摘要
目的旨在探讨胆固醇和27-羟基胆固醇对食管鳞癌增殖、侵袭和迁移能力的生物学行为及对细胞因子MCP-1分泌的影响。方法动物体内实验:通过建立裸鼠食管鳞癌动物模型,给予高胆固醇饮食(高胆固醇饮食组,n=4)和正常饮食(对照组,n=4),观察胆固醇在体内对食管鳞癌肿瘤生长的影响,第5周实验终止时,对两组瘤体体积进行测量并计算抑瘤率。细胞实验:观察胆固醇(浓度分别为0、0.123、0.148、0.185、0.269、0.370 mg/mL)和27-羟基胆固醇(浓度分别为0、1、5、10、20μmol/mL)对正常食管鳞癌细胞(ECA109)和基因cyp27a1、cyp7b1敲除后的ECA109细胞增殖能力的影响,采用细胞计数试剂盒(CCK-8)检测不同药物浓度干预下细胞的增殖活性。采用划痕实验和侵袭实验(胆固醇0.185 mg/mL,27-羟基胆固醇1μmol/mL)检测肿瘤细胞的侵袭效应。利用慢病毒转染敲除27-羟基胆固醇的上游基因cyp27a1和下游基因cyp7b1,重复上述实验,探讨27-羟基胆固醇合成或代谢基因敲除对ECA109细胞增殖、侵袭和迁移能力的影响,并通过ELISA实验检测基因敲除后对肿瘤细胞单核细胞趋化因子-1(MCP-1)分泌的影响。两组不同时间瘤体体积和CCK-8吸光度值采用两因素方差分析,交互作用有统计学意义时进一步行简单效应分析。多组间数据均值差异比较采用单因素方差分析,两两多重比较采用LSD法。结果动物实验结果表明:裸鼠异种移植肿瘤体积在高胆固醇饮食组与对照组分别为(5.055±0.774)cm3和(1.866±0.618)cm3,抑瘤率为-170.79%,差异有统计学意义(P<0.05)。细胞实验结果表明:(1)胆固醇可以促进ECA109细胞和上游基因cyp27a1敲除后的ECA109细胞增殖,胆固醇低浓度下促进上游基因cyp27a1敲除后的ECA109细胞增殖(P均<0.05);27-羟基胆固醇可以抑制ECA109细胞增殖,但促进下游基因cyp7b1敲除后的ECA109细胞增殖(P均<0.05);(2)胆固醇和27-羟基胆固醇对于ECA109细胞迁移能力无影响(F=2.418,P=0.170),27-羟基胆固醇的上游基因cyp27a1和下游基因cyp7b1敲除对ECA109细胞迁移能力无影响(F=0.602,P=0.578);(3)基因敲除后细胞与对照组相比,细胞侵袭能力发生改变(F=3.992,P=0.047),其中下游基因cyp7b1敲除后,侵袭能力受到抑制(P<0.05);上游基因cyp27a1敲除对ECA109细胞侵袭能力无影响(P>0.05);(4)与正常ECA109细胞相比(151.883±2.948),基因敲除可以使肿瘤细胞MCP-1因子的分泌发生改变(F=553.538,P<0.001)。上游基因cyp27a1敲除后,细胞的MCP-1因子分泌增多(213.823±4.572),下游基因cyp7b1敲除后,细胞的MCP-1因子分泌减少(107.240±4.121)(P均<0.001)。结论胆固醇在体内外均可刺激ECA109细胞的增殖;27-羟基胆固醇抑制ECA109细胞增殖,胆固醇促进ECA109细胞的增殖,并且具有浓度依赖性;上游基因cyp27a1敲除可以增强胆固醇在低浓度下对细胞增殖能力的影响,但对细胞侵袭能力无影响;下游基因cyp7b1敲除可以改变27-羟基胆固醇对细胞增殖活性的影响,并抑制细胞的侵袭能力;27-羟基胆固醇上游基因cyp27a1敲除可促进细胞MCP-1因子的分泌,下游基因cyp7b1敲除抑制MCP-1因子的分泌。
Objective To investigate the effects of cholesterol and 27-hydroxycholesterol on the proliferation,invasion and migration of esophageal squamous cell carcinoma(ESCC)and on the secretion of cytokine MCP-1.Methods In vivo animal experiments:After animal models of ESCC were established,they were given high cholesterol diet(high cholesterol diet group,n=4)or normal diet(control group,n=4),to observe the effects of cholesterol on the growth of ESCC in vivo.At the end of the 5 th week,the tumor volume of the two groups was measured and tumor inhibition rate was calculated.Cell experiments:Effects of cholesterol(0,0.123,0.148,0.185,0.269,0.370 mg/mL)and 27-hydroxycholesterol(0,1,5,10,20μmol/mL)on the proliferation of normal ECA109 cells,and ECA109 cells with cyp27 a1 and cyp7 b1 knockout were observed.The cell proliferation at different concentrations was detected with CCK-8.Cell invasion was determined with scratch test and invasion test(cholesterol 0.185 mg/mL,27-hydroxycholesterol 1μmol/mL).The upstream gene cyp27 a1 and downstream gene CYP7 B1 of 27-hydroxycholesterol were transfected with lentivirus.The above experiments were repeated to observe the effects of gene