CN109200193A - 铁皮枫斗对糖尿病肾病及肾小球系膜细胞信号检测方法 - Google Patents
铁皮枫斗对糖尿病肾病及肾小球系膜细胞信号检测方法 Download PDFInfo
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Abstract
本发明属于中药检测技术领域,公开了一种铁皮枫斗对糖尿病肾病及肾小球系膜细胞信号检测方法,以链脲佐菌素诱导建立糖尿病大鼠模型,给予不同剂量的铁皮枫斗颗粒,共8周,观察各组大鼠的生化指标及肾脏病理的变化。体外培养HMC,分别加入正常浓度的葡萄糖、甘露醇、高糖及不同浓度的铁皮枫斗,观察p‑38MAPK的活性及下游TGF‑β、CTGF的表达。本发明通过建立糖尿病大鼠模型,使用不同浓度的铁皮枫斗进行治疗,铁皮枫斗又能够明显降低HMC细胞中TGF‑β和CTGF的表达水平,能够通过影响p‑38MAPK信号通路的活性调节下游TGF‑β和CTGF的表达而发挥抗纤维化作用。
Description
技术领域
本发明属于中药检测技术领域,尤其涉及一种铁皮枫斗对糖尿病肾病及肾小球系膜细胞信号检测方法。
背景技术
目前,业内常用的现有技术是这样的:糖尿病肾病是导致糖尿病死亡的主要原因之一,高糖是糖尿病肾病重要的致病因素,糖尿病肾病最主要的病症就是出现纤维化,p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路是细胞内信号传递的共同通路,转化生长因子β(TGF-β)和结缔组织生长因子(CTGF)是重要的促纤维化因子,在糖尿病肾病(DN)纤维化过程中发挥了重要作用,目前对糖尿病肾病的治疗主要依靠于西药进行控制,西药的副作用较大,研究一种中药对糖尿病肾病进行治疗是非常有必要的。
综上所述,现有技术存在的问题是:
目前对糖尿病肾病的治疗主要依靠于西药进行控制,西药的副作用较大,需要研究一种中药对糖尿病肾病进行治疗。目前在ND发病后的治疗有:药物控制高血压和控制高血糖、血液净化、干细胞移植、肾移植等,较为热点的是移植免疫学研究、肾脏保存、排异反应的诊断和防治,但这些治疗都有很多不足,如副作用多、价格昂贵、肾源难以寻找等缺点。
解决上述技术问题的难度和意义:中医药治疗肾病在我国已有二千多年的历史,国际上兴起的“天然植物药热”为我国中医药的研究提供了良好的契机。中医药物在该病的治疗上取得了一定的疗效,铁皮枫斗系列制剂的研究为新药开发树立了榜样。近年来运用铁皮枫斗TG从调节免疫和抗炎角度治疗糖尿病肾病(DN)已逐渐成为一种新的治疗方法,从分子生物学水平及病理生理学角度研究使人们对其有新认识。
发明内容
针对现有技术存在的问题,本发明以链脲佐菌素诱导建立糖尿病大鼠模型,给予不同剂量的铁皮枫斗颗粒,共8周。观察各组大鼠的生化指标及肾脏病理的变化。体外培养HMC,分别加入正常浓度的葡萄糖、甘露醇、高糖及不同浓度的铁皮枫斗,观察p-38MAPK的活性及下游TGF-β、CTGF的表达,提供了一种铁皮枫斗对糖尿病肾病及肾小球系膜细胞信号检测方法。
本发明是这样实现的,一种铁皮枫斗的药物用途,所述铁皮枫斗作为成分或唯一成分用于制备治疗糖尿病肾病的药物。
进一步,所述药物为铁皮枫斗系列制剂。
本发明的另一目的在于提供一种验证所述铁皮枫斗的药物作用的动物模型,所述动物模型为:
动物分组:将体重为180-200g的健康雄性大鼠70只,随机分为正常对照组和糖尿病组,正常组10只,糖尿病组60只;
