【作者】 周丹；

【导师】 江冰；

【作者基本信息】 中南大学 ， 眼科学， 2009， 硕士

【摘要】 外伤性视神经病变（traumatic optic neuropathy,TON）病情复杂,预后不良,常遗留永久性视力损害。因此应积极处理以挽救视力。目前TON的治疗方法有药物治疗、手术治疗以及药物和手术联合治疗,但各种治疗方法的疗效差别很大。近期有学者提出采用移植干细胞治疗视神经损伤的构想,并已在动物实验中取得了一定的进展,因此该方向很可能成为TON治疗中的一条有效的、崭新的途径。干细胞是指一类具有高度增值和自我更新能力,并具有多项分化潜能的原始细胞,随着干细胞技术的不断发展及干细胞本身所具有的生物学特性,体外培养某些干细胞,定向诱导分化为所需的组织细胞以治疗某些疾病已成为当前研究的热点。脐血是干细胞的主要来源,作为外周血,其取材方便,来源广泛,加之脐血中富含的间充质干细胞具有免疫调节及加速造血恢复等特点,因此,脐血的移植成功率高,移植反应弱,移植物抗宿主病少见。视神经作为视网膜神经节细胞（retinal ganglion cells,RGCs）的轴突,其损伤与RGCs的凋亡密切相关。本实验用夹持法制作了大鼠外伤性视神经损伤模型,并采用闪光视觉诱发电位（flash visual evoked potentials,F-VEP）、苏木精-伊红（hematomylin-eosin,HE）染色、脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling,TUNEL）以及RT-PCR等方法,对损伤后视神经和RGCs的存活情况,以及人脐血干细胞移植对外伤所致的大鼠RGCs是否具有保护作用及其可能机制进行了较为详细的实验研究。第一部分人脐血干细胞移植对大鼠外伤性视神经部分损伤闪光视觉诱发电位的影响目的观察大鼠外伤性视神经部分损伤模型中F-VEP的改变;研究人脐血干细胞移植对大鼠外伤性视神经部分损伤F-VEP修复的影响。方法健康成年96只SD大鼠随机分为视神经钳夹30s（中度损伤）组及60s（严重损伤）组,每组48只,所有大鼠的左眼为损伤眼,右眼为正常对照眼。两组按视神经损伤后处理方式的不同随机分为损伤组（A组）和治疗组（B、C、D组）,每组均为12只,A组不予以药物治疗,治疗组分别予以玻璃体腔内注射神经营养因子（B组）、人脐血干细胞（C组）、人脐血干细胞+神经营养因子混合液（D组）。大鼠的损伤眼均以夹持力为40g的特制视神经夹,在大鼠眼球后2mm处夹持视神经30s或60s,制成大鼠外伤性视神经部分损伤模型。所有大鼠均在损伤后1小时、1周、2周、3周和4周分别进行损伤眼和正常对照眼的F-VEP检查,记录波幅及峰潜时,并进行统计分析。结果1.正常对照眼的F-VEP:所有大鼠正常对照眼的F-VEP的波形稳定,随时间延长均无明显变化。其波幅波动于12.7±0.6μV:峰潜时波动于73.8±3.2ms。2.损伤组（A组）的F-VEP:所有大鼠损伤眼的F-VEP的潜伏期明显延长,振幅明显降低。钳夹30s组,在损伤后1小时、1周、2周、3周和第4周,其波幅分别为6.41±0.6μV、5.38±0.6μV、4.56±0.76μV、3.78±0.53μV和3.17±0.75μV,P1峰潜时依次为86.5±1.2ms、100.6±3.3ms、109.6±7.2ms、111.2±8.1ms和104.2±4.9ms;钳夹60s组,依上述时间点,其波幅各自为6.32±0.4μV、5.27±0.4μV、4.41±0.56μV、3.60±0.32μV和2.99±0.51μV,P1峰潜时分别为81.4±1.0ms、95.5±3.1ms、104.5±7.0ms、106.1±8.3ms和99.3±3.9ms。3.神经营养因子移植组（B组）的F-VEP:钳夹30s组,在损伤后1小时、1周、2周、3周和第4周,其波幅分别为6.46±0.9μV、6.00±0.7μV、5.89±0.8μV、5.29±0.65μV和4.27±0.57μV,P1峰潜时依次为86.9±1.5ms、95.7±2.7ms、97.2±6.9ms、102.6±4.4ms和98.1±4.5ms;钳夹60s组,依上述时间点,其波幅各自为6.37±0.7μV、5.89±0.6μV、5.74±0.60μV、5.11±0.44μV和4.09±0.38μV,P1峰潜时分别为82.0±1.2ms、90.7±2.5ms、92.3±6.5ms、97.5±4.3ms和94.2±3.5ms。4.人脐血干细胞移植组（C组）的F-VEP.钳夹30s组,在损伤后1小时、1周、2周、3周和第4周,其波幅分别为6.54±1.2μV、6.08±1.0μV、5.97±1.1μV、5.37±0.9μV和4.39±0.65μV,P1峰潜时依次为87.2±2.5ms、91.8±1.8ms、93.2±6.6ms、98.6±3.8ms和94.1±3.8ms;钳夹60s组,依上述时间点,其波幅各自为6.45±1.0μV、5.97±0.8μV、5.82±0.90μV、5.20±0.85μV和4.23±0.55μV,P1峰潜时分别为82.7±2.0ms、86.2±1.8ms、88.7±6.3ms、93.6±3.5ms和89.8±2.7ms。5.混合药物移植组（D组）的F-VEP:钳夹30s组,在损伤后1小时、1周、2周、3周和第4周,其波幅分别为6.61±1.4μV、6.15±1.2μV、6.04±1.3μV、5.67±1.1μV和4.82±0.95μV,P1峰潜时分别为87.5±2.0 ms、89.5±1.6 ms、91.2±6.0 ms、96.6±3.2 ms和91.9±3.0ms:钳夹60s组,依上述时间点,其波幅各自为6.52±1.2μV、6.04±1.0μV、5.89±1.1μV、5.51±1.0μV和4.66±0.85μV,P1峰潜时分别为82.9±2.2 ms、84.5±1.6 ms、86.6±6.0 ms、91.6±3.0 ms和85.5±2.0ms。6.损伤组与正常对照眼的比较:无论钳夹30s组还是钳夹60s组,与正常对照眼相比,损伤眼的F-VEP的波形均在损伤后1小时即出现明显变化:各时间点波幅和峰潜时与正常对照眼相比,差异均有统计学意义（P＜0.01）。钳夹30s组和钳夹60s组的波幅和峰潜时比较,各个时间点的差异均有统计学意义（P＜0.05）。随损伤后观察时间的推移,F-VEP的波幅逐渐降低,潜伏期明显延长,两者均于第3周达到高峰。7.损伤组与治疗组之间的比较:波幅:无论是钳夹30s组还是钳央60s组,在损伤后各个时间点,损伤组与各治疗组之间的两两比较,差异均有统计学意义（1小时和1周时P＜0.05,余时间点P＜0.01）;损伤后第3周,损伤组与各治疗组之间波幅差异最大,钳夹30s组中,A、B、C、D各组的波幅分别为3.78±0.53μV、5.29±0.65μV、5.37±0.9μV、5.67±1.1μV;钳夹60s组中,A、B、C、D各组的波幅依次为3.60±0.32μV、5.11±0.44μV、5.20±0.85μV、5.51±1.0μV。