体外星形胶质细胞损伤后炎症模型的建立及应用

Initial Studies on an in vitro Neruoinflammation Model in Astrocytes with Post-traumatic Injury

目的建立胶质细胞损伤后体外炎症模型,探讨轻度低温、槲皮素及清蛋白对白细胞黏附及胶质细胞损伤的影响。方法利用原代培养的成熟星型胶质细胞采用体外划伤方式造成细胞损伤,然后与白细胞共培养建立星形胶质细胞损伤后体外炎症模型。将共培养的细胞放入CO2孵箱中,将温度控制在32℃的亚低温状态,探讨轻度低温、槲皮素及清蛋白对白细胞黏附及胶质细胞损伤的影响。结果白细胞主要黏附于损伤边缘的胶质细胞,并对其有损伤作用。32℃亚低温可明显降低白细胞对胶质细胞的黏附和损伤,但无白细胞存在时其作用不明显。槲皮素和清蛋白对白细胞的黏附无显著影响,对胶质细胞无保护作用。结论低温主要通过降低白细胞黏附达到保护胶质细胞作用,对单纯的细胞机械损伤无明显保护作用,提示胶质细胞损伤后的反应性增生不是介导白细胞黏附的关键因素。

Objective To establish a new in vitro model of post-traumatic neuroinflammation and study on the effects of hypothermia, quercetin and albumin on leukocytes adherence and astrocyte survival with post-traumatic injury. Methods An in vitro post traumatic neuroinflammation model was created by the coculture of leukocytes, which were freshly separated from rat whole blood, and primary cell culture of astrocytes, which had been traumatized by the scratch method. The scratch injury to astrocytes was produced by scratching a confluent culture with a 200 L sterile plastic pipette tip according to a grid. Co-cultures were placed on a slow speed shaker for 30 min, and then gently washed to remove floating leukocytes. Quantitation of leukocytes adhesion was done by labeling leukocytes with Calcin-AM at 37℃ for 30 min. The number of adherent cells was estimated from 10 fields along versus away from the edge of the injury using an inverted fluorescent microscope. Cell viability was estimated by Lactic dehydrogenase （LDH） release and live/dead assay. Results Leukocytes preferentially adhered to injured [（187.3＋31.5） cells/10 hpf] compared to uninjured astrocytes [（18.3＋11.4） cells/10 hpf]. Optimal adhesion along scratch wound edge occurred 12 and 24 hours post-injury. Leukocytes induced injury to astrocytes if co culture time more than 2 days. Under hypothermia at 32℃, LDH release and leukocytes adhesion were reduced by 54% and 37%. However, no any similar effects were found after treatments with quercetin and albumin although quercetin inhibited hypertrophic cell process after astrocytes traumatic injury. Conclusions In our new in vitro neuroinflammation model, leukocytes preferentially adhered to injured astrocytes, enhanced injury and increased cell death. Hypothermia at 32℃ reduced leukocyte adherence and secondary injury to injured astrocytes. Quercetin and albumin had no any effects on leukocytes adherence and astrocytes survival, Together with our previous finding these results showed that hypertrophic cell processes were not key factors in leukocytes adherence.