抑制miR-142-3p对奶山羊化学诱导乳腺上皮细胞泌乳功能的影响

doi:10.16431/j.cnki.1671-7236.2023.01.006

摘要/Abstract

摘要： 【目的】探索miR-142-3p对奶山羊化学诱导乳腺上皮细胞（chemical induced mammary epithelial cells，CiMECs）体外泌乳功能的调节作用，旨在为促进转基因乳腺生物反应器的发展以及"培养皿奶"的商业化生产奠定基础和提供新的思路。【方法】选取奶山羊耳缘成纤维细胞（GEFs）为研究材料，经单一小分子化合物TGFβR-1/ALK5的抑制剂（RepSox）诱导8 d后，分别从细胞的形态变化、特异性标记物免疫荧光染色以及油红O染色对其进行鉴定。之后运用脂质体转染技术在诱导成功的奶山羊CiMECs中分别转染miR-142-3p的抑制物（miR-142-3p inhibitor）及抑制物对照（inhibitor-NC），不转染的细胞作为空白对照（BC），转染24 h后，采用实时荧光定量PCR检测miR-142-3p的表达水平，CCK-8法检测细胞的增殖率，Western blotting检测β-酪蛋白（β-casein）的表达量，甘油三酯酶法测定甘油三酯的含量。【结果】GEFs诱导8 d后形成岛屿状多核仁样的上皮样聚集；免疫荧光染色结果显示，诱导后的GEFs表达乳腺上皮细胞（MECs）的特异性标记物细胞角蛋白14（CK14）和CK18；油红O染色结果显示，诱导后的GEFs细胞质内弥散分布着大小不等被染上红色的脂滴，表明成功获得了具有泌乳功能的奶山羊CiMECs。奶山羊CiMECs转染试验结果显示，与空白对照组相比，miR-142-3p inhibitor组miR-142-3p的相对表达量显著降低（P<0.05），inhibitor-NC组miR-142-3p表达量无显著差异（P>0.05）；miR-142-3p inhibitor组奶山羊CiMECs的增殖率、β-casein表达量及甘油三酯分泌量均显著增加（P<0.05），inhibitor-NC组奶山羊CiMECs的增殖率、β-casein表达量及甘油三酯分泌量均无显著差异（P>0.05）。【结论】GEFs经RepSox诱导8 d可成功转变为具有泌乳功能的奶山羊CiMECs，抑制miR-142-3p可显著促进奶山羊CiMECs的增殖并增加β-casein表达量及甘油三酯的分泌量，从而影响泌乳功能。

关键词: 奶山羊; 乳腺上皮细胞(MECs); miR-142-3p

Abstract: 【Objective】 The aim of this study was to explore the regulatory effect of miR-142-3p on the lactation function of chemical induced mammary epithelial cells (CiMECs) of dairy goats in vitro, in order to promote the development of transgenic mammary gland bioreactor and lay a foundation and provide new ideas for the commercial production of "petri dish milk".【Method】 Milk goat ear fibroblasts (GEFs) were selected as the study material.After 8 days of induction by a single inhibitor of small molecule compound TGFβR-1/ALK5 (RepSox), the GEFs were identified by morphological changes, immunofluorescence staining of specific markers and oil red O staining.After that, liposome transfection technology was used to transfect miR-142-3p inhibitor (miR-142-3p inhibitor) and inhibitor control (inhibitor-NC) respectively in CiMECs of dairy goats with successful induction, and the cells without transfection were used as blank control (BC).24 hours after transfection, the expression of miR-142-3p was detected by Real-time quantitative PCR, the cell proliferation rate was detected by CCK-8 method, the expression of β-casein was detected by Western blotting, and the content of triglyceride was determined by triglyceride enzyme method.【Result】 After 8 days of GEFs induction, insular polynucleolus-like epithelioid aggregation was formed.Immunofluorescence staining showed that the induced GEFs expressed CK14 and CK18, the specific markers of mammary epithelial cells (MECs).The results of oil red O staining showed that lipid droplets of different sizes stained red were dispersed in the cytoplasm of induced GEFs, indicating that CiMECs of dairy goats with lactation function were successfully obtained.CiMECs transfection test results of dairy goats showed that compared with blank control group, the relative expression of miR-142-3p in miR-142-3p inhibitor group was significantly decreased (P<0.05), but there was no significant difference in miR-142-3p expression in inhibitor-NC group (P>0.05).The proliferation rate, β-casein expression and triglyceride secretion of CiMECs in dairy goats with miR-142-3p inhibitor group were significantly increased (P<0.05).There were no significant differences in the proliferation rate, β-casein expression and triglyceride secretion of CiMECs in dairy goats with inhibitor-NC group (P>0.05).【Conclusion】 GEFs could be successfully transformed into dairy goat CiMECs with lactation function after 8 days of induction by RepSox.Inhibition of miR-142-3p could significantly promote the proliferation of dairy goat CiMECs and increase the secretion of β-casein and triglyceride, thus affecting the lactation function.

Key words: dairy goats; mammary epithelial cells (MECs); miR-142-3p

中图分类号:

S827

潘思尧, 张丹丹, 刘权辉, 王国栋, 吕丹薇, 黄奔. 抑制miR-142-3p对奶山羊化学诱导乳腺上皮细胞泌乳功能的影响[J]. 中国畜牧兽医, 2023, 50(1): 58-66.

PAN Siyao, ZHANG Dandan, LIU Quanhui, WANG Guodong, LYU Danwei, HUANG Ben. Effects of Inhibition of miR-142-3p on Lactation Function of Chemical Induced Mammary Epithelial Cells in Dairy Goats[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 58-66.

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