摘要目的：研究排卵期阴道脱落细胞的雌、孕激素受体。方法：自然排卵妇女、注射人绒毛膜促性腺激素（human chorionic gonadotropin, hCG）促排卵妇女排卵期阴道脱落细胞，应用免疫组织化学ABC法和图像分析技术进行雌激素受体（estrogen receptor, ER）和孕激素受体（progesterone receptor, PR）的定位和定量研究。结果：两组组内排卵前、后阴道脱落细胞ER比较均无差异；注射hCG后阴道脱落细胞的ER在排卵前和排卵后均较自然排卵组有明显增高；自然排卵组PR在排卵后较之排卵前明显升高。结论：排卵前后阴道上皮细胞ER无明显变化； hCG可提高阴道上皮细胞ER水平；排卵后阴道上皮细胞PR增加。

中国图书资料分类法分类号R339.22

Study of estrogen receptors and progesterone receptors in epithelial cells of vagina in natural ovulation and ovulation by injection of human chorionic gonadotropin

JIAO Li-Ya； YANG Jin-Jin； LIU Bin

(Department of Embryology and Histology, Beijin Medical University, Beijing100083 China)

MeSHOvulation/drug effGonadotropins, chorionic/pharmacolReceptors, estrogen/analReceptors, progesterone/analVagina/cytol

ABSTRACTObjective: To study the estrogen receptors(ER) and progesterone receptors(PR) in vagina epithelial cells during ovulation phase.Methods: Immunohistochemical ABC method and Q-500 image analysis system were used to locate and quantitative determinate of ER and PR in vagina epithelial cells in the nature ovulation women and injection of human chorionic gonadotropin (hCG) to induce ovulation women.Results: ER and PR positive immunostaining was found in the cytoplasm and nuclei of the vagina epithelial cells . ER level in vagina epithelial cells within the two groups had no difference in pre-ovulation and post-ovulation respectively; the ER level in the vagina epithelial cells in of hCG injection group was higher than that in natural ovulation group no matter which was in pre-ovulation or post-ovulation. The PR level in the vagina epithelial cells increased in post-ovulation.Conclusion: hCG could increase the ER level in the vagina epithelial cells; After ovulation, the PR level in vagina epithelial cells increased.

（J Beijing Med Univ, 1999,31:415-417）

生育期妇女阴道上皮的生长、发育、成熟直接受性激素的影响。通过对阴道脱落细胞的检测，可间接反映卵巢功能。以往人们常采用阴道脱落细胞巴氏染色或绍氏染色来了解体内性激素水平。但近年来，免疫组织化学方法被用来检测组织中雌激素受体（estrogen receptors, ER）和孕激素受体（progesterone receptors, PR）水平^［1］，具有方便、快捷，定位准确的特点，且方法成熟。本实验对自然排卵妇女注射人绒毛膜促性腺激素(human chorionic gonadotropin, hCG)促排卵妇女排卵期阴道脱落细胞，应用免疫组织化学方法和图像分析技术进行ER与PR的定位和定量研究，旨在揭示排卵前、后阴道上皮细胞中ER和PR的变化，及hCG对阴道上皮的ER的影响。

1材料与方法

1.1试剂

鼠抗人ER抗体、鼠抗人PR抗体由孙素莲教授惠赠(我校临床肿瘤学院自制，经检验抗体效价符合标准)；ABC药盒系美国Vector 公司产品。

1.2材料

收集1994～1997年来我室进行人工授精的育龄妇女（年龄25～42岁）44例，其中自然排卵妇女36例、注射hCG 5 000 IU促排卵妇女8例分别进行ER测定，自然排卵组8例进行了PR测定。用刮片法采集阴道脱落细胞，均匀涂布于载玻片上。

1.3免疫组织化学反应程序

阴道脱落细胞涂片即刻放入4℃丙酮固定30 min后，再入体积分数为0.3%的H₂O₂甲醇，室温，30 min，然后按ABC法进行免疫组织化学反应^［1］。第一抗体为鼠抗人ER或鼠抗人PR单克隆抗体（ER体积比1∶100稀释；PR为原液），DAB显色。每张涂片均设阴性对照，用磷酸缓冲液(phosphatic balance solution, PBS)代替一抗。

1.4阴道脱落细胞ER及PR原位定量

自然排卵组、注射hCG促排卵组ER测定病例，及自然排卵组PR测定病例各取6例，经上述免疫组织化学染色后，用Q500图像分析系统（Leica公司）测定每例ER或PR 的灰度值。光镜下（100×）每张涂片随机测量8个单位面积，每组排卵前后分别测48个单位面积。各组灰度值+s组间和组内各以48个样本数，采用ANOVA继以Newman-Keuls检验进行统计学处理。

2结果

2.1ER和PR的定位

光镜下观察，ER或PR阳性细胞，其胞质内或胞核内有黄色或棕黄色颗粒，强度明显高于底色。对照及阴性细胞胞质内或胞核内无黄色颗粒，呈均一淡黄色或与背景相同（图1～3）。

图1自然排卵组排卵前阴道脱落细胞ER免疫组化反应。胞质和胞核内见棕黄色阳性颗粒。免疫组化ABC法×330

Figure 1ER positive immunostaining was found in the cytoplasm and nuclei of the vagina epithelial cells preovulation in nature ovulation group. Immunohislochemical ABC method×330

图2注射hCG促排卵组排卵前阴道脱落细胞ER免疫组化反应。胞质和胞核内见棕黄色阳性颗粒,左上及右下为阳性细胞 ，右上为阴性细胞。免疫组化ABC法×330

Figure 2ER positive immunostaining was found in the cytoplasm and nuclei of the vagina epithelial cells preovulation in injection hCG ovulation group. top left corner and right lower is positive cell, top right corner is negative cell. Immunohislochemical ABC method×330

图3自然排卵组排卵后阴道脱落细胞PR免疫组化反应。胞质和胞核内见棕黄色阳性颗粒，左侧为阳性细胞，右侧为阴性细胞。免疫组化ABC法×330

Figure 3PR positive immunostaining was found in the cytoplasm and nuclei of the vagina epithelial cells preovulation in nature ovulation group. left cell is positive cell, right cell is negative cell. Immunohislochemical ABC method×330

2.2ER和PR的相对含量

自然排卵组、hCG促排卵组阴道脱落细胞的ER，和自然排卵组PR的免疫组织化学定量见表1。自然排卵组的阴道脱落细胞的ER排卵前与排卵后比较无差异；hCG促排卵组阴道脱落细胞的ER在排卵前与排卵后也无差异；与自然排卵组排卵前的阴道脱落细胞的ER比较，hCG促排卵组阴道脱落细胞的ER在排卵前有明显增高（P＜0.05）；与自然排卵组的排卵后的阴道脱落细胞的ER比较，hCG促排卵组阴道脱落细胞的ER在排卵后亦有明显增高（P＜0.05）；自然排卵组阴道脱落细胞PR在排卵后较之排卵前明显升高（P＜0.05）。

表1各组排卵前后阴道脱落细胞ER及PR免疫反应阳性物质灰度值(+s)

Table 1The immunopositive substance value of grey degree of ER and PR in vagina epithelial cells (+s)

Group Number of

case Number of

measured

field ER PR Before After Before After Nature