【作者】 范焕芳；

【导师】 陈志强；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2007， 博士

【摘要】 目的:系膜细胞(Mesangial cells, MCs)是肾小球中最活跃的固有细胞成分。MCs的过度增殖或凋亡减少导致层粘连蛋白(Laminin, LN)、Ⅳ型胶原(CollagenⅣ, Col-Ⅳ)和纤维连接蛋白(Fibronectin,FN)等细胞外基质(Extracellular matrix, ECM)成分分泌增多。MCs过度增殖还可分泌多种细胞因子和炎症介质,从而导致ECM的过量生成和多种炎症细胞的聚积,形成肾脏纤维化。肾纤维化是肾小球硬化、肾间质纤维化和肾小管萎缩的简称。研究表明,MCs能表达基质金属蛋白酶(Matrix metalloproteinases, MMPs)及其组织抑制物(Tissue inhibitor of metalloproteinase, TIMPs)。MMPs/TIMPs对肾小球基质的降解起着重要的调控作用。MMPs是ECM降解的关键酶。在MMPs家族中MMP-9是重要成员之一,是降解Ⅳ型胶原的最主要成分。TIMP-1是MMP-9的特异性抑制因子,在ECM积聚和降解的生理平衡中起关键作用,其过度表达必然导致ECM合成过剩并大量沉积于肾小球内导致肾小球硬化的发生。转化生长因子β(Transforming Growth Factor beta, TGF-β)在调控MCs增殖尤其是ECM的积聚中起着重要作用,其中TGF-β1是重要的致肾纤维化因子之一。因此,抑制MCs的过度增殖、减少ECM合成、纠正MMP-9/TIMP-1的失衡、抑制TGF-β1的表达是延缓肾纤维化的关键环节。随着对肾脏病发病机制的深入研究,尤其是细胞、分子学的研究,针对ECM代谢的不同环节,确立了多种对抗肾纤维化的途径。抗肾纤维化研究得到了很大发展,但其效果并不理想。中医药可以从不同环节进行干预,延缓肾纤维化的发展,具有良好的应用前景。鳖甲煎丸为东汉末年医圣张仲景所创,具有气血同治、寒热并用、攻补兼施之特点,是治疗“疟母”的名方。现在多用于治疗肝炎、肝纤维化等,我们课题组将其用于治疗肾纤维化,取得了一定成绩。为了从细胞和分子水平探讨鳖甲煎丸对肾小球MCs干预的作用机制,本实验利用脂多糖(LPS)作为一种刺激因子,将其作用于体外培养的MCs中,同时以缬沙坦作对照,探讨鳖甲煎丸对MCs增殖、凋亡、ECM及相关基因的干预作用。方法1药物血清制备选年龄、体重相近的成年健康志愿者3名,其中2名分别口服鳖甲煎丸、缬沙坦,鳖甲煎丸每次4g,每日2次;缬沙坦每次80mg,每日1次,连续3d,末次用药1h后无菌采静脉血20ml,分离血清,56℃30min灭活处理后用0.22um微孔滤膜过滤,-70℃冰箱保存备用。不服用药物的1名与前2名同时采血,分离血清备用。2 MCs的培养及鉴定MCs取自5月大的水囊引产胎肾,由河北医科大学附属医院提供,迅速无菌取其肾脏,剥除肾包膜,将肾皮质剪成碎屑,研磨依次通过80目、140目、220目筛网,收集肾小球,Ⅳ型胶原酶消化6~7min,接种于25cm2培养瓶,培养液为含20%FCS、Hepes、青霉素、链霉素的RPIM-1640液,并用5%NaHCO3调PH值至7.2~7.4。5d左右,肾小球贴壁,周围长出细胞,首次换培养液,以后每2d换液一次。当MCs长至80%融合时,用胰蛋白酶消化传代。亚培养的MCs生长很快,36~48h布满瓶底,采用相差显微镜、透射电镜及免疫组织化学方法鉴定为MCs,继续培养进行实验。3分组及指标观察3.1分组及培养测增殖以及细胞上清液中各成分含量时,将MCs以1×104/孔接种于96孔板内,24h后换成含1%胎牛血清的培养液孵育24h,使细胞同步于静止期,弃上清;96孔板从左至右依次分为空白对照组(简称“空白组”,加入5%正常人血清)、鳖甲煎组(加入5%鳖甲煎血清)、缬沙坦组(加入5%缬沙坦血清)、LPS组(加入LPS10ug/ml和5%正常人血清)、LPS鳖甲煎组(简称“脂甲组”,加入LPS10ug/ml和5%鳖甲煎血清)、LPS缬沙坦组(简称“脂坦组”,加入LPS10ug/ml和5%缬沙坦血清)。共6个组,每组设有复孔(测增殖时设18个复孔;ELISA法测LN、Col-Ⅳ、FN、MMP-9、TIMP-1时设6个复孔)。各组均加入含胎牛血清的RPMI-1640培养液,使血清终浓度为10%,每孔培养液总量为200ul,37℃、5%CO2培养箱中孵育48h,准备指标检测。测流式及基因表达时,取第6~7代MCs以密度1×106/ml种于25cm2培养瓶中,加入1% FCS RPMI-1640培养液孵育24h,使之转入G0期,然后分为6组,具体分组同前。各组均加入含有胎牛血清的RPMI-1640培养液,使血清终浓度为10%,继续培养48h后,进行流式细胞检测、提取MCs总RNA。3.2 MCs增殖的检测各组培养24h,48h后,用四甲基偶氮唑盐(MTT)法检测各组MCs的增殖情况。3.3 MCs增殖、凋亡和细胞周期的检测采用流式细胞检测方法测定MCs增殖、凋亡和细胞周期。3.4细胞上清液中各成分含量的检测培养48小时结束后,采用酶联免疫吸附试验(ELISA)法定量测定细胞上清夜中LN、Col-Ⅳ、FN、MMP-9、TIMP-1的含量。3.5 FNmRNA、TGF-β1mRNA的检测采用逆转录—多聚酶链反应(RT-PCR)对FNmRNA、TGF-β1mRNA的相对含量进行检测。4统计学方法数据采用SPSS11.5 for window统计分析软件进行处理,所有数据以均数±标准差(mean±s)表示,采用单因素方差分析进行检验。结果1肾小球MCs的培养与鉴定1.1肾小球的分离经不同目数的不锈钢筛网提取的肾小球用倒置显微镜观察显示:95%以上的肾小球均脱去囊,视野中基本上无肾小管存在。1.2形态学特征原代培养后第4d,将细胞培养瓶取出于倒置显微镜下观察,有部分肾小球贴壁。第6d时有50~80%肾小球贴壁,肾小球上皮细胞及MCs开始爬出,因不适合此培养条件,第12d左右上皮细胞基本死亡,而MCs从肾小球中移出生长增多,细胞多呈星状或三角形并伴有不规则的突起,第16d时细胞生长密集,80%布满瓶底,胞核位于细胞中央呈清晰的卵圆形,胞质向外伸出数个长短不一的突起。传代的细胞一般36~48 h全部贴壁,生长迅速,形态为星形或梭形。1.3透射电镜亚培养的肾小球MCs透射电镜下观察:细胞形态多样,细胞质中富含大量线粒体、粗面内质网、游离核糖体,并可见少量分泌颗粒。细胞内有大的圆形或椭圆形核,含1个或2个核仁,核膜清晰;核染色质分布稀疏,核周围胞质及胞浆内含丰富的束状微丝结构。1.4免疫组织化学结果显微镜下观察:培养细胞均呈Desmin染色阳性,证明培养的细胞为MCs。2鳖甲煎丸对人肾小球MCs增殖、凋亡及细胞周期的影响2.1 MTT法测定鳖甲煎丸药物血清对正常培养条件及LPS刺激下MCs增殖的影响结果显示:空白组在培养24h、48h时均有表达;LPS组24h、48h与空白组比较均明显增强,经统计学处理有显著性差异(P<0.01);鳖甲煎组、缬沙坦组24h、48h与空白组比较MCs增殖均明显降低,有显著性差异(P<0.01)和明显差异(P<0.05);脂甲组、脂坦组24h MCs增殖与LPS组比较均明显降低,有显著性差异(P<0.01);脂甲组48h与LPS组比较均明显降低,有显著性差异(P<0.01);脂坦组48h与LPS组比较无明显差异(P>0.05);但数值有所降低。