输卵管组织中内皮素-1基因表达的实验研究
作者：李文林 石小玉 熊丽霞 周莹 奚明 郑坚
单位：李文林 熊丽霞(江西医学院病理生理学教研室， 江西 南昌 330006)；石小玉 周莹(组织学与胚胎学教研室)；奚明(生理学教研室)；郑坚(口腔医院病理室)
关键词：内皮缩血管肽类；输卵管；杂交；基因表达；兔
中国病理生理杂志000605
[摘 要] 目的：为了探明内皮素(ET)在输卵管组织中的分布及来源，以便进一步探讨内皮素-1(ET-1)与输卵管功能调节之间的关系。方法：用SABC免疫组织化学法和地高辛精标记的ET-1cRNA探针核酸原位杂交组织化学法，观察了兔输卵管组织ET-1的分布和ET-1 mRNA的表达。结果：输卵管粘膜上皮细胞内可见被染成红色的ET-1颗粒，主要分布在细胞核的上方，靠近游离面，在肌层可见少量的ET-1颗粒。输卵管粘膜上皮细胞内可见呈深兰色的ET-1 mRNA阳性杂交信号，阳性杂交信号强且密集，主要分布在细胞核的上方，靠近游离面，在肌层未见ET-1 mRNA阳性杂交信号。结论：表明兔输卵管粘膜上皮细胞自分泌ET-1。
, http://www.100md.com
[中图分类号] Q786 [文献标识码] A
[文章编号] 1000-4718(2000)06-0498-03
Experimental study of endothelin-1 gene expression in uterine tube tissue
LI Wen-lin, XIONG Li-xia
(1Department of Pathophysiology)
SHI Xiao-yu, ZHOU Ying
(Department of Histology and Embryology)
XI Ming
, 百拇医药
(Department of Physiology)
ZHEN Jian
(Department of Pathology of Stomaological Hospital, Jiangxi Medical College, Nanchang 330006, China)
[Abstract] AIM: The distribution of endothelin-1(ET-1) in uterine tube and its source were investigated in order to find out relation between endothelin-1 and function of uterine tube.METHODS: The distribution of endothelin-1 and the expression of ET-1 mRNA in the rabbits uterine tubes were studied using SABC immunohistochemistry and in situ hybridization histochemistry with digoxigenin-labelled rat ET-1 cRNA probe. RESULTS: ET-1 granules with red color were seen in the uterine tube epithelial cells and they were located mainly above the cell nucleus and under the cell surface membrane. Less ET-1 granules were seen in the myometrium of uterine tube. ET-1 mRNA positive hybridization signals with deep blue were distributed in the uterine tube epithelial cells. These signals were strong and dense. They were distributed mainly above the nucleus and near the cell surface membrane. ET-1 mRNA positive hybridization signal was not seen in the myometrium of uterine tube.CONCLUSION: Our results indicate that epithelial cells of rabbit's uterine tube epithelial cells secrete ET-1.
, http://www.100md.com
[MeSH] Endothelins; Fallopian tubes; Hybridization; Gene expression; Rabbits
自1988年Yanagisawa[1]从猪主动脉内皮细胞分离纯化出21个氨基酸组成的多肽-内皮素(endothelin, ET)以来，受到各国学者的关注。国内外对内皮素在女性生殖器官的分布和作用的研究主要集中在子宫和卵巢，本文采用免疫组织化学法和核酸原位杂交组织化学法，探讨内皮素-1(ET-1)在输卵管组织中的分布及来源。
材 料 和 方 法
一、动物处理
实验用动物为健康新西兰白兔(BK公司)6只，体重(2.75±0.25) kg，雌性。戊巴比妥ip麻醉后，剖腹取输卵管，生理盐水洗净血迹，4%多聚甲醛固定，脱水，透明，石蜡包埋，切片，裱片后经脱蜡，水化，进行免疫组化研究。冰冻切片，4%多聚甲醛固定，0.4% Triton X-100洗涤和蛋白酶K(1 μg/mL)消化，乙酸处理后，进行核酸原位杂交组织化学。
, 百拇医药
二、SABC免疫组织化学
0.25%胰蛋白酶消化，37℃，30 min，PBS洗4次；封闭液室温孵育5 min，PBS洗4次；鼠抗ET-1单克隆抗体(ABR，USA，深圳晶美生物工程公司)，1∶250稀释，4℃过夜，PBS洗4次；生物素标记羊抗鼠IgG(Neomarker, USA, 深圳晶美生物工程公司)，室温孵育10～15 min，PBS洗4次；链霉亲和素—碱性磷酸酶(Neomarker, USA, 深圳晶美生物工程公司)，室温孵育10～15 min，PBS洗4次；底物(fast red)，显色10～15 min，PBS洗4次；Harris苏木精复染，甘油封片。阴性对照为一抗由正常兔血清替代。
