白蒺藜醇甙对混合培养鼠视网膜神经节细胞的作用

The effect of tribulus fruit alcohol glycoside on rat retinal ganglion cells in vitro

目的 :观察刺蒺藜单体成分白蒺藜醇甙 (tribulusfruitalcoholglycoside ,TFAG)对体外混合培养的鼠视网膜神经节细胞 (retinalganglioncells,RGCs)存活的影响 ,并筛选出其有效的药物浓度 ,同时与脑源性神经营养因子BDNF和碱性成纤维生长因子bFGF比较。方法 :采用胰酶消化法将 12只生后 2～ 3天的Sprague Dawley(SD)乳鼠视网膜制成细胞悬液 ,接种于鼠尾胶原包被的 96孔培养板中 (5× 10 4 个细胞 /孔 )。分为A组 (对照组 )和各种浓度梯度的TFAG实验组 :B组 (1× 10 -8g/L)、C组 (1× 10 -7g/L)、D组 (1× 10 -6g/L)、E组 (1× 10 -5g/L)、F组 (1× 10 -4 g/L)、G组 (1× 10 -3 g/L) ,在 37℃、5 %CO2 恒温培养箱中培养 ,于 1、3、5天 ,用MTT微量自动比色法测量存活细胞的吸光度A值。用Thy 1抗体、NSE抗体和GFAP抗体进行细胞免疫化学检查以鉴定RGCs。结果 :培养在鼠尾胶原上的细胞生长良好 ,大部分伸出突起。上述浓度的TFAG对培养细胞形态无影响。培养 1d ,C和D组吸光度 (A)值高于对照组 ,差异具有显著性意义 (P <0 .0 5 ,P <0 .0 1) ;培养 3d ,仅D组吸光度 (A)值高于对照组 ,差异具有显著性意义 (P <0 .0 5 )。结论 :TFAG具有类似神经营养因子的作用 ,能促进混合培养RGCs在体外的存活。

Objective:To study the effects of tribulus fruit alcohol glycoside(TFAG), one of the main ingredients in tribuls terrestris L.(TTL), on the survival of rat retinal ganglion cells(RGCs) in vitro and to screen for their effective concentration.Methods:RGCs were isolated 2～3 days postnatally in Sprague Dawley rat eyes and cultured. RGCs in cell culture were identified by morphologic criteria and immunocytochemical staining. Various concentrations(1×10 -8 g/L～1×10 -3 g/L) of TFAG were added to individual cultures. MTT was added to each well culture at 1, 3 and 5 days, respectively; then the surviving RGCs were evaluated according to absorbance(A) values using an ELISA Reader. The A values in wells to which TFAG had been added were compared to those from control wells.Results:The cells cultured in the wells by murine tail collagen grew very well. Most of them stained positively for NSE and Thy 1 and negatively for GFAP, indicating they were RGCs. At d 1, the A values of the TFAG groups(1×10 -6 g/L～1×10 -7 g/L) were significantly higher than for those in control group(P<0.01,P<0.05). At d 3, only the A value of the TFAG group(1×10 -6 g/L) was significantly higher than that for the control group(P<0.05).Conclusion:TFAG improves survival of rat RGCs in vitro.