6个组48h时MCs增殖与各自24h相比明显增强,经统计学处理有明显差异(P<0.05)和显著性差异(P<0.01)。2.2鳖甲煎丸药物血清对正常培养条件及LPS刺激下MCs周期的影响空白组有27.93%的MCs被阻滞在G0/G1期;LPS组与空白组比较,被阻滞在G0/G1期的MCs明显减少(P<0.01);鳖甲煎组、缬沙坦组被阻滞在G0/G1期的MCs与空白组相比明显增多,经统计学处理有显著性差异(P<0.01);鳖甲煎组与缬沙坦组之间无明显差异(P>0.05),但鳖甲煎组G0/G1期的百分数较高;脂甲组、脂坦组被阻滞在G0/G1期的MCs与LPS组相比明显增多,有显著性差异(P<0.01);脂甲组与脂坦组之间无明显差异(P>0.05),但脂甲组G0/G1期的百分数较高。2.3鳖甲煎丸药物血清对正常条件及LPS刺激条件下人MCs增殖指数及凋亡率的影响空白组增殖指数为72.07%,凋亡率为3.99%;LPS组增殖指数与空白组相比明显升高,经统计学处理有显著性差异(P<0.01);LPS组凋亡率与空白组相比无明显差异(P>0.05);鳖甲煎组、缬沙坦组与空白组比较,增殖指数明显降低,凋亡率明显升高,有显著性差异(P<0.01);在降低增殖指数方面鳖甲煎组与缬沙坦组之间无明显差异(P>0.05);在促进凋亡方面鳖甲煎组凋亡率高于缬沙坦组,有显著性差异(P<0.01);脂甲组、脂坦组与LPS组比较,增殖指数明显降低,凋亡率明显升高,有显著性差异(P<0.01);脂甲组与脂坦组之间无明显差异(P>0.05)。3鳖甲煎丸药物血清对ECM的影响3.1 ELISA法测定LN、Col-Ⅳ、FN结果结果表明,LPS组LN、Col-Ⅳ及FN含量明显升高,与空白组比较有显著性差异(P<0.01);鳖甲煎组、缬沙坦组与空白组比较,LN、Col-Ⅳ及FN含量均明显降低,经统计学处理有明显差异(P<0.05)和显著性差异(P<0.01);鳖甲煎组与缬沙坦组之间无明显差异(P>0.05)。但鳖甲煎组的含量低于缬沙坦组;脂甲组、脂坦组与LPS组比较,LN、Col-Ⅳ及FN含量均明显降低,经统计学处理有显著性差异(P<0.01);脂坦组与脂甲组之间无明显差异(P>0.05)。但脂甲组的含量低于脂坦组。3.2 MCs总RNA提取以及FN RT-PCR结果3.2.1 MCs总RNA提取结果所提取细胞总RNA,经核酸分析仪测定:OD260/OD280在1.8~2.0之间,琼脂糖凝胶电泳,28S/18S≥2:1,说明所提总RNA纯度较高,且较完整,没有降解,适于进行进一步分析。3.2.2 RT-PCR结果逆转录产物用FN引物进行PCR扩增,出现346bp特异性扩增带。其中LPS组的扩增带最亮,其次为空白对照组。鳖甲煎组、缬沙坦组与空白组相比,扩增带较暗;脂甲组、脂坦组与空白组相近。各组FNmRNA表达用目的基因扩增片断的积分光密度与β-actin扩增片断的积分光密度的比值表示。结果显示:LPS组FNmRNA/β-actinmRNA与空白组相比其比值明显上升,经统计学处理有显著性差异(P<0.01);鳖甲煎组、缬沙坦组与空白组比较,FNmRNA/β-actinmRNA比值均明显下降,经统计学处理有显著性差异(P<0.01)和明显差异(P<0.05);鳖甲煎组与缬沙坦组比较有显著性差异(P<0.01),鳖甲煎组低于缬沙坦组;脂甲组、脂坦组与LPS组比较,FNmRNA/β-actinmRNA比值均明显降低,经统计学处理有显著性差异(P<0.01);脂甲组与脂坦组之间有明显差异(P<0.05),脂甲组低于脂坦组。4鳖甲煎丸对人MCs MMP-9、TIMP-1及MMP-9/TIMP-1表达的影响结果表明,LPS组MMP-9的含量与空白组比较无明显差异(P>0.05),但LPS组的含量低于空白组;LPS组TIMP-1的含量与空白组比较明显升高,两者含量比较有显著性差异(P<0.01);LPS组MMP-9/TIMP-1比值与空白组比较明显降低,两者比较有显著性差异(P<0.01)。鳖甲煎组、缬沙坦组与空白组比较,MMP-9含量无明显差异(P>0.05),但鳖甲煎组、缬沙坦组的含量高于空白组;鳖甲煎组与缬沙坦组之间无明显差异(P>0.05)。鳖甲煎组与空白组比较,TIMP-1含量明显降低,经统计学处理有明显差异(P<0.05);缬沙坦组与空白组比较,TIMP-1含量无明显差异(P>0.05);鳖甲煎组、缬沙坦组与空白组比较MMP-9/TIMP-1比值明显升高,经统计学处理有显著性差异(P<0.01);鳖甲煎组与缬沙坦组之间无明显差异(P>0.05)。脂甲组、脂坦组与LPS组比较,MMP-9的含量无明显差异(P>0.05),但脂甲组、脂坦组的含量高于LPS组;脂甲组与脂坦组之间无明显差异(P>0.05);脂甲组、脂坦组TIMP-1表达与LPS组比较明显降低,经统计学处理有显著性差异(P<0.01);脂甲组与脂坦组之间无明显差异(P>0.05)。但脂甲组的含量低于脂坦组。脂甲组、脂坦组MMP-9/TIMP-1与LPS组比较明显升高,经统计学处理有显著性差异(P<0.01);脂甲组与脂坦组之间无明显差异(P>0.05)。但脂甲组的含量高于脂坦组。5鳖甲煎丸对人肾小球MCs TGF-β1mRNA表达的影响5.1 MCs总RNA提取结果所提取细胞总RNA,经核酸分析仪测定:OD260/OD280在1.8~2.0之间,琼脂糖凝胶电泳,28S/18S≥2:1,说明所提总RNA纯度较高,且较完整,没有降解,适于进行进一步分析。5.2 RT-PCR结果取逆转录产物用TGF-β1引物进行PCR扩增,出现321bp特异性扩增带。其中LPS组的扩增带较亮,其次为空白对照组。鳖甲煎组、缬沙坦组与空白组相比,扩增带较暗;脂甲组、脂坦组与空白组相近。各组TGF-β1mRNA表达用目的基因扩增片断的积分光密度与β-actin扩增片断的积分光密度的比值表示。结果显示:空白组TGF-β1mRNA/β-actinmRNA有基础表达;LPS组与空白组相比其比值明显上升,经统计学处理有显著性差异(P<0.01);鳖甲煎组、缬沙坦组与空白组比较,TGF-β1mRNA/β-actinmRNA比值均明显下降,经统计学处理有显著性差异(P<0.01);鳖甲煎组明显低于缬沙坦组(P<0.01)。脂甲组、脂坦组与LPS组比较,TGF-β1mRNA/β-actinmRNA比值均明显降低,经统计学处理有显著性差异(P<0.01);脂甲组明显低于脂坦组(P<0.01)。结论:本实验利用LSP作为一种刺激因子,将其作用于体外培养的MCs中,用鳖甲煎丸进行干预,同时以缬沙坦作为对照进行了研究,可以得出以下结论:1 LPS具有促进MCs增殖的作用,但无明显抑制细胞凋亡作用;鳖甲煎丸及缬沙坦药物血清能明显抑制正常培养条件及LPS刺激条件下MCs增殖,促进MCs凋亡。鳖甲煎丸略有优势。2 LPS具有促进ECM分泌、促进FNmRNA表达的作用;鳖甲煎及缬沙坦药物血清能明显抑制正常培养条件及LPS刺激下ECM的分泌,抑制FNmRNA表达。鳖甲煎丸优于缬沙坦。3 LPS具有促进TIMP-1表达的作用,但对MMP-9表达较弱,其结果使MMP-9/TIMP-1比值降低;鳖甲煎丸及缬沙坦药物血清具有抑制正常培养条件及LPS刺激下TIMP-1表达的作用。鳖甲煎丸及缬沙坦药物血清促进MCs MMP-9表达作用较弱。4 LPS具有促进MCs TGF-β1mRNA表达的作用;鳖甲煎丸及缬沙坦药物血清能明显抑制正常培养条件及LPS刺激条件下MCs TGF-β1mRNA的表达,且鳖甲煎丸优于缬沙坦。5鳖甲煎丸可能通过抑制TIMP-1的表达及ECM的分泌,与TGF-β1的作用相对抗,从而减少ECM的积聚。更多还原