三、地高辛精标记的ET-1 cRNA探针制备[2，3]
含大鼠ET-1 cDNA 5’编码区序列(480 bp)的PCR 1 000质粒，经限制性内切酶EcoR Ⅰ线性化后，在T7 RNA聚合酶作用下，合成ET-1 cRNA探针，地高辛精标记。
, http://www.100md.com
四、核酸原位杂交组织化学[3]
乙酸处理的切片与地高辛精标记的ET-1 cRNA探针43℃杂交16 h。杂交结束后用6×SSC、1×SSC和0.1×SSC(1×SSC内含150 mmol/L氯化钠和15 mmol/L柠檬酸三钠)溶液洗涤，过剩的ET-1 cRNA探针用RNA酶(20 μg/mL)消化30 min。切片与碱性磷酸酶标记的抗地高辛抗体在室温下反应4 h，用NBT和BCIP室温避光显色3 h，封片。
结 果
一、ET-1在兔输卵管组织中的分布
输卵管粘膜、肌层和浆膜结构正常，ET-1被染成红色，粘膜上皮细胞内可见呈颗粒状或弥散状的ET-1(图1)，ET-1主要分布在细胞核的上方，靠近细胞游离面，粘膜上皮细胞游离面细胞膜表面粘附有ET-1，在肌层可见少量的ET-1(图2)。
, http://www.100md.com
Fig 1 SABC Immunohistochemistry staining. ET-1 granules with red color were seen in the uterine tube epithelial cells and they were located mainly above cell nucleus and under cell surface membrane
图1 SABC免疫组织化学染色，粘膜上皮细胞内可见ET-1被染成红色，呈颗粒状或弥散状，ET-1主要分布在细胞核的上方，靠近细胞游离面
Fig 2 SABC immunohistochemistry staining. ET-1 was stained with red. ET-1 was seen at the surface membrane of epithelial cells of mucosa. Less ET-1 granules were seen in the myometrium of uterine tube
, http://www.100md.com
图2 SABC免疫组织化学染色，ET-1被染成红色，在粘膜上皮细胞游离面细胞膜表面可见ET-1，在肌层可见少量的ET-1
二、ET-1 mRNA在兔输卵管组织中的表达
输卵管粘膜上皮细胞胞浆内可见ET-1 mRNA阳性杂交信号，呈深兰色，阳性杂交信号强且密集，该阳性杂交信号主要分布在细胞核的上方，靠近游离面，在肌层未见ET-1 mRNA阳性杂交信号(图3，图4)。
Fig 3 Hybridization histochemistry in situ. ET-1 mRNA positive hybridization signals with deep blue were distributed in the epithelial cells of the uterine tube. These signals were strong and dense. They were distributed mainly above nucleus and near cell surface membrane
, 百拇医药
图3 核酸原位杂交组织化学染色，输卵管粘膜上皮细胞胞浆内可见ET-1 mRNA阳性杂交信号，呈深兰色，阳性杂交信号强且密集，该阳性杂交信号主要分布在细胞核的上方，靠近游离面
Fig 4 Hybridization histochemistry in situ. ET-1 mRNA positive hybridization signals were stained with deep blue. ET-1 mRNA positive hybridization signals were seen in the epithelial cells of uterine tube. ET-1 mRNA positive hybridization signals were not seen in the myometrium of uterine tube
, http://www.100md.com 图4 核酸原位杂交组织化学染色，ET-1 mRNA阳性杂交信号为深兰色，输卵管粘膜上皮细胞胞浆内可见ET-1 mRNA阳性杂交信号，在肌层未见ET-1 mRNA阳性杂交信号
讨 论
ET是目前已知最强烈缩血管多肽之一，它对各器官的作用近年来引起了人们高度重视。1994年，Rosselli报道[4]，在牛输卵管离体粘膜上皮细胞培养液中存在ET。本研究以兔为实验对象，用SABC免疫组织化学法和地高辛精标记ET-1 cRNA探针核酸原位杂交组织化学法，探讨ET-1在输卵管组织中的分布和来源。SABC免疫组织化学法敏感度高，背景染色低，避免了内源性生物素的非特异性结合。用该方法清晰地观察到在输卵管粘膜上皮细胞内有ET-1，在肌层有少量ET-1。为进一步了解输卵管粘膜上皮细胞和肌层平滑肌细胞是否自分泌ET-1，用地高辛精标记的大鼠ET-1 cRNA探针核酸原位杂交组织化学法，观察到在输卵管粘膜上皮细胞内可见ET-1 mRNA阳性杂交信号，而在肌层未见ET-1 mRNA阳性杂交信号。我们的实验结果表明，输卵管粘膜上皮细胞内和肌层平滑肌细胞内有ET-1，粘膜上皮细胞有自分泌ET-1的功能。输卵管是运送生殖细胞和受精的场所，对精子的获能、受精卵的正常卵裂和生存都很重要。输卵管粘膜上皮细胞合成、分泌ET-1，以自分泌的方式作用粘膜上皮细胞，促进粘膜上皮细胞的生长，有利于受精卵的运送和正常卵裂。SABC免疫组化结果显示，ET-1主要分布在细胞游离面，表明ET-1参加输卵管液的组成，这对精子在输卵管内的运动，精子的获能以及受精卵的正常运行有重要作用。从我们的结果观察到，在输卵管肌层可见少量的ET-1颗粒，但未见ET-1 mRNA 阳性杂交信号，肌层平滑肌细胞无自分泌ET-1的功能，肌层的ET-1来自旁分泌或内分泌，ET-1可促进平滑肌细胞收缩[5]，有利于受精卵的正常运行。
, 百拇医药
