【作者】 屠迪；

【导师】 崔燕；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 为了建立牦牛子宫内膜腺上皮和基质细胞的体外分离及培养体系,并确定二者的特异性标记物,本研究首次对泛角蛋白(CKs)、角蛋白7(CK7)、波形蛋白(Vimentin)和平滑肌肌动蛋白(Actin)在未妊娠和妊娠30天左右的牦牛子宫的表达及牦牛子宫内膜腺上皮和基质细胞的分离培养体系进行了研究。研究结果显示:未妊娠时,泛角蛋白在子宫内膜腺上皮细胞、腔上皮细胞内表达,波形蛋白在子宫内膜基质细胞内表达,平滑肌肌动蛋白在子宫平滑肌和血管平滑肌内表达,牦牛子宫任何部位均不表达角蛋白7;妊娠30天左右时,泛角蛋白在子宫内膜腺上皮细胞、子宫内膜腔上皮细胞、滋养层细胞、内胚层细胞和尿囊细胞内表达,波形蛋白在子宫内膜基质细胞和内胚层细胞内表达,平滑肌肌动蛋白在子宫平滑肌和血管平滑肌内表达,角蛋白7在尿囊细胞内表达,偶尔在腔上皮细胞的细胞核边缘表达;消化法进行原代培养时,组织经胶原酶消化并通过100目和400目筛网组合可以有效地分离原代子宫内膜基质细胞和子宫内膜腺上皮细胞;分离得到的子宫内膜基质细胞活率达90%以上,并可在体外传代7次以上;分离得到的子宫内膜腺上皮细胞活率可达85%以上,并可在体外传代5次以上;RPMI1640培养基最适合子宫内膜基质细胞和子宫内膜腺上皮细胞的生长,维持子宫内膜基质细胞正常生长的FBS添加量为20%,维持子宫内膜腺上皮细胞正常生长的胎牛血清添加量为30%。对分离得到的子宫内膜基质细胞和子宫内膜腺上皮细胞进行免疫组织化学标记的结果显示在体外子宫内膜基质细胞表达泛角蛋白,子宫内膜腺上皮细胞表达波形蛋白,并且这一特性不因为传代而发生丢失。更多还原

【Abstract】 To establish a culture system and get the specific markers for Yak Endometrial Glandular cell and Stromal cell,the distribution of Cytokeratins , CK7 , vimentin and Actin in the normal uterus and the gravid uterus of Yak were investigated immunohistochemically. In the normal uterus, Cytokeratins immunolabelling were detected in glandular cell, luminal epithelial cell, Vimentin immunolabelling were detected in stromal cell and endoblastic cell; CK7 immunolabelling were not detected in any tissue of the yak utenus. In the gravid uterus, The CKs immunolabelling were detected in glandular cell, luminal epithelial cell, traphoblast cell, endoblastic cell and allantoic cell; Vimentin immunolabelling were detected in stromal cell and endoblastic cell; CK7 immunolabelling were not detected in any tissue of the yak utenus but in endoblastic cell and some luminal epithelial cell. It is an effective way to separate the stromal cell from the glandular cell that the tissue was digested by 0.1% collagenase, and then tissue digest was passed over a stacked sterile wire sieve assembly with number 100 wire sieve, followed by a number 400 wire sieve. The viability of stromal cell was 90%, The viability of glandular cell was 85%. YSC and YGE was cultured and propagated in RPMI1640 for at least 7 and 10 population doublings. There is at least 20% and 30% FBS in culture media for YSC and YGE growing normally. The CKs was detected in YGE, and the Vimentin was dected in YSC; and the charactorzation was reminded by propagating, in vitro. There were not CK7 in YGE and YSC.更多还原