锋潜时:钳夹30s组和60s组中,损伤组与各治疗组之间的两两比较,除损伤后1小时组的差异无统计学意义（P＞0.05）外,其余各时间点的差异均有统计学意义（1周P＜0.05,2、3、4周P＜0.01）;损伤后第2周,损伤组与各治疗组之间的峰潜时差异最大,钳夹30s组中,A、B、C、D各组的峰潜时分别为109.6±7.2ms、97.2±6.9ms、93.2±6.6ms、91.2+6.0ms钳夹60s组,A、B、C、D各组的峰潜时分别为104.5±7.0ms、92.3±6.5ms、88.7+6.3ms、86.6±6.0ms。损伤后第4周,30s组和60s组的峰潜时均较前缩短。8.钳夹30s组和钳夹60s组之间的比较:无论是波幅还是峰潜时,在损伤后相同时间点的两两比较,二者的差异均具有统计学意义（P＜0.01）,与30s组相比,60s组的波幅明显降低,潜伏期延长。9.各个治疗组之间的比较:钳夹30s组和60s组,自第1周开始后的各个时间点,D组（混合药物移植组）的波幅均高于B组（神经营养因子移植组）,锋潜时的峰值较之缩短,差异均有统计学意义（P＜0.05）:其余各治疗组之间在相同时间点的两两比较,差异均无统计学意义（P＞0.05）。结论1.正常大鼠的视神经用夹持力为40g的视神经夹夹持30s或60s后,可造成视神经的部分损伤,表现为被夹持视神经F-VEP的波幅明显降低,峰潜时明显延长;视神经被夹持的时间越长,其受损程度越重,F-VEP的改变越明显;伤后观察时间越长,F-VEP的潜伏期延迟越显著,波幅降低程度越大,F-VEP的熄灭越早。2.神经营养因子、人脐血干细胞和人脐血干细胞+神经营养因子混合液对大鼠外伤性视神经部分损伤后视神经传导功能的恢复有促进作用,人脐血干细胞和神经营养因子的联合使用,可以提高其促进神经传导功能恢复的作用。第二部分人脐血干细胞移植对大鼠外伤性视神经部分损伤保护作用机制的探讨目的观察大鼠外伤性视神经部分损伤后其RGCs形态学以及内质网应激相关因子GRP78和CHOP表达的改变;探讨人脐血干细胞对外伤所致的大鼠RGCs是否具有保护作用及其可能机制。方法102只健康成年SD大鼠建立外伤性视神经部分损伤模型（视神经钳夹60s）,所有大鼠左眼均为损伤眼,右眼为正常对照眼。随机分为三组:A组（HE染色组）30只,B组（TUNEL检测组）36只,C组（RT-PCR检测组）36只。A组又随机分为A₁、A₂两组,各15只,A₁组为损伤组,视神经钳夹60s,仅制作视神经部分损伤模型,A₂组为人脐血干细胞移植组（治疗组）,即在制作视神经部分损伤模型后立即予以玻璃体下腔注射脐血干细胞;两组均在制模后3d、7d、14d、21d和28d处死动物,并应用光镜观察大鼠RGCs形态学的改变。B组和C组又分别分为两组:B₁、B₂组和C₁、C₂组,每组18只大鼠,B₁组和C₁组为损伤组,仅制作模型而不进行药物治疗;B₂组和C₂组为治疗组,制作视神经部分损伤模型后立即予以玻璃体下腔注射人脐血干细胞。在损伤后3h,12h,24h,48h,72h和1w,B组行TUNEL,C组行RT-PCR,以检测凋亡细胞和内质网应激相关蛋白的表达。结果RGCs的数量（个/10个视野）:正常对照组:随损伤后观察时间的延长,A组正常对照眼RGCs的数量无明显变化,在术后3d、7d、14d、21d和28d,RGCs数量分别为46.56±1.87、45.38±0.65、46.35±0.96、45.98±1.02和45.65±0.97,相邻各时间点两两比较,RGCs数量的差异无统计学意义（P＞0.05）;损伤组（A₁组）:钳夹视神经60s后,A₁组RGCs的数量严重减少,在术后3d、7d、14d、21d和28d,RGCs数量分别为30.50±1.31、27.03±1.22、20.16±0.87、18.16±0.84和15.68±0.79。在损伤后14天内,RGCs数量减少较快。与正常对照组相比,术后第14天时RGCs的数量减少50%左右:第14天以后,RGCs数量缓慢减少;第28天时,RGCs数量较正常组丢失60%左右。与正常对照组相比,在损伤后各个时间点,RGCs数量的差异均有统计学意义（P＜0.01）;治疗组（A₂组）:A₂组RGCs数量降低趋势较缓和,损伤后3d、7d、14d、21d和28d,RGCs数量分别为40.13±1.31、36.22±2.67、32.26±1.43、31.61±0.76和30.31±0.51;治疗组与正常对照组,或损伤组相比,在损伤后5个不同时间点RGCs数量的差异均有统计学意义（P＜0.01）。TUNEL阳性细胞率:损伤组（B₁组）:损伤后3h、12h、24h、48h、72h和1w时TUNEL阳性细胞率依次为4.23%±1.19、6.21±1.03%、15.33%±2.78%、39.41±1.86%、58.83%±8.36%和41.15%±6.37%。相邻各时间点的两两比较,其凋亡细胞数量的差异有统计学意义（P＜0.01）;治疗组（B₂组）:损伤后3h、12h、24h、48h、72h和1w时,其TUNEL阳性细胞率分别为0、0、0、12.33±2.63%、34.23%±1.98%和22.13±1.93%。自损伤后24h开始,相邻各时间点的两两比较,其凋亡细胞数量的差异有统计学意义（P＜0.01）;损伤组在伤后3h内核层即可见少量凋亡细胞,治疗组在伤后48h方可见凋亡细胞,两组的凋亡细胞数量均在伤后72h达高峰,并在1w时逐渐减少。在相同时间点的两两比较,损伤组与治疗组的TUNEL阳性细胞数量的差异有统计学意义（P＜0.01）。GRP78与CHOP的表达:GRP78:在损伤后3h、12h、24h、48h、72h和1w,损伤组（C₁组）GRP78mRNA的OD值依次为1.221±0.12、1.313±0.22、1.226±0.18、1.220±.14、1.197±0.10和1.190±0.09;治疗组（C₂组）GRP78mRNA的OD值分别为1.732±0.21、2.370±0.18、2.251±0.22、2.038±0.15、1.945±0.09和1.835±0.12;在同一时段,治疗组与损伤组相比,GRP78的差异均有统计学意义（P＜0.01）;CHOP:在损伤后3h、12h、24h、48h、72h和1w,损伤组（C₁组）CHOPmRNA的OD值依次为2.251±0.21、2.31±0.15、2.370±0.14、2.038±0.09、1.945±0.12和1.835±0.12:治疗组（C₂组）CHOPmRNA的OD值分别为1.226±0.22、1.262±0.14、1.313±0.21、1.220±0.18、1.197±0.11和1.190±0.09;在相同时间点,治疗组与损伤组相比,CHOPmRNA的差异均有统计学意义（P＜0.01）;损伤后GRP78和CHOP的表达在损伤组和治疗组均出现上升,GRP78的表达在损伤后12h达高峰,其在治疗组的变化高于损伤组;CHOP的表达在伤后24h达高峰,其在治疗组的表达较损伤组微弱。结论1、大鼠外伤性视神经部分损伤后,其RGCs的丧失以凋亡为主,且随大鼠存活时间的推移,RGCs呈渐进性丧失。2、内质网应激机制参与了外伤所致大鼠视神经部分损伤引起的RGCs凋亡。3、人脐血干细胞可明显延缓外伤所致大鼠视神经部分损伤引起的RGCs凋亡,对RGCs损伤提供保护部分作用。更多还原

