中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2015年
5期
871-876
,共6页
田甜%张金娟%罗新华%谢汝佳%韩冰%杨婷%陈腾祥%杨勤
田甜%張金娟%囉新華%謝汝佳%韓冰%楊婷%陳騰祥%楊勤
전첨%장금연%라신화%사여가%한빙%양정%진등상%양근
肝星状细胞%组蛋白修饰%肝纤维化
肝星狀細胞%組蛋白脩飾%肝纖維化
간성상세포%조단백수식%간섬유화
Hepatic stellate cells%Histone modifications%Liver fibrosis
目的：观察体外培养大鼠肝星状细胞（ HSCs）活化过程中组蛋白修饰的改变以及与 HSCs活化指标α-平滑肌肌动蛋白（α-SMA）的关系，探讨组蛋白修饰在HSCs活化过程中可能的作用。方法：体外分离、鉴定、培养大鼠HSCs，光镜观察HSCs活化过程中的形态变化，细胞免疫荧光染色和Western blotting检测desmin和α-SMA的表达，比较静止型HSCs和激活型HSCs中H4K12乙酰化、H3K9乙酰化、H3K4二甲基化和H3K9二甲基化的变化。结果：（1）细胞形态学观察结果表明HSCs在培养过程中形态由静息状态向高度分化的肌成纤维细胞转化。细胞免疫荧光染色及Western blotting检测结果显示，分离培养24 h的HSCs有desmin表达，但几乎不表达α-SMA；随着培养时间延长，HSCs内α-SMA和desmin表达逐步增加，至15 d时达到最大。（2）根据HSCs细胞形态变化及HSCs活化标志蛋白检测结果，确定培养24 h的HSCs为静止型HSCs，培养15 d 的HSCs为激活型HSCs，分别检测其组蛋白修饰变化。结果显示，与静止型HSCs比较，激活型HSCs中H4K12乙酰化、H3K9乙酰化和H3K9二甲基化修饰水平明显降低（P＜0.01），而H3K4二甲基化修饰水平明显增加（P＜0.01），且H3K4二甲基化修饰水平变化与α-SMA表达变化一致。结论：在体外培养HSCs活化过程中，组蛋白修饰发生明显异常，提示组蛋白修饰改变有可能参与了HSCs活化以及肝纤维化的发生。
目的：觀察體外培養大鼠肝星狀細胞（ HSCs）活化過程中組蛋白脩飾的改變以及與 HSCs活化指標α-平滑肌肌動蛋白（α-SMA）的關繫，探討組蛋白脩飾在HSCs活化過程中可能的作用。方法：體外分離、鑒定、培養大鼠HSCs，光鏡觀察HSCs活化過程中的形態變化，細胞免疫熒光染色和Western blotting檢測desmin和α-SMA的錶達，比較靜止型HSCs和激活型HSCs中H4K12乙酰化、H3K9乙酰化、H3K4二甲基化和H3K9二甲基化的變化。結果：（1）細胞形態學觀察結果錶明HSCs在培養過程中形態由靜息狀態嚮高度分化的肌成纖維細胞轉化。細胞免疫熒光染色及Western blotting檢測結果顯示，分離培養24 h的HSCs有desmin錶達，但幾乎不錶達α-SMA；隨著培養時間延長，HSCs內α-SMA和desmin錶達逐步增加，至15 d時達到最大。（2）根據HSCs細胞形態變化及HSCs活化標誌蛋白檢測結果，確定培養24 h的HSCs為靜止型HSCs，培養15 d 的HSCs為激活型HSCs，分彆檢測其組蛋白脩飾變化。結果顯示，與靜止型HSCs比較，激活型HSCs中H4K12乙酰化、H3K9乙酰化和H3K9二甲基化脩飾水平明顯降低（P＜0.01），而H3K4二甲基化脩飾水平明顯增加（P＜0.01），且H3K4二甲基化脩飾水平變化與α-SMA錶達變化一緻。結論：在體外培養HSCs活化過程中，組蛋白脩飾髮生明顯異常，提示組蛋白脩飾改變有可能參與瞭HSCs活化以及肝纖維化的髮生。
목적：관찰체외배양대서간성상세포（ HSCs）활화과정중조단백수식적개변이급여 HSCs활화지표α-평활기기동단백（α-SMA）적관계，탐토조단백수식재HSCs활화과정중가능적작용。방법：체외분리、감정、배양대서HSCs，광경관찰HSCs활화과정중적형태변화，세포면역형광염색화Western blotting검측desmin화α-SMA적표체，비교정지형HSCs화격활형HSCs중H4K12을선화、H3K9을선화、H3K4이갑기화화H3K9이갑기화적변화。결과：（1）세포형태학관찰결과표명HSCs재배양과정중형태유정식상태향고도분화적기성섬유세포전화。세포면역형광염색급Western blotting검측결과현시，분리배양24 h적HSCs유desmin표체，단궤호불표체α-SMA；수착배양시간연장，HSCs내α-SMA화desmin표체축보증가，지15 d시체도최대。（2）근거HSCs세포형태변화급HSCs활화표지단백검측결과，학정배양24 h적HSCs위정지형HSCs，배양15 d 적HSCs위격활형HSCs，분별검측기조단백수식변화。결과현시，여정지형HSCs비교，격활형HSCs중H4K12을선화、H3K9을선화화H3K9이갑기화수식수평명현강저（P＜0.01），이H3K4이갑기화수식수평명현증가（P＜0.01），차H3K4이갑기화수식수평변화여α-SMA표체변화일치。결론：재체외배양HSCs활화과정중，조단백수식발생명현이상，제시조단백수식개변유가능삼여료HSCs활화이급간섬유화적발생。
AIM:To investigate the changes of histone modifications during the activation of primarily cultured rat hepatic stellate cells ( HSCs) and the relationship between histone modification patterns andα-smooth muscle actin (α-SMA) expression, and to explore the roles of histone modifications in the activation of HSCs.METHODS:The rat HSCs were isolated by in situ perfusion of collagenase combined with density gradient centrifugation, cultured in vitro and identi-fied by immunofluorescence staining.The morphological features of the cells were observed under inverted microscope.The changes of desmin and α-SMA during the activation of HSCs were detected by immunofluorescence staining and Western blotting.The levels of histone 3 lysine 4 dimethylation (H3K4me2), histone 3 lysine 9 dimethylation (H3K9me2), his-tone 3 lysine 9 acetylation (acH3K9) and histone 4 lysine 12 acetylation (acH4K12) in quiescent HSCs and activated HSCs were determined by Western blotting.RESULTS: The morphology of HSCs shifted from a quiescent phenotype to highly activated myofibroblast during the culture.Immunofluorescence staining and Western blotting showed that the expres-sion levels of α-SMA and desmin were increased over time and reached maximum at 15 d.According to the results of cell morphology and immunofluorescence staining, the cells cultured for 24 h and 15 d were quiescent and activated HSCs, re-spectively.Compared with quiescent HSCs, there were higher H3K4me2 and lower H3K9me2, acH3K9 and acH4K12 modification levels in activated HSCs ( P<0.01 ) .CONCLUSION: Histone modifications show anomalous expression during the activation of primarily cultured rat HSCs.Histone modifications may contribute to the transdifferentiation of HSCs and the development of hepatic fibrosis.
