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| 绵羊瘤胃上皮细胞的体外分离培养、冻存及复苏方法 |
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金鑫1,2, 张曼1,2, 王云鹤1,2, 魏方1,2, 温婧怡1,2, 杨银凤1,2
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| (1.内蒙古农业大学 兽医学院, 呼和浩特 010018;2.农业部动物疾病临床诊疗技术重点实验室, 呼和浩特 010018) |
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| 摘要: |
| 本研究旨在建立绵羊瘤胃上皮细胞（Ovine rumen epithelial cells，ORECs）的分离培养、冻存和复苏方法，为反刍动物瘤胃功能的研究和相关瘤胃疾病的发病机制研究提供功能性的细胞模型。以绵羊瘤胃为研究对象，采用胰蛋白酶连续消化法对瘤胃组织进行不同时间（40、30、20、10、10、8和5 min）的消化，并对每次消化后的瘤胃组织和消化液分别进行H.E染色分析和显微镜观察，以确定细胞开始收集及终止消化的时间，最后将收集的瘤胃上皮细胞进行原代培养。在传代培养时，运用差时消化法和差速贴壁法对ORECs进行纯化，并从细胞形态学、细胞生长曲线、角蛋白18（CK-18）免疫荧光染色和上皮细胞标志物E-钙黏蛋白和碱性磷酸酶基因表达等方面对ORECs进行鉴定。然后设置不同体积配方的冻存液，将传代细胞进行冻存，通过检测复苏后的细胞活力和24 h贴壁率确定最佳冻存液配方。结果表明：第4次消化后就基本消化到瘤胃组织的棘层，此时即可开始收集细胞；第7次消化完后瘤胃上皮的基底层细胞基本被消化掉，此时即可终止消化细胞。经纯化后传代的细胞呈现均一的"铺路石"形态，生长曲线明显呈"S"形，且经CK-18免疫荧光染色后鉴定为阳性，同时PCR检测到上皮细胞特异性标志蛋白E-钙黏蛋白和碱性磷酸酶均有表达。此外，ORECs经不同体积配方冻存液冻存复苏后发现，冻存液配方为VFBS∶VDMEM∶VDMSO=9∶0∶1时ORECs的细胞活力及贴壁率最高，且传代后的细胞生长状况良好。因此本试验成功建立了ORECs的体外分离培养、冻存和复苏方法。 |
| 关键词: 绵羊 瘤胃上皮细胞 原代培养 传代培养 冻存 复苏 |
| DOI：10.11841/j.issn.1007-4333.2019.05.07 |
| 投稿时间：2018-10-11 |
| 基金项目:国家自然科学基金项目（31560682）；内蒙古自治区研究生教育创新计划资助项目（B20171012921） |
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| In vitro isolation,culture,cryopreservation and resuscitation of ovine rumen epithelial cells |
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JIN Xin1,2, ZHANG Man1,2, WANG Yunhe1,2, WEI Fang1,2, WEN Jingyi1,2, YANG Yinfeng1,2
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| (1.College of Veterinary, Inner Mongolia Agricultural University, Hohhot 010018, China;2.Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease of Ministry of Agriculture, Hohhot 010018, China) |
| Abstract: |
| The aims of this study were to establish a method for isolation,culture,cryopreservation and resuscitation of ovine rumen epithelial cells (ORECs) and provide a functional cell model to study rumen function and the pathogenesis of related rumen diseases in ruminants.The rumen of sheep was used as the research object,and it was digested by trypsin continuous digestion at different times (40,30,20,10,10,8 and 5 min,respectively).H.E staining analysis and microscopic observation were then performed on each rumen tissue and digestive juice after each digestion to determine the time when the cells began to collect and terminate the digestion,and finally the collected ORECs were primary cultured.In subculture,ORECs were purified by differential digestion and differential adherence,and the ORECs were identified from cell morphology,cell growth curve,cytokeratin 18 (CK-18) immunofluorescence staining and the gene expression of epithelial cell marker E-cadherin and alkaline phosphatase.Then,the frozen solution of different volume formulas was set,the passage cells were frozen,and the optimal cryopreservation formula was determined by detecting the cell viability after resuscitation and the 24 h adherence rate.The results showed that:After the fourth digestion,the spinous layer of rumen tissue was basically digested,and the cells were collected at this time;After the seventh digestion,the basal cells of the rumen epithelium were basically digested,and the digested cells were terminated;After purification,the passaged cells showed a uniform "paving stone" morphology,the growth curve was obviously "S" shape,and the CK-18 immunofluorescence staining was positive.Meanwhile,the expression of epithelial cell-specific marker proteins E-cadherin and alkaline phosphatase were detectable by PCR.In addition,ORECs were cryopreserved by cryopreservation at different volume formulas and it is found that when the cryopreservation formula was VFBS:VDMEM:VDMSO=9:0:1,the cell viability and adherence rate of ORECs was the highest,and the cells after passage were well grown.In conclusion,the in vitro isolation,culture,cryopreservation and resuscitation methods for ORECs were successfully established in this study. |
| Key words: sheep rumen epithelial cells primary culture subculture cryopreservation resuscitation |
