CAJ | 학술논문

目的依次建立大鼠急性肝衰竭模型,D-氨基半乳糖(D-gal)急性肝损伤模型和氯化铵(NH4Cl)诱导急性肝损伤模型,探索肝衰竭中高血氨再次诱导肝细胞损伤机制.方法 (1)大鼠急性肝衰竭动物模型建立:将雄性SD大鼠36只分为等渗盐水对照组(n=10),12h急性肝衰竭模型组(n=10),24h急性肝衰竭模型组(n=16)；D-gal 400 mg/kg+脂多糖50μg/kg混合剂量给予各组大鼠腹腔注射,分别在给药后12h或24h处死各组大鼠.(2) NH4C1所致肝脏损伤动物模型的建立:将雄性SD大鼠40只分为3组:NH4Cl模型组(n=20),D-gal肝脏损伤组(n=10)和等渗盐水对照组(n=10)；NH4C1模型组每8h给予NH4C1 10 ml/kg灌胃,急性D-gal肝脏损伤组则于大鼠腹腔每6d注射1次D-gal (800 mg/kg),对照组为等渗盐水10 ml/kg灌胃,30d后处死各组大鼠.(3)分别检测大鼠血氨,血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AS2)；凝血酶原时间活动度比率(PT-A),甲胎蛋白(AFP),γ-谷氨酰转移酶(GGT)；肝脏HE染色后观察病理变化和细胞凋亡指数；统计学分析采用秩和检验.结果在大鼠急性肝衰竭动物模型中,12h后出现转氨酶升高[ALT和AST分别为(1202.51±282.00) U/L和(1560.14±298.98) U/L],血氨随之升高[(165.9±23.6)μmol/L],24h检测ALT、AST和血氨分别为(774.40±207.65) U/L、(967.60±121.94)U/L和(143.4±18.1)μmol/L,与等渗盐水对照组比较,P值均＜0.05.24h后转氨酶和血氨下降,但未发现AFP(除急性肝衰竭24h组外)、GGT变化；肝脏病理检查显示各组肝细胞出现片状弥漫性出血性坏死,淤血,并有灶状出血,伴随细胞凋亡指数逐渐上升.但在NH4C1所致急性肝脏损伤中,6h后即检测到血氨升高,ALT和AST同比增加,30d后检测血氨,ALT和AST进一步升高,但AFP,GGT与对照组相比,无明显差异;HE染色未见肝细胞明显变性、坏死和淋巴细胞浸润,无肝细胞再生或纤维组织增生.另外,30 d NH4Cl模型组和D-gal肝损伤组内肝组织均见较多的凋亡细胞,但D-gal肝损伤组可见明显炎性细胞侵润.除血氨外,其余检测指标与NH4C1组差异均无统计学意义.结论在急性肝衰竭模型中,血氨随着肝细胞坏死而升高,相同血氨浓度可以导致大鼠肝脏再次损伤,但这种损伤与D-gal诱导肝损伤明显不同:不是通过肝细胞坏死,炎症细胞侵润实现,而是可能通过细胞凋亡途径,抑制肝细胞再生,进一步导致肝细胞功能受阻. 目的依次建立大鼠急性肝衰竭模型,D-氨基半乳糖(D-gal)急性肝損傷模型和氯化銨(NH4Cl)誘導急性肝損傷模型,探索肝衰竭中高血氨再次誘導肝細胞損傷機製.方法 (1)大鼠急性肝衰竭動物模型建立:將雄性SD大鼠36隻分為等滲鹽水對照組(n=10),12h急性肝衰竭模型組(n=10),24h急性肝衰竭模型組(n=16)；D-gal 400 mg/kg+脂多糖50μg/kg混閤劑量給予各組大鼠腹腔註射,分彆在給藥後12h或24h處死各組大鼠.(2) NH4C1所緻肝髒損傷動物模型的建立:將雄性SD大鼠40隻分為3組:NH4Cl模型組(n=20),D-gal肝髒損傷組(n=10)和等滲鹽水對照組(n=10)；NH4C1模型組每8h給予NH4C1 10 ml/kg灌胃,急性D-gal肝髒損傷組則于大鼠腹腔每6d註射1次D-gal (800 mg/kg),對照組為等滲鹽水10 ml/kg灌胃,30d後處死各組大鼠.(3)分彆檢測大鼠血氨,血清丙氨痠轉氨酶(ALT)、天鼕氨痠轉氨酶(AS2)；凝血酶原時間活動度比率(PT-A),甲胎蛋白(AFP),γ-穀氨酰轉移酶(GGT)；肝髒HE染色後觀察病理變化和細胞凋亡指數；統計學分析採用秩和檢驗.結果在大鼠急性肝衰竭動物模型中,12h後齣現轉氨酶升高[ALT和AST分彆為(1202.51±282.00) U/L和(1560.14±298.98) U/L],血氨隨之升高[(165.9±23.6)μmol/L],24h檢測ALT、AST和血氨分彆為(774.40±207.65) U/L、(967.60±121.94)U/L和(143.4±18.1)μmol/L,與等滲鹽水對照組比較,P值均＜0.05.24h後轉氨酶和血氨下降,但未髮現AFP(除急性肝衰竭24h組外)、GGT變化；肝髒病理檢查顯示各組肝細胞齣現片狀瀰漫性齣血性壞死,淤血,併有竈狀齣血,伴隨細胞凋亡指數逐漸上升.但在NH4C1所緻急性肝髒損傷中,6h後即檢測到血氨升高,ALT和AST同比增加,30d後檢測血氨,ALT和AST進一步升高,但AFP,GGT與對照組相比,無明顯差異;HE染色未見肝細胞明顯變性、壞死和淋巴細胞浸潤,無肝細胞再生或纖維組織增生.另外,30 d NH4Cl模型組和D-gal肝損傷組內肝組織均見較多的凋亡細胞,但D-gal肝損傷組可見明顯炎性細胞侵潤.除血氨外,其餘檢測指標與NH4C1組差異均無統計學意義.結論在急性肝衰竭模型中,血氨隨著肝細胞壞死而升高,相同血氨濃度可以導緻大鼠肝髒再次損傷,但這種損傷與D-gal誘導肝損傷明顯不同:不是通過肝細胞壞死,炎癥細胞侵潤實現,而是可能通過細胞凋亡途徑,抑製肝細胞再生,進一步導緻肝細胞功能受阻.
