人孕中期胎儿结膜上皮干细胞定位特征的初步研究

发布时间：2018-11-06 18:51

【摘要】：人孕中期胎儿结膜上皮干细胞定位特征的初步研究目的本文参考有关文献，应用免疫组化方法研究孕中期不同胎龄胎儿结膜上皮细胞中，k7，k14，k19，P63和PCNA的表达及分布情况，探讨人胎儿结膜上皮干细胞的定位特征。材料和方法 1．材料选择取正常引产六小时内的胎儿。17周两例，19周两例，20周两例，23周两例，合计8例16支眼。所有病例无遗传病史和孕期感染史，且取材经过胎儿家属及医院的许可。 2．组织标本制备连眼睑取出完整眼球，除去皮肤及皮下软组织(睑缘保留)剪除后部巩膜，取出玻璃体及色素膜，沿眼球矢状面剪取眼球组织块(呈窄条状，包含睑缘、全部睑结膜、球结膜、穹隆部结膜和部分角膜组织)。40g／L多聚甲醛固定，常规石蜡包埋。将组织蜡块制成3-5μm的切片。 3．SP免疫组化染色采用两步法，严格按照SP试剂盒(购自福建迈新公司)说明书程序操作，k7，k14，k19，P63和PCNA抗体(购自迈新公司)的工作浓度为1∶100。取相应切片按免疫组化SP法染色，切片脱蜡至水，新鲜配制30ml／LH_2O_2室温孵育10min以灭活内源性过氧化物酶：修复抗原，将切片放入柠檬酸缓冲液，微波炉加热10min。待其自然冷却后用正常兔血清室温封闭20min，除去多余血清，滴加一抗(1∶150)，室温孵育70min，PBS洗3次。滴加过氧化物酶标记的链霉亲和素，室温孵育15min，，PBS洗3次。DAB显色4min，苏木素复染，切片脱水，透明，封闭，空白对照以PBS代替一抗。K7、K14、K19
[Abstract]:Preliminary study on the localization of human fetal conjunctival epithelial stem cells in the second trimester of pregnancy objective to refer to relevant literature. Immunohistochemical method was used to study the expression and distribution of p63 and PCNA in human fetal conjunctival epithelial cells at different gestational ages, and to investigate the localization of human fetal conjunctival epithelial stem cells. Materials and methods 1. Materials were selected for fetuses within 6 hours of normal induction of labor. Two fetuses at 17 weeks, two cases at 19 weeks, two cases at 20 weeks, two cases at 23 weeks, Total 8 cases 16 eyes. All cases had no history of hereditary disease and infection during pregnancy, and the materials were obtained with the permission of fetal family members and hospitals. 2. Preparation of tissue specimen, removal of intact eyeball with eyelid, removal of skin and subcutaneous soft tissue (eyelid margin preservation), removal of posterior sclera, removal of vitreous body and pigment membrane, The ocular tissue mass was cut along the sagittal plane of the eyeball (narrow stripe, including eyelid rim, all eyelid conjunctiva, bulbar conjunctiva, fornix conjunctiva and part of cornea tissue). 40g/L polyformaldehyde was fixed, and conventional paraffin embedded. The tissue wax block was made into 3-5 渭 m slice. 3.SP immunohistochemical staining was performed by two-step method, and the procedure of SP kit (purchased from Fujian Mai Xin Company) was strictly followed. The working concentration of p63 and PCNA antibodies was 1: 100. The corresponding sections were stained with immunohistochemical SP method. The sections were dewaxed to water. 10min was incubated with 30ml/LH_2O_2 at room temperature to inactivate endogenous peroxidase: repair antigen. The slices were added to citric acid buffer solution and heated in microwave oven for 10 minutes. After natural cooling, the rabbit serum was blocked at room temperature for 20 min, and the superfluous serum was removed. The first antibody was added (1: 150), and incubated at room temperature for 70 min, the PBS was washed for 3 times. Streptavidin labeled by peroxidase was dripped and incubated at room temperature for 15 mins. DAB developed for 4 mins, hematoxylin was stained again, slice was dehydrated, transparent and closed. PBS was used to replace the first antibody. K7K14 K19 in blank control
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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