内皮祖细胞在大鼠外伤性视神经损伤中作用的研究
내피조세포재대서외상성시신경손상중작용적연구
Role of endothelial progenitor cells in optic nerve injury of rats
  • 万方数据
    中华眼科杂志 중화안과잡지
    Chinese Journal of Ophthalmology
    2011年 12期 1089-1095 ,共7页

    刘玉梅%胡艳滨%赫天耕%颜华

    류옥매%호염빈%혁천경%안화

    视神经损伤%内皮细胞%干细胞%流式细胞术%大鼠 視神經損傷%內皮細胞%榦細胞%流式細胞術%大鼠
    시신경손상%내피세포%간세포%류식세포술%대서
    Optic nerve injuries%Endothelial cells%Stem cells%Flow cytometry%Rats
    目的 探讨内皮祖细胞(EPC)在大鼠外伤性视神经损伤中的作用.方法 实验研究.采用液压冲击颅脑损伤仪制作外伤性视神经损伤动物模型,108只大鼠(108只眼)采用随机数字表法随机分为视神经损伤组和假手术对照组,每组54只；两组按照损伤前24h及损伤后3、12、24、48、72 h、1、2、3周时间点随机分为9个亚组,每个亚组6只.测定各组上述时间点外周血EPC数量并观察视神经HE染色、血管内皮标志物CD31免疫组织化学染色及闪光视觉诱发电位(F-VEP)变化.视神经损伤组与假手术对照组同一时间点的比较采用独立样本t检验；不同检测项目间的相关性分析采用Pearson积差相关检验.结果 正常大鼠外周血EPC数量为(46 ～52)个/200 000单个核细胞,大鼠外伤性视神经损伤后3、12、24、48、72 h及1、2、3周外周血EPC计数分别为(34±4、34±5、69±9、76±6、107±9、69±7、58 ±6、56±4)个/200 000单个核细胞,视神经损伤组与假手术对照组比较,3、12、24、48、72 h、1、2周时间点差异有统计学意义(t=5.29,2.90,-4.30,-7.61,- 14.17,-5.74,-2.79;P ＜0.05).正常大鼠视神经及周围组织CD31+细胞数为(7～9)个/5个高倍视野,视神经损伤后创伤区CD31+细胞计数分别为(8.36±1.52、7.17±1.10、10.41±1.92、11.43 ±1.58、14.29±2.03、17.33±1.47、17.86±1.22、18.13±1.40)个/5个高倍视野,视神经损伤组与假手术对照组比较,48、72 h、1、2、3周时间点差异均有统计学意义(t=4.31,-7.61,-8.17,- 10.08,- 10.79；P＜0.05).正常大鼠视神经及周围组织微血管数量为(6～9)个/5个高倍视野,视神经损伤后损伤区微血管数量分别为(7.54±2.01、8.52±2.21、11.02±1.62、15.40±2.04、18.39±1.96、23.21±1.50、22.78±2.40、24.13 ±2.51)个/5个高倍视野,视神经损伤组与假手术对照组比较,48、72 h、1、2、3周时间点差异均有统计学意义(t=4.25,-7.74,-8.26,- 10.28,-11.49；P＜0.05).视神经损伤后F-VEP中P波潜伏期于损伤后3h降低,24h反弹增高到正常水平以上,并趋于稳定,视神经损伤组与假手术对照组相比,3、12、24、48、72 h、1、2、3周差异均有统计学意义(=4.15,3.74,5.84,6.08,6.40,6.52,6.53,6.61；P ＜0.05)；F-VEP中振幅于3h降低,12 h升高到接近基础水平,24h后逐渐降低至基础水平以下,实验组与对照组比较,3、24、48、72 h、1、2、3周差异有统计学意义(t=3.95,4.14,5.26,5.78,6.49,6.72,6.23；P＜0.05).外周血EPC数量变化与创伤区周围CD31+细胞、微血管及F-VEP变化存在相关性(r=0.43,0.41,0.43；P＜0.01).结论 外伤性视神经损伤后外周血内皮祖细胞明显增加,归巢到创伤区,参与了创伤区血管新生和组织损伤修复.
    목적 탐토내피조세포(EPC)재대서외상성시신경손상중적작용.방법 실험연구.채용액압충격로뇌손상의제작외상성시신경손상동물모형,108지대서(108지안)채용수궤수자표법수궤분위시신경손상조화가수술대조조,매조54지；량조안조손상전24h급손상후3、12、24、48、72 h、1、2、3주시간점수궤분위9개아조,매개아조6지.측정각조상술시간점외주혈EPC수량병관찰시신경HE염색、혈관내피표지물CD31면역조직화학염색급섬광시각유발전위(F-VEP)변화.시신경손상조여가수술대조조동일시간점적비교채용독립양본t검험；불동검측항목간적상관성분석채용Pearson적차상관검험.결과 정상대서외주혈EPC수량위(46 ～52)개/200 000단개핵세포,대서외상성시신경손상후3、12、24、48、72 h급1、2、3주외주혈EPC계수분별위(34±4、34±5、69±9、76±6、107±9、69±7、58 ±6、56±4)개/200 000단개핵세포,시신경손상조여가수술대조조비교,3、12、24、48、72 h、1、2주시간점차이유통계학의의(t=5.29,2.90,-4.30,-7.61,- 14.17,-5.74,-2.79;P ＜0.05).정상대서시신경급주위조직CD31+세포수위(7～9)개/5개고배시야,시신경손상후창상구CD31+세포계수분별위(8.36±1.52、7.17±1.10、10.41±1.92、11.43 ±1.58、14.29±2.03、17.33±1.47、17.86±1.22、18.13±1.40)개/5개고배시야,시신경손상조여가수술대조조비교,48、72 h、1、2、3주시간점차이균유통계학의의(t=4.31,-7.61,-8.17,- 10.08,- 10.79；P＜0.05).정상대서시신경급주위조직미혈관수량위(6～9)개/5개고배시야,시신경손상후손상구미혈관수량분별위(7.54±2.01、8.52±2.21、11.02±1.62、15.40±2.04、18.39±1.96、23.21±1.50、22.78±2.40、24.13 ±2.51)개/5개고배시야,시신경손상조여가수술대조조비교,48、72 h、1、2、3주시간점차이균유통계학의의(t=4.25,-7.74,-8.26,- 10.28,-11.49；P＜0.05).시신경손상후F-VEP중P파잠복기우손상후3h강저,24h반탄증고도정상수평이상,병추우은정,시신경손상조여가수술대조조상비,3、12、24、48、72 h、1、2、3주차이균유통계학의의(=4.15,3.74,5.84,6.08,6.40,6.52,6.53,6.61；P ＜0.05)；F-VEP중진폭우3h강저,12 h승고도접근기출수평,24h후축점강저지기출수평이하,실험조여대조조비교,3、24、48、72 h、1、2、3주차이유통계학의의(t=3.95,4.14,5.26,5.78,6.49,6.72,6.23；P＜0.05).외주혈EPC수량변화여창상구주위CD31+세포、미혈관급F-VEP변화존재상관성(r=0.43,0.41,0.43；P＜0.01).결론 외상성시신경손상후외주혈내피조세포명현증가,귀소도창상구,삼여료창상구혈관신생화조직손상수복.