목적 의차건립대서급성간쇠갈모형,D-안기반유당(D-gal)급성간손상모형화록화안(NH4Cl)유도급성간손상모형,탐색간쇠갈중고혈안재차유도간세포손상궤제.방법 (1)대서급성간쇠갈동물모형건립:장웅성SD대서36지분위등삼염수대조조(n=10),12h급성간쇠갈모형조(n=10),24h급성간쇠갈모형조(n=16)；D-gal 400 mg/kg+지다당50μg/kg혼합제량급여각조대서복강주사,분별재급약후12h혹24h처사각조대서.(2) NH4C1소치간장손상동물모형적건립:장웅성SD대서40지분위3조:NH4Cl모형조(n=20),D-gal간장손상조(n=10)화등삼염수대조조(n=10)；NH4C1모형조매8h급여NH4C1 10 ml/kg관위,급성D-gal간장손상조칙우대서복강매6d주사1차D-gal (800 mg/kg),대조조위등삼염수10 ml/kg관위,30d후처사각조대서.(3)분별검측대서혈안,혈청병안산전안매(ALT)、천동안산전안매(AS2)；응혈매원시간활동도비솔(PT-A),갑태단백(AFP),γ-곡안선전이매(GGT)；간장HE염색후관찰병리변화화세포조망지수；통계학분석채용질화검험.결과 재대서급성간쇠갈동물모형중,12h후출현전안매승고[ALT화AST분별위(1202.51±282.00) U/L화(1560.14±298.98) U/L],혈안수지승고[(165.9±23.6)μmol/L],24h검측ALT、AST화혈안분별위(774.40±207.65) U/L、(967.60±121.94)U/L화(143.4±18.1)μmol/L,여등삼염수대조조비교,P치균＜0.05.24h후전안매화혈안하강,단미발현AFP(제급성간쇠갈24h조외)、GGT변화；간장병리검사현시각조간세포출현편상미만성출혈성배사,어혈,병유조상출혈,반수세포조망지수축점상승.단재NH4C1소치급성간장손상중,6h후즉검측도혈안승고,ALT화AST동비증가,30d후검측혈안,ALT화AST진일보승고,단AFP,GGT여대조조상비,무명현차이;HE염색미견간세포명현변성、배사화림파세포침윤,무간세포재생혹섬유조직증생.령외,30 d NH4Cl모형조화D-gal간손상조내간조직균견교다적조망세포,단D-gal간손상조가견명현염성세포침윤.제혈안외,기여검측지표여NH4C1조차이균무통계학의의.결론 재급성간쇠갈모형중,혈안수착간세포배사이승고,상동혈안농도가이도치대서간장재차손상,단저충손상여D-gal유도간손상명현불동:불시통과간세포배사,염증세포침윤실현,이시가능통과세포조망도경,억제간세포재생,진일보도치간세포공능수조.
Objective To establish an accurate new rat model ofhyperammonemia-induced liver injury for use in studies of the molecular mechanisms underlying acute liver failure (ALF).Methods Twenty-six Sprague-Dawley rats were administered D-galactosamine (400 mg/kg) and endtoxin (50 μg/kg) via intraperitoneal injection to induce ALF and sacrificed at 12 h post-injection (ALF-12 group,n =10) or 24 h post-injection (ALF-24 group,n =16).Ten rats adminstered physiological saline served as the control group.In addition,20 rats were given serial oral administrations of 10％ NH4C1 solution (10 ml/kg,every 8 hrs) to establish the hyperammonemia-induced liver