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虫草肾茶胶囊对高糖培养下人肾小球系膜细胞影响的实验研究
Effects of Chongcaoshencha Capsule on Human Mesangial Cells Cultured in Vitro under High Glucose Condition
【作者】 金丽霞；
【导师】 宋立群；
【作者基本信息】 黑龙江中医药大学 ， 中医内科学， 2009， 博士
【摘要】 目的：观察虫草肾茶胶囊对高糖培养条件下人肾小球系膜细胞(HMC)的影响，并探讨虫草肾茶胶囊对糖尿病肾病(DN)肾脏保护作用的机制。方法：首先采用血清药理学的方法制备虫草肾茶胶囊及福辛普利药物血清，再进行药物血清干预HMC的研究。将常规培养的HMC分为正常对照组、高糖组、虫草肾茶高、中、低剂量组、福辛普利组，采用高糖模型(即高糖刺激体外培养的系膜细胞)进行研究。正常对照组予10％正常大鼠FCS改良型RPMI-1640培养液培养干预，高糖组予10％正常大鼠FCS改良型RPMI-1640高糖培养液培养干预，虫草肾茶高、中、低剂量组分别予10％虫草肾茶胶囊高、中、低剂量含药血清改良型RPMI-1640高糖培养液培养干预，福辛普利组予10％福辛普利含药血清改良型RPMI-1640高糖培养液培养干预。分别在相应干预因素处理后，于培养的24h、48h及72h用MTT法检测各组细胞增殖的情况；于相应干预因素作用48h及72h后，用ELISA法检测细胞培养液上清中转化生长因子-β1(TGF-β1)、纤维连接蛋白(FN)、Ⅳ型胶原(Col-Ⅳ)、层粘连蛋白(LN)的含量；用实时定量PCR方法检测人肾小球系膜细胞转化生长因子-β1mRNA(TGF-β1mRNA)、纤维蛋白溶解酶原激活物抑制剂-1mRNA(PAI-1mRNA)的表达。结果：(1)虫草肾茶胶囊高剂量药物血清对高糖(30mmol／L)刺激下24h、48h和72h的HMC的增殖均有抑制作用(p＜0.05，p＜0.01)，其作用优于福辛普利药物血清。虫草肾茶胶囊中、低剂量药物血清对高糖刺激下48h和72h的HMC的增殖有抑制作用(p＜0.05)。(2)在48h和72h时段，虫草肾茶胶囊高、中剂量组TGF-β1的分泌明显低于高糖组(p＜0.05，p＜0.01)，而虫草肾茶胶囊低剂量药物血清及福辛普利药物血清对72h时段TGF-β1的分泌有抑制作用(p＜0.05)。在抑制TGF-β1的分泌方面，虫草肾茶胶囊优于福辛普利。(3)虫草肾茶胶囊高、中、低剂量组及福辛普利组48h及72h HMCⅣ型胶原的合成均低于高糖组(p＜0.05，p＜0.01)。(4)虫草肾茶胶囊高剂量组细胞培养上清中FN、LN在48h时段和72h时段的分泌均低于高糖组(p＜0.05，p＜0.01)，而虫草肾茶胶囊中、低剂量药物血清及福辛普利药物血清在72h时段可减少细胞培养上清中FN、LN的分泌(p＜0.05，p＜0.01)。(5)虫草肾茶胶囊高、中剂量组及福辛普利组TGF-β1mRNA和PAI-1mRNA表达均低于高糖组，且以虫草肾茶胶囊高剂量组表达最低(p＜0.05)。结论：虫草肾茶胶囊可以抑制高糖刺激下HMC的增殖，降低高糖刺激下TGF-β1及细胞外基质(ECM)主要成分FN、LN、Col-Ⅳ的合成和分泌，下调高糖刺激下HMC TGF-β1mRNA及PAI-1mRNA的表达，起到延缓DN进展的作用。
【Abstract】 Objective:To investigate the effects of Chongcaoshencha capsule on humanmesangial cells cultured in vitro under high glucose condition and explorethe nephroprotective mechanism of Chongcaoshencha capsule for DN.Methods:We first get the rats serum with different concentration of Chongcaoshenchacapsule and fosinopril, and then study the effects of the serum onHMCs. The cultured HMCs were divided into normal control group, highglucose group, High-dose, Middle-dose and Low-dose Chongcaoshenchacapsule group and fosinopril group. The HMCs induced by high glucose wereadopted to study. The normal control group HMCs were cultured with normalrat serum RPMI-1640 culture solution under normal glucose condition, highglucose group HMCs were cultured with normal rat serum high glucoseRPMI-1640 culture solution, the high-dose, middle-dose and low-doseChongcaoshencha group HMCs were incubated with different concentrationof Chongcaoshencha capsule rat serum RPMI-1640 culture solution underhigh glucose condition.the fosinopril group HMCs were incubated withfosinopril rat serum RPMI-1640 culture solution under high glucosecondition. The FCS concentration of every group is 10％. After therespective intervention of 24 hours, 48 hours and 72 hours, the HMCsproliferation was assessed by MTT assay. After culturing 48 and 72 hours byadding different serum respectively, transforming growth factor-betal(TGF-β1) , Fibronectin (FN) , Collagen-Ⅳ(Col-Ⅳ) , laminin (LN)secretion was determined by ELISA. The real time PCR was used to detectthe expression of transforming growth factor beta 1mRNA (TGF-β1mRNA)and plasminogen activator inhibitor-1 mRNA (PAI-1 mRNA)Result:(1) The high-dose Chongcaoshencha capsule serum can inhibit theproliferation of mesangial cells under high glucose condition (30mmol/L) at 24h、48h and 72h (p＜0.05, p＜0.01) , and showed better effect than thefosinopril serum. The middle-dose and low-dose Chongcaosheneha capsulecan inhibit the proliferation of mesangial cells under high glucose condition(30mmol/L) at 48h and 72h (p＜0.05) . (2) TheTGF-β1 secretion of thehigh-dose and middle-dose Chongcaoshencha capsule group is lower thanthat of the high glucose group at 48h and 72h (p＜0.05, p＜0.01) . Thelow-dose Chongcaoshencha capsule and fosinopril can decrease the secretionof TGF-β1 at 72h (p＜0.05) . Chongcaoshencha capsule has better effect. (3)The secretion of Col-Ⅳof Chongcaoshencha capsule group and thefosinopril group is lower than that in the high glucose group at 48h and 72h(p＜0.05, p＜0.01) . (4) The secretion of FN and LN of the high-doseChongcaoshencha capsule group is lower than that in the high glucose groupat 48h and 72h (p＜0.05, p＜0.01 ) . The middle-dose and low-doseChongcaoshencha capsule serum and fosinopril serum can decrease thesecretion of FN、LN at 72h (p＜0.05, p＜0.01) . (5) The expression ofTGF-β1mRNA and PAI-1mRNA of the high-dose and middle-doseChongcaoshencha capsule group and the fosinopril group are lower than thatof the high glucose group, and the expression of high-dose Chongcaoshenehacapsule group is the lowest (p＜0.05)Conclusion:Chongcaoshencha capsule not only can inhibit high glucose-inducedproliferation of HMCs, but also can decrease the secretion of TGF-β1, FN,Col-Ⅳ, LN, and degrade the expression of TGF-β1mRNA and PAI-1mRNA inthe high glucose cultured condition, so to delay the progression of DN.