摘要
目的探究来源于肝细胞的内质网应激(ERS)信号对小鼠原代肝星状细胞(p-HSCs)的影响。方法在体外利用ERS激动剂衣霉素(TM)刺激小鼠肝细胞系AML1212 h,随后更换正常培养基培养16 h,收集细胞培养上清离心(ERS传递条件培养基),加入有p-HSCs的细胞培养皿中继续培养24 h。显微镜下观察细胞形态变化。采用Real-time PCR(qPCR)和蛋白质印迹法检测ERS相关指标GRP78、IRE1α、PERK、CHOP的表达,并进一步利用qPCR检测纤维化相关指标Ⅰ型胶原(CollagenⅠ)、Ⅲ型胶原(CollagenⅢ)、转化生长因子β(TGFβ)的表达;以及凋亡相关基因Bax、Fas和Bcl 2的表达。结果显微镜下观察细胞形态,发现加有TM的AML12细胞与对照组相比,出现细胞皱缩变圆,胞质浓缩,细胞生长受到抑制;加有相应ERS传递条件培养基的p-HSCs细胞形态未发生明显变化,细胞生长轻微受抑制。与正常对照相比,经TM刺激后的AML12细胞,ERS相关基因GRP78、PERK分别升高12.4倍和1.5倍,同时ERS相关蛋白GRP78、PERK、P-PERK的表达也增加2.0、1.8、2.0倍,差异有统计学意义(P<0.05)。接受ERS传递的p-HSCs与未接受ERS信号的对照组相比,GRP78、IRE1α、PERK、CHOP基因表达分别升高1.3、1.3、1.18和1.3倍,GRP78、IRE1α、ATF6蛋白表达水平也升高1.6、2.0、1.8倍,差异有统计学意义(P<0.05)。接受ERS传递的p-HSCs促凋亡基因Bax、Fas的表达显著上调(分别升高2.2、1.2倍),抑制凋亡基因Bcl 2表达下调(为对照组的60%),差异有统计学意义(P<0.05);而HSC纤维化相关基因TGFβ、CollagenⅠ、CollagenⅢ的表达两组之间未见明显差异。结论ERS可以从肝细胞传递至HSCs,并可诱导HSCs凋亡。
Objective To investigate the effects of endoplasmic reticulum stress(ERS)signals from hepatocytes on mice primary hepatic stellate cells(p-HSCs)through ERS transmission.Methods After stimulation for 12 h by ERS activator tunicamycin(TM),mice liver cell line AML12 was incubated with normal medium for 16 h.The AML12 cell culture supernatant(ERS conditional medium)was collected and centrifuged,and then added into primary hepatic stellate cells from mice(p-HSCs)for further 24 h.Cell morphology was observed under a microscope.Real-time PCR(qPCR)and Western blotting were used to detect the expression of ERS markers:GRP78,IRE1α,PERK and CHOP.Fibrosis markers typeⅠcollagen(CollagenⅠ),typeⅢcollagen(CollagenⅢ),transforming growth factor beta(TGFβ)and apoptosis markers Bax,Fas and Bcl (2)were further detected with qPCR.Results Compared with the control group,the AML12 cells stimulated by TM were wrinkled and round,and the cytoplasm was concentrated.The cell growth was inhibited after TM stimulation.The p-HSCs cells incubated with the corresponding ERS conditional medium had no significant change in cell morphology,but the cell growth was slightly inhibited.Compared with control group,the expressions of GRP78 and PERK were increased by 12.4 and 1.5 times respectively in TM stimulated AML12 cells,meanwhile the expressions of ERS associated protein GRP78,PERK,P-PERK were also increased by 2.0,1.8,2.0 times,and the differences were statistically significant(P<0.05).The expressions of GRP78,IRE1α,PERK and CHOP of p-HSCs receiving ERS transmission increased by 1.3,1.3,1.18,and 1.3 times respectively compared with the control group,the expression of GRP78,IRE1α,ATF6 protein was also increased by 1.6,2.0,1.8 times,and the differences were statistically significant(P<0.05).The expressions of pro-apoptotic genes Bax and Fas were significantly up-regulated in p-HSCs subjected to ERS transmission(2.2 and 1.2 times respectively),and the expression of anti-apoptotic genes Bcl 2 was down-regulated(to 60% of the control group)with statistical significance(P<0.05).However,there were no significant differences between HSCs with and without ERS transmission groups in terms of the expressions of fibrosis related genes TGFβ,CollagenⅠand CollagenⅢ.Conclusion Endoplasmic reticulum stress signals can be transmitted to HSCs from hepatocytes and induce apoptosis of HSCs.
作者
王晓凡
谢超
刘天会
王萍
范旭
刘琳
丛敏
WANG Xiao-fan;XIE Chao;LIU Tian-hui(Liver Research Center,Beijing Friendship Hospital,Capital Medical University,Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis,National Clinical Research Center of Digestive Diseases,Beijing 100050,China)
出处
《临床和实验医学杂志》
2022年第5期449-453,共5页
Journal of Clinical and Experimental Medicine
基金
国家自然科学基金(编号:81570542,82170614)
王宝恩肝纤维化基金(编号:CFHPC 2021042)。
关键词
肝细胞
星状细胞
内质网应激
Hepatocytes
Hepatic stellate cells
Endoplasmic reticulum stress
