SPARC 敲除通过调节肝脏脂质代谢影响代谢相关性脂肪肝相关肝癌发展的研究

摘要/Abstract

摘要： 目的探究富含半胱氨酸型酸性蛋白(SPARC)敲除通过调节肝脏脂质代谢影响代谢相关性脂肪肝相关肝癌发展。方法研究样本选取17只成年健康大鼠(对照组)及17只SPARC 基因敲除大鼠(研究组),两组各选取1只提取肝细胞进行体外实验,剩余个体均使用链脲佐菌素注射并给予高脂肪饮食诱导代谢相关性脂肪肝相关肝癌模型。观察两组大鼠病理变化及其肝细胞脂质沉积、脂质合成、脂质代谢、细胞活力相关指标及AMPK、α-Tubulin蛋白表达。结果在第16周时,两组所有大鼠均发展为肝癌,研究组肝癌平均直径(1.39±0.14)mm显著高于对照组(1.01±0.09)mm;NAS评分显示,研究组大鼠脂肪变性评分(2.71±0.25)分显著高于对照组(1.55±0.19)分,研究组小叶炎症评分(0.74±0.09)分显著低于对照组(1.27±0.11)分。HE染色结果显示,研究组大鼠门静脉炎症减弱;天狼星红染色结果显示,研究组大鼠肝脏纤维化病变高于对照组;体外研究中,油红O染色结果显示,研究组细胞脂质沉积增加,且GL-TAG(23.15±5.39比5.73±1.47)、GPL(0.29±0.03比0.23±0.02)表达显著高于对照组;研究组肝细胞中[U-14C]-甘油表达显著高于对照组;研究组PPARγ2(1.95±0.20比1.01±0.11)、PPARα(1.27±0.12比0.98±0.09)、CD36(2.24±0.26比0.98±0.09)、FABP4(1.92±0.19比0.99±0.10)、FABP5(2.31±0.27比0.97±0.10)、ACC1(2.52±0.27比1.01±0.11)、FASN(1.28±0.13比0.99±0.09)、SREBP1(3.95±0.43比1.02±0.11)、LXRα(1.52±0.16比0.97±0.09)、CPT1(2.76±0.29比1.02±0.11)、LIPIN(2.49±0.26比1.01±0.11)、ACOX1(2.69±0.28比1.01±0.11) mRNA相对表达显著低于对照组(P<0.05),两组ACADSB mRNA表达差异无统计学意义(P>0.05);吖啶橙-溴化乙锭染色结果显示,研究组细胞活力高对照组,细胞存活率(97.89±1.44)%显著高于对照组的(48.47±1.21)%;免疫荧光检测结果显示,研究组细胞增加肝细胞中SREBP核定位,蛋白印迹实验结果显示,研究组AMPK、α-Tubulin蛋白表达低于对照组。结论 SPARC敲除可通过调节大鼠脂质代谢、细胞活力及肝细胞内脂质积累增加等途径影响代谢相关性脂肪肝相关肝癌发展。

关键词: SPARC, 脂质代谢, 代谢相关性脂肪肝, 肝癌

Abstract: Objective To investigate the impact of secreted protein acidic and rich in cysteine (SPARC) gene knockout on the development of metabolic fatty liver related liver cancer by regulating liver lipid metabolism.Methods The study samples were selected from 17 adult healthy rats as the control group and 17 SPARC knockout rats as the study group. Hepatocytes were extracted from one rat each from both groups for in vitro experiments. The remaining animals were treated with streptozotocin Injection and high-fat diet to induce metabolic fatty liver related liver cancer. The pathological changes, the lipid deposition, lipid synthesis, lipid metabolism, cell viability related indicators and the protein expression of AMPK and α-Tubulin in the livers of two groups of rats were studied.Results At the 16th week, all rats from both groups developed liver cancer. The average diameter of liver cancers in the study group was (1.39±0.14) mm, which was significantly larger than that of (1.01±0.09) mm in the control group; the NAS score showed that the fatty degeneration score of the study group was (2.71±0.25), which was significantly higher than that of (1.55±0.19) of the control group. The score of lobular inflammation in the study group was (0.74±0.09), which was significantly lower than that of (1.27±0.11) in the control group. The results of H&E staining showed that the portal vein inflammation of the rats in the study group was weaker; the results of Sirius red staining showed that the liver fibrosis of the rats in the study group was more severe than that of the control group; in the in vitro study, the results of oil red O staining showed that the lipid deposits in the cells of the study group increased, and the expression of GL-TAG (23.15±5.39 vs. 5.73±1.47) and GPL (0.29±0.03 vs. 0.23±0.02) were significantly higher than those of the control group; the expression of [U-14C]-glycerol in the liver cells of the study group was significantly higher than that of the control group; The relative expressions of PPARγ (1.95±0.20 vs. 1.01±0.11), PPARα(1.27±0.12 vs. 0.98±0.09), CD36 (2.24±0.26 vs. 0.98±0.09), FABP4 (1.92±0.19 vs. 0.99±0.10), FABP5 (2.31±0.27 vs. 0.97±0.10), ACC1 (2.52±0.27 vs. 1.01±0.11), FASN (1.28±0.13 vs. 0.99±0.09), SREBP1 (3.95±0.43 vs. 1.02±0.11), LXRα (1.52±0.16 vs. 0.97±0.09), CPT1 (2.76±0.29 vs. 1.02±0.11), LIPIN (2.49±0.26 vs. 1.01±0.11), ACOX1 (2.69±0.28 vs. 1.01±0.11) mRNAs in rat livers of the study group were significantly lower than that of the control group (all P<0.05). There was no statistically significant difference in ACADSB mRNA expression in rat livers from these two groups (P>0.05); the results of acridine orange-ethidium bromide staining showed that cells from the study group had higher viability than those from the control group, and had a significantly higher cell survival rate than those from the control group {(97.89±1.44)% vs. (48.47±1.21)%}; Immunofluorescence detection results showed that cells in the study group had increased nuclear localization of SREBP in hepatocytes. By Western blot analysis it was shown that the expression of AMPK and α-Tubulin protein in the study group was lower than that of the control group.Conclusion SPARC knockout can affect the development of metabolic fatty liver-related liver cancer by regulating lipid metabolism, cell viability and lipid accumulation in the liver cells in rats.

Key words: secreted protein acidic and rich in cysteine, liver, lipid metabolism, metabolic fatty liver, liver cancer

常业飞, 郑盛, 胡滔, 洪颖, 钟勇, 张晓珺. SPARC 敲除通过调节肝脏脂质代谢影响代谢相关性脂肪肝相关肝癌发展的研究[J]. 肝脏, 2022, 27(12): 1309-1313.

CHANG Ye-fei, ZHENG Sheng, HU Tao, HONG Ying, ZHONG Yong, ZHANG Xiao-jun. SPARC knockout affects the development of metabolic fatty liver related liver cancer by regulating liver lipid metabolism[J]. Chinese Hepatolgy, 2022, 27(12): 1309-1313.

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