补肾活血中药对TGF-β_2及高糖状态下体外培养的视网膜Müller细胞活力的影响

Effect of Bushen Huoxue Decoction on retinal Müller cells cultured in TGF-β_2 and high glucose conditions

目的探讨高糖及转化生长因子-β2(Transforming growth factor,TGF-β2)和高糖同时存在条件对体外培养的Müller细胞活力的影响,以及补肾活血中药的干预作用。方法以改良酶消化法体外培养大鼠Müller细胞,将P3细胞分别置于模拟正常、高糖、TGF-β2及TGF-β2和高糖同时存在条件下进行培养,并以补肾活血中药复方含药血清进行干预。实验分为正常对照组、TGF-β2组、高糖组、TGF-β2+高糖组、正常中药干预组、TGF-β2+高糖中药干预组;分别在试验干预12h、24h及48h后,以490型酶标仪测定各组细胞外液中乳酸脱氢酶(lactate dehydrogenase,LDH)的漏出量。结果 TGF-β2组和高糖组12h和24h的LDH漏出量与正常对照组比较均无明显差异(P>0.05),但48h的LDH漏出量均较正常对照组增多(P<0.05);TGF-β2组24h的LDH漏出量较高糖组增多(P<0.05);TGF-β2+高糖组24h和48h的LDH漏出量均较TGF-β2组减少(P<0.05);TGF-β2+高糖组48h的LDH漏出量较高糖组减少(P<0.05);补肾活血中药血清能降低24h时TGF-β2和高糖同时存在条件下LDH的漏出量,降低各时段正常条件下的LDH漏出量(P<0.05)。结论 Müller细胞对短期高糖状态以及浓度为150pg/ml的TGF-β2短期干预可能有一定的耐受性,但当高糖(50mol/l)以及TGF-β2(150pg/ml)干预48h后Müller细胞活力明显降低;补肾活血中药复方含药血清能提高正常及高糖和TGF-β2同时存在条件下视网膜Müller细胞膜的稳定性、增强Müller细胞活力可能是补肾活血中药复方防治糖尿病性视网膜病变的另一药物干预途径。

OBJECTIVE To observe the change of activity in vitro retinal Müller cells cultured in high glucose condition and high glucose condition intervened with transforming growth factor-β2（TGF-β2）,and to investigate the effect of the traditional Chinese medicine named Bushen Huoxue Decoction.METHODS Modified enzyme-digesting method was used to culture retinal Müller cells,P3 cells were then cultured in normal control conditions,high glucose conditions,TGF-β2 conditions and high glucose conditions intervened with TGF-β2,Bushen Huoxue medicine-containing serum was added to the culture medium.The Experiment was assigned into normal control group,TGF-β2 group,high glucose group,TGF-β2 ＋ high glucose group,TCM intervening group;Detected the lactate dehydrogenase（LDH） content of extracellular fluid by 490-enzyme-labeled instrument.RESULTS Compared with normal group,the LDH leakage was significantly increased in TGF-β2 group and high glucose group at 48h（P0.05）,whereas there was no difference at 12h and 24h（P0.05）;compared with high glucose group at 24h the LDH leakage of TGF-β2 group was increased（P0.05）;compared with TGF-β2 group（at 24h,48h） and high glucose group（at 48h）,the LDH leakage of TGF-β2 ＋ high glucose group was decreased（P 0.05）;Bushen Huoxue medicine-containing serum could significantly decrease the LDH leakage both in high glucose conditions intervened with TGF-β2 at 24h and normal control conditions at each period（P0.05）.CONCLUSIONS Retinal Müller cells displayed tolerability to high glucose condition and TGF-β2（150pg/ml） condition in short period,whereas its activity was obviously decreased after 48h,the effect of TGF-β2 on retinal Müller cells was different depending on the intervening time and condition.Bushen Huoxue medicine-containing serum can stabilize cells＇ membrane and enhance the activity of retinal Müller cell,in normal control conditions and high glucose conditions intervened with TGF-β2,accordingly this might be another important mechanism of Bushen Huoxue Decoction to prevent and cure diabetic retinopathy.