山羊乳腺上皮细胞的分离与体外培养

【作者】 任芳丽；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2002， 硕士

【摘要】 本试验研究了山羊乳腺组织细胞的分离及原代培养方法，建立了山羊乳腺上皮细胞系，对乳腺上皮细胞的一些生长特性作了总结；并探索了乳腺特异表达载体的构建，结果如下： 1．用组织块培养法可获得良好的山羊乳腺细胞原代培养物；培养的山羊乳腺成纤维细胞比上皮细胞对胰蛋白酶更敏感，据此可在传代过程中将二者分离纯化，获得纯细胞系；混合培养物先用0.25％胰蛋白酶在37℃消化5～7min所收集的细胞主要为成纤维细胞；再加0.15％胰蛋白酶-0.02％EDTA消化液在37℃继续消化5～6min所回收的细胞绝大多数为上皮细胞，经过2～3代，即可得到纯化的乳腺上皮细胞系。 2．用含10％（v／v）胎牛血清（FCS）的RPMI1640分别溶解150U／ml Ⅰ型胶原酶和100U／ml透明质酸酶，37℃消化组织块4h，可得到足够数量的分散单细胞用以接种，是获得山羊乳腺组织分离单细胞的适宜方法。 3．山羊乳腺组织在铺鼠尾胶原的培养皿中5～0天均长出了乳腺细胞原代培养物，而培养皿中直接培养的组织块在相应的时间内没有乳腺上皮细胞长出。对于传代的乳腺上皮细胞或酶消化的乳腺细胞，鼠尾胶原对其增殖生长没有影响。 4．以RPMI1640为基础培养液，山羊乳腺上皮细胞在含血清、双抗的同时，添加氢化考的松、Insulin、IGF-1、E₂、EGF及ITS的不同组合，以细胞生长群体倍增值为参考标准，评估不同组合的生物活性因子对山羊乳腺上皮细胞生长的影响。结果表明：培养液中添加氢化考的松、IGF-1、EGF及ITS对细胞生长有较明显的促进作用，其倍增时间是34h。 5．分别以RPMI1640、DMEM／F₁₂为基础培养液，添加最佳组合的一组生长因子，作两种培养液的生长曲线。从生长曲线可以看出，传代的乳腺上皮细胞接种后有2天的滞留期，之后进入对数生长期，持续3-4天，第7-8天进入平台期。山羊乳腺上皮细胞在添加氢化考的松、IGF-1、EOF及ITS的DMEM／F₁₂培养液中生长更好。 6．用添加血清和双抗的DMEM／F₁₂为基础培养液，分别添加15％甘油与10％DMSO冻存山羊乳腺上皮细胞，解冻后48h贴壁率分别为71.95％与82.21％，两种冻存液的效果无显著差异；以含10％DMSO的DMEM／F₁₂为基础培养液，分别添加10％、30％与50％胎牛血清的冻存液冻存山羊乳腺上皮细胞，解冻后48h贴壁率分别为72.53％、82.21％和90.37％。结果表明：用添加30％、50％胎牛血清的10％DMSO冻存液冻存效果没有显著差异，均可满足细胞长期保存的需要。 7．用DMEM／F₁₂体外培养山羊乳腺上皮细胞，传代至第7代时其生长在光镜下仍 山羊乳腺上皮细胞的分离与体外培养 正常，没有凋亡迹象。透射电镜观察发现，第6代和第7代细胞没有大的区别： 细胞核结构完整，呈圆形或椭圆形，有些细胞核表面有突起和凹陷，核仁较大， 染色质丰富；细胞质中有粗面内质网；线粒体数目较多，线粒体膜层次清晰。 胞质内有脂滴及高尔基小泡。细胞有较旺盛的分泌活动，胞内物质向外运转， 有大量的小泡向细胞表面移动，部分小泡与细胞膜接触、融合，小泡内物质向 胞外吐出。细胞超微结构表明：山羊乳腺上皮细胞在体外传代到第7代仍增殖 旺盛。8．以自制抗山羊酪蛋白血清为标准抗体，以山羊酪蛋白为标准抗原，用免疫琼脂 双扩散法检测细胞上清液、细胞裂解液中的酪蛋白。结果表明：细胞裂解液中 含有山羊酪蛋白，证明体外培养的山羊乳腺上皮细胞具有合成蛋白质的功能。9．将在乳腺细胞中特异分泌的信号肽序列连接到EZ基因，PCR得到了目的片段， 再将pGFP( 载体上的Kana基因切下与在乳腺细胞中特异表达的P22载体连 接，使其带有筛选标记，然后将带有信号肽的EZ插入到P22中，试图构建山羊 乳腺上皮细胞的特异性表达载体。探索了构建的含目的基因的乳腺特异性表达 载体调控成分的有效性，为在体外培养的乳腺细胞中定位重组目的基因一体细 胞克隆～动物乳腺生物反应器工作的开展作准备工作。更多还原

