作者：许茜, 袁志兰 戈应滨
作者单位：中国江苏省卫生厅招标课题资助项目(No.H200750) 1(210029)中国江苏省南京市，南京医科大学生理教研室;2(210029)中国江苏省南京市，江苏省人民医院眼科
【摘要】 目的:研究尾静脉注射骨髓细胞对于链脲佐菌素(streptozotocin,STZ)诱导的糖尿病(diabetes mellitus,DM)小鼠视网膜振荡电位(oscillatory potentials,OPs)和视网膜新生血管密度的影响。方法: 40只C57BL/6J小鼠随机分成正常对照(NC)组和DM造模组。DM造模组给予多性腹腔注射，造模成功后再随机分为DM对照组和DM骨髓治疗组，饲养16wk，罗兰视觉电生理仪检测其OPs波，视网膜铺片联合免疫荧光染色检测视网膜新生血管密度。DM骨髓治疗组给予小鼠骨髓细胞悬液尾静脉注射，注射2wk和8wk后再次分别检测OPs波及视网膜新生血管密度。结果:高血糖的影响使得DM小鼠造模成功后OPs波振幅呈明显降低趋势，OPs峰潜时明显延长，与对照组小鼠各时期相比有显著差异(P<0.05)。骨髓治疗2wk和8wk后即小鼠26wk及32wk时，DM骨髓治疗组较DM组OPs波幅明显升高，OPs峰潜时明显缩短，有显著差异(P<0.05)。而26wk及32wk时NC组及DM骨髓治疗两组OPs振幅及峰潜时相比无显著差异(P>0.05)。视网膜铺片正常对照组血管发育成熟，血管网结构清晰，DM对照组血管迂曲阻塞，大量结构异常新生血管，丧失了血管网结构，DM骨髓治疗组见血管网结构清晰，仍有少部分深层血管阻塞，形态接近正常小鼠。结论:骨髓细胞移植可以有效抑制视网膜新生血管形成。
【关键词】 高血糖;糖尿病视网膜病变;骨髓移植;视网膜振荡电位;小鼠
Therapeutic effect of retinal neovascularization in diabetic mice by injection of isolated bone marrow cell through vena caudalis
Qian Xu1, ZhiLan Yuan2, YingBin Ge1
Foundation item:Funded Tender of Jiangsu Health Department, Jiangsu Province, China(No.H200750)
1Department of Physiology, Nanjing Medical University,Nanjing 210029, Jiangsu Province, China; 2Department of Ophthalmology, Jiangsu Province Peoples Hospital, Nanjing 210029, Jiangsu Province, China
Abstract
AIM: To investigate the change of the oscillatory potentials(OPs) and dense of retinal neovascularization(RNV) of diabetes mellitus(DM) rats after injection of isolated bone marrow cell through vena caudalis.
METHODS: Forty C57BL/6J rats were divided at random to receive streptozotocin(50mg/kg) intraperitoneally(diabetes) or cit rate buffer alone (normal control). One week after injections, only streptozotocintreated rats with plasma glucose concentrations above 16.7mmol/L were considered to be diabetic and were subsequently divided into two DM group and bone marrow(BM)treated group. At the end of the 16th week, the OPs of the rats in three groups were detected by Roland Electrophysiological Instrument and the dense of RNV measured by immunohistochemical examination.Afterward the BMtreated group received injection of BM suspension of rats via vena caudalis and the OPs and dense of RNV were reexamined at the end of 18th and 24th week,respectively.
RESULTS: After 16 weeks from models, the amplitude of ΣOP(sum of OP1,OP2,OP3 and OP4) and OP TTP (sum of P1,P2,P3 and P4) were significantly lower than those in normal control group(P<0.05),which recovered after injection of isolated bone marrow(P<0.05).There was no statistical difference between BMtreated group and control group(P>0.05).Immunohistochemical examination showed that the vasculature was distinctly displyed in normal control group; a lot of curved blocked vessels and retinal neovascularization make the retina lose its original structure in DM group;while vasculature was clearly demonstrated but there was still blockage of vessels in BMtreated group.
CONCLUSION: Vena caudalis injection of isolated bone marrow cell can inhibit retinal neovascularization.