动物建模:糖尿病组按60mg/kg一次性腹腔注射l%链脲佐菌素溶液,对照组注射等体积的枸橼酸缓冲溶液0.1mol/L,72h小时后尾取血测定血糖浓度,当浓度≥16.7mmol/L时建模成功;
建模成功动物分组:将建模成功的糖尿病大鼠随机分为模型对照组DM和DC治疗组,DC治疗组分为低剂量治疗组DC 0.2g/kg、中剂量治疗组DC 0.4g/kg和高剂量治疗组DC0.8g/kg;
分组后培养:建模成功后DC治疗组分别按规定剂量每日灌胃一次,持续8周,正常对照组和模型对照组给予相同体积的生理盐水。
本发明的另一目的在于提供一种验证所述铁皮枫斗的药物作用的铁皮枫斗对肾小球系膜细胞信号检测方法,所述铁皮枫斗对肾小球系膜细胞信号检测方法包括:
步骤一:HMC常规培养于37℃、5%CO2含15%胎牛血清的DMEM培养基中;
步骤二:将细胞分为6组,分别为对照组Control,加入5.6mmol/L的葡萄糖;甘露醇组加入5.6mmol/L的葡萄糖和24.4mmol/L的甘露醇;高糖组HG加入30mmol/L的葡萄糖;DC干预1组,加入30mmol/L的葡萄糖和50mg/L的DC;DC干预2组,加入30mmol/L的葡萄糖和100mg/L的DC;DC干预3组,加入30mmol/L的葡萄糖和300mg/L的DC;
所述大鼠标本采集及生化指标的测定方法为:8周末,称量小鼠体重,并收集小鼠24h尿液用于测定24h尿蛋白UP,然后用水合氯醛腹腔麻醉后静脉取血并分离血清,置于-20℃保存,用于测定血肌酐Scr、尿素氮BUN,取双肾,充分灌洗、称重,肾重指数KI=右肾重量/体重,右肾取肾皮质固定于4%的聚甲醛溶液中,剩余肾皮质置于液氮中保存待用,收集细胞用于蛋白和总RNA的提取。
本发明的另一目的在于提供一种验证所述铁皮枫斗的药物作用的大鼠肾皮质组织和细胞中MDA-1、SOD的测定方法,所述大鼠肾皮质组织和细胞中MDA-1、SOD的测定方法为:采用硫代巴比妥酸TBA法检测各组脂肪过氧化物MDA含量,黄嘌嘌呤氧化酶法检测超氧化物歧化酶SOD活性。
本发明的另一目的在于提供一种验证所述铁皮枫斗的药物作用的大鼠肾皮质组织中TGF-β、CTGF的测定方法,所述大鼠肾皮质组织中TGF-β、CTGF的测定方法为:采用免疫组化法测定肾皮质组织中TGF-β、CTGF的含量。分别加TGF-β一抗、CTGF一抗,孵育,加二抗,显色并复染,设置对照,高倍显微镜下(×400)每张切片随机选取5个不同的视野,测定光密度值并计算平均值。
本发明的另一目的在于提供一种验证所述铁皮枫斗的药物作用的p-38MAPK的活性,TGF-β、CTGF蛋白的含量检测方法,所述p-38MAPK的活性,TGF-β、CTGF蛋白的含量检测方法为:采用Western印迹法分别提取大鼠肾组织和细胞中的蛋白并测定浓度;取裂解蛋白50μg,经8%SDS-PAGE电泳、转膜封闭,分别加入抗p-38MAPK抗体、抗TGF-β抗体、抗CTGF抗体,4℃孵育过夜,用TBST洗涤3次后加入用HRP标记的二抗,室温封闭2h,TBST洗涤后,加入ECL试剂,用Image J 1.48软件对各蛋白条带进行灰度值定量分析,各自重复3次。
本发明的另一目的在于提供一种验证所述铁皮枫斗的药物作用的大鼠肾皮质及细胞中TGF-βmRNA和CTGF mRNA表达水平的检测方法,所述大鼠肾皮质及细胞中TGF-βmRNA和CTGF mRNA表达水平的检测方法为:采用RT-PCR法进行检测,首先采用Trizol法抽取总RNA,使用逆转录试剂盒将总RNA逆转录为cDNA,在ABI 7500实时荧光定量PCR仪上进行定量分析,然后应用2-△△ct法对结果进行分析;