【Abstract】 Objective:Mesangial cells(MCs) are the most active cell component in glomeruli. The excessive proliferation or less apoptosis of MCs leads to more secrete of extracellular matrix (ECM), such as laminin (LN),collagen-Ⅳand fibronectin (FN) and all kinds of cell factors and mediators of inflammation. The deposit of massive ECM and many of the mediators of inflammation will result into kidney fibrosis. Kidney fibrosis is the shortened name of glomerular sclerosis and renal interstitium fibrosis and renal tubule wither. MMPs was the key enzyme of ECM degration. MMP-9 was a important member in MMPs family, which was the most important composition of degradation collagenⅣ. TIMP-1 was specificity inhibiting factor of MMP-9, and it had a key effect on the nutritive equilibrium of ECM accumulation and degradation, if MMP-9 overexpression, the reason was that ECM composition was surplus and deposited in glomcrulus to reduce to glomerular sclerosis eventually.The investigation discovered that transforming growth factor-β(TGF-β) played an important part in controlling proliferation of MCs, especially accumulation of ECM. TGF-β1 was one of the factors inducing kidney fibrosis. Accordingly, the criticality links of delaying kidney fibrosis is to inhibit proliferation of MCs, reduce ECM composition, retrieve matrix metalloproteinase disequilibrium and inhibit the expression of TGF-β1.With further investigation about the pathogenesy of kidney fibrosis, especially the investigation about cell and molecular biology, many pathways of anti-renal interstitium fibrosis were established in the different links of ECM metabolism. The anti-renal interstitium fibrosis investigation was developed, however, the result was not satisfactory. Traditional Chinese medicine had a good prospective because it could intervent and delay kidney fibrosis advancement. BJJ pill was founded by Zhang Zhongjing of Han dynasty. Its feature was healing QI-blood, apploying chills and fever medicine, and simultaneous application of purging-tonifying therapy, which was a distinguished prescription of treating NUEMU. The prescription was applied in treating hepatitis and liver fibrosis and so on. We applied it into kidney fibrosis and gained some achievement. We found that liver and kidney fibrosis had autoploidy by the summary of literature report. For approaching BJJ pill mechanism of action from the level of cell and molecule, lipopolysaccharide (LPS) as a kind of stimulating factor was applied for MCs in out-of-body cultivation in the experiment. We study the intervention of BJJ pill in MCs proliferation, apoptosis, ECM and cell factor.Methods1 Medicamentum blood-serum preparationThree healthy volunteers of similar age and weight were selected. Among the total, two persons respectively took medicine BJJ pill and Valsartan for three days, the former 4g each time, twice every day; the latter 80mg each time , once every day. After three days, venous blood was taken