【Abstract】 Traumatic optic neuropathy（TON） is the complex disease to those who suffered with permanent optic damage even after cure.This need positive treatments to saving eyesight.At present,the main treatments of TON are medication,surgical treatment and combination therapy with drug and operation,while the efficacy of various treatment methods vary widely.Recently some experts have proposed the concept with transplanted stem cells in treating of the optic nerve injury,and have made some progress in animal experiments,which perhaps lead to an effective and innovative direction to the TON treatment.Stem cell is a kind of original cells with high value-added and self-renewal abilities that can possess a number of differentiation potential.With the continuous development of stem cell technology and by its own biological characteristics,it has become a hot research that some stem cells in vitro be induced to differentiate into the required orientation of tissue cells to the treatment of certain diseases.Umbilical cord blood is the main source of stem cells.As peripheral blood,it is easy to gain and contains plenty of mesenchymal stem cells with immune modulation and accelerated hematopoietic recovery,etc.Therefore,the successful transplantation of cord blood is high,the response to transplantation is weak,and the graft-versus-host disease is rare.As the axons of retinal ganglion cells（RGCs）,the optic nerve’s damnification is closely related to witherer of RGCs.In this experiment,the rule was made by clamping rat models of traumatic optic nerve injury,and the use of flash visual evoked potentials（F-VEP）,hematomylin-eosin（HE） staining,terminal deoxy nucleotidyl transferase-mediated DUTP-biotin nick end labeling（TUNEL） method and the RT-PCR method,and so on,which carried on a detailed experimental study on the survival of injured optic nerve and RGCs,as well as the protective effect of human umbilical cord blood stem cells transplantation for trauma-induced rat RGCs and its possible mechanism. The First Part The impact of human umbilical cord blood stem cell transplantation in rat to flash visual evoked potentials of the Traumatic part-injury optic nerveObjective:（1） Observe the changes of F-VEP in traumatic rat model of optic nerve which was part damaged.