[基金项目] 国家自然科学基金资助(No.39560028)
[参 考 文 献]
[1] Yanagisawa M, Kutihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endotholial cells[J]. Nature, 1988, 332: 411～415.
[2] Sakurai T, Yanagisawa M, Inoue A, et al. cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA[J]. Biochem Biophys Res Commun, 1991, 175:44～47.
, http://www.100md.com
[3] 蒋应明，孙庆文， 向正华.大鼠下丘脑ET-1 mRNA阳性表达神经元的分布[J]. 中国神经科学，1998， 14(3)：151～154.
[4] Rosselli M, Imthurn B, Macas E, et al. Endothelin production by bovine oviduct epithelial cells[J]. J Reprod Fertil, 1994, 101:27～30.
[5] Rosselli M, Imthurn B, Macas E, et al. Endogenous nitric oxide modulates endothelin-1 induced contraction of bovine oviduct[J]. Biochem Biophys Res Commun, 1994, 201:143～148.
[收稿日期]1999-07-21 [修回日期]1999-11-18
, 百拇医药
单位：李文林 熊丽霞(江西医学院病理生理学教研室， 江西 南昌 330006)；石小玉 周莹(组织学与胚胎学教研室)；奚明(生理学教研室)；郑坚(口腔医院病理室)
关键词：内皮缩血管肽类；输卵管；杂交；基因表达；兔
中国病理生理杂志000605
[摘 要] 目的：为了探明内皮素(ET)在输卵管组织中的分布及来源，以便进一步探讨内皮素-1(ET-1)与输卵管功能调节之间的关系。方法：用SABC免疫组织化学法和地高辛精标记的ET-1cRNA探针核酸原位杂交组织化学法，观察了兔输卵管组织ET-1的分布和ET-1 mRNA的表达。结果：输卵管粘膜上皮细胞内可见被染成红色的ET-1颗粒，主要分布在细胞核的上方，靠近游离面，在肌层可见少量的ET-1颗粒。输卵管粘膜上皮细胞内可见呈深兰色的ET-1 mRNA阳性杂交信号，阳性杂交信号强且密集，主要分布在细胞核的上方，靠近游离面，在肌层未见ET-1 mRNA阳性杂交信号。结论：表明兔输卵管粘膜上皮细胞自分泌ET-1。
, http://www.100md.com
[中图分类号] Q786 [文献标识码] A
[文章编号] 1000-4718(2000)06-0498-03
Experimental study of endothelin-1 gene expression in uterine tube tissue
LI Wen-lin, XIONG Li-xia
(1Department of Pathophysiology)
SHI Xiao-yu, ZHOU Ying
(Department of Histology and Embryology)
XI Ming
, 百拇医药
(Department of Physiology)
ZHEN Jian
(Department of Pathology of Stomaological Hospital, Jiangxi Medical College, Nanchang 330006, China)
[Abstract] AIM: The distribution of endothelin-1(ET-1) in uterine tube and its source were investigated in order to find out relation between endothelin-1 and function of uterine tube.METHODS: The distribution of endothelin-1 and the expression of ET-1 mRNA in the rabbits uterine tubes were studied using SABC immunohistochemistry and in situ hybridization histochemistry with digoxigenin-labelled rat ET-1 cRNA probe. RESULTS: ET-1 granules with red color were seen in the uterine tube epithelial cells and they were located mainly above the cell nucleus and under the cell surface membrane. Less ET-1 granules were seen in the myometrium of uterine tube. ET-1 mRNA positive hybridization signals with deep blue were distributed in the uterine tube epithelial cells. These signals were strong and dense. They were distributed mainly above the nucleus and near the cell surface membrane. ET-1 mRNA positive hybridization signal was not seen in the myometrium of uterine tube.CONCLUSION: Our results indicate that epithelial cells of rabbit's uterine tube epithelial cells secrete ET-1.
