牦牛IGFBP4对肝细胞增殖的调控及功能分析

doi:10.11843/j.issn.0366-6964.2022.05.009

摘要/Abstract

摘要： 旨在探究牦牛胰岛素样生长因子结合蛋白4(Bos grunniens insulin-like growth factor binding protein 4, BgIGFBP4)在牦牛肝细胞增殖及小鼠生长中的作用。本研究构建了pET-28a-BgIGFBP4原核表达载体，对其进行表达、鉴定，用终浓度为0.002、0.02、0.2、2、20 μg·mL^-1的BgIGFBP4蛋白处理肝细胞，采用CCK8法、细胞集落形成试验检测BgIGFBP4蛋白对肝细胞增殖能力的影响。再根据试验结果选取浓度蛋白处理肝细胞，检测24、48、72 h时肝细胞活性、肝细胞上清生长类激素和肝细胞PI3K-Akt信号通路的变化。选取30日龄((18±1)g)健康雄性KM小鼠，试验组每2 d灌喂100 μL 50 μg·mL^-1 BgIGFBP4蛋白，对照组用等量0.9%的生理盐水进行灌喂，每组40只，共80只。试验前进行饥饿处理12 h，试验周期为28 d，在试验28 d时检测各项生长性能指标、血清生长类激素水平和肝PI3K-Akt信号通路的变化。结果显示，获得大小约28.86 ku的BgIGFBP4蛋白，并纯化鉴定得到该目的蛋白。2 μg·mL^-1 BgIGFBP4蛋白可促进牦牛肝细胞增殖及集落形成。与对照组相比，试验组(2 μg·mL^-1BgIGFBP4蛋白处理)肝细胞上清GH、VEGF含量显著升高(P<0.05)，肝细胞中PI3K-Akt信号通路细胞增殖相关基因ERBB2、IRS1、PIK3R1、AKT1、RAF1、MAPK3转录水平显著升高(P<0.05)。与对照组相比，试验组(100 μL 50 μg·mL^-1 BgIGFBP4蛋白处理)小鼠平均日增重显著增加、料重比显著降低，肝、脾、肺、肾、小肠器官指数显著增加(P<0.05)。试验组小鼠血清GH、ISN、VEGF含量显著升高(P<0.05)，小鼠肝中PI3K-Akt信号通路相关基因ERBB2、IRS1、PIK3R1、AKT1、RAF1、MAPK3的转录水平显著升高(P<0.05)。综上表明，BgIGFBP4可通过调控PI3K-Akt信号通路促进牦牛肝细胞增殖、并能促进小鼠生长。这为深入研究IGFBP4在牦牛肝生长发育过程中的作用提供了参考。

关键词: IGFBP4, 牦牛, 肝细胞, 肝

Abstract: This study aimed to explore the function of BgIGFBP4 in the proliferation of yak hepatocyte and the development of mice. The prokaryotic expression vectors pET-28a-BgIGFBP4 was constructed, induced and identified. Hepatocytes were treated with 0.002, 0.02, 0.2, 2, 20 μg·mL^-1 of BgIGFBP4 protein. The effects of BgIGFBP4 protein concentrations on the proliferation of hepatocytes were detected by CCK8 kit and colony forming assay. According to the results of the previous experiment, the optimal concentration of BgIGFBP4 was selected to treat hepatocytes for different time, and the changes of hepatocyte activity, growth-like hormone of hepatocyte supernatant and PI3K-Akt signaling pathway were detected at 24, 48 and 72 h. Healthy male KM mice aged 30 days ((18 ±1)g) were selected and divided into two groups (40 mice per group). The experimental group were fed with 100 μL 50 μg·mL^-1 BgIGFBP4 protein every two days. And the control group was fed with the same amount of 0.9% sterile saline. Before the experiment, the mice were starved for 12 h. The test period was 28 days. The growth performance indexes, serum growth-like hormone and liver PI3K-Akt signaling pathway were detected at 28 days. Prokaryotic expression indicated that a 28.86 ku recombinant protein was obtained and identified. The activities of hepatocytes and the colony formation of hepatocytes could be optimized by 2 μg·mL^-1 BgIGFBP4 protein. Compared with the control group, the GH and VEGF concentrations in supernatant of yak hepatocytes in the experimental group were significantly increased (P<0.05), and the transcription levels of cell proliferation related genes ERBB2, IRS1, PIK3R1, AKT1, RAF1 and MAPK3 in PI3K-Akt signaling pathway were significantly increased (P<0.05). In comparison with the control group, the average daily gain of the experimental group mice was significantly increased, the feed to weight ratio was significantly decreased, and the liver, spleen, lung, kidney and intestine organ indexes were significantly increased (P<0.05). The contents of serum GH, ISN and VEGF were significantly higher than those of control (P<0.05), and the transcription levels of cell proliferation related genes ERBB2, IRS1, PIK3R1, AKT1, RAF1 and MAPK3 in PI3K-Akt signaling pathway were significantly increased (P<0.05). In conclusion, BgIGFBP4 can promote the proliferation of yak hepatocytes and the growth of mice. This study provides reference for further study of the role of IGFBP4 in yak liver growth and development.

Key words: IGFBP4, yak, hepatocyte, liver

中图分类号:

S823.85

傅芳, 唐小凤, 王利, 李键, 官久强. 牦牛IGFBP4对肝细胞增殖的调控及功能分析[J]. 畜牧兽医学报, 2022, 53(5): 1409-1419.

FU Fang, TANG Xiaofeng, WANG Li, LI Jian, GUAN Jiuqiang. Regulation on Hepatocyte Proliferation and Function Analysis of IGFBP4 in Yak[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1409-1419.

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