结缔组织生长因子体外调控奶牛乳腺上皮细胞生长和泌乳分化

doi:10.11843/j.issn.0366-6964.2024.08.018

摘要/Abstract

摘要：

旨在探究结缔组织生长因子在体外培养体系中对奶牛乳腺上皮细胞生长和泌乳分化的调控作用。本研究主要通过人为添加结缔组织生长因子(connective tissue growth factor, CTGF)重组蛋白和过表达CTGF基因分析其对细胞增殖、凋亡、乳脂和乳蛋白合成分泌及信号通路的影响。使用EdU试剂盒和流式细胞仪分别检测细胞增殖凋亡情况；应用尼罗红染料和甘油三酯试剂盒检测细胞内脂滴数量和细胞外培养液乳脂含量；实时荧光定量PCR和蛋白免疫印迹检测细胞增殖凋亡，β酪蛋白，乳脂和乳蛋白合成关键信号分子以及细胞外基质细胞表面受体β1整联蛋白的表达；免疫共沉淀分析CTGF和β1整联蛋白之间的蛋白质相互作用。结果表明：体外培养时，无论CTGF重组蛋白还是CTGF过表达均能通过增强增殖、抑制凋亡促进贴壁细胞生长；实时荧光定量PCR和蛋白免疫印迹结果表明，CTGF在转录和翻译水平增强促增殖的CyclinD1、MAPK和Bcl2表达，抑制促凋亡的Caspase3和Bax表达；CTGF一方面上调乳脂合成关键基因FASN、SREBP1、PPARγ表达水平，促进细胞内脂滴合成和胞外甘油三酯分泌；另一方面上调乳蛋白合成关键基因PRLR、STAT5和p-STAT5表达水平，进而显著促进β酪蛋白合成；CTGF上调β1整联蛋白表达水平，Co-IP也证实了CTGF和β1整联蛋白之间存在蛋白质相互作用。综上所述，CTGF促进体外培养的奶牛乳腺上皮细胞生长及泌乳能力；CTGF与β1整联蛋白共存于黏附复合物，可以通过影响β1整联蛋白信号途径实现其部分生物学作用。

关键词: 奶牛, 乳腺上皮细胞, CTGF, 细胞生长, 乳脂, β-酪蛋白

Abstract:

This research aimed to explore how connective tissue growth factor influences the growth and lactation differentiation of mammary epithelial cells in dairy cows within an in vitro culture setting. In this study, the effects of CTGF recombinant protein and overexpression of CTGF gene on cell proliferation, apoptosis, milk fat and milk protein synthesis and secretion and signaling pathways were analyzed. The EdU kit and flow cytometry were used to identify cell proliferation and apoptosis; the Nile red dye and triglyceride kit were used to count intracellular lipid droplets and milk fat levels in the culture solution; qRT-PCR and Western blot were used to detect the expression of β-casein, crucial signaling molecules in milk fat and protein synthesis, and the extracellular matrix cell surface receptor β1 integrin. Furthermore, the protein interaction between CTGF and β1 integrin was examined using co-immunoprecipitation. In vitro culture, both CTGF recombinant protein and overexpression of CTGF were found to enhance the growth of adherent cells by promoting proliferation and inhibiting apoptosis. Real-time quantitative PCR and Western blot results demonstrated that CTGF upregulated the expression of pro-proliferative CyclinD1, MAPK, Bcl2, while downregulating the expression of pro-apoptotic Caspase3 and Bax at both transcriptional and translational levels. Furthermore, CTGF upregulated the expression levels of FASN, SREBP1, PPARγ key genes involved in milk fat synthesis-leading to increased intracellular lipid droplets synthesis and extracellular triglyceride secretion. Additionally, it also upregulated the expression levels of PRLR, STAT5, p-STAT5, the key genes involved in milk protein synthesis-significantly promoting β-casein synthesis. Moreover, CTGF was found to upregulate the expression level of β1 integrin with Co-IP confirming a protein interaction between CTGF and β1 integrin. Ultimately, CTGF promoted the growth and lactating ability of cultured dairy cow mammary epithelial cells; CTGF coexists with β1 integrin in the adhesion complex and can achieve some of its biological effects by influencing the β1 integrin signaling pathway.

Key words: dairy cow, mammary epithelial cells, CTGF, cell growth, milk fat, β-casein

中图分类号:

S823.2

王若薇, 许曦瑶, 汤晓娜, 王春梅, 赵锋. 结缔组织生长因子体外调控奶牛乳腺上皮细胞生长和泌乳分化[J]. 畜牧兽医学报, 2024, 55(8): 3446-3459.

Ruowei WANG, Xiyao XU, Xiaona TANG, Chunmei WANG, Feng ZHAO. Connective Tissue Growth Factor Regulates the Growth and Differentiation of Cows Mammary Epithelial Cells in Vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3446-3459.

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基因 Gene	引物序列(5′→3′) Primer sequence
PPARγ	F: CTTCACCACCGTTGACTTCTCCAG R: CAGGCTCCACTTTGATTGCACTTTG
FASN	F: CTGAGTCGGAGAACCTGGAGGAG R: CTTCCACCGCCTGTCATCATCTG
SREBP1	F: GCCACTGGTAGTAGACACTGACAAG R: TTGATGGAAGAGCGGTAGCGTTTC
CSN2	F: TGTACCTGGTGAGATTGTGGAAAGC R: GTTCATCCTCTGTTTGCTGCTGTTC
PRLR	F: ACCAAGCAGAGGGAGTCAGAAGG R: CCAAGGGTTTAGCAGAGATCAAGGG
STAT5a	F: GCGGAAGCAGCAGACCATCATC R: GCCAACTTCTCACACCAGGACTG
STAT5b	F：GCGGAAGCAGCAGACCATCATC R：GCCAACTTCTCACACCAGGACTG
ITGB1	F: CGCCTGTGACTGTTCTCTGGATAC R: CCAAGGCAGGTCTGACACATCTC
CyclinD1	F: GCCGAGGAGAACAAGCAGATCATC R: AGGGCGGGTTGGAAATGAACTTC
MAPK	F：AACACCTCAGCAACGACCACATC R：TGTTGAGCAGCAGGTTGGAAGG
Bcl-2	F: GCTTCAGGGTTTCATCCAGGATCG R: CAGACACTCGCTCAGCTTCTTGG
Bax	F: GCTTCAGGGTTTCATCCAGGATCG R: CAGACACTCGCTCAGCTTCTTGG
Caspase3	F: TTGCAGAAGTCTGACTGGAAAACCC R: CAGCACCACTGTCTGTCTCAATACC
β-Actin	F: TGCGGCATTCACGAAACTAC R: ACAGCACCGTGTTGGCGTA

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