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大鼠鞘内泵入吗啡对大鼠细胞免疫功能的影响

时间:2010-08-24 11:36:23  来源:  作者:

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Effect of intrathecal pumping morphine on spleen T lymphocyte proliferation and NK cell activity in rats

 

郭曲练  邹望远  王锷  蔡进

GUO QulianZOU WangyuanWANG Eet al.Department of AnesthesiologyXiangya HospitalCentral South Universitychangsha 410008China

 

Abastrat

  ObjectiveTo evaluate the effct of intrathecal pumping different dosage morphine on cellmediated immunity.

  MethodsForty SpragueDawley rats290±30gwere randomly divided into 8 groupsn8 in each group):saline groupNS),sham controlF),morphine groupsM1group 10μg/h)、M2 group 5μg/h)、M3 group2.5μg/h.Which were anesthetized with 10 Chlroral Hydrate 300350mg/kg.A microspinal catheterinner diameter 0.12mmouter diameter 0.35mm was inserted into the lumbar subarachnoid space. After five daysan Alzet? osmotic minipump was connected tightly with a polyurethane catheter which inner dimeter is about 0.36mm. 5 formalin50μlwas injected <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />s.c. into plantar suface of left hindpaw of the rats using a microsyringe with a 26gauge needlepain intensity scoringPISwas utilized to assess antinociceptive effect of morphine. And spleens were aseptically removed to obtain splenic cells. T lymphocyte function was evaluated based on ConcanavalinAConAinduced splenocyte proliferation. A modified lactic acid dehydrogenaseLDHrelease assay was to assess NK cell activity.

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  Result1Compare with NS and sham groupmorphine groupsPIS is decreased markedlyP<0.01 in the early and late phase of formalin painand showing dosedependent;(2After pumping morphine 7 daysspleen index splenocyte proliferation induced by ConA and NK cell activity were significantly suppressed by intrathecal pumping morphine.

  ConclusionThere was significant antinociception of intrathecal pumping morphine suppressed T lymphocyte proliferation and NK cell activity dosedependently.

  key wordsMorphineIntrathecal injectionT lymphocyte proliferationNatural killer cell activityImmunesuppression

 

  吗啡是阿片样镇痛药的代表,研究表明吗啡对机体免疫有抑制作用,如抑制T淋巴细胞增殖水平、调节T淋巴细胞表面抗原的表达,抑制NK细胞活性等[12]。但这些多是通过全身(腹腔、皮下)给药下得到的研究结果。吗啡鞘内给药是一个临床广泛使用的镇痛方式,但其对免疫功能的影响尚无定论。本研究拟在大鼠鞘内以不同速率持续泵入吗啡后,构建福尔马林炎性疼痛模型,观察其镇痛效果及对免疫功能的影响。 

 

材料与方法

  主要仪器和试剂  Alezt泵(200μlModel2001 美国),Microspinal导管(内径0.12mm,外径0.35mm,美国),β液体闪烁计数仪(LS6500BECKMAN,美国),酶联免疫检测仪(ELx800UV,美国),二氧化碳培养箱(Queue,日本)。ConA(刀豆蛋白)(Vector,美国),3HTDR(甲基-3H胸像嘧啶核苷)(中国科学院上海原子核研究所),LDH细胞毒试剂盒(Bivision 美国),胎牛血清(杭州四季青生物工程材料有限公司),Dhanks RPMI1640GIBCO,美国)。细胞株:小鼠淋巴瘤(YAC1)细胞株(本学院免疫学教研室燕美玉老师惠赠)。

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  方法  40250300g纯种清洁级雄性SD大鼠,随机分为5组(n8):假手术组(F组)、生理盐水组(NS组),吗啡组按给药速率不同分为三个亚组,给药速率分别为2.5μg/h5μg/h10μg/h,记为M1M2M3组。大鼠腹腔注射10%的水合氯醛300350mg/kg麻醉后,按改良Yaksh[3]鞘内置入Microspinal 导管至脊髓腰椎部(插入深度约8.5cm),逐层缝合伤口。F组即暴露寰枕膜并挑开,看到清亮的脑脊液,但并不置管。置管5天后Alzet泵注射吗啡、生理盐水,将泵容量调节器与聚乙烯管紧密相接,植入到大鼠背部皮下合适位置,鞘内持续泵入7d。大鼠处死前1h参照文献[4]的方法进行福尔马林实验评估镇痛效果  将大鼠置于透明玻璃缸内,适应环境30min,用100μl微量注射器在大鼠左足跖皮下注射5%的福尔马林50μl。双盲法观察行为学变化,每5min记录一次,持续一小时。将大鼠炎性痛分为四级,0级:两后脚掌平放在地面,活动无异常;1级:注射脚掌轻微接触地面,活动时有跛行;2级:注射脚掌抬起,不接触地面;3级:大鼠舔咬或摇动注射脚掌。痛级(PIS)评分计算方法[4]PIST12×T2+3×T3/5×60,其中T1T2T3分别是5分钟内出现123级的时间。PIS评分后处死大鼠,用电子天平称取体重及脾脏重量,计算脾脏指数=脾脏重量(mg/体重(g)。

  脾脏单个核细胞悬液的制备  大鼠处死后,无菌取脾置于80目不锈钢筛网中,剪碎脾后,轻轻捻磨脾脏,使分散的细胞滤过金属网进入无菌平皿中;用200目的钢筛过滤;离心弃上清;双蒸水破解红细胞;Dhanks液洗涤细胞23次,用含1mM/L的谷氨酰胺、100IU/ml青霉素G100μg/ml链霉素、10%的胎牛血清的RPMI1640重悬细胞,调节细胞浓度为5×106/ml,台盼兰染色检测细胞存活率大于95%;接种至培养板中。

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