摘要
目的 探讨环孢素（CsA）与胆酸减轻受a-鹅膏毒肽（a—AMA）攻击人肝细胞损伤的作用及机制。方法根据本课题前期研究确定的人肝细胞生存的最小a-AMA攻击质量浓度（1．4g／L）将实验分为5组：空白对照组、损伤组、甘氨鹅脱氧胆酸组、CsA组、CsA联合胆酸组（CsA＋牛磺胆酸、CsA＋鹅脱氧胆酸、CsA＋甘氨胆酸、CsA＋甘氨鹅脱氧胆酸和CsA＋牛磺鹅脱氧胆酸），每个组均设立3个时间点：攻击24h、48h和72h，在攻击24h后采用CsA、甘氨鹅脱氧胆酸和CsA联合胆酸保护损伤肝细胞，在倒置相差显微镜下观察细胞的形态学改变，使用3-（4，5-二甲基噻唑-2）_2，5-二苯基四氮唑溴盐（M1T）法进行活细胞计数，生化法检测培养上清液中天冬氨酸氨基转移酶（AST）和丙氨酸氨基转移酶（ALT）水平。结果与空白对照组比较，损伤组肝细胞生长明显受到抑制，细胞进行性凋亡，MTr法吸光度值明显下降（0．345±0．021）；AST、ALT活性逐渐升高，最高值在损伤72h[分别为（98．4±6．7）U／L和（116．2±9．5）U／L]。与损伤组比较，甘氨鹅脱氧胆酸组、CsA组和CsA联合胆酸组破碎细胞器明显减少，吸光度值增高，且CsA＋鹅脱氧胆酸组在各时间点吸光度值最高（0．656±0．014）（P〈0．05）；同时AST、ALT升高不明显，CsA＋鹅脱氧胆酸组在各时间点数值最低[（22．3±6．2）U／L和（20．2±5．4）U／L，P〈0．05]。结论CsA与胆酸一样对a-AMA攻击人肝细胞有保护作用，作用机制是CsA作为有机阴离子转运多肽（OATP1B1和OATP1B3）的抑制剂抑制a—AMA的吸收。CsA＋鹅脱氧胆酸的联合应用优于单一使用OATP的底物或抑制剂。
Objective To explore the effect and mechanism of Cyclosporin A（CsA） and cholic acid on redu- cing the human hver cell damage induced by a - amanitin（AMA）. Methods According to the previous research results, the minimum hepatocellular survival concentration against a - AMA （ 1.4 g/L）, the experiment was conducted in 5 groups：control group, damage group, glycochenodeoxycholic acid group, CsA group, and CsA combined with cholic acid group （ CsA＋ taurocholic acid,CsA＋ chenodeoxycholic acid,CsA＋ glycocholic acid,CsA＋ glycochenodeoxycholie acid,and CsA＋ taurochenodeoxycholic acid）. For each group,there were 3 time points for observation （24 h,48 h and 72 h after attacking） ,CsA and CsA＋ glycochenodeoxycholic acid was used to protect hepatocytes, respectively, and morphological changes in cells were observed with inverted phase contrast microscope, and the live cells were counted by 3 - （4,5 - dimethyl - 2 - thiazolyl） - 2,5 - diphenyl - 2 - H - tetrazolium bromide （MTT） method, and aspertate aminot ransferase （AST） and alanine aminotransferase （ALT） in the culture supernatant were detected by biochemical method. Results Compared with the control group, hepatocellular growth in the injury group was obviously suppressed, with progressive cel- lular apoptosis and significantly decreased absorbance value of M＇IT（0. 345 ± 0. 021 ） ;the activity of AST and ALT in- creased gradually,reaching the highest after 72 h [ （98.4 ±6.7） U/L and （ 116.2 ±9.5） U/L,respectively]. Compared with injury group, broken organelles decreased significantly and absorbance value elevated in glycochenodeoxycholic acid group,CsA group and CsA combined with cholic acid group,and at each time point,the highest absorbance value in the CsA＋ tanrochenodeoxycholic acid group [ the highest was （0.656 ± 0.014）, P 〈 0.05 ] ; at the same time, the activity of AST and ALT didn＇t increase obviously, at each time point, the lowest in CsA ＋ tanrochenodeoxycholic acid group [ the lowest was （22.3 ± 6.2） U/L and （20.2 ± 5.4） U/L, P 〈 0. 05, respectively 1- Conclusions CsA, as well as cholic acid, can protect human liver cells from the attack of a - AMA. The mechanism may be CsA, as an organic anion transfer peptide in humans （OATP1 B1 and OATP1 B3 ） suppressant,inhibits the absorption of a -AMA. The joint application of CsA and taurochenodeoxycholic acid is superior to the single OATP substrate or inhibitor.
出处
《中华实用儿科临床杂志》
CSCD
北大核心
2016年第7期503-506,共4页
Chinese Journal of Applied Clinical Pediatrics
基金
四川省卫生厅课题（070098）
关键词
环孢素
胆酸
人肝细胞
鹅膏毒肽
损伤
Cyclosporine A
Cholic acid
Human liver cell
Amanitin
Damage
