中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
7期
986-988
,共3页
姚长江%苏长辉%戈应滨%曹晓建%眭涛
姚長江%囌長輝%戈應濱%曹曉建%眭濤
요장강%소장휘%과응빈%조효건%휴도
成纤维细胞%丝裂霉素C%脱噬作用
成纖維細胞%絲裂黴素C%脫噬作用
성섬유세포%사렬매소C%탈서작용
Fibroblast%Mitomycin C%Apoptosis
目的 观察丝裂霉素C(MMC)对成纤维细胞诱导凋亡的作用及机制.方法 体外培养成纤维细胞并传至5～8代,分别用含有0～0.3g/L MMC的MEM培养液处理成纤维细胞12h后CCK-8试剂盒检测不同浓度MMC对成纤维细胞的生长抑制效果;以0～0.2g/L MMC处理细胞爬片后用Hoechest33342细胞核染色,荧光显微镜下观察凋亡细胞核形态并用image pro-plus软件计数凋亡细胞;按不加药培养细胞12 h作对照组、0.2g/L.MMC培养细胞12h、P13K/Akt抑制剂LY294002 100 nmol/L与MEK/ERK抑制剂PD98059 100nmol/L分别处理细胞1h后换新鲜培养基培养11h分为4组,提取胞质蛋白,Western blot法检测Caspase-3、p-ERK1/2、p-Akt表达量.结果 MMC对成纤维细胞的抑制率与药物浓度呈正相关性,0.2g/L MMC对细胞活性抑制率达50.21%;Hoeeheat33342细胞核染色随MMC浓度升高出现凋亡细胞核特征的染色质凝聚,边集,呈亮蓝色核的比率逐渐增高,凋亡细胞计数显示0.05、0.1、0.2g/L MMC处理组凋亡率显著高于其他低浓度组,差异有统计学意义(P<0.05).Western blot检测0.2 g/L MMC组、PD98059组、LY294002组C8spase-3表达均显著高于对照组,p-ERK1/2、p-Akt表达均较对照组降低.结论 一定浓度的MMC可诱导成纤维细胞发生凋亡,其效应途径部分是通过降低ERK1/2、Akt的磷酸化水平使胞质内凋亡执行蛋白Caspase-3的表达水平增加,诱导成纤维细胞由正常增生状态转而发生凋亡减少胶原纤维生成,从而抑制瘢痕形成.
目的 觀察絲裂黴素C(MMC)對成纖維細胞誘導凋亡的作用及機製.方法 體外培養成纖維細胞併傳至5～8代,分彆用含有0～0.3g/L MMC的MEM培養液處理成纖維細胞12h後CCK-8試劑盒檢測不同濃度MMC對成纖維細胞的生長抑製效果;以0～0.2g/L MMC處理細胞爬片後用Hoechest33342細胞覈染色,熒光顯微鏡下觀察凋亡細胞覈形態併用image pro-plus軟件計數凋亡細胞;按不加藥培養細胞12 h作對照組、0.2g/L.MMC培養細胞12h、P13K/Akt抑製劑LY294002 100 nmol/L與MEK/ERK抑製劑PD98059 100nmol/L分彆處理細胞1h後換新鮮培養基培養11h分為4組,提取胞質蛋白,Western blot法檢測Caspase-3、p-ERK1/2、p-Akt錶達量.結果 MMC對成纖維細胞的抑製率與藥物濃度呈正相關性,0.2g/L MMC對細胞活性抑製率達50.21%;Hoeeheat33342細胞覈染色隨MMC濃度升高齣現凋亡細胞覈特徵的染色質凝聚,邊集,呈亮藍色覈的比率逐漸增高,凋亡細胞計數顯示0.05、0.1、0.2g/L MMC處理組凋亡率顯著高于其他低濃度組,差異有統計學意義(P<0.05).Western blot檢測0.2 g/L MMC組、PD98059組、LY294002組C8spase-3錶達均顯著高于對照組,p-ERK1/2、p-Akt錶達均較對照組降低.結論 一定濃度的MMC可誘導成纖維細胞髮生凋亡,其效應途徑部分是通過降低ERK1/2、Akt的燐痠化水平使胞質內凋亡執行蛋白Caspase-3的錶達水平增加,誘導成纖維細胞由正常增生狀態轉而髮生凋亡減少膠原纖維生成,從而抑製瘢痕形成.
목적 관찰사렬매소C(MMC)대성섬유세포유도조망적작용급궤제.방법 체외배양성섬유세포병전지5～8대,분별용함유0～0.3g/L MMC적MEM배양액처리성섬유세포12h후CCK-8시제합검측불동농도MMC대성섬유세포적생장억제효과;이0～0.2g/L MMC처리세포파편후용Hoechest33342세포핵염색,형광현미경하관찰조망세포핵형태병용image pro-plus연건계수조망세포;안불가약배양세포12 h작대조조、0.2g/L.MMC배양세포12h、P13K/Akt억제제LY294002 100 nmol/L여MEK/ERK억제제PD98059 100nmol/L분별처리세포1h후환신선배양기배양11h분위4조,제취포질단백,Western blot법검측Caspase-3、p-ERK1/2、p-Akt표체량.결과 MMC대성섬유세포적억제솔여약물농도정정상관성,0.2g/L MMC대세포활성억제솔체50.21%;Hoeeheat33342세포핵염색수MMC농도승고출현조망세포핵특정적염색질응취,변집,정량람색핵적비솔축점증고,조망세포계수현시0.05、0.1、0.2g/L MMC처리조조망솔현저고우기타저농도조,차이유통계학의의(P<0.05).Western blot검측0.2 g/L MMC조、PD98059조、LY294002조C8spase-3표체균현저고우대조조,p-ERK1/2、p-Akt표체균교대조조강저.결론 일정농도적MMC가유도성섬유세포발생조망,기효응도경부분시통과강저ERK1/2、Akt적린산화수평사포질내조망집행단백Caspase-3적표체수평증가,유도성섬유세포유정상증생상태전이발생조망감소효원섬유생성,종이억제반흔형성.
Objective To elucidate the mitomycin C (MMC) -induced apoptosis of fibroblasts and the mechanism. Methods The cultured fibroblasts were exposed for 12 h to 0-0. 3 g/L MMC, respectively. The cell proliferation was measured by CCK-8 assay and apoptotic cell death was analyzed by Ho-echst33342 stain on the fisherbrand covergla. The expression of Caspase-3 and the activation of p-ERK1/2, p-Akt were detected by Western blotting. Results Treatment of fibroblasts with MMC induced a significant decrease in proliferation. With the increases of MMC from 0. 001-0. 2 g/L MMC, Hoechst 33342 staining displayed nuclei with abnormal morphologies: "bean"-shaped nuclei with highly condensed chromatin or nuclei with irregular clumps of dense chromatin. 0.2g/L MMC, 100 nmol/L PD98059 (ERK kinase inhibitor) , 100 nmol/L LY294002 (AKT kinase inhibitor) induced a significant increase of Caspase-3 as compared with control group, accompanied by an increase in cell apoptosis. 0.2 g/L MMC, PD98059, LY294002 caused decrease of p-ERKl/2 and p-Akt. Conclusion 0. 2 g/L MMC can induce fibroblast apoptosis by reducing the phosphorylation of p-ERKl/2 and p-Akt, resulting in the increase in the Caspase-3 expression. MMC can inhibit fibroblast proliferation and induce its apoptosis, which may be one of mechanisms by which MMC prevents peridural adhesion after laminectomy.
