褪黑素对鞘内泵入吗啡致免疫功能抑制大鼠免疫功能的影响

目的本实验采用ALZET泵建立鞘内持续泵入吗啡所致的免疫功能抑制大鼠模型,腹腔注射褪黑素观察其对鞘内泵入吗啡大鼠免疫功能的影响。方法 32只SD大鼠随机分为4组：生理盐水组（NS组,n=8）、吗啡组（M组,10μg/h,n=8）,吗啡组＋褪黑素组（M＋MLT组,n=8）,吗啡组＋生理盐水组（M＋NS组,n=8）。用microspinal导管根据改良Yaksh法进行鞘内置管,连接ALZET泵,持续泵入吗啡、生理盐水,MLT组连续7 d腹腔注射褪黑素,每次时间点为7：00-9：00 am,5 mg/kg。M＋NS组连续7 d腹腔注射生理盐水。7 d后原代分离脾脏单个核细胞进行培养,采用3H-TDR（甲基3H胸腺嘧啶核苷）掺入法检测脾T淋巴细胞对刀豆蛋白（ConA）刺激诱导的增殖水平;乳酸脱氢酶（LDH）释放法检测脾NK细胞活性;流式细胞仪检测脾T淋巴细胞亚群（CD3＋、CD3＋CD4＋、CD3＋CD8＋、CD4＋/CD8＋）和NK细胞（CD161＋）表型变化。结果与NS组相比较,泵入吗啡7 d后M组T淋巴细胞增殖水平、NK细胞活性降低（P〈0.05）,M组CD3＋、CD3＋CD4＋、CD3＋CD8＋细胞数量及百分率降低,CD4＋/CD8＋降低,CD161＋降低（P〈0.05）;同M＋NS组比较,M＋MLT组T淋巴细胞增殖水平、NK细胞活性升高（P〈0.05）,M＋MLT组CD3＋、CD3＋CD4＋、CD3＋CD8＋数量及百分率升高,CD4＋/CD8＋升高,CD161＋升高（P〈0.05）。结论鞘内泵入吗啡（10μg/h）可明显抑制大鼠细胞免疫功能;褪黑素可改善鞘内泵入吗啡所致的免疫功能抑制大鼠细胞免疫功能。

Effect of Melatonin on Immune Function in Immunosuppressed Rats Induced by Intrathecal Pumping Morphine

Objective To evaluate the effect of melatonin on immune function in immunosuppressed rats caused by intrathecal pumping morphine.Methods Thirty-two Sprague-Dawley rats were randomly divided into 4 groups(each n=8): saline(NS) group,morphine(M,10 μg/h) group,morphine plus melatonin(M+MLT) group and morphine plus saline(M+NS) group.The rats were anesthetized with 10% intraperitoneal chlroral hydrate 300~350 mg/kg.A microspinal catheter(inner diameter 0.12 mm,outer diameter 0.35 mm) was inserted into the lumbar subarachnoid space(8 cm).And after five days,an ALZET osmotic minipump was connected tightly with a polyurethane catheter whose inner diameter was about 0.36 mm.Melatonin(5 mg/kg) was intraperitoneally injected for 7 days.Spleens were aseptically removed to obtain splenic cells.T lymphocyte function was evaluated based on concanavalin-A(ConA) induced splenocyte proliferation.A modified lactic acid dehydrogenase release assay was used to assess NK cell activity.And phenotypic expression of cell surface markers of T lymphocyte subsets(CD3+,CD3+CD4+,CD3+CD8+,CD4+/CD8+) and NK cell(CD161+) in the spleen was analyzed by flow cytometry.Results As compared with NS group,after pumping morphine 7 days,spleen indexes and splenocyte proliferation induced by ConA and NK cell activity were significantly suppressed by intrathecal pumping morphine.Phenotypic expression of T lymphocyte subsets and NK cell in all morphine groups assessed by flow cytometry were different from control group.The results showed that 5 mg/kg(ip) melatonin was able to recover the lowered splenocyte proliferation,NK cell activity and T lymphocyte subsets and NK cell phenotype in immunosupressed rats.Conclusions Intrathecal pumping morphine can significantly inhibit lymphocyte proliferation and NK cell activity,decrease the number of splenic lymphocyte,and alter phenotypic expression of cell surface markers dose-dependently.Melatonin can recover NK cell activity,lymphocyte proliferative capacity and phenotypic expression of T lymphocyte subsets and NK cell,but the mechanism deserves further investigation.