
1．The CFbs culture and identification．
(1)CFbs were obtained by the method of enzymatic dispersion culture．
Under the microscope：the shape of the CFbs Was spindle，it's boundary Was
clear,and the nucleus was transparent and bright．After culturing 2～3 days，
the CFbs grew confluent and subconfconfluent with the proliferation of the cell，
and cells call be passed．The 2～5 generation cells were used for this
experiment．
(2)The CFbs were identified by immunocytochemistry．CFbs Was
stained with anati-vimentin antibody by means of S-ABC．The plasma was
stained positively by the color of brown yellow,but the nucleus of the cells
‘wasnegative．
(3)Under transmission electronic microscope，the size of the CFbs was
varied，most of them were large with irregular shape．There were many micro
villis on the surface of this cell．The plasma Was darkly stained，in which
vacuole increased，mitochondrion’S volume decreased and RER dilated．
2．The effect ofNE on synthesizing collagen ofthe CFbs．
(1)The effect ofNE on Type I collagen synthesis ofCFbs
We measured the HYP content in the culture medium of CFbs by HYP
detection reagent kit．The HYP Can indicate collagen content of the culture
medium．Type I content in culture medium Was examined using direct ELISA
to observe the effectS of NE in collagen synthesis of CFbs．B0th of them
increased significantly in the group NE compared to the control group
(P<0．05)．
(2)The effect of NE were observed on the expression of 醄I]collagen
mRNA of CFbs using RT-PCR technology．The expression of mRNA in the
group NE Was significantly higher than
fibrosis)。心肌纤维化临床表现多无特征性，主要造成难治性心衰及严重 的心律失常。
心脏间质中各种纤维及无定型物质，统称为心脏的细胞外基质 (ECM)。胶原是ECM中最丰富的结构成分。心肌间质胶原成分主要为I型及Ⅲ型胶原，I型胶原约占80％，Ⅲ型约12％。心肌成纤维细胞(CFbs)是合成心肌胶原的主要细胞。研究表明，CFbs能感受、结合机械性刺激并产生功能性应答，还可以合成I、III型胶原、蛋白胶原酶及其他细胞外基质成分。I型胶原以前胶原(procollagen)的形式释放到培养基质中。
儿茶酚胺(CA)对CFbs有很强的促增殖作用。去甲肾上腺素(NE)通过
激活肾上腺素能受体，导致心肌细胞肥大和CFbs增殖。NE可促进大鼠I型前胶原 1[I]链及转化生长因子- (TGF- 1)的mRNA表达增加。临床上给予心肌梗死病人硒(Se)和维生素E(VE)作为辅助治疗，能够促进心功能指标的改善。李保玉等发现，心肌损伤后心肌内胶原蛋白含量增加，并与低Se、低VE相关，补Se、补VE后，心肌胶原蛋白含量下降，说明体内低Se、低VE状态易促进心肌胶原过度蓄积。低Se、低VE造成体内脂质过氧化物增多，不但加重细胞损伤，同时促进前胶原羟化和胶原蛋白的交联，致使心肌内胶原过度蓄积。说明二者与心肌纤维化具有很重要的关系。
本研究通过体外培养CFbs，移入6孔和24孔培养板，加入0．4％小
牛血清DMEM培养液、0．2g．μg／ml NE、不同浓度的Na2Seo3(0．05～0．2
μg／ml)和VE(1～100μg／m1)，培养48小时后，收集细胞及上清。测定上清液中的HYP和I型胶原含量、CFbs中的醄I]型胶原mRNA的表
达，以确定Se和VE对胶原合成的作用情况。实验内容如下：
一、CFbs的培养与鉴定．
(一)酶消化法体外培养CFbs，倒置显微镜下见贴壁的CFbs呈梭形，
境界清楚，核透亮，随着细胞的增殖，2～3天细胞渐至亚融合及融合状
态，此时行传代培养。实验用细胞为2～5代细胞。
(二)免疫组化法鉴定CFbs，S-ABC法对培养的CFbs进行波形蛋白单
克隆抗体(virnentin)染色。染色结果：光镜下见胞浆围绕核周围呈现棕
黄色反应，呈颗粒状，胞核不着色，阳性率达95％以上。
(三)透射电镜下见CFbs大小不等，细胞表面有较多的微绒毛，细胞
质基质深染，线粒体小，胞质空泡增多，可见髓样小体以及粗面内质网扩张。
二、NE对I型CFbs胶原合成的影响：
(一)NE对CFbs培养液I型胶原含量的影响
羟脯氨酸检测试剂盒测定培养液的羟脯氨酸(HYP)含量，间接EI,ISA．
法测定I型胶原蛋白的含量。结果如下，0．2μg／ml NE作用48小时后上清中：HYP和I型胶原含量增加，与对照组比较有显著性差异(P<0．01)。
(二)NE对CFbs醄αl [I]型胶原mRNA的表达的影响
用RT-PCR法对体外培养的CFbsαl [I]型胶原mRNA进行半定量分
析，记录醄I]型胶原和内参β-actin电泳条带强度×面积的比值。可见
NE上调Cfbsalαl [I]型胶原mRNA的表达，与对照组比较有显著性差异
(P<0．01)。表明NE可通过上调CFbsαl[I]型胶原mRNA的表达，使
CFbs I 型胶原合成与分泌明显增加。
三、Se和VE对细胞培养上清中胶原含量的影响
(一)收集0．2μg／ml NE与不同浓度Se和’VE作用48小时后的细胞和
培养上清液，各组培养上清中HYP含量随着Se浓度的增加(0．05、0．1、
0．2μg /ml)而逐渐降低，呈剂量依赖性，表明补Se可抑制胶原蛋白的合
成与分泌。NE和1~100lμg／ml VE作用48小时后，上清中的HYP含量与对照组、NE组比较，有显著性降低(p<0．01)，但无量效关系。联合补充0．05μg／ml Se与10μg／ml VE可降低培养上清中的HYP含量，较对照组、NE组、O．05μg /ml Se和10μg／ml VE组显著降低(p
