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目的探讨凝血酶增高大鼠的小脑颗粒细胞N-甲基-D-天冬氨酸受体(NMDA)敏感性是否与细胞内组织型谷氨酰胺转移酶(tTG)变化有关。方法以0·01U/ml浓度凝血酶单独或与tTG抑制剂单丹磺酰尸胺(MDC)0·5×10-4mol/L处理原代培养的Wistar大鼠小脑颗粒细胞30min后,以0·1×10-4mol/L、0·25×10-4mol/L和0·5×10-4mol/L3种浓度NMDA作用3h诱导上述处理的细胞凋亡,比较流式细胞仪检测到的细胞凋亡率差异以判断NMDA受体敏感性的变化;RT-PCR和Gelpro图像分析软件检测各NMDA浓度有/无凝血酶预处理的细胞内tTG mRNA变化差异。结果凝血酶预处理细胞后上述3种浓度NMDA诱导的细胞凋亡率均升高[(8·3±0·5~15·1±0·8)%,(14·1±1·1~23·8±0·7)%,(26·8±1·9~33·7±2·1)%]。预处理细胞时加入MDC可阻断凝血酶对NMDA受体的这种影响,细胞凋亡率降至(5·8±1·2)%、(11·5±1·5)%和(19·0±1·7)%;凝血酶预处理后各浓度NMDA处理细胞内tTG mRNA量均同一浓度单独NMDA处理的细胞高。结论凝血酶通过增高细胞内tTG使小脑颗粒细胞NMDA受体敏感性增高。
Objective To investigate whether the sensitivity of N-methyl-D-aspartate receptor (NMDA) to cerebellar granule cells in thrombin-induced rats is related to the changes of intracellular tissue transglutaminase (tTG). Methods Primary cultures of Wistar rat cerebellar granule cells were treated with 0 · 01U / ml thrombin alone or with 0.5 × 10-4mol / L tanshinone (MDC) The apoptosis of the above-treated cells was induced by 1 × 10-4 mol / L, 0 · 25 × 10-4 mol / L and 0 · 5 × 10-4 mol / L of the concentration of NMDA for 3h, and the cells detected by flow cytometry Apoptotic rate to determine the change of NMDA receptor sensitivity; RT-PCR and Gelpro image analysis software to detect each NMDA concentration with / without thrombin pretreatment of intracellular tTG mRNA differences. Results After thrombin preconditioning, the cell apoptosis rate induced by the above three concentrations of NMDA increased from (8.3 ± 0.5 to 15.1 ± 0.8)%, (14.1 ± 1.1 ~ 23.8 ± 0.7%, (26.8 ± 1.9 · 33.7 ± 2.1)%]. Pretreatment of cells with MDC blocked the effect of thrombin on NMDA receptors, and the rates of apoptosis were decreased to (5 · 8 ± 1 · 2)%, (11 · 5 ± 1 · 5)% and (19 · 0 ± 1 · 7)%. The amount of tTG mRNA in NMDA-treated cells after thrombin pretreatment was higher than that in NMDA-treated cells alone. Conclusion Thrombin increases the sensitivity of NMDA receptors in cerebellar granule cells by increasing intracellular tTG.