在ABI 7500实时荧光定量PCR仪的反应条件为:预变性95℃5min;95℃15s,退火60℃30s,40个循环;终末延伸72℃5min。
综上所述,本发明的优点及积极效果为:本发明通过建立糖尿病大鼠模型,使用不同浓度的铁皮枫斗进行治疗,DC治疗组大鼠的肾重指数、血Scr、血BUN、24h UP等评价肾功能相关的指标均显著改善,DC治疗组大鼠的肾脏病理学改变较轻,铁皮枫斗能够降低损伤组织MDA水平、增加抗氧化物SOD-1水平,对糖尿病肾组织有抗氧化作用,能够减少肾脏的损伤;通过体外模拟糖尿病模型,以不同浓度的铁皮枫斗干预高糖诱导的HMCs,铁皮枫斗能够降低HMC细胞中p-38MAPK的活性,同时铁皮枫斗又能够明显降低HMC细胞中TGF-β和CTGF的表达水平,能够通过影响p-38MAPK信号通路的活性调节下游TGF-β和CTGF的表达而发挥抗纤维化作用。
本发明验证了铁皮枫斗通过降低p-38MAPK的活性,降低TGF-β、CTGF蛋白和mRNA的表达,进而抵抗肾脏组织纤维化,发挥对DN的保护作用。
本发明的基金项目:河南省商丘市科技局自然科学基础研究计划项目(135004)
附图说明
图1是本发明实施例提供的铁皮枫斗对糖尿病肾病及肾小球系膜细胞信号检测方法流程图。
图2是本发明实施例提供的铁皮枫斗对肾小球系膜细胞信号检测方法流程图。
图3是本发明实施例提供的不同组大鼠肾组织MDA-1和SOD的表达水平;
图中:1:对照,2:DM,3:DM+DC 0.2g·kg-1,4:DM+DC 0.4g·kg-1,5:DM+DC 0.8g·kg-1。
图4是本发明实施例提供的大鼠HE染色后肾脏病理改变图;
图中:A:对照,B:DM组,C:DM+DC 0.2g·kg-1组,D:DM+DC 0.4g·kg-1组,E:DM+DC0.8g·kg-1组。
图5是本发明实施例提供的TGF-β1和CTGF在大鼠肾组织中的表达示意图;图中:A,B:通过免疫染色检测TGF-β1和CTGF的IOD;C,D:Western印迹检测TGF-β1和CTGF蛋白的表达;E,F:TGF-β1mRNA和CTGF mRNA的相对表达。1:对照,2:DM,3:DM+DC 0.2g·kg-1,4:DM+DC0.4g·kg-1,5:DM+DC 0.8g·kg-1。
图6是本发明实施例提供的各组间HMC细胞中MDA-1和SOD的表达示意图;图中:1:对照,2:甘露醇,3:HG,4:HG+DC 50mg/L,5:HG+DC 100mg/L,6:HG+DC 300mg/L。
图7是本发明实施例提供的各组间p-38MAPK在HMC细胞中的表达示意图;图中:1:对照,2:甘露醇,3:HG,4:HG+DC 50mg/L,5:HG+DC 100mg/L,6:HG+DC 300mg/L。
图8是本发明实施例提供的TGF-β和CTGF在HMC细胞中的表达示意图;图中:A,B:Western blot检测TGF-β1和CTGF蛋白的表达;C,D:TGF-β1mRNA和CTGF mRNA的相对表达。1:对照,2:甘露醇,3:HG,4:HG+DC 50mg/L,5:HG+DC 100mg/L,6:HG+DC 300mg/L。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
下面结合附图对本发明的应用原理做详细描述。