knockout on proliferation,invasion and migration of cells,and the effect on the secretion of MCP-1 was detected with ELISA.The tumor volume and absorbance measured by CCK-8 in the two groups at different times were analyzed with two-factor ANOVA.When the interaction was statistically significant,simple effect analysis was performed.One-way ANOVA was used for the comparison of mean differences between groups,and LSD method was used for pairwise multiple comparison.Results Animal experiments showed the tumor volume was(5.055±0.774)cm^3 in the high cholesterol diet group and(5.055±0.774)cm^3 in the control group,and the tumor inhibition rate was-170.79%,with statistically significant difference(P<0.05).Cell experiments showed:(1)Cholesterol promoted the proliferation of ECA109 cells and ECA109 cells with cyp27 a1 knockout at low concentration(P<0.05).27-hydroxycholesterol inhibited the proliferation of ECA109 cells,but promoted the proliferation of ECA109 cells with CYP7 b1 knockout(P<0.05).(2)Cholesterol and 27-hydroxycholesterol had no effects on the migration of ECA109 cells(F=2.418,P=0.170),and no effects on the migration of ECA109 cells with cyp27 a1 and CYP7 b1 knockout(F=0.602,P=0.578).(3)After gene knockout,the cell invasion changed(F=3.992,P=0.047).Cells with CYP27 b1 knockout had inhibited invasion(P<0.05),while cells with CYP7 a1 knockout had unchanged invasion(P>0.05).(4)Gene knockout affected the secretion of MCP-1(F=553.538,P<0.001).CYP27 A1 knockout increased the secretion of MCP-1(213.823±4.572),while CYP7 B1 knockout decreased the secretion of MCP-1(107.240±4.121)(P<0.001).Conclusion Cholesterol can stimulate the proliferation of ECA109 cells both in vivo and in vitro.27-hydroxycholesterol can inhibit the proliferation of ECA109 cells while cholesterol can promote the proliferation of ECA109 cells,both in a concentration-dependent manner.The knockdown of upstream gene cyp27 a1 can enhance the effect of cholesterol on the proliferation but does not affect the cell invasion.The knockdown of downstream gene cyp7 b1 can enhance the effect of 27-hydroxycholesterol on cell proliferation and inhibit cell invasion.The secretion of MCP-1 is increased by cyp27 a1 knockout and inhibited by cyp7 b1 knockout.
作者
李昌达
史永军
林彦良
LI Changda;SHI Yongjun;LIN Yanliang(Department of Gastroenterology,Shandong Provincial Hospital Affiliated to Shandong University,Jinan 250021,Shandong,China)
出处
《山东大学学报（医学版）》
CAS
北大核心
2020年第11期45-52,58,共9页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金(81201778)
山东省重点研发计划(2016GSF201002)
济南临床医学科技创新计划(201602166)
关键词
27-羟基胆固醇
胆固醇
人食管癌细胞
增殖
侵袭
27-Hydroxylcholesterol
Cholesterol
Human esophageal cancer cell
Proliferation
Invasion
