【作者】 刘丽娟；

【导师】 赵瑞芝；

【作者基本信息】 广州中医药大学 ， 药剂学， 2009， 硕士

【摘要】 目的通过引经药醋柴胡水提液对大鼠肝膜通透性、细胞内pH值、膜成分、膜形态、p-糖蛋白的表达等变化的研究,从药物作用的不同环节探讨醋柴胡引经的作用的可能机理。方法以MTT法检测确定醋柴胡水提液对大鼠肝细胞试验的最佳剂量;应用流式细胞仪测定细胞内荧光强度,以确定醋柴胡对大鼠肝细胞膜通透性影响;通过荧光探针法研究醋柴胡水提液对火鼠肝细胞内pH值的变化,运用荧光分光光度计测定大鼠肝细胞内的荧光强度比以计算细胞内pH值;测定细胞膜蛋白和胆固醇含量运用自动生化分析仪,测定细胞膜磷脂含量应用薄层扫描法;观察醋柴胡水提液对大鼠肝细胞膜的形态的影响应用电子镜显微镜;醋柴胡水提液对大鼠肝细胞膜上p-糖蛋白的表达,应用流式细胞仪法。结果醋柴胡水提液对大鼠肝细胞有增殖作用,且在0.16mg/ml-4mg/ml这个浓度范围内对大鼠肝细胞的增殖作用最强,本实验选用10 mg/mL,2 mg/mL,O.4 mg/mL三个浓度组;醋柴胡对细胞膜通透性的影响表明,醋柴胡水提液对大鼠肝细胞膜通透性影响随着药物作用时间的延长,其膜的通透性逐渐增加,且药物组与空白组比较有显著差异,其p<O.01;醋柴胡水提液对细胞质内pH值影响随时间、浓度不同而不同,24h各药物剂量组细胞质中pH与空白组细胞相比有统计意义,其p<O.01;加药组囊泡膜内pH值呈现下降的趋势,药物作用18h的组囊泡膜内pH值与空白相比有统计意义,其p<O.05,但不同浓度组间无统计意义,其他各组细胞内及囊泡膜内pH值均与对照组没有显著差异;醋柴胡水提液对大鼠肝细胞组分的结果表明,药物对细胞膜组分的影响随时间、浓度不同而不同。12h各药物浓度组与24h高浓度组使膜蛋白总含量均降低,与空白组相比有统计意义,其p(O.01。24h低浓度组与48h低中浓度组使细胞膜蛋白总量增加,与空白组相比有统计意义,其p<O.01。药物作用3小时,除磷脂酰丝氨酸以外,低浓度醋柴胡使细胞膜内的鞘磷脂、磷脂酰胆碱与磷脂酰乙醇胺含量均增加,与空白组相比有统计学意义,其p<O.05;而中高浓度组使细胞膜内的鞘磷脂、磷脂酰胆碱与磷脂酰乙醇胺含量都有所下降,具有统计学上的显著差异,其p<O.05。而其它时间点药物组与空白组相比没有显著差异;醋柴胡对大鼠肝细胞膜表面形态影响不大,其中高浓度组的细胞膜表面特化部分如桥粒、微绒毛遭到轻微的破化,细胞膜表面变得比较光滑;醋柴胡对p-糖蛋白的结果表明,醋柴胡可降低p-糖蛋白在大鼠肝细胞膜上的表达,特别是中低浓度组对p-糖蛋白影响较大。结论醋柴胡水提液在O.16mg/m1-4mg/ml范围内对大鼠肝细胞有增殖作用,能增加大鼠肝细胞膜的通透性和降低大鼠肝细胞膜p-糖蛋白的表达,醋柴胡对大鼠肝细胞内pH值影响和膜形念影响不显著,醋柴胡不同浓度在不同时间点对大鼠肝细胞膜组分有着不同的影响,在药物作用早期对细胞膜脂有影响,随着药物作用时间的延长,药物对细胞膜总蛋白又有影响,其具体的影响规律及机理还有待进一步研究和探讨。以上结果提示,醋柴胡有可能通过影响细胞膜组成及表面P糖蛋白等因素影响细胞膜的通透性,进而影响药物的肝摄取。更多还原

