压力对大鼠纯化视网膜神经节细胞中Fas/FasL mRNA表达的影响

Pressure influence on mRNA expression of Fas/FasL in purified retinal ganglion cells of rats

目的 通过检测不同压力及时间纯化培养的大鼠视网膜神经节细胞 (retinalgangal cells,RGCs)中 Fas/ Fas L m RNA的表达 ,探讨 Fas/ Fas L在 RGCs凋亡中的作用。方法 用 SD系大鼠脑源性星型胶质细胞同化培养液双界面法培养羊抗鼠 Thy1.1单克隆抗体纯化的 SD系大鼠 RGCs。分别在 0 m m Hg(对照组 )和 2 0、4 0、6 0 and80 mm Hg(1k Pa=7.5 m m Hg)的压力下培养 2 4 h及 4 8h后 ,用免疫组化和原位杂交对 RGCs中 Fas/ Fas L及其 m RNA的变化进行定性及半定量观察 ;TU NEL 法检测各组细胞的凋亡。结果纯化的 RGCs培养 2 4 h用羊抗鼠 FITC- Thy- 1.1单克隆抗体进行鉴定阳性 ,纯度为 97% .免疫组化和原位杂交检测 Fas/ Fas L及其 m RNA,结果培养 2 4 h对照组无表达 ,2 0 mm Hg组有较弱的表达信号 ,4 0、6 0及 80 mm Hg组表达逐渐增强 ;与对照组比较差异均有显著性 (P<0 .0 5 ) ;培养 4 8h对照组弱表达 ,压力≥ 2 0 mm Hg各组较相同压力培养 2 4 h各组表达增强 ,差异有显著性 (P<0 .0 5 )。 TU NEL 法检测细胞凋亡 ,培养 2 4 h对照组未见细胞凋亡20 m m Hg见少量细胞凋亡 ,随着压力的增高细胞凋亡数增多 ,凋亡指数增高 ;与对照组相比均有显著性差异。培养 4 8h组压力≥ 2 0 m m Hg各组较相同压力培养 2 4 h组?

Objective To study the Fas/FasL mRNA expression in rats purified retinal ganglion cells(RGCs) that cultured in different pressures. Methods To culture RGCs that from Sprague Dawley(SD) neonatal rats(postnatal 1~5d) on two planes in assimilative culture solution in vitro , RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured under pressures of 0?20? 40? 60 and 80mmHg(1kPa=7.5mmHg) for 24 hours and 48 hours, respectively. The changes of Fas and FasL mRNA expression in RGCs under different pressures were demonstrated qualitatively and quantitatively by immunohistochemistry and in situ hybridization method and their apoptosis were detected by TUNEL method, respectively. Results After cultured 24 hours in vitro , the purification rate of RGCs in the experiment arrived at 97 percent. After cultured 24 hours, the expression of apoptotic cells in RGCs became higher and higher with the pressure increased. There were a few apoptotic cells expression in the control group (0mmHg) and 20?40?60 and 80mmHg groups had a significant difference from the control group, respectively( P <0.05). After cultured 48 hours, had a significant difference each of 20?40?60 and 80mmHg groups than which of cultured 24 hours, respectively ( P <0.05). If the pressure equal or higher than 20mmHg, the expression of Fas, FasL and their mRNA in RGCs became higher and higher as the pressure was increased and had a significant difference from control group, respectively( P <0.01). Conclusion Pressure can evoke the expression of Fas and FasL mRNA became higher in purified retinal ganglion cells in vitro and induce or aggravate RGCs to apoptosis.