如图1所示,本发明实施例提供的铁皮枫斗对糖尿病肾病及肾小球系膜细胞信号检测方法为:
S101:动物分组:将体重为180-200g的健康雄性大鼠70只,随机分为正常对照组和糖尿病组,正常组10只,糖尿病组60只;
S102:动物建模:糖尿病组按60mg/kg一次性腹腔注射l%链脲佐菌素溶液,对照组注射等体积的枸橼酸缓冲溶液(0.1mol/L),72h小时后尾取血测定血糖浓度,当浓度≥16.7mmol/L时建模成功;
S103:建模成功动物分组:将建模成功的糖尿病大鼠随机分为模型对照组(DM)和DC治疗组,DC治疗组分为低剂量治疗组(DC 0.2g/kg)、中剂量治疗组(DC 0.4g/kg)和高剂量治疗组(DC 0.8g/kg);
S104:分组后培养:建模成功后DC治疗组分别按规定剂量每日灌胃一次,持续8周,正常对照组和模型对照组给予相同体积的生理盐水;
S105:8周后,分别对大鼠进行标本采集及生化指标、肾皮质组织和细胞中MDA-1、SOD、肾皮质组织中TGF-β、CTGF、p-38MAPK的活性,TGF-β、CTGF蛋白的含量、大鼠肾皮质及细胞中TGF-βmRNA和CTGF mRNA表达水平进行检测。
如图2所示,本发明实施例提供的铁皮枫斗对肾小球系膜细胞信号检测方法为:
S201:HMC常规培养于37℃、5%CO2含15%胎牛血清的DMEM培养基中;
S202:将细胞分为6组,分别为对照组(Control),加入5.6mmol/L的葡萄糖;甘露醇组加入5.6mmol/L的葡萄糖和24.4mmol/L的甘露醇;高糖组(HG)加入30mmol/L的葡萄糖;DC干预1组,加入30mmol/L的葡萄糖和50mg/L的DC;DC干预2组,加入30mmol/L的葡萄糖和100mg/L的DC;DC干预3组,加入30mmol/L的葡萄糖和300mg/L的DC。
本发明实施例提供的大鼠标本采集及生化指标的测定方法为:8周末,称量小鼠体重,并收集小鼠24h尿液用于测定24h尿蛋白(UP),然后用水合氯醛腹腔麻醉后静脉取血并分离血清,置于-20℃保存,用于测定血肌酐(Scr)、尿素氮(BUN),取双肾,充分灌洗、称重,肾重指数(KI)=右肾重量/体重,右肾取肾皮质固定于4%的聚甲醛溶液中,剩余肾皮质置于液氮中保存待用,收集细胞用于蛋白和总RNA的提取。
本发明实施例提供的大鼠肾皮质组织和细胞中MDA-1、SOD的测定方法为:采用硫代巴比妥酸(TBA)法检测各组脂肪过氧化物(MDA)含量,黄嘌嘌呤氧化酶法检测超氧化物歧化酶(SOD)活性。
本发明实施例提供的大鼠肾皮质组织中TGF-β、CTGF的测定方法为:采用免疫组化法测定肾皮质组织中TGF-β、CTGF的含量。分别加TGF-β一抗、CTGF一抗,孵育,加二抗,显色并复染,设置对照,高倍显微镜下(×400)每张切片随机选取5个不同的视野,测定光密度值并计算平均值。
本发明实施例提供的p-38MAPK的活性,TGF-β、CTGF蛋白的含量检测方法为:采用Western印迹法分别提取大鼠肾组织和细胞中的蛋白并测定浓度。取裂解蛋白50μg,经8%SDS-PAGE电泳、转膜封闭,分别加入抗p-38MAPK抗体、抗TGF-β抗体、抗CTGF抗体,4℃孵育过夜,用TBST洗涤3次后加入用HRP标记的二抗,室温封闭2h,TBST洗涤后,加入ECL试剂,用Image J 1.48软件对各蛋白条带进行灰度值定量分析,各自重复3次。
