苦参碱对冻存人胎肝细胞p38MAPK和JNK通路表达的影响.docVIP

摘 要 目的：使用不同浓度的苦参碱对人肝细胞的冻存过程进行干预, 观察肝细胞、、及的表达，明确苦参碱对人肝细胞冻存的保护作用，并从分子水平其作用机制。为肝细胞的高效冻存方法提供新思路，为生物型反应器的构建、肝细胞库的建立及肝细胞移植。 方法：本实验将5组不同浓度的苦参碱加入人肝细胞的统冻存液中进行超低温冻存。同时设立一组常规冻存方法组为对照组。对复苏后的人肝细胞进行ALBALT、LDH检测，比较冻存前后肝细胞功能状况。检测冻存前后苦参碱组及常规方法冻存组肝细胞的活。采用免疫印迹法(WesternBlot法)检测人肝细胞冻存前后的表达。 结果：64mg/L组外, 各苦参碱冻存组复苏后的胎肝细胞的活力、、 ALBALT、LDH的表达均优于对照组。 其中8mg/L组效果最好, 有明显统计学意义(P0.01)。利用免疫印迹法观察到复苏后的各组人胎肝细胞p38M APK和JNK的含量均比冻存前增加, 冻存液中加入苦参碱后, 复苏后p38M APK和JNK的含量均比对照组有所下降, 8mg/L组尤为明显, 有明显统计学意义(P0.01), 结果提示冻存增加肝细胞JNK 和p38MAPK的表达, 而加入苦参碱后, JNK 和p38M APK的表达明显比对照组下降, 提示苦参碱对人胎肝细胞的保护作用可能是通过抑制JNK和p38MAPK的转导通路激活来完成。然而, 64mg/L组无论肝细胞的活性、功能、p38M APK和JNK表达都差于对照组, 提示苦参碱对胎肝细胞冻存的保护作用可能有剂量依赖性, 64mg/L组可能抑制p38MAPK和JNK通路的表达。 结论：苦参碱对冻存人肝细胞有好的保护作用, 其中8mg/L组效果最好。剂赖, 苦参碱浓64mg/L时, 对冻存人胎肝细胞可能有抑制作用。苦参碱通过抑制JNK 和p38转导通路保护人肝细胞超低温冻存时的损伤。关键词：苦参碱；冻存；人肝细胞；JNK ；p38ABSTRACT Objective: The different concentration matrine was added to human fetal hepatocytes from cryopreservation. By detecting p38 MAPK and JNK, to find protection mechanism of the human fetal hepatocytes on molecule level. Just to find way to preserve human fetal hepatocytes from cryopreservation effectively, and to establish platform for biological reactor, hepatocyte library and transplantation of hepatocyte. Methods: Human fetal hepatocytes were stored from cryopreservation in five different matrine concentration, with a normal cryopreservation as control. Expression of p38 MAPK and JNK in human fetal hepatocytes before and after cryopreservation were measured by Western-Blot. Results： livability, adhesion, morphological manifestation and cell function, and in 8mg/L matrine was the most obvious. Expressions of p38 MAPK and JNK after thawing were higher than before thawing, but lower when added with Matrine. It hints that cryopreservation prompts the expression of p38MAPK and JNK in human fetal hepatocytes, and matrine inhibits the activation of conduction pathway. Conclusions: Matrine at some concertration has a well protective effect on the hepatocytes and 8mg/L