中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2014年
11期
737-741
,共5页
陶丽丽%丁笛%张继君%刘秀萍%张锦生%尹为华
陶麗麗%丁笛%張繼君%劉秀萍%張錦生%尹為華
도려려%정적%장계군%류수평%장금생%윤위화
肝硬化%肝星状细胞%CCAAT增强子结合蛋白类
肝硬化%肝星狀細胞%CCAAT增彊子結閤蛋白類
간경화%간성상세포%CCAAT증강자결합단백류
Liver cirrhosis%Hepatic stellate cells%CCAAT-enhancer-binding proteins
目的 检测CCAAT/增强子结合蛋白-α(C/ EBP-α)基因诱导肝纤维化小鼠体内肝细胞和肝星状细胞(HSC)凋亡的差异.方法 将60只BALB/c小鼠均分为正常组、造模组、治疗组、空白对照组、阴性对照组,每组12只.除正常组外,其余各组腹腔注射四氯化碳(CCl4)建立小鼠肝纤维化模型.治疗组、空白对照组、阴性对照组均于造模第1周经尾静脉分别注射表达C/EBP-α的腺病毒载体(Ad-C/EBP-α)、PBS、腺病毒空载体.免疫组织化学法检测小鼠肝组织中C/EBP-α和α-平滑肌肌动蛋白(α-SMA)的表达.经透射电子显微镜观察小鼠肝组织标本中汇管区周围和远离汇管区部位肝血窦内皮结构.用TUNEL法检测小鼠肝组织的细胞凋亡情况.通过天狼星红染色和肝组织羟脯氨酸含量测定来检测小鼠的肝纤维化情况.多组间比较行单因素方差分析,两组间比较行t检验.结果 正常组C/EBP-α主要表达于肝细胞核内.造模组、空白对照组、阴性对照组C/EBP-α表达量均减少.治疗组C/EBP-α表达量升高,主要表达于肝血窦间隙和血管周细胞.造模组、空白对照组、阴性对照组肝血窦结构扭曲,血管壁增厚,肝细胞变性,出现大量脂滴.治疗组内皮细胞血管壁增厚程度较造模组有所降低.正常组、造模组、治疗组、空白对照组、阴性对照组天狼星红阳性染色细胞所占比例分别为(0.10±0.03)％、(5.81±0.32)％、(2.32±0.45)％、(6.34±0.81)％、(6.10±0.92)％,羟脯氨酸含量分别为(0.07±0.00) μg/mg、(0.69±0.10) μg/mg、(0.19±0.06) μg/mg、(0.56±0.03) μg/mg、(0.64±0.08) μg/mg,α-SMA阳性染色细胞所占比例分别为(0.50±0.03)％、(5.30±0.52)％、(2.15±0.29)％、(5.53±0.43)％、(5.42±0.25)％,TUNEL阳性染色细胞数分别为0.25±0.08、0.15±0.02、7.10±1.53、0.13±0.03、0.18±0.07,各指标组间比较差异有统计学意义(F=113.74、148.29、292.43、140.25,P均＜0.05),其中正常组与造模组、造模组与治疗组、治疗组与空白对照组、治疗组与阴性对照组各指标比较,差异均有统计学意义(t天狼星红阳性染色细胞=-52.54、-16.20、-10.60、-7.99,t羟脯氨酸含量=-168.00、11.53、11.07、12.54,tα-SMA阳性染色细胞=-24.77、-13.82、15.94、18.37,tTUNEL阳性染色细胞=3.26、-11.91、-11.95、-11.88,P均＜0.05),造模组与空白对照组、造模组与阴性对照组比较则差异均无统计学意义(P均＞0.05).小鼠肝组织中TUNEL阳性染色细胞主要位于肝血窦间隙和血管周围,与α-SMA阳性染色细胞的分布较一致.结论 C/EBP-α基因能有效缓解CCl4诱导的小鼠肝纤维化,主要与诱导HSC凋亡有关,而对肝细胞无明显促凋亡作用.
