CAJ | 학술논문

背景：以往采用胰酶冷消化法培养脐带间充质干细胞的研究较少。目的：比较两种方法体外培养人脐带间充质干细胞的生物学特性。<br>　　方法：采用胰酶冷消化法和组织块法从人脐带中分离、纯化和传代培养人脐带间充质干细胞，记录首次出现贴壁细胞时间及原代培养周期，倒置相差显微镜观察脐带间充质干细胞的形态及生长情况，制作第3代脐带间充质干细胞生长曲线，流式细胞仪分析检测第3代脐带间充质干细胞表面标志的表达，第3代脐带间充质干细胞加入成神经诱导培养基诱导分化第3天行荧光免疫化学染色检测Nestin的表达。<br>　　结果与结论：使用上述两种方法均可获得脐带间充质干细胞，胰酶冷消化法首次出现贴壁细胞时间早于组织块法(P <0.05)；原代培养周期差异无显著性意义(P >0.05)。倒置相差显微镜下细胞均为贴壁生长，形态为成纤维细胞样，但胰酶冷消化法培养的少数原代细胞呈多角形，不规则，细胞的体积较组织块法大。组织块法获得第3代细胞的增殖速率显著快于胰酶冷消化法，在进入对数生长期后各个时间点细胞数量差异有显著性意义(P<0.05)。两种细胞具有均一的细胞表型，均表达CD29，CD105，不表达CD34，CD45。两种方法培养出来的第3代脐带间充质干细胞经诱导后，免疫荧光均显示神经干细胞特征性标志物nestin阳性表达。结果表明胰酶冷消化法与组织块法均能培养出脐带间充质干细胞，但组织块法更适合于此类细胞的培养，对细胞的伤害更低。
배경：이왕채용이매랭소화법배양제대간충질간세포적연구교소。목적：비교량충방법체외배양인제대간충질간세포적생물학특성。<br>　　방법：채용이매랭소화법화조직괴법종인제대중분리、순화화전대배양인제대간충질간세포，기록수차출현첩벽세포시간급원대배양주기，도치상차현미경관찰제대간충질간세포적형태급생장정황，제작제3대제대간충질간세포생장곡선，류식세포의분석검측제3대제대간충질간세포표면표지적표체，제3대제대간충질간세포가입성신경유도배양기유도분화제3천행형광면역화학염색검측Nestin적표체。<br>　　결과여결론：사용상술량충방법균가획득제대간충질간세포，이매랭소화법수차출현첩벽세포시간조우조직괴법(P <0.05)；원대배양주기차이무현저성의의(P >0.05)。도치상차현미경하세포균위첩벽생장，형태위성섬유세포양，단이매랭소화법배양적소수원대세포정다각형，불규칙，세포적체적교조직괴법대。조직괴법획득제3대세포적증식속솔현저쾌우이매랭소화법，재진입대수생장기후각개시간점세포수량차이유현저성의의(P<0.05)。량충세포구유균일적세포표형，균표체CD29，CD105，불표체CD34，CD45。량충방법배양출래적제3대제대간충질간세포경유도후，면역형광균현시신경간세포특정성표지물nestin양성표체。결과표명이매랭소화법여조직괴법균능배양출제대간충질간세포，단조직괴법경괄합우차류세포적배양，대세포적상해경저。
BACKGROUND:Cold trypsin digestion is rarely reported to culture umbilical cord mesenchymal stem cells. OBJECTIVE:To compare the biological characteristics of umbilical cord mesenchymal stem cells cultured by cold trypsin digestion and tissue explant method. METHODS:Human umbilical cord mesenchymal stem cells were isolated, purified and passaged using cold trypsin digestion and tissue explant method, and then the first adhesion time and cellcycle were recorded. Morphology of umbilical cord mesenchymal stem cells was observed under inverted phase contrast microscope to draw growth curve of cells at passage 3. Flow cytometry was used to detect the surface markers of passage 3 umbilical cord mesenchymal stem cells, and Nestin expression was detected in passage 3 cells after 3 days culture in neural induction medium by fluorescence immunochemistry staining. RESULTS AND CONCLUSION:These two methods were both successful to harvest umbilical cord mesenchymal stem cells, but the first adhesion time was earlier in cells cultured by cold trypsin digestion than tissue explant method (P<0.05), and there was no difference in primary cellcycle (P>0.05). Under the inverted microscope, cells grew adherently and presented fibroblast-like shape. However, the minority of primary cells under induction of cold trypsin digestion was polygonal, irregular, and had larger cellvolume than those cultured by tissue explant method. Passage 3 cells cultured by tissue explant method showed faster proliferation rate than those cultured by cold trypsin digestion, and at logarithmic growth phase, cells cultured by these two methods were significant different in cellnumber (P<0.05). Two types of cells had a uniform cellphenotype, both of which expressed CD29, CD105, but did not express CD34, CD45. Under induction, passage 3 cells cultured by these two methods were both positive for nestin. These findings indicate that these two methods can both be used to culture umbilical cord mesenchymal stem cells, but the tissue explant method is more suitable for culture of umbilical cord mesenchymal stem cells and exhibits less damage to cells.