基于胰岛素样生长因子-Ⅰ信号通路体外研究丁酸钠促进羔羊瘤胃上皮细胞增殖的机理

Mechanism of Sodium Butyrate Promoting Rumen Epithelial Cell Proliferation in Vitro Based on Insulin-Like Growth FactorⅠSignaling Pathway

本试验基于胰岛素样生长因子-Ⅰ(IGF-Ⅰ)信号通路通过体外试验研究丁酸钠促进羔羊瘤胃上皮细胞增殖的机理。试验选取4头健康、胎次一致、体重相近的49日龄公羔羊,屠宰采集瘤胃上皮组织,利用胰酶消化获取原代瘤胃上皮细胞。待原代瘤胃上皮细胞贴壁后,将细胞分为4组,分别添加0、2、4、8 mmol/L丁酸钠。为了进一步研究IGF-Ⅰ信号通路在添加丁酸钠促进瘤胃上皮细胞增殖中的作用,将细胞分为3组,丁酸钠组添加4 mmol/L丁酸钠,抑制剂组添加4 mmol/L丁酸钠+2.5μmol/L胰岛素样生长因子-Ⅰ受体(IGF-ⅠR)抑制剂,对照组加入相同体积的DMEM。所有细胞试验重复4次。细胞处理24 h后收集细胞液用于测定IGF-Ⅰ浓度,收集细胞检测细胞周期以及增殖和凋亡相关基因的表达。结果表明:1)0和8 mmol/L丁酸钠组G0/G1期细胞比例显著高于2和4 mmol/L丁酸钠组(P<0.05),且8 mmol/L丁酸钠组显著高于0 mmol/L丁酸钠组(P<0.05)。4 mmol/L丁酸钠组S期细胞比例显著高于0、2和8 mmol/L丁酸钠组(P<0.05)。8 mmol/L丁酸钠组G2/M期细胞比例显著低于0、2和4 mmol/L丁酸钠组(P<0.05)。2)8 mmol/L丁酸钠组的细胞周期蛋白A2(Cyclin A 2)mRNA相对表达量显著低于0、2和4 mmol/L丁酸钠组(P<0.05),8 mmol/L丁酸钠组的细胞周期蛋白依赖性激酶Ⅰ(CDKⅠ)mRNA相对表达量显著低于0和2 mmol/L丁酸钠组(P<0.05),2和4 mmol/L丁酸钠组的细胞周期蛋白DⅠ(Cyclin DⅠ)mRNA相对表达量显著高于0和8 mmol/L丁酸钠组(P<0.05),4 mmol/L丁酸钠组的细胞周期蛋白依赖性激酶4(CDK4)mRNA相对表达量显著高于0、2和8 mmol/L丁酸钠组(P<0.05),2和4 mmol/L丁酸钠组的细胞周期蛋白依赖性激酶6(CDK6)mRNA相对表达量显著高于8 mmol/L丁酸钠组(P<0.05)。3)8 mmol/L丁酸钠组的含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)和B淋巴细胞瘤-2相关X蛋白(Bax)的mRNA相对表达量显著高于0、2和4 mmol/L丁酸钠组(P<0.05)。4)丁酸钠组的IGF-ⅠR mRNA相对表达量和细胞培养液中IGF-Ⅰ浓度显著高于对照组和抑制剂组(P<0.05)。丁酸钠组Cyclin DⅠ和CDK4 mRNA相对表达量显著高于对照组和抑制剂组(P<0.05)。由此可见,添加4 mmol/L丁酸钠可以促进羔羊瘤胃上皮细胞增殖,并且主要是通过IGF-Ⅰ信号通路来发挥作用,说明丁酸钠促进瘤胃上皮生长的作用机制与IGF-Ⅰ信号通路密切相关。

This experiment was conducted to study the mechanism of sodium butyrate promoting rumen epithelial cell proliferation in vitro based on insulin-like growth factorⅠ(IGF-Ⅰ)signaling pathway.Four healthy 49-day-old male lambs with similar parity and body weight were slaughtered to collect rumen epithelial tissue,which was digested with trypsin to obtain primary rumen epithelial cells.After the cells adhered to the wall,they were divided into four groups,and supplemented 0,2,4 and 8 mmol/L sodium butyrate,respectively.In order to further study the role of IGF-Ⅰsignaling pathway in the promotion of rumen epithelial cell proliferation by sodium butyrate addition,the cells were divided into three groups,the sodium butyrate group supplemented 4 mmol/L sodium butyrate,the inhibitor group supplemented 4 mmol/L sodium butyrate+2.5μmol/L insulin-like growth factorⅠreceptor(IGF-ⅠR)inhibitor,and the control group supplemented the same volume of DMEM.All cell experiments were repeated 4 times.When the cells were treated for 24 h,the cell fluid was collected for determinate IGF-Ⅰconcentration,and the cells were collected to analyses the cell cycle and expression of genes related to proliferation and apoptosis.The results showed as follows:1)the proportion of cells in G0/G1 phase in 0 and 8 mmol/L sodium butyrate groups was significantly higher than that in 2 and 4 mmol/L sodium butyrate groups(P<0.05),and the proportion of cells in G0/G1 phase in the 8 mmol/L sodium butyrate group was significantly higher than that in the 0 mmol/L sodium butyrate group(P<0.05).The proportion of cells in S phase in the 4 mmol/L sodium butyrate group was significantly higher than that in 0,2 and 8 mmol/L sodium butyrate groups(P<0.05).The proportion of cells in G2/M phase in the 8 mmol/L sodium butyrate group was significantly lower than that in 0,2 and 4 mmol/L sodium butyrate groups(P<0.05).2)The mRNA relative expression level of cyclin protein A2(Cyclin A2)in the 8 mmol/L sodium butyrate group was significantly lower than that in 0,2 and 4 mmol/L sodium butyrate groups(P<0.05),the mRNA relative expression level of cyclin dependent protein kinasesⅠ(CDKⅠ)in the 8 mmol/L sodium butyrate group was significantly lower than that in the 0 and 2 mmol/L sodium butyrate group(P<0.05),the mRNA relative expression level of cyclin protein DⅠ(Cyclin DⅠ)in 2 and 4 mmol/L sodium butyrate groups was significantly higher than that in 0 and 8 mmol/L sodium butyrate groups(P<0.05),the mRNA relative expression level of cyclin dependent protein kinases 4(CDK4)in the 4 mmol/L sodium butyrate group was significantly higher than that in the 0,2 and 8 mmol/L sodium butyrate groups(P<0.05),and the mRNA relative expression level of cyclin dependent protein kinases 6(CDK6)in 2 and 4 mmol/L sodium butyrate groups was significantly higher than that in 8 mmol/L sodium butyrate group(P<0.05).3)The mRNA relative expression levels of cysteinyl aspartate specific proteinase-3(Caspase-3)and B-lymphoma-2-associated X protein(Bax)in the 8 mmol/L sodium butyrate group were significantly higher than those in 0,2 and 4 mmol/L sodium butyrate groups(P<0.05).4)The IGF-ⅠR mRNA relative expression level and cell culture fluid IGF-Ⅰconcentration in sodium butyrate group were significantly higher than those in control group and inhibitor group(P<0.05).The mRNA relative expression levels of Cyclin DⅠand CDK4 in sodium butyrate group were significantly higher than those in control group and inhibitor group(P<0.05).These results suggest that adding 4 mmol/L sodium butyrate promotes the rumen epithelial cell proliferation,mainly through the IGF-Ⅰsignaling pathway,it shows that the mechanism of sodium butyrate promoting rumen epithelial growth is closely related to IGF-Ⅰsignaling pathway.