KEYWORDS: hyperglycemia;diabetic retinopathy; bone marrow transplantation; retinal oscillatory potential; mice
0引言
糖尿病视网膜病变(diabetic retinopathy，DR)是指在视网膜内异常增生的新生血管侵袭至玻璃体腔而造成的严重病变，是糖尿病最常见的微血管并发症之一[1]。新生血管在形成与发展过程中易出血、机化，引发牵拉性视网膜脱离，严重损害视功能[2]。目前，激光及手术治疗虽然都取得了一定疗效,但其局限性依然存在。完善微血管功能，减少血管空灌是改善视网膜缺血缺氧状态的有效途径。本研究通过观察DM小鼠以及骨髓移植后小鼠视网膜振荡电位(oscillatory potentials, OPs)及视网膜血管网新生血管密度的变化，探讨骨髓细胞移植对DM小鼠视网膜新生血管病变的防治作用。
1材料和方法
1.1材料 8周龄健康成年近交系C57BL/6J小鼠40只(扬州大学实验动物中心提供)，雄性，体质量约18～20g。予以12h明暗交替光照，不限食水，室温18～23℃条件下饲养。外眼和检眼镜检查屈光间质清晰，眼底无改变。试剂：链脲霉素(streptozotocin，STZ)美国Sigma公司产品，盐酸氯胺酮(2ml：0.1g)为江苏恒瑞医药股份有限公司产品，美多丽(复方托吡卡胺)、倍诺喜(盐酸奥布卡因)为参天株式会社产品，GSIB4(货号FL1201)为VACTOR实验室产品，One Touch血糖仪为美国Life scan公司产品，尿糖试纸带为广州市越秀东方新科技研究所研制，德国罗兰视觉电生理仪(RETIport)。
1.2方法
1.2.1 STZ诱导的糖尿病小鼠模型的建立 小鼠随机分成两组:正常对照(NC)组和糖尿病造模组。造模组小鼠禁食10h，STZ溶于0.1mmol/L柠檬酸柠檬酸钠缓冲液(pH4.5)，腹腔注射(50mg/kg),连续5d，共造模30只,其余10只注射以等量生理盐水[3]。给药后1wk后剪尾法取血测其血糖和尿糖，血糖>16.7mmol/L，尿糖+++以上，持续1wk以上的小鼠被认为建模成功，共30只。以后测血糖及尿糖1次/wk。随机分为DM对照组(10只)和DM骨髓治疗组(20只)。
1.2.2小鼠骨髓细胞制备与回输 4～6周龄C57BL/6J雄性小鼠断颈处死后，无菌条件下取出小鼠双侧股骨、胫骨，反复用含100mL/L胎牛血清的LDMEM培养基冲洗骨髓腔，搜集冲洗液，离心后PBS洗涤、混悬，制成细胞悬液。
1.2.3小鼠电生理检测 将小鼠用氯胺酮按照80mg/kg麻醉后，固定于自制实验台上，实验小鼠用美多丽散瞳，暗适应30min以上，倍诺喜滴眼液表面麻醉，记录电极固定于角膜表面，参考电极位于同侧外眦部，接地电极置于头部，采用RETIport系统(Roland Consult，德国)，根据国际临床视觉电生理学会标准化方案进行振荡电位OPs的检查，OPs闪烁反应光强为3.0cd·s/m2，记录OPs的通频带为200～500Hz，刺激之间间隔15～60s，叠加9次以获得均值。应用RETIport系统进行波形幅值和潜伏期的测量。以OPs波总峰潜时(OP TTP=P1+P2+P3+P4)及总波幅(ΣOP=OP1+OP2+OP3+OP4)进行评价[4]。
1.2.4小鼠视网膜铺片联合免疫荧光染色 以30g/L戊巴比妥钠深度麻醉处死小鼠后,行全身循环灌注，约灌注生理盐水200mL，至眼球变苍白，再灌注40g/L多聚甲醛 表1骨髓细胞回输对糖尿病小鼠OPs振幅的影响表2骨髓细胞回输对糖尿病小鼠OPs峰潜时的影响
约50mL作血管内固定，取双眼以40g/L多聚甲醛固定24h。自睫状体后部近锯齿缘剪开巩膜，去掉眼前段和玻璃体，显微镜下剥取视网膜。0.01mol/L PBS漂洗3次,每次10min，10g/L BSA孵育15min后PBS冲洗10min。GSIB4(1∶50稀释,20g/L)避光室温染色30min，PBS冲洗10min[5]。视网膜铺片，荧光封片剂封片，激光共聚焦显微镜下观察视网膜血管网形态。
统计学分析:所得数据以±s的形式表示, 用SPSS 10. 0 统计软件进行配对t检验。
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