（2）Research the impact of human umbilical cord blood stem cell transplantation in rat to F-VEP of the optic nerve which was part-injury.Methods:96 physical fitness adult Sprague-Dawley rats were divided into the optic nerve 30s clamped-group（moderate injury） and the 60s clamped-group（serious injury） randomly,as 48 rats in each group,and the left eyes was injury group and the right eyes normal control group in all rats.The two groups were randomly divided into injury group（A group） and treatment group（B,C,D group） according to the different treatment after optic nerve injury,and there were 12 rats in every group. The A group has not medication.The treatment group were injected neurotrophic factor to the vitreous cavity（B group）,human umbilical cord blood stem cells（C group）,the mixture of human umbilical cord blood stem cells and neurotrophic factor（D group）.Clamp the optic nerve which was 2mm after the eyeball for 30s or 60s by the clamping force which was 40g in injury eyes in all rats to making the rat traumatic optic part of damage model.All rats were examed the F-VEP in injury and control eyes at 1 hour,1 week,2 weeks,3 weeks and 4 weeks after injury,recorded amplitude and peak latency,and made statistical analysis.Results:1.The F-VEP of normal control eyes:the F-VEP waveform of normal control eyes in all rats were Stable,with no significant change with the time.The wave amplitude fluctuated in 12.7±0.6μV and the wave latency 73.8±3.2ms.2.The F-VEP of injury group（A group）:In All rats,the latency was significantly prolonged,the amplitude decreased.In the moderate injury group,1 hour,1 week,2 weeks,3 weeks and 4 weeks after injury,The wave amplitude were 6.41±0.6μV,5.38±0.6μV,4.56±0.76μV, 3.78±0.53μV and 3.17±0.75μV respectively,the wave latency were 86.5±1.2ms,100.6±3.3ms,109.6±7.2ms,111.2±8.1ms and 104.2±4.9ms. In the serious injury Group,according to the same time point,the wave amplitude were 6.37±0.7μV,5.89±0.6μV,5.74±0.60μV,5.11±0.44μV and4.09±0.38μV respectively,the wave latency were 81.4±1.0ms, 95.5±3.1ms,104.5±7.0ms,106.1±8.3ms and 99.3±3.9ms.3.The F-VEP of B group:In the moderate injury group,1 hour,1 week,2 weeks,3 weeks and 4 weeks after injury,The wave amplitude were 6.46±0.9μV,6.00±0.7μV,5.89±0.8μV,5.29±0.65μV and 4.27±0.57μV, the P1 wave latency were 86.9±1.5ms,95.7±2.7ms,97.2±6.9ms, 102.6±4.4ms and 98.1±4.5ms.In the serious injury Group,according to the same time point,the wave amplitude were 6.37±0.7μV,5.89±0.6μV, 5.74±0.60μV,5.11±0.44μV and 4.09±0.38μV,the Plwavelatency were 82.0±1.2ms,90.7±2.5ms,92.3±6.5ms,97.5±4.3ms和94.2±3.5ms.4.The F-VEP of C group:In the moderate injury group,1 hour,1 week,2 weeks,3 weeks and 4 weeks after injury,The wave amplitude were 6.54±1.2μV,6.08±1.0μV,5.97±1.1μV,5.37±0.9μV and 4.39±0.65μV,the P1 wave latency were 87.2±2.5ms,91.8±1.8ms,93.2±6.6ms,98.6±3.8ms and 94.1±3.8ms.In the serious injury Group,according to the same time pointthe above point in time,the wave amplitude were 6.45±1.0μV, 5.97±0.8μV,5.82±0.90μV,5.20±0.85μV and 4.23±0.55μV,the P1 wave latency were 82.7±2.0ms,86.2±1.8ms,88.7±6.3ms,93.6±3.5ms和89.8±2.7ms.5.The F-VEP of D group:In the moderate injury group,1 hour,1 week,2 weeks,3 weeks and 4 weeks after injury,The wave amplitude were 6.61±1.4μV,6.15±1.2μV,6.04±1.3μV,5.67±1.1μV和4.82±0.95μV,the P1 wave latency