摘要
目的观察不同时间点2种剂量重组人粒细胞集落刺激因子（rhG—CSF）干预对缺氧缺血性脑损伤（HIBD）新生大鼠脑组织Nogo受体（NgR）表达的影响，揭示rhG—CSF的神经保护作用。方法将新生7日龄SD大鼠通过抽签法随机分成4组：假手术组、模型组、小剂量组、大剂量组，每组24只。每组分别于手术后1d、3d、7d、14d处死大鼠，每个时间点各6只。小剂量组、大剂量组分别于造模后即刻颈部皮下注射rhG-CSF50μg／kg、100μg／kg，每日1次，连续7d；模型组造模后颈部皮下注射等量9g／L盐水；假手术组不予任何药物。各组新生大鼠于不同时间点取脑组织，采用免疫组织化学法及实时荧光定量PCR法检测脑组织NgR蛋白及NgRmRNA的表达。结果免疫组织化学：假手术组各时间点大脑皮质均可见NgR蛋白呈基础性表达；与假手术组相比，模型组各时间点NgR蛋白的表达均明显增加（135．67±16．63、173．98±17．82、234．00±14．70、319．59±25．22），差异均有统计学意义（P均〈0．01）；与模型组相比，小剂量组（134．35±8．89、109．04±12．62、75．99±13．39）和大剂量组（81．38±12．25、80．14±10．50、72．58±13．66）3d、7d、14d大脑皮质NgR蛋白表达均明显下降，差异均有统计学意义（P均〈0．01）。大剂量组比小剂量组起效快，大剂量组3d及7d的NgR蛋白表达较小剂量组明显下降，差异均有统计学意义（P均〈0．05）。实时荧光定量PCR：与假手术组相比，模型组各时间点NgRmRNA表达量呈增加趋势（1．34±0．24、1．88±0．27、2．88±0．84、4．26±0．86），差异均有统计学意义（P均〈0．05），小剂量组7d（1．08±0．30）、14d（0．93±0．26）及大剂量组3d（0．61±0．10）、7d（0．56±0．28）、14d（0．47±0．12）与模型组相比差异均有统计学意义（P均〈0．05）；小剂量组、大剂量组随时间的增加NgRmRNA表达量逐渐下降。大剂量组3d的NgRmRNA表达较小剂量组下降明显（P〈0．05）。结论rhG—CSF干预可降低新生大鼠HIBD模型脑组织NgR的表达，小剂量rhG—CSF干预也可发挥神经保护作用，但其作用可能较弱。
Objective To observe the different effects of 2 doses recombinant human granulocyte colony - stimulating factors （ rhG - CSF） on Nogo receptor（NgR） expression in the brain tissue of neonatal rats after hypoxic - ischemic brain damage（ HIBD）at different times in order to reveal the neuroprotective effects of rhG- CSF. Methods Seven - day neonatal Sprague - Dawley （SD） rats were randomly divided into 4 groups by drawing method ： sham opera- tion group, model group,low - dose rhG - CSF group and high - dose rhG - CSF group, 24 rats in each group. Then each group was divided into 4 subgroups（6 rats in each subgroup）and all rats were exterminated at different times after HIBD（ 1 d ,3 d,7 d and 14 d）. In the low -dose rhG -CSF group and high -dose rhG -CSF group,the rats were given daily doses of rhG - CSF 50 μg/kg, 100 μg/kg respectively for 7 days by subcutaneous injection immediately after the molding（ total 7 injections）. In model group, rats received an injection of same amount of 9 g/L saline. In sham operation group, rats received no special treatment. Brain tissues of rats from each group were collected at different time points. The expressions of NgR protein and NgR mRNA in the left brain tissue were detected by immunohistochemistry and real - time fluorescent quantitative polymerase chain reaction （PCR）. Results Immunohistochemistry ： NgR proteins were constitutively expressed in the cerebral cortex in sham operation group at each time point;compared with sham operation group,the expressions of NgR in model group were increased markedly at each time point （135.67 ± 16.63, 173.98 ± 17.82, 234.00 ± 14.70,319.59 ±25.22）, and the differences were statistically significant （ all P 〈 0. 01 ） ; compared with model group, the expressions of NgR in the cerebral cortex in low - dose rhG - CSF group （ 134. 35 ±8.89,109.04 ±12.62,75.99 ±13.39 ） and high - dose rhG - CSF group （ 81. 38 ± 12.25,80.14 ±10.50, 72.58 ±13. 66 ） on the 3^rd, 7^th, 14^th day were reduced significantly （ all P 〈 0. 01 ）. Compared with low - doserhG - CSF group, the protein expressions of NgR in the high - does rhG - CSF group were decreased faster, and had the marked difference on the 3^rd ,7^th day （P 〈 0.05 ）. Real -time fluorescent quantitative PCR： compared with the sham operation group, the expressions of NgR mRNA increased gradually in the cerebral cortex in the model group （ 1.34 ±0. 24,1.88 ± 0.27, 2.88 ±0. 84,4.26 ± 0.86）, the differences in NgR mRNA expression were statistically significant at different times（ all P 〈 0.05） ; compared with model group, the expressions of NgR mRNA in low - dose rhG - CSF group on the 7th（ 1.08 ± 0.30）, 14^th day （0.93 ±0.26） and high -dose rhG- CSF group on the 3^rd（0.61 ± 0.10）, 7^th （0.56 ±0. 28 ）, 146th day （ 0.47 ±0. 12 ） were significantly different （ all P 〈 0. 05 ）. The expressions of low - dose group and high - dose group were reduced gradually. The NgR mRNA expression reduced more quickly in the high - dose group than in the low - dose rhG - CSF group and had substantial difference between two groups in 3 days （ P 〈 0. 05 ）. Conclusions The findings suggest that rhG - CSF intervention can reduce the expressions of NgR in the brain tissues of neonatal rats after HIBD, and low - dose rhG - CSF also has neuroprotective effect, but it could be weaker than high - dose rhG - CSF.
出处
《中华实用儿科临床杂志》
CAS
CSCD
北大核心
2015年第6期456-460,共5页
Chinese Journal of Applied Clinical Pediatrics
基金
贵州省优秀科技教育人才省长专项资金项目[黔省专合字（2009）35号]
关键词
重组人粒细胞集落刺激因子
缺氧缺血性脑损伤
NOGO受体
Recombinant human granulocyte colony -stimulating factor
Hypoxic ischemic brain damage
Nogo receptor
作者简介
通信作者：何志旭，Email：hzx@gmc．edu．cn
