CAJ | 학술논문

背景以往视网膜血管消化铺片法仅能够获得部分视网膜血管,无法客观评价全视网膜血管的病理改变,因此进一步获得完整的视网膜血管网是相关疾病实验研究和临床研究的基础. 目的在国外对全视网膜血管消化铺片技术的基础上进行改良,建立全视网膜血管的铺片方法. 方法 30只健康Wistar 大鼠随机数字表法分为模型组和对照组,模型组20只大鼠用溶于0.05 mmol/L柠檬酸缓冲液的质量分数1.25％链脲佐菌素(STZ)腹腔内注射法制作糖尿病模型,对照组10只大鼠以同样的方法注射等容量柠檬酸缓冲液.注射后8个月经左心室注射PBS 100 ml,剪开右心房使血液及PBS流出后注射质量分数4％多聚甲醛100 ml,摘除眼球,将完整剥离的大鼠视网膜先进行胰蛋白酶消化,然后剥离玻璃体及内界膜,去除视网膜神经组织,得到完整的视网膜毛细血管网,平铺于载玻片上,将视网膜毛细血管网以视盘为中心划为内、中、外3个区域,采用盲法在光学显微镜下判定和计数中间区域影周细胞及无细胞血管. 结果视网膜铺片显示,大鼠的视网膜血管为全血管型,可见放射状的视网膜中央动脉与视网膜中央静脉主干及逐级分支结构、小动脉与小静脉之间表浅层和深层的毛细血管网.周细胞略突出于血管管壁,在其形成影周细胞时,原有的细胞核缺失.无细胞血管的管径为邻近毛细血管管径的20％以上,并且在整个血管上无细胞核和影周细胞.结论全视网膜消化铺片技术能够提供实验所需的完整视网膜毛细血管网,配合细胞计数方法,能够较好地评价视网膜的形态改变,并为视网膜血管和管壁细胞的定量分析提供有利条件.
배경 이왕시망막혈관소화포편법부능구획득부분시망막혈관,무법객관평개전시망막혈관적병리개변,인차진일보획득완정적시망막혈관망시상관질병실험연구화림상연구적기출. 목적 재국외대전시망막혈관소화포편기술적기출상진행개량,건립전시망막혈관적포편방법. 방법 30지건강Wistar 대서수궤수자표법분위모형조화대조조,모형조20지대서용용우0.05 mmol/L저몽산완충액적질량분수1.25％련뇨좌균소(STZ)복강내주사법제작당뇨병모형,대조조10지대서이동양적방법주사등용량저몽산완충액.주사후8개월경좌심실주사PBS 100 ml,전개우심방사혈액급PBS류출후주사질량분수4％다취갑철100 ml,적제안구,장완정박리적대서시망막선진행이단백매소화,연후박리파리체급내계막,거제시망막신경조직,득도완정적시망막모세혈관망,평포우재파편상,장시망막모세혈관망이시반위중심화위내、중、외3개구역,채용맹법재광학현미경하판정화계수중간구역영주세포급무세포혈관. 결과 시망막포편현시,대서적시망막혈관위전혈관형,가견방사상적시망막중앙동맥여시망막중앙정맥주간급축급분지결구、소동맥여소정맥지간표천층화심층적모세혈관망.주세포략돌출우혈관관벽,재기형성영주세포시,원유적세포핵결실.무세포혈관적관경위린근모세혈관관경적20％이상,병차재정개혈관상무세포핵화영주세포.결론 전시망막소화포편기술능구제공실험소수적완정시망막모세혈관망,배합세포계수방법,능구교호지평개시망막적형태개변,병위시망막혈관화관벽세포적정량분석제공유리조건.
Background Previous retinal vascular mounting only obtained part of retinal vessels for the study of retinal diseases,and thus it is difficult to comprehensively assess these diseases.So optimizing the trypsin digestion method to show the complete retinal capillary network is very important for the study of retinal diseases.Objective This study was to modify the preparing way of trypsin digested whole retinal capillary network and offer a basis for a quantitative analysis of cells and capillaries.Methods Thirty Wistar rats were divided into model group (20 rats) and control group (10 rats).Streptozotocinum(STZ) of 1.25％ dissolved in 0.05 mmol/L sodium citratehydrochloric acid buffer was intraperitoneally injected to establish diabetes models in the model group,and the equal volume of solvent was used in the same way in the control group.Eight months after injection,100 ml PBS was injected via ventriculus sinister and released via cut right atrium,and then 100 ml 4％ paraformaldehyde was injected into the ventriculus sinister.The rat retinas containing part of the optic nerve were entirely isolated,and digested by trypsin,and vitreous,inner limiting membrane and neural retinal tissue were removed.The whole retinal capillaries network was mounted on the slide.The ghost pericytes and acellular capillaries in the middle retinal area were identified and counted under the optical microscope.Results The retinal mount exhibited that the retinal vessels were stained by hematoxylin and periodic acid schiff.The vessels network presented with the entire type in shape with the radical central retinal arteries and veins and their branches.The capillary showed the shallow-and deep-layer networks between the small arteries and veins.Pericytes distributed and protruded vessel wall and formed the ghost cells without nuclei.The diameter of acellular capillaries was 20％ or more than that of near capillaries,and no cellular nuclei or ghost cell was found through the vessel.Conclusions The experimental technique for setting-up of cleaned vasculature and mounting vessels on glass microscopic slide provides intact vessels,which is helpful for the evaluation of retinal vascular morphology and quantitative analysis.