2,5-己二酮对坐骨神经及运动神经元瘤细胞VSC4.1神经生长因子水平的影响

目的 研究2,5-己二酮(2,5-hexanedione,2,5-HD)对大鼠坐骨神经和运动神经元神经生长因子(nerve growth factor,NGF)水平的影响.方法 应用随机数字表法将50只Wistar大鼠分为400 mg·kg~(-1)·d~(-1),5-HD染毒0、7、14、21-,28 d组,每组10只,采用免疫组织化学显色和荧光定量PCR检测不同时间坐骨神经横断面NGF水平和坐骨神经NGF mRNA水平.选用0、2.5、5.0、10.0、20.0 mmol/L 2,5-HD染毒神经元瘤细胞VSCA.1,应用免疫荧光法观察NGF水平的改变;并选用10.0 mmol/L2,5-HD作染毒剂量,观察0、1、3、6、12、24、48 h NGF水平的变化.结果 随染毒时间延长,坐骨神经NGF呈先增高后降低的趋势;坐骨神经NGF mRNA水平在染毒14 d(2~(-△△Ct)=3.46)、21 d(2~(-△△Ct)=5.28)和28 d(2~(-△△Ct)=3.10)高于染毒0d(2~(-△△Ct)=1)和7 d(2~(-△△Ct)=0.78),差异有统计学意义.各染毒剂量组VSCA.1细胞NGF水平差异有统计学意义(F=188.88,P<0.01);5.0、10.0、20.0 mmol/L组NGF平均荧光强度值(分别为43.24±7.52、43.48±10.86、63.13±10.68)高于0 mmol/L组(16.32±4.20)(q值分别为19.92、19.72、32.78,P值均<0.01)和2.5 mmol/L组(19.78±2.66)(q值分别为17.50、17.42、30.63,P值均<0.01);20.0 mmol/L组高于5.0、10.0 mmol/L组(q值分别为13.04、11.71,P值均<0.01).10.0 mmol/L 2,5-HD染毒不同时间VSCA.1细胞NGF水平差异有统计学意义(F=75.69,P<0.01);染毒6、12、24、48 h NGF平均荧光强度值(分别为18.66±2.89、23.14±6.08、27.66±6.11、17.25±3.05)高于染毒0 h(10.18±1.81)(q值分别为9.64、15.74、21.76、8.50,P值均<0.01)、染毒1 h(9.31±1.28)(q值分别为10.28、16.17、21.95、9.20,P值均<0.01)和染毒3 h(10.44±2.13)(q值分别为9.25、15.24、21.17、8.10,P值均<0.01);染毒12、24 h NGF平均荧光强度值高于染毒6 h(q值分别为5.24、10.77,P值均<0.01)和染毒48 h(q值分别为7.31、13.26,P值均<0.01).结论一定时间内,2,5-HD可导致大鼠坐骨神经和运动神经元NGF的水平升高,有剂量(时间)依赖关系.

The effect of 2,5-hexanedione on nerve growth factor in sciatic nerve of rats and VSC4. 1 cell

Objective To explore the effect of 2,5-hexanedione (2,5-HD) on the levels of nerve growth factor (NGF) in sciatic nerve of rats and motor-neurons. Method A total of 50 Wistar rats were randomly designed into five groups and intoxicated with 400 mg·kg~(-1)·d~(-1) 2,5-HD for 0,7,14,21,28 d.Immunohiscbemistry and real-time PCR were used to detect the levels of NGF and NGF mRNA. Motorneuron VSCA. 1 cells were administrated with 0,2. 5,5.0,10. 0,20. 0 mmol/L 2,5-HD for 24 h and 10. 0 mmol/L 2,5-HD was chosen to intoxicated VSCA. 1 cells for 0,1,3,6,12,24,48 h respectively. Immunofluorescence technique was selected to detect the levels of NGF. Results The NGF level in sciatic nerve of rats administrated with 400 mg·kg~(-1)·d~(-1) 2,5-HD showed increase tendency at begin and then decrease after exposure. The NGF mRNA level in 14 d (2~(-△△Ct) =3.46),21 d (2~(-△△Ct)=5. 28) and 28 d (2~(-△△Ct)=3. 10)were higher than those in 0 d (2~(-△△Ct) =1) and 7 d (2~(-△△Ct)=0. 78). In vitro tests of VSCA. 1 cells showed that NGF levels in 5.0 mmol/L ( 43.24 ±7.52), 10. 0 mmol/L ( 43.48 ±10. 86) and 20. 0 mmol/L(63. 13±10.68) were higher than those in 0 mmol/L (16.32±4.20) (q values were 19.92,19.72,32.78,respectively,P<0.01) and 2.5 mmol/L (19.78±2.66) (q values were 17.50,17.42,30.63,respectively,P <0. 01) in 24 h and the NGF level in 20. 0 mmol/L was higher than those in 5. 0 mmol/L( q= 13.04,P < 0. 01) and 10. 0 mmol/L ( q = 11.71, P < 0. 01). The NGF levels of VSC4. 1 cells with 10.0 mmol/L2,5-HD in 6 h (18.66±2.89),12 h (23. 14±6.08) ,24 h (27.66±6. 11) and 48 h(17.25±3.05) were increased compared with that in 0 h (10.18 ± 1.81) (q values were 9.64,15.74,21.76,8. 50,respectively, P< 0.01), 1 h (9.31 ±1.28) (q values were 10.28, 16.17,21.95,9.20,respectively ,P <0. 01) and 3 h ( 10. 44 ±2. 13) ( q values were 9. 25,15.24,21.17,8. 10,respectively,P <0. 01), and NGF levels in 12 h and 24 h increased compared with those in 6 h ( q values were 5.24,10. 77,respectively,P< 0.01) and 48 h (q values were 7.31, 13.26, respectively, P< 0.01). Conclusion 2,5-HD could increase NGF levels in sciatic nerve of rats and motor-neurons ,and the dose or time dependent effects were observed in this study.