CAJ | 학술논문

背景:干细胞移植为慢性肾脏病的治疗提供了新的视角,特异性标记干细胞并体内示踪是这一领域研究的基础,目前尚无一种公认的适用于所有细胞的标记方法.目的:观察DAPI及绿色荧光蛋白标记细胞在阿霉素肾病大鼠体内的定位及分化,探讨便捷的大鼠胚胎后肾间充质细胞的体外标记方法.设计、时间及地点:分组对比观察,于2007-04/12在中南大学湘雅二医院完成.材料:清洁级雌性Sprague-Dawley(SD)大鼠60只,体质量180～220 g,用于制备阿霉素肾病大鼠模型.方法:分别将DAPI、DAPI体外标记后肾间充质细胞、绿色荧光蛋白体外标记后肾间充质细胞,通过尾静脉注入阿霉素肾病大鼠体内:于细胞移植后1,3,5周行肾组织冰冻切片,观察DAPI及绿色荧光蛋白荧光分布,并ABC免疫酶染色法检测肾组织中绿色荧光蛋白表达.主要观察指标:比较DAPI及绿色荧光蛋白两种不同方法标记后肾间充质细胞在阿霉素慢性肾病大鼠肾内的定植情况,以及绿色荧光蛋白标记细胞不同时间点移植在肾内的定植改变.结果:①DAPI标记细胞及单纯DAPI尾静脉注射后1周,均可在肾小管上皮细胞中观察到DAPI阳性细胞,至移植后5周仍可观察到,但随时间延长荧光有减弱趋势.②绿色荧光蛋白标记细胞移植后1周,可在肾小管上皮细胞中观察到绿色荧光蛋白阳性细胞,至移植后5周仍可观察到,荧光无明显减弱.③ABC免疫酶染色法检测,显示绿色荧光蛋白阳性细胞主要表达于近端肾小管中,肾小球系膜区亦可观察到少最表达:绿色荧光蛋白阳性产物主要表达于胞浆.绿色荧光蛋白表达半定量评分显示,标记细胞的早期应用阳性率大于晚期应用者,且随观察时间延长,阳性率有升高趋势.结论;后肾间充质细胞的脂质体感染法绿色荧光蛋白标记是一便捷,有效的体外标记、示踪方法,适于移植细胞的短期内示踪、观察. 揹景:榦細胞移植為慢性腎髒病的治療提供瞭新的視角,特異性標記榦細胞併體內示蹤是這一領域研究的基礎,目前尚無一種公認的適用于所有細胞的標記方法.目的:觀察DAPI及綠色熒光蛋白標記細胞在阿黴素腎病大鼠體內的定位及分化,探討便捷的大鼠胚胎後腎間充質細胞的體外標記方法.設計、時間及地點:分組對比觀察,于2007-04/12在中南大學湘雅二醫院完成.材料:清潔級雌性Sprague-Dawley(SD)大鼠60隻,體質量180～220 g,用于製備阿黴素腎病大鼠模型.方法:分彆將DAPI、DAPI體外標記後腎間充質細胞、綠色熒光蛋白體外標記後腎間充質細胞,通過尾靜脈註入阿黴素腎病大鼠體內:于細胞移植後1,3,5週行腎組織冰凍切片,觀察DAPI及綠色熒光蛋白熒光分佈,併ABC免疫酶染色法檢測腎組織中綠色熒光蛋白錶達.主要觀察指標:比較DAPI及綠色熒光蛋白兩種不同方法標記後腎間充質細胞在阿黴素慢性腎病大鼠腎內的定植情況,以及綠色熒光蛋白標記細胞不同時間點移植在腎內的定植改變.結果:①DAPI標記細胞及單純DAPI尾靜脈註射後1週,均可在腎小管上皮細胞中觀察到DAPI暘性細胞,至移植後5週仍可觀察到,但隨時間延長熒光有減弱趨勢.②綠色熒光蛋白標記細胞移植後1週,可在腎小管上皮細胞中觀察到綠色熒光蛋白暘性細胞,至移植後5週仍可觀察到,熒光無明顯減弱.③ABC免疫酶染色法檢測,顯示綠色熒光蛋白暘性細胞主要錶達于近耑腎小管中,腎小毬繫膜區亦可觀察到少最錶達:綠色熒光蛋白暘性產物主要錶達于胞漿.綠色熒光蛋白錶達半定量評分顯示,標記細胞的早期應用暘性率大于晚期應用者,且隨觀察時間延長,暘性率有升高趨勢.結論;後腎間充質細胞的脂質體感染法綠色熒光蛋白標記是一便捷,有效的體外標記、示蹤方法,適于移植細胞的短期內示蹤、觀察.
