CAJ | 학술논문

背景：文献报道人羊膜间充质干细胞提取方法各不一致，得到的细胞数量各不相同。目的：探索人羊膜间充质干细胞在体外分离培养的最适方法。 　　方法：无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片，分别通过4个实验7种方法体外培养人羊膜间充质干细胞。①实验一：分别采用3种方法：0.05 g/L胰蛋白酶消化10 min后，再加0.75 g/L胶原酶Ⅰ消化60 min；0.75 g/L胶原酶Ⅰ直接消化120 min；0.05 g/L胰蛋白酶与0.75 g/L胶原酶Ⅰ同时消化60 min。②实验二：采用0.05 g/L的胰蛋白酶消化30 min后，再加0.75 g/L胶原酶Ⅰ消化30 min。③实验三：分别采用2种方法：0.05 g/L的胰蛋白酶连续2次消化30 min后，再加入0.75 g/L的Ⅰ型胶原酶消化60 min；0.05 g/L的胰蛋白酶连续2次消化40 min后，再加0.75 g/L胶原酶Ⅰ消化60 min。④实验四：采用0.05 g/L的胰蛋白酶连续2次消化30 min后，再加1 g/L的Ⅰ型胶原酶消化60 min。显微镜下观察其形态，研究人羊膜间充质干细胞在体外分离培养的最适方法。 　　结果与结论：用0.05 g/L的胰蛋白酶连续消化2次每次消化30 min，然后用1 g/L的胶原酶消化60 min是最合适的体外分离培养条件。细胞成细长梭形或星形，胞质丰富，细胞核呈圆形，1-3个核仁。说明实验四采用的胰酶和胶原酶消化的时间合适，而且胶原酶的浓度合适，得到的细胞数量最多。
배경：문헌보도인양막간충질간세포제취방법각불일치，득도적세포수량각불상동。목적：탐색인양막간충질간세포재체외분리배양적최괄방법。 　　방법：무균조건하취정상족월부복산태인적양막전성쇄편，분별통과4개실험7충방법체외배양인양막간충질간세포。①실험일：분별채용3충방법：0.05 g/L이단백매소화10 min후，재가0.75 g/L효원매Ⅰ소화60 min；0.75 g/L효원매Ⅰ직접소화120 min；0.05 g/L이단백매여0.75 g/L효원매Ⅰ동시소화60 min。②실험이：채용0.05 g/L적이단백매소화30 min후，재가0.75 g/L효원매Ⅰ소화30 min。③실험삼：분별채용2충방법：0.05 g/L적이단백매련속2차소화30 min후，재가입0.75 g/L적Ⅰ형효원매소화60 min；0.05 g/L적이단백매련속2차소화40 min후，재가0.75 g/L효원매Ⅰ소화60 min。④실험사：채용0.05 g/L적이단백매련속2차소화30 min후，재가1 g/L적Ⅰ형효원매소화60 min。현미경하관찰기형태，연구인양막간충질간세포재체외분리배양적최괄방법。 　　결과여결론：용0.05 g/L적이단백매련속소화2차매차소화30 min，연후용1 g/L적효원매소화60 min시최합괄적체외분리배양조건。세포성세장사형혹성형，포질봉부，세포핵정원형，1-3개핵인。설명실험사채용적이매화효원매소화적시간합괄，이차효원매적농도합괄，득도적세포수량최다。
BACKGROUND:Extraction methods of human amniotic mesenchymal stem cells are inconsistent in the number of cells. OBJECTIVE:To explore the optimal method to in vitro isolate and culture human amniotic mesenchymal stem cells. METHODS:Under sterile conditions, ful-term cesarean fetal amniotic membrane was cut into pieces, then to isolate human amniotic mesenchymal stem cells by seven methods in four experiments. In experiment 1, human amniotic mesenchymal stem cells were isolated by the fol owing three methods:(1) 0.05 g/L trypsin digestion for 10 minutes fol owed by 0.75 g/L col agenase digestion for 60 minutes;(2) 0.75 g/L col agenase I for 120 minutes;(3) co-digestion with 0.05 g/L trypsin and 0.75 g/L col agenase for 60 minutes. In experiment 2, the samples were digested with 0.05 g/L trypsin digestion for 30 minutes fol owed by 0.75 g/L col agenase digestion for 30 minutes. In experiment 3, the samples were digested by two methods:(1) 0.05 g/L trypsin digestion for 30 minutes×2, fol owed by 0.75 g/L col agenase digestion for 60 minutes;(2) 0.05 g/L trypsin digestion for 40 minutes×2, fol owed by 0.75 g/L col agenase digestion for 60 minutes. In experiment 4, the samples were digested with 0.05 g/L trypsin digestion for 30 minutes×2, fol owed by 1 g/L col agenase digestion for 60 minutes. Fol owing morphology observation under a microscope, we studied the most suitable method for isolating human amniotic mesenchymal stem cells. RESULTS AND CONCLUSION:Digestion with 0.05 g/L trypsin for 30 minutes twice fol owed by 1 g/L of col agenase digestion of 60 minutes was the most suitable isolation and culture condition in vitro. cells became elongated fusiform or star-shaped with rich cytoplasm, and nuclei were round with 1-3 nuts. We can harvest the most number of human amniotic mesenchymal stem cells using the method described in experiment 4.