+VE(10μg／m1)和NE+Se、VE作用48小时后CFbs上清液中I型胶原含量(A值)，结果为：联用组较对照组、NE组、0．05μg／ml Se组A值显著性降低(p<0．01)，较10μg／ml VE组降低，说明o．05ugμg／ml Se和10ugμg／ml VE联用对于I型胶原合成有协同抑制作用。
(三)Se和VE对NE所致CFbs I型胶原mRNA表达的影响：记录 [I]型胶原和内参13-actin电泳条带强度×面积的比值，可见：0．05μg／ml Se和
10μg／mlVE组与对照组和NE组比较mRNA表达降低(P<0．01)，0．05μg／ml Se和10μg／ml VE联用组αl [I]型胶原mRNA表达与NE组、0．05μg／ml Se和10μg／mlVE组比较显著性降低(p<0．01)，二者具有协同效应。
根据研究结果得出结论：
1．一定浓度NE(0．2μg／m1)可促进培养上清中的HYP和I型胶原含
量的增加，并可促进αl [I]型胶原mRNA的表达。说明NE有促进胶原合
成的作用。
2．Se和VE可以抑制胶原蛋白的合成，Se在0．05,---,0．2μg／ml浓度范围内有剂量依赖性效应，一定浓度Se(0．05μg／ml)和VE(10μg／m1)联合应用对抑制I型胶原产生具有协同效应。
3．Se和VE降低对αl [I]型胶原mRNA表达，一定浓度Se(0．05μg／m1)和VE(10μg／m1)联用对αl [
Cardiac fibrosis means excessive fibril collagen deposffs inter
myocardiocytes disproportionately．Usually patients with cardiac tibrosis have
no symptoms，but it can lead to incurable heart failure and serious arrhythmia．
All types of collagen and unformed material are called extmcellular
matrix(ECM)．Type I collagen is the most abundant material in ECM．The
Type I collagen is the main component of secreted ECM by CFbs，about 80％．
Cardiac fibroblasts(CFbs)are the primary cells，which can synthesize
collagen．Investigations suggest that CFbs are sensitive to mechanical
stimulation and Can produce functional responses．CFbs Can synthesize Type I
and III collagens，collagenase and other ECM．Type I collagen is released to
the out of cell in the form of procollagen．Catecholamine Can make CFbs
proliferate significantly．Norepinephrine(NE)Can make myocardiocytes
hypertrophia and CFbs proliferate by exciting receptor of NE，and it can
increase the expression of mRNA of collagen al[I]and TGF．p1．
Selenium(Se)and Vitamin E(VE)have been used in the myocardial
‘infarction patients as assistant treatment，which can improve the heart index．
Bao Yu Li et al，found that when the myocardiocytes are injured，collagen in
the matrix increased，which was associated、with low Se and low VE．After Se
and VE were added，the activity of GSH：．Px ascended and the content of
collagen protein in the heart increased，which suggests that low Se and Low
VE Can lead to collagen deposition．Low-level Se and VE Can increase lipid
peroxide in the body，which Can not only injure the cell seriously,but also
enhance the procollagen hydroxidation and collagen crosslinking and then
lead to the excessive accumulation of the collagen inter the myocardiocytes．It
could be concluded that Se and VE have important relationship with the
cardiac nbrosis．
The purpose of this study was to investigate the effect of Se and VE On
collagen I synthesizing metabolism of cultured CFbs treated with NE at
protein and gene level．Cell and culture medium were collected after 48h
culture．Then we examined the hydroxyproline(HYP)and Type I content as
well as the expression of 51[I]I collagen mRNA．The experiment suggested
that there is a close relationship between collagen production and Se and VE．
The major results are as follows：
1．The CFbs culture and identification．
(1)CFbs were obtained by the method of enzymatic dispersion culture．
Under the microscope：the shape of the CFbs Was spindle，it's boundary Was
clear,and the nucleus was transparent and bright．After culturing 2～3 days，
the CFbs grew confluent and subconfconfluent with the proliferation of the cell，
