Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports....Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.展开更多
目的观察大麻素2型受体(CB2R)激动剂JWH-015对阿尔茨海默病(AD)小鼠认知功能下降的改善作用,并探讨其作用机制是否与脑内小胶质细胞激活表型转化密切相关。方法 20只成年雄性C57BL/6J小鼠随机分为溶剂对照组、JWH-015对照组、AD模型组和...目的观察大麻素2型受体(CB2R)激动剂JWH-015对阿尔茨海默病(AD)小鼠认知功能下降的改善作用,并探讨其作用机制是否与脑内小胶质细胞激活表型转化密切相关。方法 20只成年雄性C57BL/6J小鼠随机分为溶剂对照组、JWH-015对照组、AD模型组和JWH-015治疗组。侧脑室注射4μg寡聚态β-淀粉样蛋白1-42(Aβ1-42)和等体积生理盐水构建AD模型和模型对照组后,分别接受连续3周腹腔注射JWH-015 0.5 mg/kg或等量溶剂处理。采用新物体识别实验检测小鼠认知功能;实时定量PCR方法检测皮质、海马脑区M1型小胶质细胞标记分子诱导型一氧化氮合酶(i NOS)和M2型小胶质细胞标志物几丁质酶3样蛋白(Ym1/2)m RNA表达水平。同时,取15只CB2R基因敲除C57BL/6J小鼠(CB2RKO)和5只同窝野生型(CB2RWT)小鼠按基因型和处理随机分为CB2RKO溶剂对照组、CB2RKO AD模型组、JWH-015处理组和CB2RWT小鼠溶剂对照组,用来研究CB2R对认知功能改善作用和小胶质细胞激活表型转化作用的特异性。结果与溶剂对照组相比,C57BL/6J AD模型组小鼠新奇物体识别指数显著降低(P<0.01),同时伴随皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05)和Ym1/2 m RNA表达显著降低(均为P<0.01);而JWH-015处理可显著提高C57BL/6J AD模型组小鼠新物体识别指数(P<0.05),下调皮质和海马区i NOS m RNA表达水平(均为P<0.05),上调Ym1/2 m RNA表达水平(均为P<0.05);与脑室注射生理盐水的CB2RKO溶剂对照组小鼠相比,CB2RKO AD模型组小鼠识别指数显著降低(P<0.05),皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05),皮质脑区Ym1/2 m RNA表达显著降低(P<0.05);与CB2RKO AD模型组小鼠相比,JWH-015处理组长时程给药对新奇物体识别指数无显著影响,并对皮质、海马脑区内i NOS及Ym1/2 m RNA的表达水平无显著影响。结论 JWH-015通过特异性激动CB2R改善Aβ诱导的AD模型小鼠的认知能力下降,其机制可能与其直接作用于相关脑区小胶质细胞CB2受体,引起小胶质细胞M1/M2表型转化,减轻脑内小胶质细胞介导的神经源性炎症反应有关。展开更多
基金supported by a grant from Army Medical Research Program of China(No.08G168)
文摘Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.
文摘目的观察大麻素2型受体(CB2R)激动剂JWH-015对阿尔茨海默病(AD)小鼠认知功能下降的改善作用,并探讨其作用机制是否与脑内小胶质细胞激活表型转化密切相关。方法 20只成年雄性C57BL/6J小鼠随机分为溶剂对照组、JWH-015对照组、AD模型组和JWH-015治疗组。侧脑室注射4μg寡聚态β-淀粉样蛋白1-42(Aβ1-42)和等体积生理盐水构建AD模型和模型对照组后,分别接受连续3周腹腔注射JWH-015 0.5 mg/kg或等量溶剂处理。采用新物体识别实验检测小鼠认知功能;实时定量PCR方法检测皮质、海马脑区M1型小胶质细胞标记分子诱导型一氧化氮合酶(i NOS)和M2型小胶质细胞标志物几丁质酶3样蛋白(Ym1/2)m RNA表达水平。同时,取15只CB2R基因敲除C57BL/6J小鼠(CB2RKO)和5只同窝野生型(CB2RWT)小鼠按基因型和处理随机分为CB2RKO溶剂对照组、CB2RKO AD模型组、JWH-015处理组和CB2RWT小鼠溶剂对照组,用来研究CB2R对认知功能改善作用和小胶质细胞激活表型转化作用的特异性。结果与溶剂对照组相比,C57BL/6J AD模型组小鼠新奇物体识别指数显著降低(P<0.01),同时伴随皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05)和Ym1/2 m RNA表达显著降低(均为P<0.01);而JWH-015处理可显著提高C57BL/6J AD模型组小鼠新物体识别指数(P<0.05),下调皮质和海马区i NOS m RNA表达水平(均为P<0.05),上调Ym1/2 m RNA表达水平(均为P<0.05);与脑室注射生理盐水的CB2RKO溶剂对照组小鼠相比,CB2RKO AD模型组小鼠识别指数显著降低(P<0.05),皮质和海马脑区i NOS m RNA表达显著升高(均为P<0.05),皮质脑区Ym1/2 m RNA表达显著降低(P<0.05);与CB2RKO AD模型组小鼠相比,JWH-015处理组长时程给药对新奇物体识别指数无显著影响,并对皮质、海马脑区内i NOS及Ym1/2 m RNA的表达水平无显著影响。结论 JWH-015通过特异性激动CB2R改善Aβ诱导的AD模型小鼠的认知能力下降,其机制可能与其直接作用于相关脑区小胶质细胞CB2受体,引起小胶质细胞M1/M2表型转化,减轻脑内小胶质细胞介导的神经源性炎症反应有关。
