论文部分内容阅读
目的 观察两种神经营养因子:脑源性神经营养因子BDNF和碱性成纤维生长因子bFGF对混合培养鼠视网膜神经节细胞(retinal ganglion cells, RGCs)的影响,并筛选出其有效浓度。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(5×10~4个细胞/孔)。分别加入各种浓度梯度的BDNF和bFGF,在37℃、5％CO_2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。培养1天、3天,各种浓度的BDNF和bFGF实验组吸光度A值与对照组比较均具有显著性差异(P<0.01,P<0.05);培养5天,BDNF(40ng/ml、50ng/ml)、bFGF(15ng/ml)和BDNF+bFGF组吸光度A值高于对照组,具有显著性差异(P<0.01,P<0.05)。结论 各种浓度的BDNF和bFGF均能促进鼠RGCs在体外的存活,BDNF具有浓度依赖性,BDNF的作用优于bFGF;同时应用BDNF和bFGF无叠加作用。
Objective To observe the effects of BDNF and bFGF on retinal ganglion cells (RGCs) cultured in mixed culture and to screen out the effective concentration of the two neurotrophic factors: brain derived neurotrophic factor BDNF and basic fibroblast growth factor bFGF. Methods Twelve postnatal days 2 to 3 of Sprague-Dawley (SD) neonatal rat retina were made into a cell suspension by trypsin digestion and inoculated into rat tail collagen coated 96-well plates (5 × 10-4 cells Cells / well). The BDNF and bFGF with different concentration gradients were added respectively and cultured in a constant temperature incubator at 37 ℃ and 5% CO 2. The absorbency value A of surviving cells was measured by MTT micro automatic colorimetric method at 1,3,5 days. Immunocytochemistry with Thy-1 antibody, NSE antibody and GFAP antibody was performed to identify RGCs. Results Cells cultured on murine collagen grew well with most of them protruding. After cultured for 1 day and 3 days, the absorbency A values of BDNF and bFGF groups were significantly different from those of the control group (P <0.01, P <0.05). After cultured for 5 days, BDNF (40ng / ml, 50ng / ml), bFGF (15ng / ml) and BDNF + bFGF group were significantly higher than those of the control group (P <0.01, P <0.05). Conclusion BDNF and bFGF at various concentrations can promote the survival of rat RGCs in vitro. BDNF has a concentration-dependent effect. BDNF is more effective than bFGF. BDNF and bFGF are not additive.