, http://www.100md.com
[MeSH] Endothelins; Fallopian tubes; Hybridization; Gene expression; Rabbits
自1988年Yanagisawa[1]从猪主动脉内皮细胞分离纯化出21个氨基酸组成的多肽-内皮素(endothelin, ET)以来，受到各国学者的关注。国内外对内皮素在女性生殖器官的分布和作用的研究主要集中在子宫和卵巢，本文采用免疫组织化学法和核酸原位杂交组织化学法，探讨内皮素-1(ET-1)在输卵管组织中的分布及来源。
材 料 和 方 法
一、动物处理
实验用动物为健康新西兰白兔(BK公司)6只，体重(2.75±0.25) kg，雌性。戊巴比妥ip麻醉后，剖腹取输卵管，生理盐水洗净血迹，4%多聚甲醛固定，脱水，透明，石蜡包埋，切片，裱片后经脱蜡，水化，进行免疫组化研究。冰冻切片，4%多聚甲醛固定，0.4% Triton X-100洗涤和蛋白酶K(1 μg/mL)消化，乙酸处理后，进行核酸原位杂交组织化学。
, 百拇医药
二、SABC免疫组织化学
0.25%胰蛋白酶消化，37℃，30 min，PBS洗4次；封闭液室温孵育5 min，PBS洗4次；鼠抗ET-1单克隆抗体(ABR，USA，深圳晶美生物工程公司)，1∶250稀释，4℃过夜，PBS洗4次；生物素标记羊抗鼠IgG(Neomarker, USA, 深圳晶美生物工程公司)，室温孵育10～15 min，PBS洗4次；链霉亲和素—碱性磷酸酶(Neomarker, USA, 深圳晶美生物工程公司)，室温孵育10～15 min，PBS洗4次；底物(fast red)，显色10～15 min，PBS洗4次；Harris苏木精复染，甘油封片。阴性对照为一抗由正常兔血清替代。
三、地高辛精标记的ET-1 cRNA探针制备[2，3]
含大鼠ET-1 cDNA 5’编码区序列(480 bp)的PCR 1 000质粒，经限制性内切酶EcoR Ⅰ线性化后，在T7 RNA聚合酶作用下，合成ET-1 cRNA探针，地高辛精标记。
, http://www.100md.com
四、核酸原位杂交组织化学[3]
乙酸处理的切片与地高辛精标记的ET-1 cRNA探针43℃杂交16 h。杂交结束后用6×SSC、1×SSC和0.1×SSC(1×SSC内含150 mmol/L氯化钠和15 mmol/L柠檬酸三钠)溶液洗涤，过剩的ET-1 cRNA探针用RNA酶(20 μg/mL)消化30 min。切片与碱性磷酸酶标记的抗地高辛抗体在室温下反应4 h，用NBT和BCIP室温避光显色3 h，封片。
结 果
一、ET-1在兔输卵管组织中的分布
输卵管粘膜、肌层和浆膜结构正常，ET-1被染成红色，粘膜上皮细胞内可见呈颗粒状或弥散状的ET-1(图1)，ET-1主要分布在细胞核的上方，靠近细胞游离面，粘膜上皮细胞游离面细胞膜表面粘附有ET-1，在肌层可见少量的ET-1(图2)。
, http://www.100md.com
Fig 1 SABC Immunohistochemistry staining. ET-1 granules with red color were seen in the uterine tube epithelial cells and they were located mainly above cell nucleus and under cell surface membrane
图1 SABC免疫组织化学染色，粘膜上皮细胞内可见ET-1被染成红色，呈颗粒状或弥散状，ET-1主要分布在细胞核的上方，靠近细胞游离面
Fig 2 SABC immunohistochemistry staining. ET-1 was stained with red. ET-1 was seen at the surface membrane of epithelial cells of mucosa. Less ET-1 granules were seen in the myometrium of uterine tube
, http://www.100md.com
图2 SABC免疫组织化学染色，ET-1被染成红色，在粘膜上皮细胞游离面细胞膜表面可见ET-1，在肌层可见少量的ET-1
二、ET-1 mRNA在兔输卵管组织中的表达
输卵管粘膜上皮细胞胞浆内可见ET-1 mRNA阳性杂交信号，呈深兰色，阳性杂交信号强且密集，该阳性杂交信号主要分布在细胞核的上方，靠近游离面，在肌层未见ET-1 mRNA阳性杂交信号(图3，图4)。
Fig 3 Hybridization histochemistry in situ. ET-1 mRNA positive hybridization signals with deep blue were distributed in the epithelial cells of the uterine tube. These signals were strong and dense. They were distributed mainly above nucleus and near cell surface membrane