from two persons one hour after the last time taking medicine, 20ml every person. The blood-serum was separated, deactivated 56℃30min and filtered through 0.22um filter membrane, conserved and reserved -70℃. At the same time, the person without taking medicine was taken venous blood, and the blood-serum was separated and reserved.2 The cultivation and appreciation of MCsMCs came from a 5-months-foetus induction deliveried kidney, which was provided by Hebei medical university affiliated hospital. The foetus’s kidney was dislodged quickly, kidney amicula was stripped, cortex of kidney was cut chipping and polished by passing from 80, 140, 220 order grit. The glomcrulus were collected and digested 6 to 7 minutes by Col-Ⅳenzyme, sequential seeded in 25 cm2 culture flask, cultivated by RPIM-1640 containing 20%FCs, Hepes, penicillin and phytomycin, and PH value was adjusted to 7.2-7.4 by 5%NaHCO3. About 5 days later, glomcrulus adherence ,cells growth, virgin fluid was changed, then once every two days. MCs were digested and passed by parenzyme when it had fused 80%. MCs in deuto-cultivation grew quickly, being covered with the bottom for 36h to 48h, MCs were accredited by contrast phase microscope, transmission electron microscope and immunohistochemical method.3 The indicatrix and method3.1 Subgroup and cultivation3.1.1 Subgroup in the time of proliferation and ELISA MCs were inoculated in 96 shadow masks 1×104 every aperture, after 24 hours, 1% fetal bovine serum was added in culture flask to incubate 24 hours, then the clear supernatant liquid was discarded; 96 shadow mask from left to right were divided into Blank Control Group (calling for short Blank Group, 5% normal anthropo-blood-serum added), BJJ Group (5% BJJ blood-serum added), Valsartan Group (5% Valsartan blood-serum added), LPS Group (LPS10ug/ml and 5% normal blood-serum added), LPS and BJJ Group (calling for short ZhJ Group, LPS10ug/ml and 5% BJJ blood-serum added), LPS and Valsartan Group (calling for short ZhT Group, LPS10ug/ml and 5% Valsartan blood-serum added). There were six groups, fukongs were added in every group (18 fukongs in generation, 6 fukongs in detecting LN, Col-Ⅳ, FN, MMP-9 and TIMP-1 by the means of ELISA). RPMI-1640 culture fluid contenting fetal bovine serum was added to every group to blood-serum concentration achieving 10%, culture fluid volume dose was 200ul, incubating 48 hours in 5%CO2 incubator, preparing for detecting indicatrixs.3.1.2 Subgroup in the time of flow cytometry and gene detectionThe 6 or 7 generation MCs were inoculated into 25cm2 culture flask with the density of 1×106/ml, 1% FCS RPMI-1640 culture fluid was added to incubate 24 hours, when MCs were changed into G0 stage, it was divided into 6 groups, the method was the same as the anterior. RPMI-1640 culture fluid contenting fetal bovine serum was added into every group to blood-serum concentration achieving 10%, after incubating 48 hours, MCs con-RNA was extracted.3.2 The detection of MCs proliferation After incubating 48 hours in every group, MCs proliferation was detected by the means of MTT.3.3 The detection of MCs