were 87.5±2.0 ms,89.5±1.6 ms,91.2±6.0 ms,96.6±3.2 ms和91.9±3.0ms.In the serious injury Group,according to the same time point,the wave amplitude were 6.52±1.2μV,6.04±1.0μV,5.89±1.1μV, 5.51±1.0μV和4.66±0.85μV,the P1 wave latency were 82.9±2.2 ms, 84.5±1.6 ms,86.6±6.0 ms,91.6±3.0 ms和85.5±2.0ms. 6.The comparison of the injury group and the normal control eyes:Whether the moderate injury group or the serious injury Group, Compared with normal control eyes,the Waveform of injury group（A group）appeared Significant changes in 1 hour after injury.At all time points,the amplitude and latency compared with normal control eyes,the difference were statistically significant（P＜0.01）.Comparison of the amplitude and the Latency between the optic nerve 30s clamped-group and the 60s clamped-group,the difference of every time points were statistically significant（P＜0.05）.With the passage of observing time, F-VEP amplitude decreased and latency prolonged,Both reached the climax at the three weeks after injury.7.The comparison between the injury group and treatment group:（1） Wave amplitude:The differences of comparison between the injured group and treatment group were statistically significant in various time points after injury both in 30s clamped-group and 60s clamped-group（P＜0.05 at 1 hour and 1 week after injury,P＜0.01 at other time point）.The difference of amplitude between the injury group and treatment group was greatest at 3 weeks after injury.At the same time,the amplitude of each group were 3.78±0.53μV,5.29±0.65μV,5.37±0.9μV,5.67±1.1μV in 30s clamped-group.the amplitude of each group were 3.60±0.32μV,5.11±0.44μV,5.20±0.85μV,5.51±1.0μV in 60s clamped-group.（2）Wave latency:The differences of comparison between the injured group and treatment group were statistically significant（P＜0.05 at1 week after injury,P＜0.01 at 2,3,4 week after injury） in all time points except 1 hour after injury,group there was no significant difference（P＞0.05）.The difference of wave latency between the two groups was biggest at2 weeks after injury.At the same time,the wave latency of each group were 109.6±7.2ms,97.2±6.9ms,93.2±6.6ms, 91.2±6.0ms in 30s clamped-group.the wave latency of each group were 104.5±7.0ms,92.3±6.5ms,88.7±6.3ms,86.6±6.0ms in 60s clamped-group.The wave latency in both groups were shorter than before at 4 weeks after injury.8.The comparisons between the 30s and 60s clamped-group:The difference between the two groups were statistically significant（P＜0.01） at the same time point after injury both the amplitude and latency.Compared with the 30s group,the amplitudes in 60s groups were significantly decreased and the incubation period were extended.9.The comparisons between the treatment groups:The amplitude of D group were higher than B group while the latency were shorter at all time points since the 1st week both in the 30s and 60s grous,and the differences were statistically significant（P＜0.05）.The