【摘要】：黑色素細(xì)胞所合成色素的種類、數(shù)量及分布與被毛顏色密切相關(guān),本試驗(yàn)對(duì)小鼠黑色素細(xì)胞進(jìn)行體外分離培養(yǎng)并研究其生物學(xué)特性,篩選出研究基因功能的理想材料。試驗(yàn)及結(jié)果如下：1.用0.25%Dispase酶Ⅱ和0.25%胰酶/0.02% EDTA分離到的小鼠黑色素細(xì)胞形態(tài)呈典型的多樹(shù)突狀或雙極形,樹(shù)突細(xì)長(zhǎng),伴有特殊生長(zhǎng)暈。2.對(duì)傳代的4代、7代、10代和13代細(xì)胞經(jīng)多巴染色后發(fā)現(xiàn),細(xì)胞的胞質(zhì)、樹(shù)突和胞核均成棕褐色;用免疫細(xì)胞化學(xué)染色對(duì)細(xì)胞內(nèi)的S-100、MITF、TYR、TYRP1和TYRP2蛋白進(jìn)行鑒定,發(fā)現(xiàn)細(xì)胞內(nèi)均有陽(yáng)性信號(hào),具有黑色素細(xì)胞的生物學(xué)特征。3.用qRT-PCR檢測(cè)不同代數(shù)細(xì)胞中MITF、TYR和TYRP1 mRNA的表達(dá)水平,結(jié)果顯示：與4代細(xì)胞相比,7、10代細(xì)胞內(nèi)基因表達(dá)量變化不大；而13代細(xì)胞中TYR和TYRP1表達(dá)量顯著升高,具有黑色素瘤細(xì)胞的特征。4.用iCelligence實(shí)時(shí)檢測(cè)不同代數(shù)細(xì)胞的貼壁和增殖,結(jié)果發(fā)現(xiàn)培養(yǎng)2h后,各代細(xì)胞均開(kāi)始貼壁增殖;培養(yǎng)40 h后,4、7、10代細(xì)胞增殖基本達(dá)頂峰,之后細(xì)胞數(shù)減少,而13代細(xì)胞可維持到100 h左右,之后細(xì)胞數(shù)維持恒定,其增殖力比其他3個(gè)代數(shù)細(xì)胞強(qiáng),具有黑色素瘤細(xì)胞的特征。5.在13代細(xì)胞培養(yǎng)基中加入a-MSH,培養(yǎng)72h, MITF、TYR和TYRP2 mRNA表達(dá)水平分別是對(duì)照組的1.528、4.623和1.489倍。結(jié)論：13代細(xì)胞具有較強(qiáng)的增殖能力且TYR基因的表達(dá)量顯著升高,可能轉(zhuǎn)化為黑色素瘤細(xì)胞。因此,10代前的小鼠黑色素細(xì)胞是研究黑色素形成過(guò)程的理想材料。
[Abstract]:The species, quantity and distribution of melanocytes were closely related to the color of the coat. In this experiment, the melanocytes of mice were isolated and cultured in vitro and their biological characteristics were studied, and the ideal materials for studying gene function were screened out. The results are as follows: 1. The melanocytes isolated from mice with 0.25%Dispase 鈪，

本文編號(hào)：2278013