本发明实施例提供的大鼠肾皮质及细胞中TGF-βmRNA和CTGF mRNA表达水平的检测方法为:采用RT-PCR法进行检测,首先采用Trizol法抽取总RNA,使用逆转录试剂盒(Thermo Scientific)将总RNA逆转录为cDNA,在ABI 7500实时荧光定量PCR仪上进行定量分析,然后应用2-△△ct法对结果进行分析。
本发明实施例提供的PCR仪采用的TGF-β上游引物为:5’-CGAGCCTGAGGCCGACTAC-3’,下游引物为:5’-TTGTGGGTTTCCACCATTAGC-3’;CTGF上游引物为:5’-TAGCTGCCTACCGACTGGAA-3’,下游引物为:5’-CTAGAACAGGCGCTCCACT-3’;GAPDH上游引物为:5’-GAAATCCCATCACCATCTTCCAGG-3’,下游引物为:5’-GAGCCCCAGCCTTCTCCATG-3’,GAPDH为内参。
本发明实施例提供的在ABI 7500实时荧光定量PCR仪的反应条件为:预变性95℃5min;95℃15s,退火60℃30s,40个循环;终末延伸72℃5min。
本发明实施例提供的计量数据均采用x±s表示。非正态分布数据经对数转换后分析,组间采用T检验或单因素方差分析,采用SPSS 19.0进行数据处理,P<0.05被认为有统计学差异。
下面结合具体实施例对本发明作进一步说明。
实施例1
DC对大鼠生理及生化指标的影响
糖尿病模型组大鼠的体重明显低于正常对照组(P<0.01),DC治疗组与模型组相比体重均有所增加(P<0.05)。与正常对照组相比,模型对照组大鼠的肾重指数显著增加(P<0.01),经DC治疗后各组的肾重指数均显著降低(P<0.01)。与正常组相比,所有糖尿病模型大鼠的血Scr、血BUN和24hUP均显著增高;在经过DC治疗之后,这些生化指标均明显降低,如下表所示。
表1组间临床和生化参数变化的比较
##P<0.01vs control group;*P<0.05,**P<0.01vs DM group
实施例2
大鼠肾皮质组织中MDA-1和SOD的表达
与正常组相比,DM组大鼠肾皮质组织中MDA-1的含量明显增加(P<0.01);与DM组相比,DC干预组MDA-1的含量明显降低,DC在0.2g/kg、0.4g/kg、0.8g/kg时具有浓度依赖性。与正常组相比,DM组大鼠肾皮质组织中SOD的含量显著降低(P<0.01);与DM组相比,DC干预组MDA-1的含量明显升高,DC在0.2g/kg、0.4g/kg时具有浓度依赖性,如图3所示。
实施例3
大鼠肾脏病理学变化
HE染色结果显示,正常对照组大鼠的肾小球结构无明显的病理改变;模型对照组大鼠肾脏有显著的病理改变,肾小球肥大,且系膜细胞增生、系膜基质增多、基底膜增厚;DC组治疗后上述病理改变明显减轻,且具有浓度依赖性,如图4所示。
实施例4
大鼠肾皮质组织中TGF-β、CTGF的表达
免疫组化结果显示,与正常组相比,DM组中TGF-β和CTGF的含量均高于正常对照组(P<0.01);与DM组相比,DC干预组TGF-β和CTGF的含量明显下降(P<0.05),(图3A、图3B)。Western blot和RT-PCR结果显示,与正常组相比,DM组中TGF-β和CTGF蛋白和mRNA的含量均高于正常对照组(P<0.01);与DM组相比,DC干预组TGF-β和CTGF蛋白和mRNA的含量明显下降(P<0.05),DC在0.2g/kg、0.4g/kg时具有浓度依赖性,如图5所示。
实施例5
