雌激素和孕酮对体外培养犬子宫内膜基质细胞增殖及孕酮受体表达的影响
发布时间：2019-02-20 20:04
【摘要】：犬子宫蓄脓(canine pyometra)是母犬发情后期发病率最高的产科疾病。已有研究表明,子宫内膜囊性增生(CEH)是本病的原发性疾病,在细菌感染和其它因素的共同作用下,最终发展为犬子宫蓄脓。在犬子宫内膜的病理变化中,雌激素(E2)和孕酮(P4)是诱发囊性增生的主要因素。本研究的主要目的是建立一个犬子宫内膜基质细胞的培养模型,探讨孕酮受体(PR)在犬子宫内膜基质细胞中的表达,并测定E2和P4对细胞增殖和PR表达的影响,以期有助于阐明E2和P4与犬子宫蓄脓发病机理的关系,为研究犬子宫蓄脓病的病因提供依据。1.选用酶消化法分离犬子宫内膜基质细胞(ESCs),经过原代培养、细胞鉴定、传代培养、消化方式的探索、血清依赖性的探索,观察犬ESCs体外培养的生长特性,探索该细胞的分离和培养的最佳方法。结果表明:37℃条件下,0.125%的胶原酶I消化3~4 h消化效果最好,易于单个细胞易分离。高浓度(20%)胎牛血清适合犬ESCs的生长。细胞呈长梭形或多角形,细胞核呈卵圆形。接种后5~6 d可长成单层,细胞间存在重叠生长现象。利用免疫组织化学染色法鉴定细胞,两种形态的细胞均表达波形蛋白,阳性率可达90%以上,角蛋白阴性,证明所得到的细胞为子宫内膜基质细胞。2.使用MTT法检测E2和P4对犬ESCs增殖的影响。结果表明,血清饥饿1 d后,无激素刺激组的细胞从第2 d开始生长速度加快,进入对数生长期,持续时间约为5 d。第6d进入平台期,随后开始脱落。第7 d~8 d细胞数量减少速度较慢。血清饥饿1 d后添加不同浓度的E2(15、30、100 pg/mL)细胞生长曲线类似于对照组。血清饥饿1 d后添加不同浓度的P4(3、15、30 ng/m L)细胞生长速度增快,平台期细胞数量增多。在细胞生长周期的三个阶段(d3:指数期,d6:平台期,d8:衰亡期),E2对ESCs的增殖无典型影响,在相同阶段,ESCs的增殖对E2浓度变化(15、30、100 pg/m L)不敏感。P4(15、30 ng/m L)对ESCs的增殖有显著的促进作用(P0.05),在同一阶段,ESCs的增殖与P4浓度变化呈正相关。3.通过细胞免疫组织化学染色的方法检测E2和P4对犬ECSs中孕酮受体表达的影响。结果显示体外培养的犬ESCs对PR抗体呈强阳性,细胞核呈棕黄色,细胞质内基本不含受体阳性物质,说明PR主要在细胞核内表达。对照组中PR的阳性率在整个培养过程中始终大于90%。培养液中添加E2(15、30、100 pg/mL)后PR的阳性率与对照组比没有显著变化。培养液中添加P4(3、15、30 ng/mL)后,第6 d起对ESCs中PR的阳性率有显著的抑制作用(P0.05),低浓度P4组(3 ng/m L)抑制程度随着作用时间延长而增强,在第8 d阳性率最低。高浓度P4组(15、30 ng/mL)阳性率第8 d与第6 d比下降不明显。低浓度P4组(3 ng/mL)第6 d、8 d的PR阳性率均低于高浓度P4组(15、30ng/m L)。以上研究表明,P4对体外培养的犬ESCs的增殖有促进作用。P4对PR的表达有抑制作用,且抑制强弱与P4浓度和作用时间有关。说明P4对PR表达可以在细胞水平进行调控。E2对犬ESCs的调节作用不显著。
[Abstract]:(canine pyometra) is the most common obstetric disease in late estrus. Studies have shown that endometrial cystic hyperplasia (CEH) is a primary disease of this disease. Estrogen (E _ 2) and progesterone (P _ 4) are the main factors inducing cystic hyperplasia in canine endometrium. The main purpose of this study was to establish a canine endometrial stromal cell culture model, to investigate the expression of progesterone receptor (PR) in canine endometrial stromal cells, and to determine the effects of E2 and P4 on cell proliferation and PR expression. It is helpful to elucidate the relationship between E _ 2 and P _ 4 and the pathogenesis of canine purulent storage, and to provide the basis for the study of the etiology of canine purulent disease. The growth characteristics of canine endometrial stromal cells (ESCs) in vitro were observed by enzyme digestion method after primary culture, cell identification, passage culture, digestion mode exploration, serum-dependent exploration, in vitro culture of canine endometrial stromal cells. To explore the best method of cell isolation and culture. The results showed that at 37 鈩，
本文编号：2427213
[Abstract]:(canine pyometra) is the most common obstetric disease in late estrus. Studies have shown that endometrial cystic hyperplasia (CEH) is a primary disease of this disease. Estrogen (E _ 2) and progesterone (P _ 4) are the main factors inducing cystic hyperplasia in canine endometrium. The main purpose of this study was to establish a canine endometrial stromal cell culture model, to investigate the expression of progesterone receptor (PR) in canine endometrial stromal cells, and to determine the effects of E2 and P4 on cell proliferation and PR expression. It is helpful to elucidate the relationship between E _ 2 and P _ 4 and the pathogenesis of canine purulent storage, and to provide the basis for the study of the etiology of canine purulent disease. The growth characteristics of canine endometrial stromal cells (ESCs) in vitro were observed by enzyme digestion method after primary culture, cell identification, passage culture, digestion mode exploration, serum-dependent exploration, in vitro culture of canine endometrial stromal cells. To explore the best method of cell isolation and culture. The results showed that at 37 鈩，
本文编号：2427213
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2427213.html