proliferation, apoptosis and cell cycle The method of detecting MCs proliferation, apoptosis and cell cycle was flow cytometry.3.4 The detection of the content of LN, Col-Ⅳ, FN, MMP-9 and TIMP-1 in MCsAfter incubating 48 hours every group, the ELISA method was applied for detecting the content of LN, Col-Ⅳ, FN, MMP-9 and TIMP-1 in cell clear supernatant liquid.3.5 The detection of FNmRNA and TGF-β1mRNAThe RT-PCR was applied to detecting the content of FNmRNA and TGF-β1mRNA.4 Statistics methodThe data was deled by the SPSS11.5 for window statistics software, all of the data were demonstrated by means±standard deviation, one-factor analysis of variance was applied in the comparison of sample mean. Results1 The cultivation and appreciation of glomcrulus MCs1.1 The glomcrulus segregation More 95% glomcrulus had no bundle in the microscope and there was no renal tubule in the different screen mesh grits.1.2 The morphology character On the fourth day of primary culture, there was a part glomcrulus grew. On the sixth day of primary culture there was 50% to 80% glomcrulus growth adherence in culture flask and glomcrulus endothelial cells and MCs were growing. Because of imadaptative cultivation, endothelial cells died on the 12 day, however MCs grew quickly, their shapes were like star and triangle and had anomal-ecphyma. On the sixteenth day, MCs grew closely, 80% MCs existed in bottle bottom, cell nucleus lied the center of cell and its shape was orbicular-ovate, kytoplasm had many of irregular ecptoma. Passage cells grew adherence at 36 to 48 hours, their shape was star or fusiform shape. 1.3 Transmission electron microscope The shape of glomcrulus MCs in the condition of deuto-cultivation was irregular, the cells borderline had many digitatio ecptoma, with plenty of rough endoplasmic reticulum and free ribosome existed in the kytoplasm. There was bigger round or elliptical pit in cells, the karyolemma was clear. The disposition of caryotin was rarefactcal, there was massive fascl microfilament in the surrounding of caryon.1.4 The result of immunohistochemical method The cells presented Desmin staining masculine. It proved that the cultivate cells were MCs.2 The effect of BJJ pill on glomcrulus MCs proliferation, apoptosis and cell cycle2.1 The effect of BJJ pill ischemic blood-serum on proliferation of MCs in the condition of normal cultivation and LPS excitationThe result displayed that Blank Group at 24h and 48h had some expression; The expression of LPS Group was more obvious than that of Blank Group, for there was significant difference(P<0.01); The proliferation of BJJ Group and Valsartan Group was more descent than that of Blank Group, for there were significant difference (P<0.01; P<0.05); The proliferation of ZhJ Group and ZhT Group at 24h was more descent than that of LPS Group, for there was significant difference (P<0.01); The proliferation of ZhJ Group at 48h was more descent than that of LPS Group, for there was significant difference (P<0.01); There was no statistics difference in ZhT Group and LPS Group at 48h about MCs proliferation (P>0.05); The proliferation of MCs in every