Leptin介导JAK/STAT信号通路对猪皮下前脂肪细胞脂类代谢调控的研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0749

摘要/Abstract

摘要：

以猪原代皮下前脂肪细胞为研究材料,检测Leptin介导JAK/STAT信号通路中基因表达水平,旨在阐明Leptin介导JAK/STAT信号通路对脂肪代谢的分子机制。用0和100 ng/mL Leptin分别处理脂肪细胞48 h,油红O染色鉴定脂肪细胞,试剂盒测定细胞中甘油三酯和游离脂肪酸含量,Real-time PCR方法检测皮下前脂肪细胞中JAK/STAT信号通路中基因表达水平。研究结果表明：皮下前脂肪细胞中,Leptin组甘油三酯含量均显著低于对照组（P<0.05）,脂肪酸含量均低于对照组;JAK/STAT信号通路中LepR、JAK2、STAT3、CPT-1、ACOX1和PGC-1α基因的表达量均显著高于对照组（P<0.05）;LepR、JAK2、STAT3、CPT-1、ACOX1和PGC-1α基因表达量均与甘油三酯和游离脂肪酸含量呈显著负相关（P<0.05）。该研究结果表明,Leptin激活皮下前脂肪细胞中JAK/STAT信号通路,上调通路相关基因的表达,促进甘油三酯分解及脂肪酸氧化,降低细胞中甘油三酯和游离脂肪酸含量。

关键词: 皮下前脂肪细胞, Leptin, JAK/STAT, 信号通路, 基因表达

Abstract:

Using in vitro cultured porcine subcutaneous preadipocytes as the study material,the expression levels of related genes were measured for investigating the molecular regulation mechanism of Leptin-mediate JAK/STAT signal pathway on lipid metabolism. The subcutaneous adipocytes were treated with 0 and 100 ng/mL Leptin for 48 h,and the dipocytes were identified by oil red O dyeing,and the contents of triglyceride and free fatty acids in the cells were measured by kit,and the mRNA expression levels of relate genes in the JAK/STAT signal pathway of subcutaneous preadipocytes were detected using real-time PCR technology. The results showed the content of the triglyceide of the subcutaneous preadipocyte that in Leptin group was significantly lower（P<0.05）than that in the control group,and the content of the free fatty acids was lower than that of the control group. The expression levels of gene lepR,JAK2,STAT3,CPT-1,ACOX1,and PGC-1 in JAK/STAT signal pathway in Leptin group were significantly higher than that in control group（P<0.05）.The expression levels of gene lepR,JAK2,STAT3,CPT-1,ACOX1,and PGC-1 were significantly negatively correlated with the content of triglyceride and free fatty acid（P<0.05）. In conclusion,the results suggested that Leptin activated JAK/STAT signal pathway in subcutaneous preadipocyte,increased the mRNA expressions of relevant genes in the JAK/STAT signaling pathway,promoted the oxidation of fatty acid and the decomposition of triglyceride,and thus decreased the content of triglycerides and free fatty acids in subcutaneous preadipocyte.

Key words: subcutaneous preadipocyte, Leptin, JAK/STAT, signal pathway, gene expression

查星琴, 杨明华, 李永能, 赵素梅, 黄英. Leptin介导JAK/STAT信号通路对猪皮下前脂肪细胞脂类代谢调控的研究[J]. 生物技术通报, 2021, 37(2): 88-95.

ZHA Xing-qin, YANG Ming-hua, LI Yong-neng, ZHAO Su-mei, HUANG Ying. Study on the Regulation of Leptin-mediated JAK/STAT Signal Pathway on Lipid Metabolism in Porcine Subcutaneous Preadipocyte[J]. Biotechnology Bulletin, 2021, 37(2): 88-95.

图/表 6

表1 内参基因与目的基因的引物序列、产物长度和登陆号

目的基因	引物序列（5'-3'）	退火温度/℃	产物大小/bp	GenBank登录号
18S rRNA	F：TAGAGTCACCAAAGCCGC	55	194	NR_002170.3
18S rRNA	R：TCAGTTATGGTTCCTTTG	55	194	NR_002170.3
LepR	F：GTGATAACTGCATTTGACTTGGC	62	271	NM001024587
LepR	R：CTGCAATGTTGTCTGCATGTACAG	62	271	NM001024587
JAK2	F：ATTTGAAGACCGAGACCC	60	272	NM_214113.1
JAK2	R：TTAGATTACGCCGACCAG	60	272	NM_214113.1
STAT3	F：CAGTAAGGAGAGGGAGCG	56	254	NM_001044580
STAT3	R：AGAGGTAGACCAGCGGAG	56	254	NM_001044580
CPT-1	F：ATGGTGGGCGACTAACT	60	321	AY181062
CPT-1	R：TGCCTGCTGTCTGTGAG	60	321	AY181062
ACOX1	F：CGGGCACGGCTATTCTCAC	56	193	NM_001101028
ACOX1	R：CTGGCTGGGCAGGTCATTC	56	193	NM_001101028
PGC-1α	F：CCCAGTCTGCGGCTATTTG	56	282	AB106108.1
PGC-1α	R：GGGATGACCGAAGTGCTTG	56	282	AB106108.1

图1 皮下前脂肪细胞油红O染色（×200） A：皮下前脂肪细胞对照组;B：皮下前脂肪细胞Leptin组

表2 甘油三酯和游离脂肪酸含量

指标	对照组	Leptin组
甘油三酯（mmol/L）	0.15±0.02^a	0.07±0.01^b
游离脂肪酸（μmol/gprot）	1.16±0.13^a	0.94±0.09^a

图2 Leptin介导JAK/STAT信号通路中基因的mRNA表达水平 A：LepR基因mRNA表达水平;B：JAK2基因mRNA表达水平;C：STAT3基因mRNA表达水平;D：CPT-1基因mRNA表达水平;E：ACOX基因mRNA表达水平;F：PGC-1α基因mRNA表达水平。不同字母表示差异显著（P<0.05）,下同

图3 JAK/STAT信号通路中基因mRNA表达水平与甘油三酯含量相关性 A：LepR基因mRNA表达水平与甘油三酯含量相关性;B：JAK2基因mRNA表达水平与甘油三酯含量相关性;C：STAT3基因mRNA表达水平与甘油三酯含量相关性;D：CPT-1基因mRNA表达水平与甘油三酯含量相关性;E：ACOX1基因mRNA表达水平与甘油三酯含量相关性;F：PGC-1α基因mRNA表达水平与甘油三酯含量相关性

图4 JAK/STAT信号通路中基因mRNA表达水平与游离脂肪酸含量相关性 A：LepR基因mRNA表达水平与游离脂肪酸含量相关性;B：JAK2基因mRNA表达水平与游离脂肪酸含量相关性;C：STAT3基因mRNA表达水平与游离脂肪酸含量相关性;D：CPT-1基因mRNA表达水平与游离脂肪酸含量相关性;E：ACOX1基因mRNA表达水平与游离脂肪酸含量相关性;F：PGC-1α基因mRNA表达水平与游离脂肪酸含量相关性

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