大片形吸虫排泄分泌产物对体外培养LO2人肝细胞的影响

摘要/Abstract

摘要：

目的初步探讨大片形吸虫排泄分泌产物（FgESP）对体外培养LO2人肝细胞的增殖及对肝细胞损伤相关的生化指标的影响。方法将LO2人肝细胞分为5个密度组（1 000、3 000、5 000、7 000、10 000个/孔）,铺于96孔板中,空白对照组只加入培养基,根据LO2细胞生长曲线选择最适细胞铺板密度。在96孔板中,以最适细胞密度铺板,分为0.02、0.10、0.20、0.40、1.00 mg/ml FgESP等5组,每组设4个重复,以PBS作为阴性对照组,在培养箱中孵育4、8、12、24、48和72 h后进行MTT细胞活性检测,测吸光度（A₄₉₀值）。在24孔板中,分为0.02、0.10、0.20、0.40、1.00 mg/ml FgESP等5组,每组设3个重复,以PBS作为阴性对照组,分别在培养4、8、12、24、48和72 h后,收集LO2细胞上清,测定碱性磷酸酶(ALP)、天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转氨酶(ALT)、白蛋白(ALB)的含量。采用GraphPad Prism 6.0 软件进行统计学分析。结果根据LO2人肝细胞生长曲线,筛选出5 000个/孔为96孔板最佳铺板密度。FgESP作用72 h后,MTT细胞活性检测结果显示,0.02、0.10、0.20、0.40、1.00 mg/ml FgESP等5组的A₄₉₀值分别为1.29 ± 0.01、1.28 ± 0.06、1.13 ± 0.08、0.97 ± 0.06、0.25 ± 0.01,均低于对照组（1.45 ± 0.05）(P < 0.01)。生化指标检测结果显示,0.40、1.00 mg/ml FgESP作用细胞LO2人肝72 h后,ALT含量分别为2.00 ± 0.00、3.67 ± 0.58,AST含量分别为5.33 ± 0.58、7.67 ± 0.58,均高于对照组的（P < 0.01）;作用48 h后,1.00 mg/ml FgESP组ALT、AST含量分别为2.00 ± 0.00、7.00 ± 0.00,高于对照组的0.33 ± 0.58、3.67 ± 0.58（P < 0.01）;在12~72 h内,0.10、0.20、0.40、1.00 mg/ml FgESP作用后ALP含量升高（P < 0.01）,而0.02 mg/ml FgESP仅在作用72 h后ALP含量升高（P < 0.01）;各实验组ALB含量与阴性对照组差异无统计学意义（P > 0.05）。结论 FgESP体外作用可抑制LO2人肝细胞增殖能力,且细胞增殖的抑制及损伤程度与FgESP的浓度相关。

关键词: 大片形吸虫, 排泄分泌产物, LO2细胞, MTT法, 生化分析, 细胞损伤

Abstract:

Objective To investigate the effect of Fasciola gigantica excretory-secretory products (FgESP) on the proliferation of LO2 hepatocytes and biochemical components related to hepatocyte injury in vitro. Methods LO2 cells were plated in a 96-well plate at 5 densities(1 000, 3 000, 5 000, 7 000 and 10 000 cells/well) while the blank control group was only added with culture medium. The growth curve was plotted to determine the optimal density for plating. FgESP was added to the wells at concentrations of 0.02, 0.10, 0.20, 0.40, and 1.00 mg/ml and incubated for 4, 8, 12, 24, 48 and 72 h followed by MTT assay. Each group had 4 replicates, and PBS was used as the negative control. Absorbance A₄₉₀ was measured in a microplate reader. In a 24-well plate, FgESP was added to the wells at concentrations of 0.02, 0.10, 0.20, 0.40, and 1.00 mg/ml and incubated for 4, 8, 12, 24, 48 and 72 h, the supernatant was then collected to assess the levels of phosphomonoesterase (ALP), alanine transaminase (ALT), glutamic oxalacetic transaminase (AST) and albumin (ALB). Each group had 3 replicates, and PBS was used as the negative control. Data were analyzed using GraphPad Prism 6.0 software. Results The optimal cell density was 5 000 cells/well based on the growth curve. After FgESP treatment for 72 h, the A₄₉₀ values for the five groups with FgESP concentrations of 0.02, 0.10, 0.20, 0.40 and 1.00 mg/ml were 1.29 ± 0.01, 1.28 ± 0.06, 1.13 ± 0.08, 0.97 ± 0.06 and 0.25 ± 0.01, respectively, which were all lower than the control group (1.45 ± 0.05) (P < 0.01). This suggested that FgESP significantly impaired the proliferation of LO2 cells in vitro, in a concentration- and time-dependent manner(P < 0.01). In addition, the contents of ALT in the 0.40 and 1.00 mg/ml FgESP group after 72 h were 2.00 ± 0.00, 3.67 ± 0.58, respectively, and the AST contents were 5.33 ± 0.58, 7.76 ± 0.58, respectively, which were higher than the control group (P < 0.01). The contents of ALT and AST in the 1.00 mg/ml FgESP group after 48 h were 2.00 ± 0.00 and 7.00 ± 0.00, significantly higher than that of control (0.33 ± 0.58, 3.67 ± 0.58 respectively) (P < 0.01). The ALP content significantly increased in comparison with the control within 12-72 hours of FgESP treatment at 0.10, 0.20, 0.40 and 1.00 mg/ml, while at 0.02 mg/ml, FgESP only increased at 72 h(P < 0.01). There was no significant difference in ALB content between the groups (P > 0.05). Conclusion FgESP could inhibit the proliferation of LO2 hepatocytes in vitro, and the extent of inhibition is positively correlated with the concentration of FgESP.