and cells call be passed．The 2～5 generation cells were used for this
experiment．
(2)The CFbs were identified by immunocytochemistry．CFbs Was
stained with anati-vimentin antibody by means of S-ABC．The plasma was
stained positively by the color of brown yellow,but the nucleus of the cells
‘wasnegative．
(3)Under transmission electronic microscope，the size of the CFbs was
varied，most of them were large with irregular shape．There were many micro
villis on the surface of this cell．The plasma Was darkly stained，in which
vacuole increased，mitochondrion’S volume decreased and RER dilated．
2．The effect ofNE on synthesizing collagen ofthe CFbs．
(1)The effect ofNE on Type I collagen synthesis ofCFbs
We measured the HYP content in the culture medium of CFbs by HYP
detection reagent kit．The HYP Can indicate collagen content of the culture
medium．Type I content in culture medium Was examined using direct ELISA
to observe the effectS of NE in collagen synthesis of CFbs．B0th of them
increased significantly in the group NE compared to the control group
(P<0．05)．
(2)The effect of NE were observed on the expression of 醄I]collagen
mRNA of CFbs using RT-PCR technology．The expression of mRNA in the
group NE Was significantly higher than
引文
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Whittaker P. Unravelling the mysteries of collagen and cicatrix after myocardial infarction. Cardiovasc Res,1995,29:758-762.
Pathak M, Sarkar S, Vellaichamy E, et al. Role of myocyte in myocardial collagen production. Hypertension,2001,37:833-840
Janet M, Schnee, Willa, A, et al. Angeotension adhension, and cardial fibrosis. Cardio research, 2000,46:264-268.
Brilla Cg, Zhou G, Marsobara L, et al. Collagen metabolism in cultured adult rat cardiac fibroblast: response to ang-Ⅱ and aldosterone. J am coll cardiol,1994,26:809-820.
Brilla Cg, Reams Gp, Maisch B, et al. Renin angiotension system and myocardial fibrosis in hypertension: regulation of the myocardial collagen matrix. Eur Heart J,1993,14(Shppl J):57-61.
Ramires Fja, Sun Y, Weber Kt, et al. Myocardial fibrosis associated with aldosterone or angiotension-Ⅱ administration: attenuation by calcium channel blockade. J Mol Cell Cardiol,1998,31:475-483.
Lanza GM, Scott MJ, Davison G, et al. Angiotensin-Ⅱ receptor blockade in Syrian hamster（TO22）cardiomyopathy does not affect microscopic cardiac material properties: implications for mechanisms of tissue remodeling. Cardiovasc Drugs Ther, 1997,11:521-529.
Bhambi B, Eghbali M. Effect of norepinephrine on myocardial collagen
gene expression and response of cardiac fibroblasts after norepinephrine treatment. Am J Pathol,1991,139:1131-1142.
李广生.低硒及相关因素与克山病. 吉林科技出版社，1997，4.
展玉涛，尤大钰，姜藻，等. 维生素E对白介素-2作用的大鼠贮脂细胞增殖及胶原合成的影响. 中华消化杂志，1999，195:358－359.
王凡，李广生，刘家骝. 我国五种主要地方病的病理学研究进展. 中华病理学杂志，1995，24: 252－254.
王凡，李广生. 克山病病理学研究新进展. 中国地方病学杂志，1989， 8: 52－55，64.
李保玉，金毅等. 硒、维生素E 对心肌胶原蛋白代谢的影响. 营养学报，1998，21：38－40.
龚素珍，潘敬远. 一种提高新生大鼠原代培养心脏成纤维细胞获率的方法. 广州医学院学报，2000，28：69－71，76.
郑恒，谢辉等. 1-（2，6二甲基苯氧基）-2-（3，4二甲氧基苯乙胺基）丙烷盐酸盐对心脏成纤维细胞胶原Ⅰ，Ⅲ mRNA表达的影响. 药学学报，2000，35：165－168.
谷伯起等，心血管病理学.人民卫生出版社，1992，373.
永井裕，藤本大三郎. 胶原蛋白实验方法.上海中医学院出版社，1992，1.
戴建国，肖华盛，张萍等. 用RT-PCR对mRNA进行半定量分析. 第四军医大学学报，1992，20：242－243，248.
李广生. 低硒及相关因素与克山病. 吉林科技出版社，1997，188.