CAJ | 학술논문

目的探索脂肪于细胞(ADSCs)的标记方法,观察增强型绿色荧光蛋白(EGFP)阳性ADSCs(EGFP-ADSCs)的体外活性、干细胞特性及体内短期活性. 方法用携带增强型绿色荧光蛋白(eGFP)基因的慢病毒载体(Lv-eGFP)在感染复数(MOI)为0、1、5、25、50、100时,转染SD大鼠ADSCs 12 h,荧光显微镜及流式细胞仪检测转染效率和荧光强度;MTT法评价转染后细胞活性;EGFP-ADSCs分别行成脂分化及油红O检测、成骨分化及茜素红染色;将EGFP-ADSCs注射到去细胞神经构建组织工程化神经,修复大鼠坐骨神经缺损,术后1周取材进行冰冻切片观察细胞在体内存活情况. 结果转染4d后,EGFP阳性率及荧光强度达到高峰;EGFP基因表达不随细胞传代而消失.MOI=0、1、5、25、50、100时,EGFP阳性转染率分别为0.13％、31.09％、75.33％、92.66％、96.70％、98.38％.实验组阳性率与对照组(MOI =0)选择相比,差异均有统计学意义(P ＜0.05);MOI =25、50、100时,组间阳性率差异无统计学意义(P＞0.05),但与MOI=1、5比时差异(P＜0.05).MTT试验观察10 d内MOI=25、50、100组细胞增值活性与非转染细胞差异无统计学意义(P＞0.05).选择MOI =25作为最佳转染滴度进行后续实验.转染后ADSCs成骨、成脂分化20 d,茜素红染色见橘红色钙沉积,油红O染色见橙红色脂滴.1周冰冻切片观察细胞于体内呈梭形,均匀分布. 结论慢病毒载体转染EGFP基因不影响ADSCs活性及成骨成脂分化能力,能为组织工程化神经修复缺损提供示踪种子细胞的方法.
목적 탐색지방우세포(ADSCs)적표기방법,관찰증강형록색형광단백(EGFP)양성ADSCs(EGFP-ADSCs)적체외활성、간세포특성급체내단기활성. 방법 용휴대증강형록색형광단백(eGFP)기인적만병독재체(Lv-eGFP)재감염복수(MOI)위0、1、5、25、50、100시,전염SD대서ADSCs 12 h,형광현미경급류식세포의검측전염효솔화형광강도;MTT법평개전염후세포활성;EGFP-ADSCs분별행성지분화급유홍O검측、성골분화급천소홍염색;장EGFP-ADSCs주사도거세포신경구건조직공정화신경,수복대서좌골신경결손,술후1주취재진행빙동절편관찰세포재체내존활정황. 결과 전염4d후,EGFP양성솔급형광강도체도고봉;EGFP기인표체불수세포전대이소실.MOI=0、1、5、25、50、100시,EGFP양성전염솔분별위0.13％、31.09％、75.33％、92.66％、96.70％、98.38％.실험조양성솔여대조조(MOI =0)선택상비,차이균유통계학의의(P ＜0.05);MOI =25、50、100시,조간양성솔차이무통계학의의(P＞0.05),단여MOI=1、5비시차이(P＜0.05).MTT시험관찰10 d내MOI=25、50、100조세포증치활성여비전염세포차이무통계학의의(P＞0.05).선택MOI =25작위최가전염적도진행후속실험.전염후ADSCs성골、성지분화20 d,천소홍염색견귤홍색개침적,유홍O염색견등홍색지적.1주빙동절편관찰세포우체내정사형,균균분포. 결론 만병독재체전염EGFP기인불영향ADSCs활성급성골성지분화능력,능위조직공정화신경수복결손제공시종충자세포적방법.
Objective To explore the labeling method of rat adipose-derived stromal cells,and observe the stem cell characteristics and the activities of EGFP-positive adipose-derived stromal cells (EGFP-ADSCs) in vitro and in vivo.Methods ADSCs were transfected for 12 h with enhanced green fluorescent protein gene (EGFP) carried by lentivirus(Lv-EGFP) vector at different value of MOI (0,5,10,25,50,100,respectively).The rate of EGFP expression and fluorescence intensity were evaluated by flow cytometric analysis and fluorescence microscopy,and cell viability was detected by MTT-test after transfection.Secondly,cells were exposed either to adipogenic medium or osteogenic medium,then stained with Oil Red O and Alizarin Red S.Cell growth was investigated on frozen longitudinal sections when EGFP-ADSCs were injected into acellular nerves to build tissue-engineered peripheral nerves repairing sciatic nerve defects in rats for 1 week in vivo.Results EGFP-positive rate and fluorescence intensity peak at 4 days after transfection.The rate of EGFP expression was 0.13％,31.09％,75.33％,92.66％,96.70％,98.38％ for MOI =0,1,5,25,50,100,respectively.The positive rate between the experimental group and control (MOI =0) existed significantly difference (P ＜ 0.05) ; the difference between MOI =1,5 groups and MOI =25,50,100 groups were also observed (P ＜ 0.05).There was no statistical difference in EGFP-positive rate and cell proliferation activity among MOI =25,50,100 groups (P ＞ 0.05).MOI =25 was chosen as best scheme to transfect ADSCs for subsequent experiments.Osteogenic and adipogenic differentiation for 20 days,orange calcium deposits,orange-red lipid droplets were seen in EGFP-ADSCs after Alizarin red and oil red O staining.At 1 week in vivo,EGFP-ADSCs evenly distributed and became fusiform on frozen longitudinal sections.Conclusion Lv-EGFP transfection does not affect the ADSCs activity and their osteogenic and adipogenic differentiation,so could be as a tracing method for ADSCs-tissue-engineered peripheral nerves repairing nerve defects.