Key words: Fasciola gigantica, Excretory-secretory product, LO2 hepatocyte, MTT assay, Biochemical analysis, Cell injury

中图分类号:

R383.26

赵文苹, 梅雪芳, 施维, 朱彬, 侯林静, 黄维义. 大片形吸虫排泄分泌产物对体外培养LO2人肝细胞的影响[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(6): 619-624.

Wen-ping ZHAO, Xue-fang MEI, Wei SHI, Bin ZHU, Lin-jing HOU, Wei-yi HUANG. In vitro effects of Fasciola gigantica excretory-secretory products on human LO2 hepatocytes[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2018, 36(6): 619-624.

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组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	0.09 ± 0.02	0.09 ± 0.02	0.09 ± 0.01	0.08 ± 0.02	0.09 ± 0.02	0.10 ± 0.03
1 000	0.14 ± 0.02^a	0.16 ± 0.02^a	0.19 ± 0.02^a	0.23 ± 0.02^a	0.38 ± 0.02^a	0.66 ± 0.04^a
3 000	0.24 ± 0.02^a	0.28 ± 0.03^a	0.36 ± 0.02^a	0.43 ± 0.02^a	0.73 ± 0.02^a	1.31 ± 0.07^a
5 000	0.34 ± 0.02^a	0.40 ± 0.02^a	0.49 ± 0.02^a	0.56 ± 0.02^a	1.01 ± 0.02^a	1.69 ± 0.05^a
7 000	0.44 ± 0.02^a	0.46 ± 0.10^a	0.60 ± 0.02^a	0.69 ± 0.02^a	1.27 ± 0.06^a	1.84 ± 0.10^a
10 000	0.58 ± 0.02^a	0.64 ± 0.07^a	0.78 ± 0.02^a	0.90 ± 0.05^a	1.56 ± 0.05^a	1.95 ± 0.03^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	0.25 ± 0.02	0.26 ± 0.02	0.30 ± 0.03	0.47 ± 0.01	0.89 ± 0.05	1.45 ± 0.05
0.02 mg/ml	0.22 ± 0.02	0.25 ± 0.01	0.37 ± 0.01	0.59 ± 0.01	1.00 ± 0.04	1.29 ± 0.01^a
0.10 mg/ml	0.21 ± 0.03	0.24 ± 0.01	0.42 ± 0.01	0.66 ± 0.01	1.01 ± 0.04	1.28 ± 0.06^a
0.20 mg/ml	0.21 ± 0.03	0.24 ± 0.01	0.44 ± 0.02	0.77 ± 0.01	0.97 ± 0.02	1.13 ± 0.08^a
0.40 mg/ml	0.23 ± 0.01	0.24 ± 0.01	0.41 ± 0.03	0.79 ± 0.01	0.90 ± 0.01	0.97 ± 0.06^a
1.00 mg/ml	0.22 ± 0.01	0.22 ± 0.03	0.35 ± 0.01	0.76 ± 0.03	0.50 ± 0.02^a	0.25 ± 0.01^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	0.33 ± 0.58
0.02 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
0.10 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	0.00 ± 0.00
0.20 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	1.00 ± 0.00	1.00 ± 0.00
0.40 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	0.33 ± 0.58	2.00 ± 0.00^a
1.00 mg/ml	0.33 ± 0.58	0.33 ± 0.58	0.67 ± 0.58	1.00 ± 0.00	2.00 ± 0.00^a	3.67 ± 0.58^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	4.33 ± 1.15	3.33 ± 0.58	3.33 ± 1.53	3.67 ± 1.15	3.67 ± 0.58	3.67 ± 0.58
0.02 mg/ml	4.67 ± 0.58	4.33 ± 1.15	3.33 ± 0.58	4.33 ± 1.15	4.33 ± 0.58	4.00 ± 0.00
0.10 mg/ml	4.67 ± 1.15	4.00 ± 0.00	4.00 ± 0.00	3.67 ± 1.15	4.00 ± 0.00	4.33 ± 0.58
0.20 mg/ml	4.00 ± 0.00	4.33 ± 1.53	4.33 ± 0.58	4.33 ± 0.58	5.00 ± 0.00	5.00 ± 1.00
0.40 mg/ml	4.00 ± 1.00	5.00 ± 1.00	4.67 ± 2.08	4.33 ± 0.58	5.33 ± 0.58^a	5.33 ± 0.58^a
1.00 mg/ml	4.67 ± 0.58	5.00 ± 0.00	5.67 ± 1.73	6.67 ± 0.58	7.00 ± 0.00^a	7.67 ± 0.58^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	6.00 ± 1.00	6.00 ± 1.00	6.00 ± 1.00	6.33 ± 0.58	7.00 ± 1.00	7.67 ± 1.15
0.02 mg/ml	6.33 ± 0.58	6.33 ± 0.58	6.00 ± 0.00	8.00 ± 2.00	11.00 ± 3.00	12.00 ± 1.00^a
0.10 mg/ml	6.00 ± 1.00	6.33 ± 0.58	9.50 ± 0.50^a	11.33 ± 0.58^a	12.00 ± 1.00^a	11.00 ± 0.00^a
0.20 mg/ml	6.33 ± 0.58	5.67 ± 0.58	9.67 ± 0.58^a	11.67 ± 0.58^a	16.00 ± 0.00^a	17.00 ± 2.00^a
0.40 mg/ml	6.00 ± 1.00	6.33 ± 0.58	11.00 ± 1.00^a	12.00 ± 0.00^a	14.67 ± 0.58^a	21.33 ± 1.53^a
1.00 mg/ml	7.00 ± 1.00	7.33 ± 0.58	12.33 ± 0.58^a	13.33 ± 1.53^a	16.68 ± 2.31^a	27.00 ± 4.36^a