补肾活血剂对高糖和(或)缺氧状态下视网膜神经节细胞活力及谷氨酰胺合成酶活性的影响  

Effects of Bushenhuoxue decoction on activities of glutamine synthetase and RGC cultured in high glucose concentration or /and hypoxic conditions

作　　者：马殿伟[1] 谢学军[2] 张梅[3] 万李[4] 

机构地区：[1]武警四川总队医院成都分院五官科,四川省成都市610041 [2]成都中医药大学临床医学院眼科,四川省成都市610075 [3]成都中医药大学临床药学院,四川省成都市610075 [4]成都中医药大学临床医学院,四川省成都市610075

出　　处：《眼科新进展》2014年第6期510-514,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30572407)~~

摘　　要：目的观察补肾活血中药对高糖和(或)缺氧状态下体外共同培养的视网膜神经节细胞(retinal ganglion cells,RGC)和Müller细胞活力以及谷氨酰胺合成酶(glutamine synthetase,GS)的影响,并对其作用机制进行探讨。方法以中药血清药理学方法获得补肾活血复方中药含药血清。建立RGC与视网膜Müller细胞共同培养模型,在体外共同培养细胞的培养液中加入葡萄糖(终浓度为50 mmol·L-1)/连二亚硫酸钠(终浓度为1.0 mmol·L-1)造成细胞高糖(缺氧)模型(高糖/缺氧组),仅加入终浓度为50 mmol·L-1葡萄糖造成细胞高糖模型(高糖组),仅加入终浓度为1.0 mmol·L-1连二亚硫酸钠造成细胞缺氧模型(缺氧组);在此基础上加入体积分数20%中药含药血清进行中药血清干预。在实验干预24 h、48 h及72 h 3个时间段分别以酶联免疫吸附法(ELISA)测定体外共同培养细胞外液中乳酸脱氢酶(lactate dehydrogenase,LDH)漏出量和GS活性。结果高糖组、缺氧组及高糖/缺氧组共同培养的细胞LDH漏出量在24 h、72 h比正常对照组均明显增加(均为P<0.05),缺氧组视网膜Müller细胞GS活性在48 h比正常对照组明显降低(P<0.05);高糖组视网膜Müller细胞GS活性在24 h、48 h和72 h三个时间点均比正常对照组增高(均为P<0.05),补肾活血中药血清能够增加缺氧条件下及高糖缺氧条件下视网膜Müller细胞GS活性,并且该作用随着时间的延长其作用逐渐加强。结论补肾活血中药血清可能通过降低高糖和(或)缺氧状态下共同培养的RGC与Müller细胞细胞膜的通透性,提高细胞活力。同时提高视网膜Müller细胞和RGC共同培养时视网膜Müller细胞GS活性,转化Glu,减轻Glu的兴奋性毒性,从而起到细胞保护作用。Objective To study the protective effects of Bushenhuoxue （BSHX） decoction on the activities of ghitamine synthetase and retinal ganglion cells （ RGC ） and retinal Mfiller cells co-cultured in vitro under high glucose concentration or/and hypoxic conditions, and study its molecular mechanism. Methods Serum with BSHX decoction was obtained by the serum pharmacology method of traditional Chinese medicine. The retinal Mtiller cells and RGC of newborn-rats were purified and cultured using the modified enzyme-digesting method by means of a two-step filtration process. RGC were then planted onto culture plates coated with retinal Mtiller cells. The two types of cells were co-cultured in a ratio of 1 ： 25 to make the co-culture model. Glucose （ final concentration of 50 mmol · L^- 1 ） or sodium dithionite （ final concentration 1.0 mmol · L^- 1 ）was added to the co-culture cells to make the high glucose concentration or hypoxic experimental models respectively （ie. high glucose or hypoxic groups）. The high glucose concentration and hypoxic group was made by adding glucose （final concentration of 50 mmol · L^- 1 ） and sodium dithionite （ final concentration 1.0 mmol · L^- 1 ） simultaneously;Each group was treated by 20% serum BSHX decoction. After 24 hours, 48 hours and 72 hours of serum BSHX decoction treatment, extracellular lactate dehydrogenase （LDH） leakage and glutamine synthetase （GS） activity of the co-culture cells were measured by enzyme-linked immunosorbent assay （ELISA）. Results Compared with the control group, the LDH leakage of co-cultured cells in the high glucose group, hypoxic group and combination of high glucose and hypoxic group after 24 hours and 72 hours treatment were significantly increased （ all P 〈 0.05 ）. Compared with the control group, the GS activity of retinal Mueller cells in the hypoxic group was significantly decreased after 48 hours treatment （P 〈 0.05 ）. Compared with the control group, the GS activity of retinal Mtiller cells in

关 键 词：糖尿病视网膜病变 补肾活血剂 高糖 缺氧 视网膜神经节细胞 

分 类 号：R775[医药卫生—眼科]