group at 48h obviously enhanced than that of at 24h,for there was significant difference (P<0.01).2.2 The effect of BJJ pill ischemic blood-serum on MCs cell cycle in the condition of normal cultivation and LPS excitationThere were 27.93% MCs in Blank Ggroup interrupted in G0/G1 stage. With comparison of Blank Group, MCs interrupted in G0/G1 stage were shorter in LPS Group(P<0.01). With comparison of Blank Group, MCs interrupted in G0/G1 stage in BJJ Group and Valsartan Group were considerable(P<0.01); for there was no significant difference between the BJJ Group and Valsartan Group (P>0.05), but the G0/G1 stage percentage of BJJ Group was higher. MCs interrupted in G0/G1 stage in ZhJ Group and ZhT Group obviously upgraded than that of LPS Group (P<0.01); for there was no significant difference between the ZhJ Group and ZhT Group (P>0.05), but the G0/G1 stage percentage of ZhJ Group was higher.2.3 The effect of BJJ pill ischemic blood-serum on proliferation index of MCs in the condition of normal cultivation and LPS excitationThe proliferation index in Blank Group was 72.07% and apoptosis rate was 3.99%. With comparison of Blank Group, the proliferation index in LPS Group was higher, for there was significant difference (P<0.01). The apoptosis rate was no significant difference between LPS Group and Blank Group (P>0.05). The proliferation index in BJJ Group and Valsartan Group was obviously descent than that of Blank Group and apoptosis rate obviously upgraded(P<0.01), for there was no significant difference between the BJJ Group and Valsartan Group(P>0.05). With comparison of LPS Group, the proliferation index in ZhJ Group and ZhT Group was obviously lower and apoptosis rate obviously upgraded (P<0.01), for there was no significant difference between the ZhJ Group and ZhT Group (P>0.05).3 The effect of BJJ pill ischemic blood-serum on ECM3.1 The result of LN, Col-Ⅳand FN by ELISAThe result proved that the contents of LN, Col-Ⅳand FN in LPS Group was obviously higher than that of Blank Group (P<0.01). The contents of LN, Col-Ⅳand FN in BJJ Group and Valsartan Group were obviously lower than that of Blank Group(P<0.05; P<0.01). There was no significant difference between the BJJ Group and Valsartan Group(P>0.05); however, the content in BJJ Group was lower than that of Valsartan Group; With comparison of LPS Group, the contents of LN, Col-Ⅳand FN in ZhJ Group and ZhT Group was lower(P<0.01). There was no significant difference between the ZhJ Group and ZhT Group (P>0.05). However, the content in ZhJ Group was lower than that of ZhT Group.3.2 MCs RNA abstraction and the result of FN RT-PCR 3.2.1 The result of MCs RNACells RNA were determined by nucleic acid analysator: The OD260/OD280 was in the middle of 1.8 to 2.0. By agarose gelatum electrophoresis, 28S/18S was larger than 2:1. It was proved that RNA purity was altus, complete and no degradation.3.2.2 The result of RT-PCRReverse transcription production was progressed PCR amplification by FN primer, 346bp specificity amplification band appeared. Amplification band of LPS Group was the brightest, sequential Blank Group. Compared