干扰HSD17B4基因对水牛乳腺上皮细胞乳脂和酪蛋白的影响

doi:10.16431/j.cnki.1671-7236.2021.09.003

摘要/Abstract

摘要： 通过将小干扰-17β-羟基固醇脱氢酶Ⅳ（si-HSD17B4）基因转染水牛乳腺上皮细胞，检测酪蛋白、甘油三酯含量及脂肪酸相关基因的表达变化，从而揭示干扰HSD17B4基因对水牛乳腺上皮细胞中乳脂和酪蛋白的影响。采用实时荧光定量PCR检测HSD17B4 mRNA在水牛各个组织中的相对表达量。合成3条水牛HSD17B4小干扰RNA （siRNA）和1条对照si-HSD17B4-nc （si-nc），并筛选干扰效果最佳的片段和时间。si-HSD17B4转染水牛乳腺上皮细胞后，通过酪蛋白试剂盒检测酪蛋白的表达情况，通过甘油三酯测定和油红O染色检测甘油三酯含量，通过实时荧光定量PCR检测干扰HSD17B4基因对甘油三酯、脂肪酸合成以及氧化相关基因表达的影响。结果表明，HSD17B4基因在水牛心脏和卵巢中相对高表达，且显著高于其他组织（P<0.05）。3条HSD17B4 siRNA片段均能有效地抑制HSD17B4基因的表达，其中si-HSD17B4-1干扰效果最佳，HSD17B4 mRNA表达量极显著低于对照组（P<0.01）；最佳干扰时间为24 h。酪蛋白检测结果显示，干扰HSD17B4基因对水牛乳腺上皮细胞中酪蛋白合成无影响。甘油三酯测定结果显示，干扰HSD17B4基因后细胞中甘油三酯含量上升，差异显著（P<0.05）。油红O染色结果显示，干扰HSD17B4基因后细胞中的脂滴明显增多。实时荧光定量PCR检测结果显示，干扰HSD17B4基因会使FABP3、ACSL1、DGAT1、DGAT2、PLIN2、BTN1A1基因表达量上调，分别上调9.28（P<0.01）、1.36（P<0.05）、1.17（P<0.05）、1.83（P<0.01）、1.60（P<0.01）和2.17（P<0.01）倍；同时使PPARA、SREBP1C、SCD、ACSS2、ACACA、AGPAT6、LPIN1、XDH、ABCD1、ACOX2、EHHADH、SCP2基因表达量下降，分别下调至50.55%（P>0.05）、61.15%（P<0.01）、84.91%（P<0.01）、89.34%（P<0.01）、21.88%（P<0.01）、86.48%（P<0.01）、25.35%（P<0.01）、27.24%（P<0.01）、41.74%（P<0.01）、22.17%（P<0.01）、62.70%（P<0.01）和70.33%（P<0.01）。结果表明，HSD17B4基因在水牛组织中广泛表达；si-HSD17B4高效抑制HSD17B4基因在水牛乳腺上皮细胞中的表达，未检测到干扰HSD17B4基因对乳腺上皮细胞酪蛋白的影响，干扰HSD17B4基因上调了FABP3、ACSL1、DGAT1、DGAT2基因的表达，从而促进甘油三酯的合成，同时降低ABCD1、ACOX2、EHHADH、SCP2基因的表达，减少脂肪酸β-氧化。

关键词: 水牛; 乳腺上皮细胞; HSD17B4; 乳脂; 酪蛋白

Abstract: The expression changes of casein content, triglyceride content and fatty acid-related genes were detected by transfecting small interfering-hydroxysteroid 17-beta dehydrogenase Ⅳ(si-HSD17B4) in buffalo mammary epithelial cell, which were revealing the effects of interfering with the HSD17B4 gene on milk fats and caseins in buffalo mammary epithelial cell. The relative expressions of HSD17B4 mRNA in various tissues of buffalo were detected by Real-time quantitative PCR. 3 HSD17B4 siRNA fragments of buffalo and one control si-HSD17B4-nc (si-nc) fragment were synthesized and then the optimal fragments and time of the interference were selected. Caseins were detected by casein kits after si-HSD17B4 transfected buffalo mammary epithelial cell. Then triglycerides were detected by triglyceride kit and oil red O staining, and the effects of HSD17B4 gene interference on the gene expression of the triglycerides synthesis and fatty acid synthesis and oxidation. The results showed that HSD17B4 gene was relatively highly expressed in buffalo heart and ovary, and was significantly higher than other tissue(P<0.05). 3 HSD17B4 siRNA fragments could effectively decrease the expression of the HSD17B4 gene, and the si-HSD17B4-1 was the best interference fragment, the relative expression of HSD17B4 mRNA was extremely significantly lower than that in the control group (P<0.01). The optimum interference time was 24 h. Casein test showed no effect of interfering HSD17B4 on casein synthesis in buffalo mammary epithelial cell. Triglyceride detections showed an increase in triglyceride content in cells after interfering with the HSD17B4 gene with significant differences (P<0.05). The oil red O tests showed a significant increase in fat droplets in cells after interfering with HSD17B4 gene. The results of Real-time quantitative PCR showed that interference with the HSD17B4 gene, the relative expressions of FABP3, ACSL1, DGAT1, DGAT2, PLIN2 and BTN1A1 gene were increased by 9.28 (P<0.01), 1.36 (P<0.05), 1.17 (P<0.05), 1.83 (P<0.01), 1.60 (P<0.01) and 2.17 (P<0.01) times, respectively. The relative expressions of PPARA, SREBP1C, SCD, ACSS2, ACACA, AGPAT6, LPIN1, XDH, ABCD1, ACOX2, EHHADH and SCP2 genes decreased to 50.55% (P>0.05), 61.15% (P<0.01), 84.91% (P<0.01), 89.34% (P<0.01), 21.88% (P<0.01), 86.48% (P<0.01), 25.35% (P<0.01), 27.24% (P<0.01), 22.17% (P<0.01), 62.70% (P<0.01) and 70.33% (P<0.01), respectively. The results showed that the HSD17B4 gene was widely expressed in buffalo tissue. si-HSD17B4 effectively reduced the expression of the HSD17B4 gene in buffalo mammary epithelial cell, and it had no effect of interfering with the HSD17B4 gene on the caseins in mammary epithelial cell, but it would increase the expressions of FABP3, ACSL1, DGAT1, DGAT2 genes to promote the synthesis of triglycerides while decrease the expression of ABCD1, ACOX2, EHADH, SCP2 genes to reduce the β-oxidation of fatty acids.

Key words: buffalo; mammary epithelial cell; HSD17B4; fatty acid; casein

中图分类号:

S823.9

段安琴, 陆杏蓉, 马小娅, 梁莎莎, 邓廷贤. 干扰HSD17B4基因对水牛乳腺上皮细胞乳脂和酪蛋白的影响[J]. 中国畜牧兽医, 2021, 48(9): 3128-3137.

DUAN Anqin, LU Xingrong, MA Xiaoya, LIANG Shasha, DENG Tingxian. Effect of HSD17B4 Gene Interference on Milk Fat and Casein in Buffalo Mammary Epithelial Cell[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3128-3137.

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