comparisons in the same point of the remaining treatment groups were not statistically significant difference（P＞0.05）. Conclusion:1.Clamp the optic nerve of the normal rays for 30s or 60s by the clamping force which was 40g can cause part injury of the optic nerve which was proved by the decreased F-VEP amplitude and prolonged latency.The clamped time was longer,the damage and the F-VEP changes was more serious.The observated time was longer,the delay of F-VEP latency was more significant and the reduction of amplitude was greater and the out of F-VEP was sooner.2.Neurotrophic factor,human umbilical cord blood stem cells and the mixture has the function to promote the optic nerve conduction after the partial optic nerve injury of traumatic optic nerve in rats.The joint use of the human umbilical cord blood stem cells and neurotrophic factor can improve the quality of the promotion of nerve conduction recovery. The Second Part:Experimental study of effect and mechanism of human umbilical cord blood cells transplantation on rat optic nerve after crush injuryObjective:（1）To observe morphology of RGCs and the expression of glucose-regulated protein78（GRP78） and chop in RGCs after endoplasmic reticulum stress.（2） To explore the effect and the mechanism of human umbilical cord blood cells（HUCBCs） on the RGCs after crush injury.Methods:102 SD rats were divided into 3 groups randomly and the details were given in the sections that follow.All the right eyes of the rats were clamped for 60s postorbital 2mm to induce the optic nerve crush injury.Group A were stained with hematoxylin and eosin;group B were used to examine the loss of RGCs by TUNEL;group C were examined by RT-PCR.Results:1.The number of RGCs:（1）Normal control group:With the passage of observing time,In A group,the number of RGCs of normal control eyes was not change significantly,At 3d,7d,14d,21d and 28d after operation,the number of RGCs were 46.56±1.87,45.38±0.65,46.35±0.96, 45.98±1.02 and 45.65±0.97.The difference of RGCs amount between the adjacent time point was not significant（P＞0.05）.（2）Injury group（A₁ group）:The number of RGCs was severely reduced at 60s after optic nerve were clamped.The number of RGCs were 30.50±1.31,27.03±1.22, 20.16±0.87,18.16±0.84 and 15.68±0.79 at 3d,7d,14d,21d and 28d after operation.The number of RGCs decreased Quickly in 14 days after injury.Compared with normal control group,the number of RGCs reduced about 50 percent at 14d after operation.The number of RGCs reduced slowly after post-surgery 14 day.The number of RGCs reduced about 60 percent at 28d after operation.Compared with normal control group,the differences of the number of RGCs were statistically significant（P＜0.01） at various time points after injury.