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当归多糖对衰老模型大鼠脾脏结构与功能的影响
Effects of angelica sinensis polysaccharide on the spleen structure and function of aging rats
目的 探讨当归多糖（ASP）对衰老模型大鼠脾脏结构与功能影响及其机制。 方法 SD大鼠随机分为正常对照组，ASP对照组，衰老模型组，ASP衰老模型组，每组10只。衰老模型组皮下注射D-半乳糖（120mg/kg）qd×42d；ASP衰老模型组注射D-半乳糖的剂量与时间同衰老模型组，第15天起腹腔注射ASP (100 mg/kg) qd×28d；正常对照组皮下注射等量生理盐水qd×42d；ASP对照组注射等量生理盐水qd×14d，第15天起腹腔注射ASP（同ASP衰老模型组)。药物注射完成后第2天，取脾脏测定脾指数，石蜡切片观察脾脏显微形态学；衰老β-半乳糖苷酶（SA-β-Gal）染色观察脾细胞百分率，CCK8测定脾细胞对刀豆蛋白A刺激的增殖能力；流式细胞术检测脾细胞周期与活性氧簇（ROS）含量； ELISA检测脾细胞分泌TNF-α、GM-CSF能力与丙二醛（MDA）、超氧化物歧化酶(SOD)含量；Western blotting 检测衰老相关蛋白P53、P21、RB蛋白表达。 结果 与正常对照组比较，D-半乳糖致衰老模型组大鼠脾指数，脾脏白髓面积比例，脾细胞对刀豆蛋白A刺激的增殖能力，S期比例及TNF-α、GM-CSF的分泌能力，SOD活性明显降低；脾细胞的SA-β-Gal阳性率，G1期与G2/M期细胞比例，ROS、MDA含量，P53、P21、RB蛋白表达显著增高。与衰老模型组比较，ASP使D-半乳糖致衰老模型大鼠脾指数，脾脏白髓面积比例，脾细胞对刀豆蛋白A刺激的增殖能力，S期比例与TNF-α、GM-CSF的分泌能力，SOD活性明显提高；脾细胞SA-β-Gal阳性率，G1期及G2/M期细胞比例，ROS、MDA含量，P53、P21、RB蛋白表达有显著抑制作用。
结论 D-半乳糖复制的衰老模型大鼠脾脏结构与功能损伤明显，当归多糖对其致衰损伤有明确的保护作用。
Objective To explore the effect of angelica sinensis polysaccharide(ASP) on the spleen structure and function of aging rats，and provide theoretical and experimental evidences for seeking the effective ingredient from natural medicine to delay immune system aging. Methods Forty SD rats were randomly divided into normal control group, ASP control group, aging model group and ASP aging model group. Aging model group were treated with D-galactose ［120 mg/(kg·d)］ for 42 days by subcutaneous injection. ASP aging group were also injected with D-galactose with the same dose and time as aging model group, and from the 14th day on, rats were given ASP (100 mg/kg) by intraperitoneal injection for 28 days. Normal control group were received saline with the same volume for 42 days. ASP control group were given saline with the same volume for 14 days, and received ASP (100mg/kg) by intraperitoneal injection for 28 days. After 2 days of finishing the treatment,the spleen index was measured, paraffin section was made to observe spleen microscopic structures；The ratio of the SA-β-Gal staining positive splenocytes were counted；the proliferative capacity of splenocytes with stimulating by concanavalin A (Con A) was detected by CCK8；the distribution of cell cycles and ROS levels was analyzed by flow cytometry(FCM)；The capability of splenocyte to secrete TNF-α, GM-CSF, malondialdehyde(MDA), superoxide dismutase (SOD)were assayed with ELISA；The aging related protein P53, P21, RB were detected by Western blotting. Results Comparing the aging rats induced by D-galactose with normal control group, the following biological features of spleens in aging model group: spleen index, splenic white pulp area proportion, the proliferative capacity of splenocytes stimulated by Con A, the ratio of S stages in cell cycle , the secretory capability of TNF-α and GM-CSF, and the active content of SOD, were obviously decreased. The percentage of SA-β-Gal positive cells, the ratio of G1 and G2/M stages, the product of ROS and MDA in splenocytes were significantly increased. The expressions of P53, P21, RB were up-regulated. Comparing ASP aging group with aging model group, ASP remarkably increased spleen index, splenic white pulp area proportion, the proliferation of splenocytes stimulated by Con A, the ratio of S stages, the secretory capability of TNF-α and GM-CSF, the active content of SOD. ASP obviously increased the percentage of SA-β-Gal positive cells, the ratio of G1 and G2/M stages, the product of ROS and MDA in splenocytes, and down-regulate the expression of P53, P21, RB. Conclusion The spleen structure and function are obviously damaged in D-galactose-induced aging model rats.ASP has definitely protective effects on the injury.
当归多糖 / D-半乳糖 / 衰老模型 / 脾脏 / 免疫印迹法 / 大鼠
Angelica polysaccharide / D-galactose / Aging model / Spleen / Western blotting / Rat
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