压力对大鼠纯化视网膜神经节细胞存活及轴突生长的影响

目的 研究压力对视网膜神经节细胞(RGC)存活和轴突生长的影响及其与神经微丝蛋白(NF)-H表达变化的关系,探讨压力引起RGC损伤的机制.方法 对照实验研究.纯化培养20只SD乳鼠的RCC.采用开放式压力控制培养系统,建立压力损伤模型,分别检测20、40、60及80 mm Hg(1 mm Hg=0.133 kPa)压力作用48 h对RGC存活数及轴突生长的影响,测定存活且有轴突的RGC数及其最长轴突的长度.采用免疫组织化学染色加图像分析系统,检测压力对RGC表达NF-H的影响,以吸光度(A)值表示.采用SPSS 13.0统计学软件对数据进行分析.组间RGC存活数和细胞轴突长度与对照组比较采用单因素方差分析(One-way ANOVA),组间进一步两两比较采用SNK法(g检验).以P<0.05作为差异有统计学意义.结果 纯化培养的SD大鼠RGC纯度可达96.24%.压力40、60及80 mm Hg作用48 h,RGC存活数和细胞轴突长度与对照组比较明显降低,差异有统计学意义(存活细胞数:P=0.001,0.000,0.000;细胞轴突长度:P=0.000,0.000,0.000),且降低程度随压力增高而增大;NF-H表达差异也有统计学意义(P=0.000,0.000,0.000),且NF-H阳性细胞分布不规则.结论 压力可影响纯化培养的RGC存活数和轴突生长,可能与其下调RGC中NF-H的表达有关,推测压力是引起青光眼视神经损害的机制之一.(中华眼科杂志,2009,45:164-167)

The influence of pressure on the cell survival and axonal growing in rat retinal ganglion cells in vitro

Objective To evaluate the effect of pressures on the changes of purified cultivation of SD suckling rats' retinal ganglion cells (RGC) by a hyperbaric cell culture model and on the expression of neurofilament protein-H (NF-H) in vitro. Methods It was a control experimental study. RGC were purified from twenty SD neonatal rots (postnatal 1-3 days) using Thy1.1 antibody and cultured under the pressures of 0, 20, 40, 60, and 80 mm Hg (1 mm Hg=0.133 kPa) respectively. The growth and survival times of RGC were observed, the numbers of processes of RGC were counted, and the processes measured under the different pressures after 48 h under phase-contrast microscope. The expression of NF-H and its distribution in primary purified RGC was investigated by an immunohistochemical technique and by semi-quantitative statistics analysis using microscopic image-analysis technique. The data was analyzed with SPSS 13.0 software. Difference among groups with One-way ANOVA and ambi-groups with SNK (q test), statistical significance was confirmed as P<0.05. Results The purification rate of RGC reached 96.24% after cultured for 12 hours. Compared with the control group, the growth and survival times of RGC were similar (P=0.595, 0.147)under the pressure of 20 mm Hg cultured for48 hours, the expression of NF-H in RGC was not different (P=0.227); but the growth and survival times of RGC were significantly different (P=0.001, 0.000, 0.000 and 0.000, 0.000, 0.000)under the pressures of 40, 60, and 80 mm Hg, respectively. The expression of NF-H in RGC were significantly (P=0.000, 0.000, 0.000) reduced under the pressures of 40, 60, and 80 mm Hg for 48 hours respectively. Conclusions The growth and survival times of cultured RGC in vitro are affected by the pressure>40 mm Hg leading to a decreased expression of NF-H indicating a pressure caused mechanism of RGC damage in glaucoma. Down-regulation of NF-H may influence the process growth of RGC. (Chin J Ophthalmol, 2009,45:164-167)