injury model; an additional 20 rats were prepared in parallel to serve as the ALF control group (n =10; D-galactosamine at 800 mg/kg every 6 d for 30 days) and the physiological saline control group (n =10).Serum samples were collected from each mouse and used to detect markers of liver function,including alanine aminotransferase (ALT),aspartate aminotransfease (AST),alpha-fetal protein (AFP),and γ-glutamyltransferrase (GGT),as well as blood ammonia (BA) level and prothrombin time activity (PT-A).Affects on liver histology was assessed by hematoxylin and eosin staining of resected liver tissues,and on apoptosis by TUNEL assay and calculating the apoptotic index (AI).Results ALF rats showed elevated levels of ALT (1202.51 ± 282.00 U/L),AST (1560.14 + 298.98 U/L),and BA (165.9 ± 23.6 μmol/L) as early as 6 hrs after model establishment; these levels peaked at 12 hrs after model establishment (ALT:774.40 ± 207.65 U/L; AST:967.60 ± 121.94 U/L; BA:143.4 ± 18.1 μmol/L; P ＜ 0.05).No significant variations were detected in the levels of AFP (except for the ALF-24 group) or GGT.Liver tissues of the ALF-12 and ALF-24 groups showed large or diffuse hemorrhagic necroses with sinusoidal congestion or spotty bleeding,as well as increased AI.Hyperammonemia-induced liver injury rats showed elevated levels of ALT and BA as early as 6 hrs after model establishment.Similar to the ALF rats,AFP and GGT were unaffected and AI increased.However,in contrast to the ALF rats,the liver tissues of the hyperammonemia-induced liver injury rats showed no signs of hepatocyte swelling,necrosis,or inflammatory cell invasion.Conclusion ALF rats and hyperammonemia-induced liver injury rats have elevated BA and marked hepatocyte necrosis.Given that reducing the level of ammonemia can improve the animal's biochemistry indexes,it is likely that hyperammonemia plays a role in acute liver injury or ALF consequent to repeated injury.The pathogenic mechanisms of repeated injury may involve promotion of hepatocyte apoptosis in conjunction with inhibition of cellular regeneration.