PACAP38对兔角膜瓣术后三叉神经节细胞及角膜神经生长的影响

Promoting effect of PACAP38 on proliferation of trigeminal ganglion cells and growth of corneal nerve after flap surgery

目的观察垂体腺苷酸环化酶激活肽(PACAP38)对培养的兔三叉神经节细胞及角膜瓣术后角膜神经生长的影响。方法从2周龄新西兰白兔中分离三叉神经节细胞,并在神经元基础培养液中进行培养。培养24h后在培养液中分别加入1μmol/LPACAP38(A组)、0.1μmol/LPACAP38(B组),C组培养液中加入PBS。1周后行抗神经纤维丝蛋白(NF200)免疫荧光染色鉴别培养的三叉神经节细胞。27只新西兰白兔右眼行角膜瓣手术,并分为3组,术后1d分别以10μmol/LPACAP38、5μmol/LPACAP38或PBS点眼,共8周,行角膜敏感度测定和NF200免疫荧光染色,评价术后不同时间角膜神经的修复情况。结果 PACAP38培养的三叉神经节细胞有较多神经细胞存活,伸出突触并交织呈网状,A组神经细胞存活最多。角膜瓣手术的3个组角膜敏感度随着术后时间的延长逐渐增加(Ftime=58.196,P=0.00),10μmol/LPACAP38点眼组角膜敏感度恢复最快。10μmol/LPACAP38点眼组和5μmol/LPACAP38点眼组角膜敏感度值明显高于PBS点眼组,差异均有统计学意义(P=0.000,P=0.005)。角膜神经染色示10μmol/LPACAP38点眼组角膜神经密度较5μmol/LPACAP38点眼组和PBS点眼组高,神经干短端多膨大,以出芽方式发出细微的新生神经纤维芽。结论 PACAP38能促进兔三叉神经节细胞增生及诱导神经突形成,可促进角膜敏感性恢复及角膜神经修复再生。

Background How to promote the repair of corneal nerve without neovascularization after corneal damage is a problem.As a neural transmitter and regulator,pituitary adenylate cyclase-activating polypeptide （PACAP38） plays a key effect in the regeneration of nerve.Research has determined PACAP38 distribute densely in cornea.Objective Present study investigated the effect of PACAP38 on cultured trigeminal ganglion cells and evaluate the efficacy of PACAP38 eye drops on rabbit corneal nerve reinnervation.Methods Trigeminal ganglion cells were isolated from 2-week-old New Zealand white rabbit and cultured in neurobasal medium.1 μmol/L,0.1 μmol/L of PACAP38 or phosphate buffered saline （PBS） was added into the medium respectively in 24 hours after culture for 1 weeks under the inhibition condition with 5-fluro-2-deoxyuridine and uridine.Cultured cells were identified by immunofluorescence.The corneal flap surgery was performed in the right eyes of 27 New Zealand white rabbits.10 μmol/L PACAP38 eye drops,5 μmol/L PACAP38 eye drops or PBS solution was topically administered in 9 rabbits 4 times a day for 8 weeks respectively after operation.Corneal sensitivity was measured with Luneau ophthalmologie tester at 1-week interval from 1 week through 8 weeks after operation.The growth of corneas nerve was evaluated with antibody staining against neurofilament.Results More nerve neuronal processes and synapsis of trigeminal ganglion cells were exhibited in 1 week after culture in 1 μmol/L PACAP38 group compared with 0.1 μmol/L of PACAP38 group or PBS group.Corneal sensitivity was gradually enhanced with the increase of time following the surgery in all of the three groups with the fastest restore of sensation in 10 μmol/L PACAP38 group （Ftime=58.196,P=0.00）.Significant differences were found in the corneal sensitivity value between 10 μmol/L PACAP38 group and PBS group （P=0.000） or 5 μmol/L PACAP38 group and PBS group （P=0.005）.Conclusion PACAP38 can promote the proliferation of rabbit trigeminal ganglion cells and improve corneal sensitivity and reinnervate corneal nerve.