HMC细胞中MDA-1和SOD的表达
与正常对照组相比,甘露醇组细胞中MDA-1的表达没有显著差异,HG组中MDA-1的表达明显增高(P<0.01);与HG组相比,DC干预组中MDA-1的表达显著下降(P<0.05),(图6A)。与正常对照组相比,甘露醇组细胞中SOD的表达没有显著差异,HG组中SOD的表达显著降低(P<0.01);与HG组相比,DC干预组中SOD的表达明显增高(P<0.05),(图6B)
实施例6
HMC细胞中p-38MAPK的活性
与正常对照组相比,甘露醇组细胞中p-38MAPK的活性无明显变化,HG组中p-38MAPK的表达明显增多(P<0.01);与HG组相比,DC干预组中p-38MAPK的表达显著下降(P<0.05),DC在50mg/L、100mg/L时具有浓度依赖性。(图7)
实施例7
HMC细胞中TGF-β、CTGF蛋白及mRNA的表达
Western blot和RT-PCR结果显示,与正常组相比,甘露醇组细胞中TGF-β、CTGF蛋白及mRNA的表达均无显著差异,HG组中TGF-β、CTGF蛋白和mRNA的含量均高于正常对照组(P<0.01);与HG组相比,DC干预组TGF-β、CTGF蛋白和mRNA的含量明显下降(P<0.05),DC在50mg/L、100mg/L时具有浓度依赖性。(图8)
铁皮枫斗具有抗氧化、耐缺氧的药理作用,对糖尿病血管病变具有一定的保护作用,DN大鼠的肾脏具有一定的保护作用,能够有效延缓糖尿病肾病的进展。DC治疗组大鼠的肾重指数、血Scr、血BUN、24h UP等评价肾功能相关的指标均显著改善,肾脏病理组织学检查也发现DC治疗组大鼠的肾脏病理学改变较轻;
铁皮枫斗能够降低损伤组织MDA水平、增加抗氧化物SOD-1水平,对糖尿病肾组织有抗氧化作用,能够减少肾脏的损伤;
在糖尿病大鼠的肾皮质中TGF-β和CTGF的含量均显著增高,而经铁皮枫斗治疗之后TGF-β和CTGF的含量有明显的下降,铁皮枫斗能够通过影响TGF-β和CTGF的表达水平发挥保护糖尿病肾脏的作用,能够降低HMC细胞中p-38MAPK的活性,同时又能够明显降低HMC细胞中TGF-β和CTGF的表达水平,能够通过影响p-38MAPK信号通路的活性调节下游TGF-β和CTGF的表达而发挥抗纤维化作用。
综上所述,铁皮枫斗有可能是通过降低p-38MAPK的活性,降低TGF-β、CTGF蛋白和mRNA的表达,进而抵抗肾脏组织纤维化,发挥对DN的保护作用。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种铁皮枫斗的药物用途,其特征在于,所述铁皮枫斗作为成分或唯一成分用于制备治疗糖尿病肾病的药物。
2.如权利要求1所述的铁皮枫斗的药物用途,其特征在于,所述药物为铁皮枫斗系列制剂。
3.一种验证权利要求1所述铁皮枫斗的药物作用的动物模型,其特征在于,所述动物模型为:
动物分组:将体重为180-200g的健康雄性大鼠70只,随机分为正常对照组和糖尿病组,正常组10只,糖尿病组60只;
动物建模:糖尿病组按60mg/kg一次性腹腔注射l%链脲佐菌素溶液,对照组注射等体积的枸橼酸缓冲溶液0.1mol/L,72h小时后尾取血测定血糖浓度,当浓度≥16.7mmol/L时建模成功;
建模成功动物分组:将建模成功的糖尿病大鼠随机分为模型对照组DM和DC治疗组,DC治疗组分为低剂量治疗组DC 0.2g/kg、中剂量治疗组DC 0.4g/kg和高剂量治疗组DC 0.8g/kg;