with Blank Group, the amplification band of BJJ Group and Valsartan Group was gloomer;the amplification band between the ZhJ Group and ZhT Group was similar. The proportionality of integral optical density in FNmRNA amplification part of every group andβ-actin amplification part was calculated. The result proved that FNmRNA in Blank Group had elementary expression; The proportionality in LPS Group upgraded obviously than that of Blank Group, there was significant difference (P<0.01). With the comparison of Blank Group, the proportionality of FNmRNA/β-actinmRNA in BJJ Group and Valsartan Group was obviously descendend, there was significant difference (P<0.05); There was significant difference between the BJJ Group and Valsartan Group(P<0.01). With the comparison of LPS Group, the proportionality of TGF-β1mRNA/β-actinmRNA in ZhJ Group and ZhT Group was obviously descendend, for there was significant difference (P<0.01). The expression in ZhJ Group was lower than that of ZhT Group, for there was significant difference(P<0.05).4 The effect of BJJ pill on human MCs MMP-9, TIMP-1and MMP-9/TIMP-1 contentsThe result proved that the contents of MMP-9 in LPS Group was no difference with Blank Group (P>0.05), but the content in LPS Group was lower that of Blank Group. The expression of TIMP-1 in LPS Group was obviously higher than that of Blank Group. The content was significant difference between two Groups(p < 0.01). The proportionality of MMP-9/TIMP-1 in LPS Group was obviously lower than that of Blank Group(p<0.01).The contents of MMP-9 in BJJ Group Valsartan Group were no difference with Blank Group (P>0.05), but the contents in BJJ Group and Valsartan Group were higher than that of Blank Group. There was no significant difference between the BJJ Group and Valsartan Group (P>0.05). The contents of TIMP-1 in BJJ Group was obviously lower than that of Blank Group (p<0.05); The contents of TIMP-1 in Valsartan Group was of no significant difference with the Blank Group (P>0.05). The proportionality of MMP-9/TIMP-1 in BJJ Group and Valsartan Group was higher than that of Blank Group(p<0.01); The proportionality of MMP-9/TIMP-1 was no significant difference between Valsartan Group and BJJ Group (P>0.05).The expression of MMP-9 in ZhJ Group and ZhT Group was no difference with LPS Group (P>0.05), but the contents in ZhJ Group and ZhT Group value was higher than that of LPS Group. There was no significant difference between the ZhJ Group and ZhT Group (P>0.05). The expression of TIMP-1 in ZhJ Group and ZhT Group was lower than that of LPS Group (P<0.01).There was no significant difference between the ZhJ Group and ZhT Group (P>0.05), however, the content in ZhJ Group was higher than that of ZhT Group. The expression of MMP-9/TIMP-1 in ZhJ Group and ZhT Group was higher than that of LPS Group (P<0.01). There was no significant difference between the ZhJ Group and ZhT Group (P>0.05), but the content in ZhJ Group was higher than that of ZhT Group.5 The effect of BJJ pill on human glomcrulus MCs TGF-β1mRNA expression5.1 The result of MCs RNACells RNA was determined by nucleic acid analysator: