4种脾虚造模法的横向对比研究

A comparative study of four types of spleen deficiency modeling method

[目的]通过对4种常见脾虚小鼠造模方法进行横向对比研究,评价各造模法脾虚损伤程度及其适用性。[方法]100只小鼠随机分为空白对照组(去离子水每只每天0.5 mL)、苦寒泻下组(100%番泻叶水浸液每只每日0.5 mL)、饮食失节组(喂饲甘蓝并每2日加喂猪脂1次)、劳倦过度组(每日捆尾力竭游泳1次)、利血平组(1%利血平,每日2 mL/kg,皮下注射),每组20只。持续造模10 d后,每组随机抽取10只取材,做相关指标检测,10只自然恢复10 d,通过对宏观体征、进食量、体质量及游泳时长的测定,观察各组宏观指标的稳定性。计算各组胸腺、脾脏指数,血液分析仪检测血液学指标,比色法测定血清D-木糖水平,碘-淀粉酶比色法测定血清淀粉酶活性,实时荧光定量PCR法检测相对mtDNA拷贝数及线粒体自噬相关蛋白Pink1、Parkin mRNA的表达,对各组小鼠进行线粒体功能评价。[结果]与空白对照组相比:1)苦寒泻下组、饮食失节组,利血平组宏观指标表现差异明显(P<0.05),以利血平组最为显著,指标稳定性以苦寒泻下组显著(P<0.05),差异均具有统计学意义。2)苦寒泻下组、劳倦过度组、利血平组胸腺指数明显降低(P<0.05),差异具有统计学意义,劳倦过度组变化显著;苦寒泻下组与利血平组脾脏指数明显降低(P<0.05),差异具有统计学意义,利血平组变化显著。3)利血平组和苦寒泻下组红细胞(RBC)、白细胞(WBC)、血红蛋白(HGB)下降明显,差异有统计学意义(P<0.05)。4)各模型组小鼠血清D-木糖水平均降低(P<0.05),差异具有统计学意义。5)苦寒泻下组、饮食失节组、利血平组血清淀粉酶活性均有所降低(P<0.05),差异具有统计学意义,利血平组最为显著。6)苦寒泻下组、饮食失节组、劳倦过度组mtDNA拷贝数均有所降低(P<0.05),差异具有统计学意义,饮食失节组最为显著;苦寒泻下组和饮食失节组线粒体自噬相关蛋白Pink1 mRNA转录水平均有所降低(P<0.05),差异具有统计学意义;利血平组Parkin mRNA转录水平有所上升(P<0.05),差异具有统计学意义。[结论]利血平组宏观指标和理化指标变化明显,但宏观指标稳定性差,其造模法为短期造模的首选。苦寒泻下组宏观指标、理化指标及线粒体功能变化明显,宏观指标稳定性好,其造模法,适合多种脾虚指标的广泛研究使用。饮食不节组理化指标和线粒体功能变化较明显,但宏观指标表现不明显,其造模法可以作为长期造模研究的脾家损伤,更贴近临床实际的一种方法。劳倦过度造模法,可用于多因素造模法复制脾虚损伤模型进行相关研究。

[Objective]Through the horizontal contrast study of four common models of spleen deficiency mice,to evaluate the damage degree and applicability of each method.[Methods]The 100 mice were randomly divided into control group(deionized water 0.5 mL/per day),bitter cold diarrhea group(100%senna leaf water extract 0.5 mL/per day),diet loss group(fed with cabbage and lard once every 2 days),fatigue group(tie the tail and swim exhausted)and reserpine group(1%reserpine for 2 mL/kg/day,subcutaneous injection)with 20 rats in each group.Ten samples were randomly selected from each group after 10 d of continuous modeling,and 10 samples were recovered naturally 10 d.The stability of macro indexes was observed by measuring macro signs,intake,weight and swimming time.The thymus and spleen indices of each group were calculated,hematological indexes were detected by blood analyzer,serum D-xylose level was determined by colorimetric method,serum amylase activity was determined by iodine-amylase colorimetric method,and the expression of relative mtDNA copy number and mitochondrial autophagy related protein Pink1,Parkin mRNA were detected by real-time fluorescence quantitative PCR method.mitochondrial function was evaluated in each group of mice.[Results]Compared with the control group:1)There were significant differences in macro indexes in the bitter cold diarrhea group,diet loss group and reserpine group(P<0.05),the most significant in the reserpine group and the significant stability in the lower group(P<0.05).2)Thymic index was decreased significantly in the bitter cold diarrhea group,fatigue group and reserpine group(P<0.05),the most significant in the reserpine group(P<0.05),and spleen index was decreased significantly in the bitter cold diarrhea group and reserpine group(P<0.05),the most significant in the reserpine group.3)The RBC,WBC,HGB of reserpine group and bitter cold diarrhea group were decreased significantly(P<0.05).4)The serum D-xylan water was decreased on average in each model group(P<0.05).5)Serum amylase activity was decreased in the bitter cold diarrhea group,diet loss group and reserpine group(P<0.05).6)The mtDNA copy number was decreased in the bitter cold diarrhea group,diet loss group and fatigue group,the most significant in the diet loss group.The transcription level Pink1 mRNA mitochondrial autophagy related protein was decreased in the bitter cold diarrhea group and the diet loss group(P<0.05),while increased in the reserpine group(P<0.05).[Conclusion]The macro indexes and physical and chemical indexes of reserpine group changed obviously,but the stability of macro indexes was poor,which could be the good choice with short time period model.The changes of macro indexes,physical and chemical indexes and mitochondrial function of the bitter cold and diarrhea group were obvious,and the stability of the macro indexes is good,which could be adopted by the most spleen deficiency model.The changes of physical and chemical indexes and mitochondrial function in diet loss group were obvious,but the macroscopic index was not obvious,which sleep damage is similar to the clinical symptom and could be the good choice with long time period model.Fatigue modeling method can be used to replicate spleen injury model by multi-factor modeling method.