目的 檢測CCAAT/增彊子結閤蛋白-α(C/ EBP-α)基因誘導肝纖維化小鼠體內肝細胞和肝星狀細胞(HSC)凋亡的差異.方法 將60隻BALB/c小鼠均分為正常組、造模組、治療組、空白對照組、陰性對照組,每組12隻.除正常組外,其餘各組腹腔註射四氯化碳(CCl4)建立小鼠肝纖維化模型.治療組、空白對照組、陰性對照組均于造模第1週經尾靜脈分彆註射錶達C/EBP-α的腺病毒載體(Ad-C/EBP-α)、PBS、腺病毒空載體.免疫組織化學法檢測小鼠肝組織中C/EBP-α和α-平滑肌肌動蛋白(α-SMA)的錶達.經透射電子顯微鏡觀察小鼠肝組織標本中彙管區週圍和遠離彙管區部位肝血竇內皮結構.用TUNEL法檢測小鼠肝組織的細胞凋亡情況.通過天狼星紅染色和肝組織羥脯氨痠含量測定來檢測小鼠的肝纖維化情況.多組間比較行單因素方差分析,兩組間比較行t檢驗.結果 正常組C/EBP-α主要錶達于肝細胞覈內.造模組、空白對照組、陰性對照組C/EBP-α錶達量均減少.治療組C/EBP-α錶達量升高,主要錶達于肝血竇間隙和血管週細胞.造模組、空白對照組、陰性對照組肝血竇結構扭麯,血管壁增厚,肝細胞變性,齣現大量脂滴.治療組內皮細胞血管壁增厚程度較造模組有所降低.正常組、造模組、治療組、空白對照組、陰性對照組天狼星紅暘性染色細胞所佔比例分彆為(0.10±0.03)％、(5.81±0.32)％、(2.32±0.45)％、(6.34±0.81)％、(6.10±0.92)％,羥脯氨痠含量分彆為(0.07±0.00) μg/mg、(0.69±0.10) μg/mg、(0.19±0.06) μg/mg、(0.56±0.03) μg/mg、(0.64±0.08) μg/mg,α-SMA暘性染色細胞所佔比例分彆為(0.50±0.03)％、(5.30±0.52)％、(2.15±0.29)％、(5.53±0.43)％、(5.42±0.25)％,TUNEL暘性染色細胞數分彆為0.25±0.08、0.15±0.02、7.10±1.53、0.13±0.03、0.18±0.07,各指標組間比較差異有統計學意義(F=113.74、148.29、292.43、140.25,P均＜0.05),其中正常組與造模組、造模組與治療組、治療組與空白對照組、治療組與陰性對照組各指標比較,差異均有統計學意義(t天狼星紅暘性染色細胞=-52.54、-16.20、-10.60、-7.99,t羥脯氨痠含量=-168.00、11.53、11.07、12.54,tα-SMA暘性染色細胞=-24.77、-13.82、15.94、18.37,tTUNEL暘性染色細胞=3.26、-11.91、-11.95、-11.88,P均＜0.05),造模組與空白對照組、造模組與陰性對照組比較則差異均無統計學意義(P均＞0.05).小鼠肝組織中TUNEL暘性染色細胞主要位于肝血竇間隙和血管週圍,與α-SMA暘性染色細胞的分佈較一緻.結論 C/EBP-α基因能有效緩解CCl4誘導的小鼠肝纖維化,主要與誘導HSC凋亡有關,而對肝細胞無明顯促凋亡作用.
목적 검측CCAAT/증강자결합단백-α(C/ EBP-α)기인유도간섬유화소서체내간세포화간성상세포(HSC)조망적차이.방법 장60지BALB/c소서균분위정상조、조모조、치료조、공백대조조、음성대조조,매조12지.제정상조외,기여각조복강주사사록화탄(CCl4)건립소서간섬유화모형.치료조、공백대조조、음성대조조균우조모제1주경미정맥분별주사표체C/EBP-α적선병독재체(Ad-C/EBP-α)、PBS、선병독공재체.면역조직화학법검측소서간조직중C/EBP-α화α-평활기기동단백(α-SMA)적표체.경투사전자현미경관찰소서간조직표본중회관구주위화원리회관구부위간혈두내피결구.용TUNEL법검측소서간조직적세포조망정황.통과천랑성홍염색화간조직간포안산함량측정래검측소서적간섬유화정황.다조간비교행단인소방차분석,량조간비교행t검험.결과 정상조C/EBP-α주요표체우간세포핵내.조모조、공백대조조、음성대조조C/EBP-α표체량균감소.치료조C/EBP-α표체량승고,주요표체우간혈두간극화혈관주세포.조모조、공백대조조、음성대조조간혈두결구뉴곡,혈관벽증후,간세포변성,출현대량지적.치료조내피세포혈관벽증후정도교조모조유소강저.정상조、조모조、치료조、공백대조조、음성대조조천랑성홍양성염색세포소점비례분별위(0.10±0.03)％、(5.81±0.32)％、(2.32±0.45)％、(6.34±0.81)％、(6.10±0.92)％,간포안산함량분별위(0.07±0.00) μg/mg、(0.69±0.10) μg/mg、(0.19±0.06) μg/mg、(0.56±0.03) μg/mg、(0.64±0.08) μg/mg,α-SMA양성염색세포소점비례분별위(0.50±0.03)％、(5.30±0.52)％、(2.15±0.29)％、(5.53±0.43)％、(5.42±0.25)％,TUNEL양성염색세포수분별위0.25±0.08、0.15±0.02、7.10±1.53、0.13±0.03、0.18±0.07,각지표조간비교차이유통계학의의(F=113.74、148.29、292.43、140.25,P균＜0.05),기중정상조여조모조、조모조여치료조、치료조여공백대조조、치료조여음성대조조각지표비교,차이균유통계학의의(t천랑성홍양성염색세포=-52.54、-16.20、-10.60、-7.99,t간포안산함량=-168.00、11.53、11.07、12.54,tα-SMA양성염색세포=-24.77、-13.82、15.94、18.37,tTUNEL양성염색세포=3.26、-11.91、-11.95、-11.88,P균＜0.05),조모조여공백대조조、조모조여음성대조조비교칙차이균무통계학의의(P균＞0.05).소서간조직중TUNEL양성염색세포주요위우간혈두간극화혈관주위,여α-SMA양성염색세포적분포교일치.결론 C/EBP-α기인능유효완해CCl4유도적소서간섬유화,주요여유도HSC조망유관,이대간세포무명현촉조망작용.
