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目的:通过体内外观察华蟾素蟾毒配基对免疫相关细胞因子的影响,分析药物对免疫系统的调节作用,为阐明免疫机制提供药效学依据。方法:无菌分离小鼠脾淋巴细胞制备淋巴细胞悬液,设置多个华蟾素蟾毒配基浓度组进行给药,MTT法检测LPS和ConA刺激小鼠脾淋巴细胞增殖;用预孵育淋巴细胞6 h后加入Con A或LPS诱导细胞活化,通过基于流式细胞术的高通量多因子检测技术对细胞上清液进行GM-CSF、TNF-α,IFN-γ,IL-1α,IL-2,IL-4,IL-5,IL-6,IL-10多细胞因子检测以评价药物体外免疫效果。建立AsP C-1胰腺癌荷瘤裸鼠动物模型,待给药一定时间后取小鼠血清,利用流式细胞仪通过相同方法对血清进行相同细胞因子检测,评价其体内免疫效果。结果:华蟾素蟾毒配基单独作用和与刺激剂协同刺激淋巴细胞结果发现,与ConA协同作用时能刺激淋巴细胞分泌INF-γ、GM-CSF、IL-1α、IL-4、IL-5和IL-6,单独作用能刺激分泌TNF-α、IL-2、IL-4和IL-5,而与LPS协同作用除了刺激TNF-α和IL-6分泌外,则抑制INF-γ、GM-CSF、IL-4和IL-5的分泌。0.05和10μg/ml为易引起药物刺激或抑制作用变化的浓度组。药物体内作用胰腺癌荷瘤裸鼠免疫效果观察发现,与吉西他滨和华蟾素注射液相比,蟾毒配基能更多的增强细胞因子的表达,1mg/kg浓度组对脾脏有一定的保护作用,而2mg/kg浓度组对脾脏的作用与华蟾素注射液和吉西他滨相当。结论:华蟾素蟾毒配基在体外可一定程度促进B淋巴细胞增殖,体内能引起多种细胞因子的分泌,增强机体的免疫功能,但作用机制以及体内免疫作用还有待进一步研究。
OBJECTIVE: To observe the influence of cinobufogenin on immune-related cytokines in vivo and in vitro and to analyze the regulatory effect of drugs on the immune system and to provide pharmacodynamic evidence for elucidating the immune mechanism. Methods: Splenic lymphocytes were aseptically isolated from mouse spleen lymphocytes, and multiple concentrations of cinobufagin were administrated. The proliferation of splenic lymphocytes was stimulated with LPS and ConA by MTT assay. Cells were treated with Con A or LPS for 6 h to induce cell activation. The supernatants of GM-CSF, TNF-α, IFN-γ, IL-1α and IL were detected by high-throughput multifactorial flow cytometry -2, IL-4, IL-5, IL-6 and IL-10 were measured to evaluate the in vitro immune response. Animal models of AsP C-1 pancreatic cancer bearing nude mice were established. After a certain period of time, the serum of mice was taken and the same cytokines were detected by flow cytometry in the same way to evaluate the immune effect in vivo. Results: The cinobuflastin alone and in combination with the stimulant stimulated lymphocytes. It was found that synergistic effect with ConA stimulated the secretion of INF-γ, GM-CSF, IL-1α, IL-4 and IL- 5 and IL-6, alone can stimulate the secretion of TNF-α, IL-2, IL-4 and IL-5, synergistic with LPS in addition to stimulating the secretion of TNF-α and IL-6, Secretion of GM-CSF, IL-4 and IL-5. 0.05 and 10μg / ml are easy to cause changes in drug stimulation or inhibition concentration group. Drugs in vivo Pancreatic cancer tumor-bearing nude mice immune effect observed and gemcitabine and cinobufotalin injection, bufaloduol can enhance the expression of cytokines, 1mg / kg concentration group has a certain protection of the spleen While the effect of 2mg / kg concentration on spleen was comparable to that of cinobufacini injection and gemcitabine. CONCLUSION: Bupivacaine can promote the proliferation of B lymphocytes in vitro to a certain extent, which can induce the secretion of various cytokines in vivo and enhance the immune function of the body. However, its mechanism of action and in vivo immune function remain to be further studied.