Abstract: Objective　This study explored the isolation and identification method of rat amniotic mesenchymal stem cells and their exosomes, laying a foundation for subsequent research on the treatment of rat amniotic mesenchymal cells and their exosomes.Methods　①For an 18-day- pregnant Sprague Dawley (SD) rat, inject 400 IU/kg heparin into the heart. Soak the uterus in 75% alcohol, and the amniotic tissue was separated in 0.9% sodium chloride solution. ②Digest step by step with 0.75 mg/mL collagenase and 0.25% trypsin, and culture in α-MEM medium (supplemented with 4 ng/mL fibroblast growth factor).③Identification was performed through morphological observation, surface marker detection, and immunofluorescence. ④Exosomes were extracted by ultra-centrifugation, and their morphology was observed by transmission electron microscopy, protein concentration was determined by the BCA method, and surface markers were measured.Results　The stem cells adhered to the wall and grew, showing a long spindle-shaped morphology, and the cells were interconnected. Flow cytometry detection of stem cell surface antigens showed that the expressions of CD29 and CD90 were positive, while the expressions of CD34 and CD45 were negative. The result of cell immunofluorescence indicated that the expression of Vimentin was positive.The results indicated that the isolated rat amniotic stem cells were mesenchymal stem cells. The exosomes isolated by ultra-centrifugation had a complete and oval shape, a bilayer membrane structure, and uniform size. The result of particle size analysis showed that the average diameter was 82.6±14.0 nm. Flow cytometry detection showed that the expressions of surface proteins CD9, CD63, and CD81 were positive. The protein concentration of exosomes determined by the BCA method was 0.995 μg/μL.Conclusion　Rat amniotic mesenchymal cells were successfully isolated by the step-by-step digestion method using collagenase and trypsin, and exosomes of rat amniotic mesenchymal cells were extracted by ultra-centrifugation, providing an experimental basis for the research and application of rat amniotic mesenchymal stem cells and their exosomes.

Key words: amnion, mesenchymal stem cells, exosomes, rat

摘要： 目的　探究大鼠羊膜间充质干细胞及其外泌体的分离与鉴定方法,为其后续治疗应用奠定基础。方法 ①妊娠18 d SD大鼠,心脏注射400 IU/kg肝素,75%乙醇浸泡子宫,在0.9%氯化钠溶液中分离羊膜组织;②用 0.75 mg/mL胶原酶和0.25%胰蛋白酶分步消化、应用α-MEM培养基(添加4 ng/mL成纤维生长因子)培养;③采 用形态观察、表面标志物检测和免疫荧光染色进行鉴定;④超速离心法提取外泌体,透射电镜观察形态、BCA法测 定蛋白浓度和流式细胞术检测表面标志物。结果　形态学观察发现干细胞贴壁生长,形态呈长梭形,细胞之间相 互连接;流式细胞术检测干细胞表面抗原显示,CD29、CD90表达呈阳性,CD34、CD45表达呈阴性;细胞免疫荧光结 果显示,Vimentin(波形蛋白)表达阳性。结果表明,分离得到的大鼠羊膜干细胞为间充质干细胞。透射电镜结果显 示,超速离心法分离得到的外泌体形态完整、呈椭圆形,具有双分子层膜结构且大小均一,粒径平均(82.6±14.0)nm; BCA测定外泌体蛋白浓度为0.995 μg/μL;流式细胞术检测表面蛋白显示CD9、CD63和CD81表达呈阳性。 结论　采用胶原酶和胰蛋白酶分步消化法成功分离出大鼠羊膜间充质细胞,通过超速离心法成功提取大鼠羊膜间 充质干细胞外泌体,为大鼠羊膜间充质干细胞及其外泌体的研究和应用奠定基础。

关键词: 羊膜, 间充质干细胞, 外泌体, 大鼠