    Objective To investigate the role of endothelial progenitor cells (EPC) in the injury of rat optic nerve.Methods An experimental study.The rat model of optic nerve injury was created by fluid percussion brain injury device (FPI).On hundred and eight rats ( 108 eyes) were divided into 2 groups randomly.Each group was further divided into 9 subgroups by the time of injury (24 h before and 3,12,24,48,72 h,1,2 and 3 weeks after the injury).The number of circulating EPCs was measured,HE staining of the optic nerve,immunohistochemistry study of CD31 (markers of vascular endothelial cells) and flash-visual evoked potential (F-VEP) were observed at every time point.Two independent sample t-test was used for the comparison between the control group and the optic nerve injury groups at the same time point.The correlation between different items was analyzed by Pearson test.P value less than 0.05 was considered significant.Results The number of EPCs in normal rats was 46 - 52/200 000 monocytes.After traumaticoptic nerve injury,the number of EPCs was (34 ±4,34 ±5,69 ±9,76 ±6,107 ±9,69 ±7,58 ±6 and 56 ±4)/200 000 monocytes at 3,12,24,48,72 h,and 1,2 and 3 weeks.The difference of number of EPCs between the experiment and control groups was significant at 3,12,24,48,72 h and 1 and 2 weeks after the injury (t =5.29,2.90,-4.30,-7.61,- 14.17,-5.74 and -2.79;P ＜0.05).The number of CD31 + cell in the optic nerve and surround tissues in normal rats was (7 -9)/5 high magnification field.After the injury,the number of CD31 + cell was 8.36 ± 1.52,7.17 ± 1.10,10.41 ± 1.92,11.43 ± 1.58,14.29 ±2.03,17.33 ± 1.47,17.86 ± 1.22 and 18.13 ± 1.40 at different time points.The difference of number of CD31 + cell between the experiment and control groups was significant at 48,72 h,and 1,2,and 3 weeks after the injury (t =4.31,-7.61,-8.17,- 10.08,and - 10.79;P ＜0.05).The number of microvessels in the optic nerve and surround tissues in normal rats was 6 -9/5 high magnification field.After traumatic optic nerve injury,the number of microvessels was 7.54 ± 2.01,8.52 ± 2.21,11.02 ±1.62,15.40 ± 2.04,18.39 ± 1.96,23.21 ± 1.50,22.78 ± 2.40 and 24.13 ± 2.51 at different time points.The difference of number of microvessels between the experiment and control groups was significant at 48,72h,1,2,and3 weeks after the injury (t=4.25,-7.74,-8.26,-10.28 and -11.49;P＜0.05).The latency period of P waves was decreased at 3 h and increased to above basic level at 24 h,and then tend to be stable.The difference of latency period of P waves between the experiment and control groups was significant at 3,12,24,48,72 h,1,2 and 3 weeks after the injury (t =4.15,3.74,5.84,6.08,6.40,6.52,6.53 and 6.61 ;P ＜0.05).The amplitude of F-VEP was decreased at 3 h and increased to the basic level at 12 h,then decreased to below the basic level gradually.The difference of the amplitude of FVEP between the experiment and control groups was significant at 3,24,48,72 h,1,2 and 3 weeks after the injury (t =3.95,4.14,5.26,5.78,6.49,6.72 and 6.23 ; P ＜ 0.05).The number of EPCs was correlated with the number of CD31 + cell,microvessels and F-VEP ( r =0.43,0.41 and 0.43 ; P ＜ 0.01 ).Conclusions The present study showed that the number of EPCs in the blood increases significantly after traumatic optic nerve injury,and the cells can arrive the traumatic area to repair injured tissue and enhance angiogenesis.
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