阿片类药物长期作用对μ-中国仓鼠卵细胞基因表达的影响及与PC12细胞线粒体膜电势之间的关系

Effects of Long- term Treatment with Opiates on μ- CHO Gene Expression and Relationship with Mitochondrial Membrane Potential in PC12 Cells

目的 从基因水平入手，筛选 DAMGO长期作用后μ-中国仓鼠卵细胞 (μ- CHO)差异表达的基因，初步研究阿片类药物与 PC12细胞线粒体膜电势之间的关系。方法 选用μ受体特异性激动剂 DAMGO长期（ 72 h）作用于μ- CHO细胞，模拟耐受成瘾。利用差异显示 PCR、克隆、 Northern印迹分析等找出表达水平有改变的基因，测序，并与 NIH Blast数据库中序列比对。利用激光共聚焦显微成像、线粒体膜电势测定等方法，观察吗啡短期与长期作用 PC12细胞线粒体膜电势的变化。结果其中一条基因在用 DAMGO长期刺激μ- CHO细胞后基因水平出现上调，并与大鼠线粒体膜上 H＋ /磷酸基同向转运蛋白编码基因同源性很高。线粒体膜电势测定发现，用 10－ 6～ 10－ 4 mol/L吗啡短时间（ 2 h）作用 PC12细胞后，细胞内线粒体膜电势部分丧失或完全丧失；长时间（ 12～ 60 h）作用后，膜电势出现从部分丧失、完全丧失到逐步恢复的现象，上述现象均能被阿片受体拮抗剂纳洛酮阻断。结论 初步提示阿片类药物长期作用含有阿片受体的细胞后，可导致细胞线粒体功能改变，推测阿片类药物可能影响细胞线粒体电子转移、 ATP合成过程，此过程可能与阿片类药物长期激活阿片受体导致的成瘾、耐受有关。

Objective To screen the specific gene expression after long- term treatment with DAMGO inμ- CHO from gene level and observe the relationship between opiates and cellular mitochondrial membrane potential in PC12 cells. Methods μ- CHO cells were exposed toμ- receptor selective agonist DAMGO for 72 hours to develop addiction and tolerance. The specific expressed genes were screened out by differential display PCR, cloning and Northern blot， and were sequenced. The sequences were compared with the database of NIH NCBI. The relationship between short- term (2 hours) and long- term (12～ 60 hours) treatment with morphine and mitochondrial membrane potential in PC12 cells was determined by TMRE staining using confocal laser scanning microscopy. Results One of the sequences was up- regulated after treatment with DAMGO. It was homologous with rat mitochondrial proton/phosphate symporter mRNA. The changes of mitochondrial membrane potential in PC12 cells after exposure to morphine were observed. The potential was partly or totally lost after short- term exposure. However, the loss of the potential induced by the short- term exposure to morphine was gradually recovered by long- term exposure. The effect of morphine induced by both short- and long- term exposure could be blocked by naloxone. Conclusions Opiates could probably lead to dysfunction of electron transport and ATP production in mitochondria. It might be associated with receptor addiction and tolerance.