배경:간세포이식위만성신장병적치료제공료신적시각,특이성표기간세포병체내시종시저일영역연구적기출,목전상무일충공인적괄용우소유세포적표기방법.목적:관찰DAPI급록색형광단백표기세포재아매소신병대서체내적정위급분화,탐토편첩적대서배태후신간충질세포적체외표기방법.설계、시간급지점:분조대비관찰,우2007-04/12재중남대학상아이의원완성.재료:청길급자성Sprague-Dawley(SD)대서60지,체질량180～220 g,용우제비아매소신병대서모형.방법:분별장DAPI、DAPI체외표기후신간충질세포、록색형광단백체외표기후신간충질세포,통과미정맥주입아매소신병대서체내:우세포이식후1,3,5주행신조직빙동절편,관찰DAPI급록색형광단백형광분포,병ABC면역매염색법검측신조직중록색형광단백표체.주요관찰지표:비교DAPI급록색형광단백량충불동방법표기후신간충질세포재아매소만성신병대서신내적정식정황,이급록색형광단백표기세포불동시간점이식재신내적정식개변.결과:①DAPI표기세포급단순DAPI미정맥주사후1주,균가재신소관상피세포중관찰도DAPI양성세포,지이식후5주잉가관찰도,단수시간연장형광유감약추세.②록색형광단백표기세포이식후1주,가재신소관상피세포중관찰도록색형광단백양성세포,지이식후5주잉가관찰도,형광무명현감약.③ABC면역매염색법검측,현시록색형광단백양성세포주요표체우근단신소관중,신소구계막구역가관찰도소최표체:록색형광단백양성산물주요표체우포장.록색형광단백표체반정량평분현시,표기세포적조기응용양성솔대우만기응용자,차수관찰시간연장,양성솔유승고추세.결론;후신간충질세포적지질체감염법록색형광단백표기시일편첩,유효적체외표기、시종방법,괄우이식세포적단기내시종、관찰.
BACKGROUND:Stem cell transplantation provides a new approach to treat chronic renal disease.Specific marking and in vivo tracing of stern cells are the basis of studies in this field.However,the marking methods appropriate for all cells remain uncertain.OBJECTIVE:To observe the in vivo location and differentiation of 4',6-diamidino-2-phenylindole (DAPI) and green fluorescence protein (GFP)-Iabeled cells in adriamycin nephrosis rats so as to explore an efficient labeling and tracing method for metanephric mesenchymal cells (MMCs) derived from embryonic rats.DESIGN,TIME AND SETTING:Grouping comparative observation was performed at the Second Xiangya Hospital of Central South University from April to December 2007.MATERIALS:A total of 60 female SD rats,weighing 180-220 g,of dean grade,were used to establish models of adriamycin nephrosis.METHODS:DAPI and MMCs infected with GFP and DAPI were respectively injected into addamycin nephrosis via the tail vein.DAPI and GFP distribution in the frozen sections was detected at 1,3,and 5 weeks,postoperatively.In addition,GFP expression in renal tissues was detected by ABC immunoenzymatic staining method.MAIN OUTCOME MEASURES:DAPI and GFP-labeled Cell grafts in adriamycin nephrosis rats were compared.The changes of GFP-transfected MMCs at different time points were observed.RESULTS:DAPI positive cells were observed in tubular structures after 1 weeks of injection of DAPI-labeled cells and DAPI alone,and remained existing at 5 weeks,but the florescence was reduced with time.GFP-transfected MMCs were able to survive and integrate into tubular structures after 1 week,and remained existing at 5 weeks.Moreover,the fluorescence was not reduced.ABC immunoanzymatic staining showed that only a few GFP-positive MMCs appeared in glomerular tufts,and mainly distributed in cytoplasm.Semi-quantitative evaluation of GFP show that the positive cell rate in rats with early application was greater than that with advanced application,and the positive rate was increased with time.CONCLUSION:Liposome mediated GFP gene transfer was an efficient labeling in vitro and suitable tracing method for cell differentiation experiment in vivo,suitable for short-term tracing and observation of transplanted cells.