【摘要】：犬子宮蓄膿(canine pyometra)是母犬發(fā)情后期發(fā)病率最高的產(chǎn)科疾病。已有研究表明,子宮內(nèi)膜囊性增生(CEH)是本病的原發(fā)性疾病,在細(xì)菌感染和其它因素的共同作用下,最終發(fā)展為犬子宮蓄膿。在犬子宮內(nèi)膜的病理變化中,雌激素(E2)和孕酮(P4)是誘發(fā)囊性增生的主要因素。本研究的主要目的是建立一個(gè)犬子宮內(nèi)膜基質(zhì)細(xì)胞的培養(yǎng)模型,探討孕酮受體(PR)在犬子宮內(nèi)膜基質(zhì)細(xì)胞中的表達(dá),并測(cè)定E2和P4對(duì)細(xì)胞增殖和PR表達(dá)的影響,以期有助于闡明E2和P4與犬子宮蓄膿發(fā)病機(jī)理的關(guān)系,為研究犬子宮蓄膿病的病因提供依據(jù)。1.選用酶消化法分離犬子宮內(nèi)膜基質(zhì)細(xì)胞(ESCs),經(jīng)過(guò)原代培養(yǎng)、細(xì)胞鑒定、傳代培養(yǎng)、消化方式的探索、血清依賴性的探索,觀察犬ESCs體外培養(yǎng)的生長(zhǎng)特性,探索該細(xì)胞的分離和培養(yǎng)的最佳方法。結(jié)果表明:37℃條件下,0.125%的膠原酶I消化3~4 h消化效果最好,易于單個(gè)細(xì)胞易分離。高濃度(20%)胎牛血清適合犬ESCs的生長(zhǎng)。細(xì)胞呈長(zhǎng)梭形或多角形,細(xì)胞核呈卵圓形。接種后5~6 d可長(zhǎng)成單層,細(xì)胞間存在重疊生長(zhǎng)現(xiàn)象。利用免疫組織化學(xué)染色法鑒定細(xì)胞,兩種形態(tài)的細(xì)胞均表達(dá)波形蛋白,陽(yáng)性率可達(dá)90%以上,角蛋白陰性,證明所得到的細(xì)胞為子宮內(nèi)膜基質(zhì)細(xì)胞。2.使用MTT法檢測(cè)E2和P4對(duì)犬ESCs增殖的影響。結(jié)果表明,血清饑餓1 d后,無(wú)激素刺激組的細(xì)胞從第2 d開(kāi)始生長(zhǎng)速度加快,進(jìn)入對(duì)數(shù)生長(zhǎng)期,持續(xù)時(shí)間約為5 d。第6d進(jìn)入平臺(tái)期,隨后開(kāi)始脫落。第7 d~8 d細(xì)胞數(shù)量減少速度較慢。血清饑餓1 d后添加不同濃度的E2(15、30、100 pg/mL)細(xì)胞生長(zhǎng)曲線類似于對(duì)照組。血清饑餓1 d后添加不同濃度的P4(3、15、30 ng/m L)細(xì)胞生長(zhǎng)速度增快,平臺(tái)期細(xì)胞數(shù)量增多。在細(xì)胞生長(zhǎng)周期的三個(gè)階段(d3:指數(shù)期,d6:平臺(tái)期,d8:衰亡期),E2對(duì)ESCs的增殖無(wú)典型影響,在相同階段,ESCs的增殖對(duì)E2濃度變化(15、30、100 pg/m L)不敏感。P4(15、30 ng/m L)對(duì)ESCs的增殖有顯著的促進(jìn)作用(P0.05),在同一階段,ESCs的增殖與P4濃度變化呈正相關(guān)。3.通過(guò)細(xì)胞免疫組織化學(xué)染色的方法檢測(cè)E2和P4對(duì)犬ECSs中孕酮受體表達(dá)的影響。結(jié)果顯示體外培養(yǎng)的犬ESCs對(duì)PR抗體呈強(qiáng)陽(yáng)性,細(xì)胞核呈棕黃色,細(xì)胞質(zhì)內(nèi)基本不含受體陽(yáng)性物質(zhì),說(shuō)明PR主要在細(xì)胞核內(nèi)表達(dá)。對(duì)照組中PR的陽(yáng)性率在整個(gè)培養(yǎng)過(guò)程中始終大于90%。培養(yǎng)液中添加E2(15、30、100 pg/mL)后PR的陽(yáng)性率與對(duì)照組比沒(méi)有顯著變化。培養(yǎng)液中添加P4(3、15、30 ng/mL)后,第6 d起對(duì)ESCs中PR的陽(yáng)性率有顯著的抑制作用(P0.05),低濃度P4組(3 ng/m L)抑制程度隨著作用時(shí)間延長(zhǎng)而增強(qiáng),在第8 d陽(yáng)性率最低。高濃度P4組(15、30 ng/mL)陽(yáng)性率第8 d與第6 d比下降不明顯。低濃度P4組(3 ng/mL)第6 d、8 d的PR陽(yáng)性率均低于高濃度P4組(15、30ng/m L)。以上研究表明,P4對(duì)體外培養(yǎng)的犬ESCs的增殖有促進(jìn)作用。P4對(duì)PR的表達(dá)有抑制作用,且抑制強(qiáng)弱與P4濃度和作用時(shí)間有關(guān)。說(shuō)明P4對(duì)PR表達(dá)可以在細(xì)胞水平進(jìn)行調(diào)控。E2對(duì)犬ESCs的調(diào)節(jié)作用不顯著。
[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，

本文編號(hào)：2427213