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目的本研究通过囊胚腔显微注射转增强型绿色荧光蛋白(EGFP)基因的脐血干细胞的方法制备嵌合体小鼠,以期为研究成体干细胞的体内分化提供一定的实验依据和理论基础。方法分离得到的表达绿色荧光的小鼠脐血干细胞用于囊胚腔显微注射。然后进行胚胎移植,出生的嵌合体小鼠首先进行毛色嵌合的观察,再对各组织进行基因组DNA和RNA水平的检测。流式细胞术检测阳性小鼠的组织中绿色荧光细胞的百分率。结果分离出的脐血干细胞,经流式检测表达绿色荧光的细胞所占的比例为80.25%。经显微注射和胚胎移植,共出生5只小鼠,均为白色,没有发生毛色嵌合。分别取心肌、肝、肺、皮肤、腿肌和脂肪共6种组织对EGFP基因进行PCR和RT-PCR检测。结果显示,有2只小鼠的腿肌和脂肪组织显示阳性,其他组织和其他3只小鼠的6种组织均为阴性。将这2只小鼠的腿肌和脂肪组织消化成单细胞悬液,进行流式细胞分析。结果显示2只嵌合小鼠腿肌和脂肪组织的平均嵌合率分别为9.87%和5.78%。结论来自成体的脐血干细胞在体内可以分化成腿部肌肉和脂肪组织的细胞。
OBJECTIVE In this study, chimeric mice were prepared by microinjection of blastocyst into green fluorescent protein (EGFP) -transfected cord blood stem cells in order to provide some experimental and theoretical basis for studying the differentiation of adult stem cells in vivo. Methods The green fluorescent-derived mouse cord blood stem cells isolated from the method were used for microinjection into the blastocoel. After embryo transfer, the chimeric mice born first observed the coat color mosaic and then detected the genomic DNA and RNA levels in each tissue. Flow cytometry was used to detect the percentage of green fluorescent cells in the tissues of positive mice. Results The percentage of umbilical cord blood stem cells isolated by flow cytometry, which expressed green fluorescence, was 80.25%. By microinjection and embryo transfer, a total of 5 mice were born, all white, without chromatic hair color. The EGFP gene was detected by PCR and RT-PCR in six kinds of tissues including heart muscle, liver, lung, skin, leg muscle and fat respectively. The results showed that two of the mice showed positive leg and adipose tissue, and the other tissues and six of the other three mice were all negative. The leg muscles and adipose tissue of these two mice were digested into single cell suspensions and analyzed by flow cytometry. The results showed that the average chimeric rates of leg muscle and adipose tissue in two chimeric mice were 9.87% and 5.78% respectively. Conclusion Adult umbilical cord blood stem cells can differentiate into cells of leg muscles and adipose tissue in vivo.