分组后培养:建模成功后DC治疗组分别按规定剂量每日灌胃一次,持续8周,正常对照组和模型对照组给予相同体积的生理盐水。
4.一种验证权利要求1所述铁皮枫斗的药物作用的铁皮枫斗对肾小球系膜细胞信号检测方法,其特征在于,所述铁皮枫斗对肾小球系膜细胞信号检测方法包括:
步骤一:HMC常规培养于37℃、5%CO2含15%胎牛血清的DMEM培养基中;
步骤二:将细胞分为6组,分别为对照组Control,加入5.6mmol/L的葡萄糖;甘露醇组加入5.6mmol/L的葡萄糖和24.4mmol/L的甘露醇;高糖组HG加入30mmol/L的葡萄糖;DC干预1组,加入30mmol/L的葡萄糖和50mg/L的DC;DC干预2组,加入30mmol/L的葡萄糖和100mg/L的DC;DC干预3组,加入30mmol/L的葡萄糖和300mg/L的DC;
所述大鼠标本采集及生化指标的测定方法为:8周末,称量小鼠体重,并收集小鼠24h尿液用于测定24h尿蛋白UP,然后用水合氯醛腹腔麻醉后静脉取血并分离血清,置于-20℃保存,用于测定血肌酐Scr、尿素氮BUN,取双肾,充分灌洗、称重,肾重指数KI=右肾重量/体重,右肾取肾皮质固定于4%的聚甲醛溶液中,剩余肾皮质置于液氮中保存待用,收集细胞用于蛋白和总RNA的提取。
5.一种验证权利要求1所述铁皮枫斗的药物作用的大鼠肾皮质组织和细胞中MDA-1、SOD的测定方法,其特征在于,所述大鼠肾皮质组织和细胞中MDA-1、SOD的测定方法为:采用硫代巴比妥酸TBA法检测各组脂肪过氧化物MDA含量,黄嘌嘌呤氧化酶法检测超氧化物歧化酶SOD活性。
6.一种验证权利要求1所述铁皮枫斗的药物作用的大鼠肾皮质组织中TGF-β、CTGF的测定方法,其特征在于,所述大鼠肾皮质组织中TGF-β、CTGF的测定方法为:采用免疫组化法测定肾皮质组织中TGF-β、CTGF的含量;分别加TGF-β一抗、CTGF一抗,孵育,加二抗,显色并复染,设置对照,高倍显微镜下(×400)每张切片随机选取5个不同的视野,测定光密度值并计算平均值。
7.一种验证权利要求1所述铁皮枫斗的药物作用的p-38MAPK的活性,TGF-β、CTGF蛋白的含量检测方法,其特征在于,所述p-38MAPK的活性,TGF-β、CTGF蛋白的含量检测方法为:采用Western印迹法分别提取大鼠肾组织和细胞中的蛋白并测定浓度;取裂解蛋白50μg,经8%SDS-PAGE电泳、转膜封闭,分别加入抗p-38MAPK抗体、抗TGF-β抗体、抗CTGF抗体,4℃孵育过夜,用TBST洗涤3次后加入用HRP标记的二抗,室温封闭2h,TBST洗涤后,加入ECL试剂,用Image J 1.48软件对各蛋白条带进行灰度值定量分析,各自重复3次。
8.一种验证权利要求1所述铁皮枫斗的药物作用的大鼠肾皮质及细胞中TGF-βmRNA和CTGF mRNA表达水平的检测方法,其特征在于,所述大鼠肾皮质及细胞中TGF-βmRNA和CTGFmRNA表达水平的检测方法为:采用RT-PCR法进行检测,首先采用Trizol法抽取总RNA,使用逆转录试剂盒将总RNA逆转录为cDNA,在ABI 7500实时荧光定量PCR仪上进行定量分析,然后应用2-△△ct法对结果进行分析;
在ABI 7500实时荧光定量PCR仪的反应条件为:预变性95℃5min;95℃15s,退火60℃30s,40个循环;终末延伸72℃5min。
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