（3）Treatment group（A₂ group）:The reduce of the number of RGCs was more moderateThe number of RGCs were 40.13±1.31,36.22±2.67,32.26±1.43,31.61±0.76 and 30.31±0.51 at 3d,7d,14d,21d and 28d after operationThe differences of the number of RGCs were statistically significant at 5 different time points after injury between the treatment group and the normal control group,or the injury group（P＜0.01）.2.The rate of TUNEL-positive cells:（1）Injury group（B₁ group）:At 3h、12h、24h、48h、72h and 1w after operation,the rate of TUNEL-positive cellswere4.23%±1.19,6.21±1.03%,15.33%±2.78%,39.41±1.86%,58.83%±8.36%and 41.15%±6.37%.The difference of the number of apoptotic cells between the adjacent time point were statistically significant（P＜0.05）.（2） Treatment group（B₂ group）:At 3h,12h,24h,48h,72h and 1w after operation,the rate of TUNEL-positive cells were 0,0,0,12.33±2. 63%,34.23%±1.98%and 22.13±1.93%.Since the beginning 24h After injury,the difference of the number of apoptotic cells between the adjacent time point were statistically significant（P＜0.05）.（3） There was a small number of apoptotic cells in the inner nuclear layer in 3h after injury in the Injured group,while 48h in the treatment group.The number of apoptotic cells reached the peak at 72h after injury in both groups and reduce gradually at 1 week after injury.The differences of the number of TUNEL-positive cells between the two groups were statistically significant（P＜0.01） at the same time point.3.The expression of GRP78 and CHOP:（1）GRP78:At 3h,12h,24h,48h, 72h and 1w after operation,the value of OD of GRP78mRNA in the Injury group（C₁ group） were 1.221±0.12,1.313±0.22,1.226±0.18, 1.220±.14,1.197±0.10and 1.190±0.09.the value of OD of GRP78mRNA in the treatment group（C₂ group） were 1.732±0.21,2.370±0.18, 2.251±0.22,2.038±0.15,1.945±0.09 and 1.835±0.12.The treatment group compared with the injured group,the differences of the number of GRP78 were statistically significant（P＜0.01） During the same period.（2）CHOP: At 3h,12h,24h,48h,72h and 1w after operation,the value of OD of CHOPmRNA in the Injury group（C₁ group） were 2.251±0.21,2.31±0.15, 2.370±0.14,2.038±0.09,1.945±0.12 and 1.835±0.12.the value of OD of CHOPmRNA in the treatment group（C₂ group） were 1.226±0.22, 1.262±0.14,1.313±0.21,1.220±0.18,1.197±0.11 and 1.190±0.09.The differences of the number of CHOP between the two groups were statistically significant（P＜0.01） at the same time point.（3）The expression of GRP78 and CHOP were increased in the injured group and treatment group After injury,The expression of GRP78 reached the peak at 12h After injury which changes higher than the injured group in the treatment group,The expression of CHOP reached the peak at 24h After injury,Compared with the Injured group,weak expression of CHOP in the treatment group.Conclusions:1.Most loss of the RGCs after the crush injury were apoptosis and the count of the loss decreased with the life span of the rat.2.endoplasmic reticulum stress may be one of the causes of the loss of RGCs after optic nerve crush injury.3.HUCBCs could extend the life span of RGCs after optic nerve crush injury.更多还原