【Abstract】 There are few reports on establishment of goat mammary epithelial cells until now. The present experiment aims to establish a goat mammary cell line for examining mammary special expression vector and other biological research. The culture methods and characteristics of goat mammary epithelial cell were also studied. The results are as follows:1. Primary culture of goat mammary gland cells could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin in Hanks’ for 5~7min at 37℃, the dispersed cells were mainly fibroblasts, then the rest treated by 0.15% trypsin-0.02% EDTA in Hanks for 5~8min at 37癈, the majority cells harvested were mammary epithelial cells. The pured mammary epithelial cell line could be obtained by 2~3 passages.2. Collegenase I and Hyaluronidase were appropriate enzymes to dissociate goat mammary gland tissue. 1mm3 goat mammary gland tissue fragments incubated in Collegenase I and Hyaluronidase solution (150IU/ml and lOOIU/ml in 10%FCS RPMI1640, especially) 4h at 37℃, could produce enough dispersed cells to explant.3. Primary culture of goat mammary gland cells could be derived by tissue of its mammary gland in culture dishs treated with rat tail collagen in 5~10d, while no mammary epithelial cells grew in plastic petri dishs without collagen treatment in the same duration. The rat tail collagen has no effect on the growth of the passaged cells or dispersed cells digested with enzyme.4. Using RPMI1640 containing 10%FCS, lOOIU/ml Penicillin and lOOIU/ml Streptomycin as basic media, several different growth factors such as insulin, hydrocortisone, insulin-like growth factor, EOF and so on, were added and selected to culture goat mammary epithelial cells. The result suggested that RPMI1640 supplemented with 10%FCS, 5ug/ml insulin, 5ng/ml hydrocortisone, 100ng/ml insulin-like growth factor, lOng/ml EGF and 5ug/ml ITS had the best protective effect to the growth of epithelial cells.5. Using RPMI1640 and DMEM/F12 as basic media, the growth curves of goat mammary epithelialcells in these media supplemented with growth factors were made. The goat mammary cells were in lag phase in first two days, then they entered log phase and persisted 5-6 days; 8 days later, they stayed at plateau phase for about 4 days. The DMEM/Fu medium added with hydrocortisone, IGF-1, EOF and ITS is fit to epithelial cells.6. The goat mammary epithelial cells were frozen in DEME/F12 with serum and antibiotics by adding with cryogen of 10% DMSO and 15% Glycerol, respectively. Attachment rates of the thawed cells after 48h were 71.95% and 82.21%, respectively. The result showed 10% DMSO and 15% Glycerol had the same protective effect. When serum content of cryogen reache 10%, 30% and 50%, their attachment rates were 72.53%, 82.21% and 90.37%, respectively. This demonstrated that the treatment with 50% PCS and 10% DMSO had the best protective effect, which can meet the needs of passaging and long-term preservation of these cells.7. The goat mammary epithelial cells keep normal characteristics by 7th passages with the medium of DMEM/Fi2 and there are no sign of apoptosis. Perspective electron microscope showed the difference between the 6th and the 7th passage is few. The nuclear structure is clear while the surface of some cells nuclear has bulges and hollows. The nucleolus are rather big. There are rough endoplasmic recticulums and mitochondria in the cytoplasm. The presence of lipid droplets and Golgi vesicles attest to the vigorous secretory function of mammary epithelial cells. Many vacuoles move toward cell membrane and some of them contact and fuse with membrane from a pore so to let out the cordents. These results suggest goat mammary epithelial cells proliferate vigorously at the 7th passage in vitro.8. Using the rabbit anti-goat-casein-serum as the standard antibody, the casein of mammary epith更多还原