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1、 摘 要 选取健康的泌乳期荷斯坦奶牛乳腺组织， 放入含双抗的 D-hanks 液带回实 验室，经胰酶消化法获得奶牛乳腺上皮细胞，用铺有鼠尾胶原的六孔板对获得的 奶牛乳腺上皮细胞进行体外培养，接种密度为 1106个/mL，培养液成分为 DMEM/F12 ，胎牛血清，胰岛素，转铁蛋白，表皮生长因子，谷氨酰胺，双抗 等。对细胞进行原代培养，收集贴壁 48h 时的细胞培养液，对细胞 -酪蛋白的 分泌特性进行检测， 鉴定贴壁的细胞是否为奶牛乳腺上皮细胞。 细胞贴壁 48h 后， 分两组对细胞进行热应激处理。第一组为单一高温处理法：将 37正常培养的 乳腺上皮细胞放置于 38、39、40、41恒温水浴锅中
2、，热处理 1h 后立即 放回 37、 95%湿度、 5% CO2环境中恢复培养， 在恢复培养 0h 、 6h、 12h、 24h 收集细胞样品，-70冻存。第二组为连续高温处理法，每隔 24h 对乳腺上皮细 胞进行一次高温处理，38、39 、40、41恒温水浴，方法同前，在每个 热处理后 24h 收集细胞样品，连续收集 3d，-70冻存。提取细胞总 RNA，经反 转录、RT-PCR、凝胶电泳、回收、与 pGEM-T 载体连接、转化、摇菌、提质粒、 酶切、测序、倍比稀释等步骤制备 HSP70 和 GAPDH 标准质粒，然后 Real time RT-PCR 制定标准曲线， 定量测定不同热应激处理
3、的样品当中 HSP70mRNA 的表 达量。 经 Western-blotting 检测，原代培养贴壁的细胞为奶牛乳腺上皮细胞。热应 激处理后，HSP70 mRNA 的表达量与温度呈正相关。且单一热应激处理后，在 恢复培养第 6h 时 HSP70 基因的表达量最高；第 12h 组与 24h 组差异不显著（P 0.05） ，这两组皆与 0h 组差异显著(P0.05)，与 6h 差异极其显著(P0.01)。 说明单一热应激恢复培养后 12h-24h 细胞基本恢复正常状态，热应激反应逐渐消 失。连续高温处理法显示，细胞热应激第 48h 组 HSP70mRNA 表达量显著高于 24h 组（P0.05）
4、, 但 72h 时 HSP70mRNA 表达量有所降低。说明细胞在连续热应激的过程中细胞损伤较大， 连续热应激后的恢复期延长。 另外， 连续热应激后， 细胞 HSP70mRNA 表达量有所下降，说明细胞可能产生了热耐受性。 热应激对奶牛影响极其严重，严重的热应激可使高产奶牛产奶量下降 80%， 给奶牛养殖小区造成了巨大的损失。 本文旨在对奶牛热应激的原理在分子领域进 行探索，研究高温应激对奶牛乳腺上皮细胞的损伤机理，为进一步的进行热应激 的营养调控提供理论基础。 原代培养的奶牛乳腺上皮细胞在细胞种类、 细胞性状、 细胞结构与细胞功能上与体内来源细胞有着极大的相似性， 又可以在培养过程中 进行处
5、理，以消除其它因素的影响，为研究提供了快捷和便利。 关键词：原代培养；HSP70； Real time RT-PCR；热耐受性；乳腺上皮细胞 The Effect of Heat Stress on the HSP70mRNA Expression of Cultured Dairy Cattle Mammary Epithelial Cell Author: Zhao Li-hong Tutor: Li Jian-guo Major: Animal Nutrition and Feed Science Abstract Select healthy lactating mammary gla
6、nd of Holstein cows, put it into the D-hanks solution with double-antibody,take back to the laboratory. we obtained the mammary epithelial cells by trypsin digestion, and cultured it in the six orifice which covered with rat tail collagen. The inoculation density was 1 106 / mL. Culture medium compo
7、sed of DMEM/F12, fetal bovine serum, insulin, transferrin, epidermal growth factor, glutamine, double anti-so. Collected the cell medium after cultureed 48h to identify of whether the cells were mammary epithelial cells. After the cells adhered 48h, divided into two groups and heat treatment ,respec
8、tively.The first group dealed with a single high-temperature processing: put the 37 normal cultured mammary epithelial cells in 38 , 39 , 40 , 41 water bath pot, immediately replace it into the 37 , 95% humidity, 5% CO2 environment for cultivation after heated 1 hour . At the resumption of cultured
9、0h, 6h, 12h, 24h to collect cell samples, -70 cryopreservation. The second group dealed with continuous heat treatment , every 24h do a high temperature for 1h, 38 , 39 , 40 , 41 water bath, the same way as before, after 24h each treatment to collect cell samples, continuous collected 3 days, -70 cr
10、yopreservation. Extraction of total cellular RNA, reverse transcription, RT-PCR, gelelectrophoresis, recovery, and pGEM-T vector, transformed, shaking bacteria, plasmid was extracted, restriction enzyme digestion, sequencing, dilution steps of HSP70 and GAPDH standard plasmid preparation and Real ti
11、me RT-PCR standard curve, quantitative determination the expression of these HSP70mRNA of the samples treated with different heat stress. By Western-blotting detection, the primary cultured cells were mammary epithelial cells. After the heat stress treatment, the expression of HSP70mRNA was positive
12、ly correlated with temperature. And after a single heat stress treatment, the highest expression of HSP70 was at he resumption of cultured 6h ; the different expression of HSP70 between the 12h and 24h group groups was not significant (P 0.05), both groups were significantly different with the 0h gr
13、oup (P 0.05) , and 6h extremely significant difference (P 0.01).Illustrated that cells returned to normal conditions after a sigle heat stess, the influence by heat stress gradually disappeared. Continuous heat treatment method showed that the expression of HSP70 mRNA of the 48h group was higher tha
14、n the 24h group (P 0.05), but the 72h group HSP70 expression decreased . It shows that cells by a continuous heat stress in the process were damaged larger. In addition, after the continuous heat stress, the cells decreased HSP70mRNA expression, indicating cell may produce a heat tolerance. Effects
15、of heat stress on dairy cows is extremely serious, serious heat stress can decrease milk production yield 80%, caused huge losses to cow breeding areas. This study was designed to the principle of heat stress on dairy cows in the field of molecular exploration,to study the heat stress mechanism of d
16、iary cow epithelial cells and to provide a theoretical basis for the nutritional regulation of heat stress.Not only were primary cultured bovine mammary epithelial cell in the cell type, cell characteristics, cell structure and cell function similar to the primary cell ,but also in the process to eliminate other influence factors, provides quick and convenience for our study. Key words: Primary culture; HSP70; Real time RT-PCR;Heat tolerance
