CAJ | 학술논문

目的：探索apelin-13对干细胞定向血管网分化的调控作用。方法采用组织块贴壁法从人脐带华通胶组织中分离间充质干细胞，流式细胞仪检测其免疫表型。取第3代人华通胶间充质干细胞，分别接种于4个培养瓶中，记为A1、A2、A3、A4组，用诱导液诱导7 d，每天向4组中分别加入浓度0、1×10－6、10×10－6、100×10－6 mol/L的apelin-1320μL，倒置显微镜下观察细胞形态及生长情况，并对其进行血管性血友病因子（vWF）抗体免疫荧光染色及CD31流式细胞学鉴定。使用水凝胶的三维培养基对诱导后的内皮细胞进行培养，并按照原A1、A2、A3、A4组的顺序重新依次编号为S1、S2、S3、S4组。每天分别向S1、S2、S3、S4组中加入浓度0、1×10－6、10×10－6、100×10－6 mol/L的apelin-1320μL。7 d后倒置显微镜下观察细胞的形态、生长情况及在三维培养基中形成血管样结构情况。结果人华通胶间充质干细胞可以诱导分化为vWF免疫荧光染色阳性的内皮样细胞。随着apelin-13蛋白浓度的增加，CD31阳性表达率也随之升高。显微镜下观察到内皮样细胞在水凝胶的三维培养基中能够形成血管样结构。结论证实apelin-13参与并调控人脐带华通胶间充质干细胞分化形成血管网。
목적：탐색apelin-13대간세포정향혈관망분화적조공작용。방법채용조직괴첩벽법종인제대화통효조직중분리간충질간세포，류식세포의검측기면역표형。취제3대인화통효간충질간세포，분별접충우4개배양병중，기위A1、A2、A3、A4조，용유도액유도7 d，매천향4조중분별가입농도0、1×10－6、10×10－6、100×10－6 mol/L적apelin-1320μL，도치현미경하관찰세포형태급생장정황，병대기진행혈관성혈우병인자（vWF）항체면역형광염색급CD31류식세포학감정。사용수응효적삼유배양기대유도후적내피세포진행배양，병안조원A1、A2、A3、A4조적순서중신의차편호위S1、S2、S3、S4조。매천분별향S1、S2、S3、S4조중가입농도0、1×10－6、10×10－6、100×10－6 mol/L적apelin-1320μL。7 d후도치현미경하관찰세포적형태、생장정황급재삼유배양기중형성혈관양결구정황。결과인화통효간충질간세포가이유도분화위vWF면역형광염색양성적내피양세포。수착apelin-13단백농도적증가，CD31양성표체솔야수지승고。현미경하관찰도내피양세포재수응효적삼유배양기중능구형성혈관양결구。결론증실apelin-13삼여병조공인제대화통효간충질간세포분화형성혈관망。
Objective To explore the role of apelin-13 in regulating stem cell differentiation into vascular net. Meth?ods Mesenchymal stem cells were isolated from human umbilical Wharton’s jelly using tissue adherence method.Their immunophenotypes were detected by flow cytometry . Passage 3 of WJ-MSCs (Wharton’s jelly-mesenchymal stem cells) were inoculated in 4 flasks, denoted as A1, A2, A3, A4 group. TwentyμL of apelin-13 at concentrations of 0, 1×10-6, 10 × 10-6 and 100 ×10-6 mol/L were added to A1, A2, A3 and A4 respectively each day. After being induced for 7 days, cell mor?phology and viability were observed under inverted microscope. Von Willebrand factor (vWF) was examined by immunofluo?rescence and CD31 was identified by flow cytometry. Upon incubating with three dimensional culture medium of hydrogel, those cultured A1, A2, A3 and A4 were renumbered as S1, S2, S3, S4. Again, twentyμL of apelin-13 at concentrations of 0, 1×10-6, 10 ×10-6and 100 ×10-6 mol/L were used to treat S1, S2, S3 and S4 respectively. After 7 days, cell morphology, via?bility and vas-like networks were observed with inverted microscope. Results Our study showed that WJ-MSCs can be in?duced by apelin 13 to differentiate into endothelial cells lineage indicated by positive of vWF staining. Moreover, CD31 expres?sion increases significantly upon apelin-13 addition in a dosage dependent manner. The endothelial cells line formed vas like networks when cultured with three-dimensional medium containing hydrogel. Conclusion This study demonstrated that ape?lin-13 could promote human umbilical cord-MSCs to differentiate into endothelium lineage then to form vascular networks.