Objective To investigate the difference of CCAAT/enhancer-binding protein α (C/EBP-α) gene induced apoptosis between hepatocytes and hepatic stellate cells (HSC) in mice with liver fibrosis.Methods Sixty BALB/c mice were evenly divided into normal group,model group,treatment group,blank control group and negative control group,12 mice in each group.Except the mice of normal control group,the mice of other groups were treated with intraperitoneal injection of CCl4 to establish liver fibrosis mice model.Mice of treatment group,blank control group and negative control group were administrated with C/EBP-α carried adenovirus (Ad-C/EBP-α),phosphate buffered solution and empty vector of adenovirus (Ad-EGFP) respectively through tail vein for the first week.The expression of C/EBP-α and α-smooth muscle actin (α-SMA) was detected by immunohistochemistry method.Sinusoidal endothelial structure of peri-portal regions and far from portal regions was observed by transmission electron microscope (TEM).Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was applied to detect apoptosis of cells in liver tissue.The degree of liver fibrosis in mice was determined with sirius red staining and hydroxyproline content measurement.Single factor variance analysis was performed for comparison among multiple groups,and t test was used for comparison between two groups.Results C/EBP-α was expressed in nucleus of hepatocyte in normal control group mice.The expression decreased in model group,blank control group and negative control group.However,the expression of C/EBP-α of treatment group increased,and mainly expressed in cells located in perisinusoidal and perivascular.Hepatic sinusoids was distorted,blood vessel wall thickened.Hepatocyte degeneration and lots of lipid droplets was found in model group,blank control group and negative control group.The thicken degree of endothelial layer of blood vessel of treatment group was lower than that of model group.The percentage of sirius red positive cells of normal group,model group,treatment group,blank control group and negative control group was (0.10±0.03)％,(5.81±0.32)％,(2.32±0.45)％,(6.34± 0.81)％ and (6.10± 0.92)％,respectively; content of hydroxyproline was (0.07±0.00) μg/mg,(0.69 ± 0.10) μg/mg,(0.19±0.06) μg/mg,(0.56±0.03) μg/mg and (0.64±0.08) μg/mg,respectively; the percentage of α-SMA positive cells was (0.50 ±0.03)％,(5.30 ± 0.52)％,(2.15 ± 0.29)％,(5.53 ± 0.43) ％ and (5.42 ± 0.25) ％,respectively; the number of TUNEL positive cells was (0.25 ± 0.08),(0.15±0.02),(7.10±1.53),(0.13±0.03) and (0.18±0.07),respectively.The differences between the groups were statistically significant (F=113.74,148.29,292.43 and 140.25,all P＜0.05).The difference between normal group and model group,between model group and treatment group,between treatment group and blank control group,between treatment group and negative control group were statistically significant (tarirus positive cell =-52.54,-16.20,-10.60 and-7.99,thydroxyproline content =-168.00,11.53,11.07 and 12.54,ta SMA pusitive cells-24.77,-13.82,15.94 and 18.37,tTUNEL positive cells =3.26,-11.91,-11.95 and-11.88,all P＜ 0.05),there was no statistically significant difference between model group and blank control group,between model group and negative control group (both P＞0.05).TUNEL positive cells mainly located in perisinusoidal and perivascular of liver in mice,which was consistent with the distribution of α-SMA-positive cells.Conclusion C/EBP-α could effectively relieve CCl4 induced liver fibrosis in mice mainly through inducing HSC apoptosis,however no apoptosis effect on hepatocytes.
