晶状体蛋白促大鼠视网膜神经节细胞存活和突起生长的体外研究

Study of cystailine promoting retinal ganglion cell survival and axonai outgrowth in rats

目的 探讨晶状体内各主要可溶性晶状体蛋白对离体培养的大鼠视网膜神经节细胞（retinal ganglion cells，RGCs）存活和突起生长的作用，为视神经损伤后再生的研究提供新的思路。方法 采用分子排阻凝胶色谱法分离纯化晶状体中各主要可溶性晶状体蛋白——α、β-H、β-L、γ晶状体蛋白，再通过SDS-PAGE电泳、肽质量指纹图谱方法鉴定其为目的蛋白质后，按组分别进行RGCs离体培养试验，观察RGCs的最长突起长度和存活细胞数。结果 RGCs的最长突起长度6d时达最大值，分别为：对照组（92．27±35．93）μm，α晶状体蛋白组（181．59±43．78）μm，β-H晶状体蛋白组（123．33±52．81）μm，β-L晶状体蛋白组（89．55±22．40）μm，γ晶状体蛋白组（86．01±39．15）μm。6d时，各组RGCs存活细胞数分别为：对照组（9．80±3．25）个／视野，α晶状体蛋白组（13．00±4．25）个／视野，β-H晶状体蛋白组（11．33±6．28）个／视野，β-L晶状体蛋白组（8．10±1．83）个／视野，γ晶状体蛋白组（5．74±2．82）个／视野。α、β-H晶状体蛋白组RGCs的最长突起长度和存活细胞数明显高于对照组，α晶状体蛋白组作用更明显，β-L晶状体蛋白组与对照组比较，差异无统计学意义，γ晶状体蛋白组存活细胞数在6d时明显少于对照组。结论 仅晶状体蛋白有明显的促进体外培养的RGCs存活和突起生长的作用，是一种晶状体源性神经保护物质。β-H晶状体蛋白也有一定的促RGCs突起生长和保护存活的作用。γ晶状体蛋白有一定的抑制RGCs存活的作用。

Objective To investigate effect of soluble crystalline proteins on retinal ganglion cells （RGCs） survival and axonal outgrowth in rats so as to find out a new path for regeneration of optic nerve after injury. Methods The size-exclusion gel-chromatograph was used to isolate the main soluble crystalline （α, β-H, β-L and γ） and SDS-PAGE and peptide mass fingerprinting to identify their protein character, Each group was added with different crystalline to observe its effect on RGCs survival and axonal outgrowth, such as the longest processes and the number of the survived cells. Results The longest processes of RGCs on six day was （92.27 ± 35.93） μm in control group, （ 123.33 ± 52.81 ） μm in β-H crystalline group, （ 89.55 ± 22.40 ） μm in β- L crystalline group and （ 86.01 ± 39. 15 ） μm in γ- crystalline group, with statistical difference （P 〈0.01 ）. While the survived number of RGCs per visual field on six day was （9.80 ±3.25） cells in control group, （ 13.00 ±4.25） cells in α crystalline group, （ 11.33 ± 6.28 ） cells in β-H crystalline group, （ 8.10 ± 1.83 ） cells in β- L crystalline group and （ 5.74 ± 2.82） cells in γ-crystalline group. The longest process and survived cells of RGCs in α crystalline group and β-H crystalline group, expecially in α crystalline group, were significantly higher than those in control group. There was no statistical difference upon the longest process and survived cells of RGCs between β-L crystalline group and control group. The survived cells in γ-crystalline group were significantly less than that in control group on 6 days. Conclusions As lens-derived neuroprotection substance, α crystalline protein has significant and direct effect on neurite outgrowth and survival of RGCs cultured in vitro, which is stronger than that of β-H crystalline protein. γ-cryatalline exerts inhibitive effect on survival of RGCs in vitro.