携带增强型绿色荧光蛋白的慢病毒载体转染大鼠脂肪干细胞

Rat ADSCs transfected by lentivirus vector-mediated enhanced green fluorescent protein

目的探索脂肪干细胞（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ADSCs12h，荧光显微镜及流式细胞仪检测转染效率和荧光强度；Mrrr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，组问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10d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20d，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。

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 eytometric analysis and fluorescence microscopy, and cell viability was detected by MTl＇-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 acellnlar 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 adipogenie differentiation for 20 days, orange calcium deposits,orange-red lipid droplets were seen in EGFP-ADSCs after Alizarin red and oil red 0 staining. At 1 week in vivo, EGFP-ADSCs evenly distributed and became fusiform on frozen longitudinal sections. Conclusion Lv-EGFP transfeetion 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.