TGF—β2干预的缺氧状态下补肾活血剂对Muller细胞及谷氨酰胺合成酶活性的影响

目的观察在缺氧和（或）转化生长因子-β2（TGF—β2）干预条件对体外培养的Muller细胞活力以及谷氨酰胺合成酶（GS）的影响。方法用出生7d的SD大鼠体外培养视网膜Muller细胞，将第2代细胞在含20％胎牛血清的DMEM中培养24h后，换用无血清的DMEM孵育24h。分组为：空白血清对照组（20％正常SD大鼠血清的DMEM）；TGF—β2组（20％正常SD大鼠血清的DMEM及终质量浓度为150ng／L的TGF—β2）；模拟缺氧组（20％正常SD大鼠血清的DMEM及终浓度1．0mmol／L的连二亚硫酸钠）；TGF—β2＋缺氧组（20％正常SD大鼠血清的DMEM液、终浓度1．0mmol／L的连二亚硫酸钠和终质量浓度为150ng／L的TGF—β2）；中药血清组（20％中药SD大鼠血清的DMEM）；中药血清＋TGF—β2＋缺氧组（20％中药SD大鼠血清的DMEM、终浓度1．0mmol／L的连二亚硫酸钠和终质量浓度为150ng／L的TGF—β2）。以透射电镜观察视网膜Muller细胞超微结构、以490型酶标仪测定细胞外液乳酸脱氢酶（LDH）释放量及GS活性。结果在缺氧条件下Muller细胞的超微结构发生明显的改变，如：细胞核变形，固缩；细胞器破坏；微绒毛内糖原明显增多。与正常对照组比较，TGF-β2干预组、缺氧组、TGF-β2＋缺氧组的LDH释放量均明显增加（P〈0．05），缺氧组、TGF—β2＋缺氧组的GS活性均明显下降（P〈0．01）；与缺氧组比较，TGF-β2＋缺氧组的LDH释放量明显增加、GS活性明显下降（P〈0．05、P〈0．01）。补肾活血中药血清能降低24h及48h时正常以及TGF—β2与缺氧同时存在条件下LDH的释放量（P〈0．05），增强12h时正常条件下以及12h和24h时TGF-β2与缺氧同时存在条件下GS的活性（P〈0．05、P〈0．01）。结论TGF—β2可加重缺氧条件下Muller细胞活力与GS活性的降低；补肾活血中药含药血清能增强视网膜Muller细胞活力及GS活性。

Effects of bushenhuoxue on activities of glutamine synthetase and retinal Muller cells under TGF-β2-intervene hypoxic condition

Background Our previous study determined that bushenhuoxue decoction can induce the reactive astrogliosis of retinal Muller cells in rat and further plays a protecting effect on retinal ganglion cells against the damage due to diabetic retinopathy（DR）. However,its mechanism is still being investigated. Objective The present study was to observe the change of activity of glutamine synthetase（GS） and retinal Muller cells in vitro under hypoxic conditions or transforming growth factor-β2 （TGF-β2）-intervened hypoxie conditions and investigate the effect of bushenhuoxue decoction. Methods Retinal Muller cells were isolated from clean 7-day-old SD rats and cultured in the DMEM containing 20% fetal bovine serum for 24 hours and then in free-serum medium for other 24 hours. The cells were grouped based on the next cultivating procedure as follows： normal group （DMEM ＋ 20% serum culture） , TGF-β2 group（ DMEM ＋ 20% serum and 150 ng/L TGF-β2 ） , hypoxia group（ DMEM ＋ 20% serum and 1.0 mmol/L sodium hydrosulfite） , TGF-β2 ＋ hypoxia group, serum with drug cultured group, serum with drug ＋ TGF-β2 ＋ hypoxia group. The uhrastructure of retinal Mailer cells was observed under the transmission electron microscope. The content of lactate dehydrogenase （LDH） in extracellular fluid was detected to evaluate the activity of retinal Mailer cells,and the activity of GS was assessed with 490-enzyme-labeled instrument. Results The uhrastructure of retinal Muller cells in hypoxia group showed obvious pathological damages in hypoxic conditions,including vacuolization of cytoplasm, deformation and agglutination of cell nucleus,destroy of cell organs and increase of glucogen in microvilli. However, these changes were mild. Compared with normal group,the release of LDH was significantly enhanced, and the activity of GS was obviously weakened in TGF-β2 group, hypoxic group and TGF-β2 ＋ hypoxic group （ P 〈 0.05 ）. Compared with hypoxic group, the LDH level was significantly increased, whereas the activity of GS was obviously decreased in TGF-β2 ＋ hypoxic group （ P 〈 0.05 ）. In comparison with TGF-β2 group or hypoxia group, the LDH content in TGF-β2 ＋ hypoxic group was considerably lower （P 〈 0. 05）. In all the groups with bushenhuoxue ＋ serum,the release of LDH was declined in comparison to without bushenhuoxue ＋ serum groups at 24 and 48 hours （P〈0.05）,and bushenhuoxue ＋ serum groups also significantly enhanced the activity of GS both in 12 hours of normal control condition and 24 - 48 hours of TGF-β2-intervened hypoxic conditions （P 〈 0.05 ）. Conclusion TGF-β2 can aggravate the activity of GS and retinal Mailer cells in hypoxic condition. Bushenhuoxue medicine-containing serum can strengthen the activity of retinal MUller cell and GS. Bushenhuoxue may be a new approach to the prevention and treatment of DR.