OD260/OD280 was in the middle of 1.8 to 2.0, by agarose gelatum electrophoresis, 28S/18S was larger than 2:1. It was proved that RNA purity was altus, complete and no degradation.5.2 The result of RT-PCRReverse transcription production was progressed PCR amplification by TGF-β1 primer, 321bp specificity amplification band appeared. Amplification band of LPS Group was brighter, sequential Blank Group. Compared with Blank Group, the amplification band of BJJ Group and Valsartan Group was gloomer. The amplification band in ZhJ Group and ZhT Group were similar with of Blank Group. The proportionality of integral optical density in TGF-β1mRNA amplification part of every group andβ-actin amplification part were calculated. The result proved that TGF-β1mRNA/β-actinmRNA in Blank Group had elementary expression. The proportionality in LPS Group was upgraded obviously than that of Blank Group, there was significant difference (P<0.01). With the comparison of Blank Group, the proportionality of TGF-β1mRNA/β-actinmRNA in BJJ Group and Valsartan Group was obviously descendend, there was significant difference (P<0.01), and the proportionality of BJJ Group was lower than that of Valsartan Group (P<0.01). With the comparison of LPS Group, the proportionality of TGF-β1mRNA/β-actinmRNA in ZhJ Group and ZhT Group was obviously descendend, there was significant difference (P<0.01). The proportionality of ZhJ Group was lower than that of ZhT Group (P<0.01).ConclusionAs a kind of incentive factor, LSP was applied in MCs in out-of-body cultivation . BJJ pill was interfered and Valsartan was applied as control at the same time. With the analysis, the conclusion was as following:1 LPS could advance MCs proliferation, but its effect on restraining apoptosis was not obvious. Metabolic blood-serum of BJJ pill and Valsartan could obviously inhibit MCs proliferation in the normal and LPS excitation condition, advance MCs apoptosis. The effect on BJJ pill was more obvious than that of Valsartan.2 LPS could advance ECM externalization and could promote the expression of MCs FNmRNA. Metabolic blood-serum of BJJ pill and Valsartan could obviously inhibit ECM externalization in the normal and LPS excitation condition and the expression of MCs FNmRNA. In the superior aspect, BJJ pill surpass Valsartan. 3 LPS could advance the expression of TIMP-1, but it was inferior to inhibit the expression of MCs MMP-9, and as a result, the proportionality of MMP-9/TIMP-1 was degraded. Metabolic blood-serum of BJJ pill and Valsartan could obviously inhibit the expression of TIMP-1 in the normal and LPS excitation condition and BJJ pill surpass Valsartan. The expression of advancing MCs MMP-9 in metabolic blood-serum of BJJ pill and Valsartan was inferior.4 LPS could advance the expression of MCs TGF-β1mRNA. Metabolic blood-serum of BJJ pill and Valsartan could obviously inhibit the expression of TGF-β1mRNA in the normal and LPS excitation condition, and BJJ pill surpass Valsartan.5 Its conceivable mechanism was that BJJ pill could inhibit the expression of TIMP-1 and oppose the the expression TGF-β1 so as to reduce ECM congestion.更多还原