CGRP预培养对肝细胞冻存损伤的保护作用及机制

Effect of Preincubation with CGRP on Impairment of Human Hepatocyte Induced by Cryopr- eservation

【目的】探讨降钙素基因相关肽（CGRP）预培养对冻存肝细胞的保护作用与机制。【方法】外科切除肝组织分成5组：①对照组（DMEM预培养0h）；②标准预培养组（DMEM预培养8h）；③CGRP三个浓度预培养组（10^-6mol／L，10^-7mol／L，10^-8mol／L；8h）。采用胶原酶灌注法分离肝细胞，经预培养后冻存5周，复苏，台盼蓝检测新鲜分离肝细胞成活率，并体外培养肝细胞48h，用MTT法观察肝细胞活力，收集细胞培养上清检测谷白蛋白（Alb），乳酸脱氢酶（LDH）水平，检测肝细胞内活性氧（ROS）及丙二醛（MDA）含量。【结果】预培养组及各CGRP（10^-6，10^-7，10^-8）处理组细胞成活率（53±5，63±6，61±6，58±7）、Alb合成（4．8±0．6，6．5±0．7，5．8±0．7，4．9±0．6）及MTT活性（0．25±0．07，0．38±0．06，0．31±0．05，0．22±0．03）较对照组（46±7，3．9±0．6；0．11±0．01）显著增加（P〈O．05），且CGRP（10^-6，10^-7）处理组细胞成活率、Alb合成及MTT活性显著高于预培养组（P〈0．05）；预培养组及各cGRP（10^-6，10^-7，10^-8）处理组肝细胞复苏后48h内LDH释放（25±7，15±4，17±5，18±6），ROS释放（107士9．5，62±4．8，70±7．1，80±5．8）及MDA水平（0．87±0．1，0．70±0．06，0．76±0．07，0．80±0．07）较对照组（31±6，120±8．5，1．31±0．04）显著减少（P〈O．05），且与预培养对照组比较，CGRP（10^-6，10^-7，10^-8）各处理组LDH及ROS释放显著减少（P〈0．05），CGRP（10^-6，10^-7 。）处理组MDA释放显著减少（P〈0．05）。【结论】cGRP预培养对冻存引起的肝细胞损伤具有保护作用，其机制与抗氧化应激损伤有关。

[Objective]To explore the effect of preincubation with calcitonin gene--related peptide （CGRP） on impairment of human hepatocyte induced by cryopreservation. [[Methods] Human liver tissue was obtained from patients undergoing partial hepatectomy. Collagenase digestion method was used to isolate human hepato- cytes. Hepatocytes were randomly divided into five groups, including control group （preincubated with DMEM for Oh）, standard group （preincubated with DMEM for 8h） and three CGRP treated groups （preineu- bated with CGRP for 8h）, then were cryopreserved. Hepatoeytes were stored at -- 140~C for 5 weeks until thawing, and the level of Alb, LDH, MDA, ROS and the viability and proliferation of hepatoeytes were deter- mined. [Results] Standard group and three CGRP treated groups （10-6 , 10-7 , 10-8 ） had significantly higher viability（53q-5. 2,62. 7±6.1,60. 5±5. 7,58±6. 8）, Alb production （4.8~0.6,6.5±0.7,5.8±0.7,4.9±0. 6） and MTT proliferation（0.25±0.07,0. 38±0.06,0. 31±0.05,0. 2210.03）than control group（46＋6.8,3. 9＋0. 6;0.11＋0. 01）（ P d0, 05）. CGRP treated groups（10-6,10 7,10-8） had significantly higher viability, Alb production and MTT proliferation than standard group（ P d0.05）. Standard group and three CGRP trea- ted groups（10-6 ,10 7,10-8） had significantly lower LDH leakage（25.1±6.9,15±3.8,16.8±5.0,17.9± 5.6） ,level of ROS （107±9.5,62±4.8,70±7.1,80±5.8）and MDA （0. 87±0.1,0. 70±0.06,0, 76±0.07, 0. 80± 0.07） than control group （31.4 ± 6. 5,120 ± 8. 5,1.31 ± 0. 04）（ P （0. 05）. CGRP treated groups （10-6 , 10-7 , 10-8） had significantly lower LDH and ROS leakage than standard group（ P〈0.05）. Compared with standard group, the level of MDA in CGRP treated groups （10-6 , 10-7） decreased markedly（ P〈0. 05）. [Conclusion]Preincubation of human hepatocyte with CGRP prior to cryopreservation can improve viability and function upon thawing through decreasing the release of MDA and ROS.