【Abstract】 ObjectiveTo explore the mechanism of TCM meridine guide drug- vinegar processed Radix Bupleuri from the different segments of drug action by investigating the effect of vinegar processed Radix Bupleuri on the permeability, intracellular pH,membrane lipid composition,Morphological study,and P-gp reversal of BRL.MethodEffect of TCM meridine guide drug vinegar processing Radix Bupleuri on the proliferation of BRL in vitro was performed by reformed MTT assay. Intracellular fluorescence intensity was detected by flow cytometry,with the purpose of the effect of vinegar processed Radix Bupleuri on the permeability of BRL.The change of pHi was determined by fluorescent probe (BCECF/AM and FIT-Dextran).The fluorescence intensity was determinated by fluorescence spectrophotometer,and pHi of BRL were calculated according to the fluorescence intensity.The membrane protein and cholesterol content of BRL was determinated by automatic biochemical analyzer,and the membrane phospholipids by TLC-scanning application. Effect of TCM meridine guide drug vinegar processed Radix Bupleuri on membrane morphology of BRL was observated by electronic microscope lens. Effect of TCM meridine guide drug vinegar processing Radix Bupleuri on membrane p-glycoprotein expression was detected by flow cytometry.ResultIt suggested that the water extract of vinegar processed Radix Bupleuri had the strongest action on proliferation of BRL with the concentration range from 0.16mg/ml to 4mg/ml.we choose 10mg/mL,2 mg/mL,0.4mg/mL in the later experiment.The effect of drug on the permeability of BRL was different in different concentration and different time.Significant changes were found in BRL with 24h 5%drug plasm effect(p＜0.05).With the time going on,The permeability of BRL became increased.The result showed the difference between vinegar processed Radix Bupleuri group and control group were significant at level 0.01.The effect of drug on cytosolic(pHcyt) in BRL was different in different concentration or different time.Low concentration vinegar processing Radix Bupleuri has less effect on cytosolic(pHcyt),but middle and high concentration drug have different degree effect on pHcyt in different time.Significant changes were found in BRL with 24h drug effect (p＜0.01),the drug has the tendency of descent apparent vesicular pH (pHves) in BRL.Significant changes were found in BRL with 18h drug effect (p＜0.05),but no significant effect was found between different drug concentrations.The effect of drug on the contents of cholsterol, phospholipid and protein membrane of BRL was different in different concentration and different time.Significant changes were found in BRL with 12h drug effect and 24h high concentration drug effect(p＜0.01),the drug has the tendency of descent the contents of protein membrane in BRL. Significant changes were found in BRL with 24h low concentration drug effect and 48h low and middle concentration drug effect(p＜0.01),the drug has the tendency of ascent the contents of protein membrane in BRL. Significant changes were found in BRL with 3h low concentration drug effect(p＜0.01),the drug has the tendency of increase the contents of SM,PC and PE in membrane of BRL.Significant changes were found in BRL with 24h middle and high concentration drug effect(p＜0.01),the drug has the tendency of descent the contents of SM,PC and PE in membrane of BRL.there were no significant changes between vinegar processed Radix Bupleuri group and control group in other time.The result showed that The membrane morphological changes of BRL was little after different drug concentration effect;but the membrane of BRL were getting smooth in middle and high concentration drug effect.The expression of P-gp in BRL were descent,especially in low and middle concentration drug effect.ConclusionThe water extract of vinegar processed Radix Bupleuri had the strongest action on proliferation of BRL with the concentration range from 0.16mg/ml to 4mg/ml.It can increase the permeability and reduce p-glycoprotein expressionl of BRL.These factors may directly affect the drug in the cell or cell-to-cell distribution.The effect of drug on the contents of cholsterol,phospholipid and protein membrane of BRL was different in different concentration and different time.Significant changes of the contents cholsterol,phospholipid were found in BRL with 3h and significant changes of the contents protein were found in BRL with 12h,24h,48h.The basic cause needed further research.Vinegar-baked Radix Bupleuri had little effect on the the morphological shape and pH value of BRL.The results of this paper showed that vinegar-baked Radix Bupleuri. increased liver uptake of other drugs may through affected the permeability and the components of the BRL memberane,the detail mechanism needs further study.更多还原