, 百拇医药
图3 核酸原位杂交组织化学染色，输卵管粘膜上皮细胞胞浆内可见ET-1 mRNA阳性杂交信号，呈深兰色，阳性杂交信号强且密集，该阳性杂交信号主要分布在细胞核的上方，靠近游离面
Fig 4 Hybridization histochemistry in situ. ET-1 mRNA positive hybridization signals were stained with deep blue. ET-1 mRNA positive hybridization signals were seen in the epithelial cells of uterine tube. ET-1 mRNA positive hybridization signals were not seen in the myometrium of uterine tube
, http://www.100md.com 图4 核酸原位杂交组织化学染色，ET-1 mRNA阳性杂交信号为深兰色，输卵管粘膜上皮细胞胞浆内可见ET-1 mRNA阳性杂交信号，在肌层未见ET-1 mRNA阳性杂交信号
讨 论
ET是目前已知最强烈缩血管多肽之一，它对各器官的作用近年来引起了人们高度重视。1994年，Rosselli报道[4]，在牛输卵管离体粘膜上皮细胞培养液中存在ET。本研究以兔为实验对象，用SABC免疫组织化学法和地高辛精标记ET-1 cRNA探针核酸原位杂交组织化学法，探讨ET-1在输卵管组织中的分布和来源。SABC免疫组织化学法敏感度高，背景染色低，避免了内源性生物素的非特异性结合。用该方法清晰地观察到在输卵管粘膜上皮细胞内有ET-1，在肌层有少量ET-1。为进一步了解输卵管粘膜上皮细胞和肌层平滑肌细胞是否自分泌ET-1，用地高辛精标记的大鼠ET-1 cRNA探针核酸原位杂交组织化学法，观察到在输卵管粘膜上皮细胞内可见ET-1 mRNA阳性杂交信号，而在肌层未见ET-1 mRNA阳性杂交信号。我们的实验结果表明，输卵管粘膜上皮细胞内和肌层平滑肌细胞内有ET-1，粘膜上皮细胞有自分泌ET-1的功能。输卵管是运送生殖细胞和受精的场所，对精子的获能、受精卵的正常卵裂和生存都很重要。输卵管粘膜上皮细胞合成、分泌ET-1，以自分泌的方式作用粘膜上皮细胞，促进粘膜上皮细胞的生长，有利于受精卵的运送和正常卵裂。SABC免疫组化结果显示，ET-1主要分布在细胞游离面，表明ET-1参加输卵管液的组成，这对精子在输卵管内的运动，精子的获能以及受精卵的正常运行有重要作用。从我们的结果观察到，在输卵管肌层可见少量的ET-1颗粒，但未见ET-1 mRNA 阳性杂交信号，肌层平滑肌细胞无自分泌ET-1的功能，肌层的ET-1来自旁分泌或内分泌，ET-1可促进平滑肌细胞收缩[5]，有利于受精卵的正常运行。
, 百拇医药
[基金项目] 国家自然科学基金资助(No.39560028)
[参 考 文 献]
[1] Yanagisawa M, Kutihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endotholial cells[J]. Nature, 1988, 332: 411～415.
[2] Sakurai T, Yanagisawa M, Inoue A, et al. cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA[J]. Biochem Biophys Res Commun, 1991, 175:44～47.
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[3] 蒋应明，孙庆文， 向正华.大鼠下丘脑ET-1 mRNA阳性表达神经元的分布[J]. 中国神经科学，1998， 14(3)：151～154.
[4] Rosselli M, Imthurn B, Macas E, et al. Endothelin production by bovine oviduct epithelial cells[J]. J Reprod Fertil, 1994, 101:27～30.
[5] Rosselli M, Imthurn B, Macas E, et al. Endogenous nitric oxide modulates endothelin-1 induced contraction of bovine oviduct[J]. Biochem Biophys Res Commun, 1994, 201:143～148.
[收稿日期]1999-07-21 [修回日期]1999-11-18
, 百拇医药