CAJ | 학술논문

目的细胞替代治疗是髓鞘化障碍疾病的可能有效的治疗方法.而细胞替代治疗必须具有相应的体外培养少突胶质前体细胞的方法.因此,本研究为了提供临床治疗所需的细胞,发展出了一种简单、经济且高效的获得人源少突胶质前体细胞(OPCs)的方法,为临床治疗提供了新的方法.方法通过磁珠分选的方法得到人源OPCs,将其接种到OPCs增殖培养基中,观察短期(2、4、6、8d)和长期体外培养中OPCs的形态,免疫荧光染色鉴定第4代细胞OPCs特异标志O4、转录因子Sox10和血小板源性生长因子α受体(PDGFαR)的表达情况及诱导后分化为少突细胞的能力,同时观察B27和N1对OPCs生长状态的影响.结果短期培养时,OPCs具有典型的双极或是三极形态且增殖状况良好.经过4代的长期培养,4代细胞均具有典型双极或三极形态;第4代OPCs免疫荧光染色鉴定90％以上细胞表达OPCs特异标志O4、Sox10和PDGFαR,诱导后,可以分化为少突胶质细胞.可见经过4代的长期培养,OPCs仍保持着初始性状且具有分化为少突细胞的能力并且仍然保持着较高纯度,表明该培养基适合人源OPCs长期培养.结论使用该培养方法,分离得到的人源OPCs在体外不仅能够稳定培养和扩增,还具有分化为少突细胞的能力.通过这种可重复的方法,可以在体外尽可能简单的稳定获得大量高纯度人源OPCs,为临床应用提供了新的选择.且该方法使用较少的细胞因子,因此为髓鞘化障碍或髓鞘损伤疾病将来的细胞治疗,提供了一种适用于临床的OPCs的稳定高效的较经济的培养方法.
목적 세포체대치료시수초화장애질병적가능유효적치료방법.이세포체대치료필수구유상응적체외배양소돌효질전체세포적방법.인차,본연구위료제공림상치료소수적세포,발전출료일충간단、경제차고효적획득인원소돌효질전체세포(OPCs)적방법,위림상치료제공료신적방법.방법 통과자주분선적방법득도인원OPCs,장기접충도OPCs증식배양기중,관찰단기(2、4、6、8d)화장기체외배양중OPCs적형태,면역형광염색감정제4대세포OPCs특이표지O4、전록인자Sox10화혈소판원성생장인자α수체(PDGFαR)적표체정황급유도후분화위소돌세포적능력,동시관찰B27화N1대OPCs생장상태적영향.결과 단기배양시,OPCs구유전형적쌍겁혹시삼겁형태차증식상황량호.경과4대적장기배양,4대세포균구유전형쌍겁혹삼겁형태;제4대OPCs면역형광염색감정90％이상세포표체OPCs특이표지O4、Sox10화PDGFαR,유도후,가이분화위소돌효질세포.가견경과4대적장기배양,OPCs잉보지착초시성상차구유분화위소돌세포적능력병차잉연보지착교고순도,표명해배양기괄합인원OPCs장기배양.결론 사용해배양방법,분리득도적인원OPCs재체외불부능구은정배양화확증,환구유분화위소돌세포적능력.통과저충가중복적방법,가이재체외진가능간단적은정획득대량고순도인원OPCs,위림상응용제공료신적선택.차해방법사용교소적세포인자,인차위수초화장애혹수초손상질병장래적세포치료,제공료일충괄용우림상적OPCs적은정고효적교경제적배양방법.
Objective Cell therapy is a possible effective way to treat myelination disorder diseases.Cell therapy needs to apply a method for culturing oligodendrocyte precursor cells.Therefore,this study was to develop a stable,efficient and economical method for obtaining human oligodendrocyte precursor cells (OPCs) in order to provide cell required for clinical treatment.And this will provide a new option for clinical applications.Methods Human OPCs were obtained through magnetic bead sorting and cultured in OPCs proliferation medium.For a short-time (2,4,6,8days) and long-time culture,morphology of OPCs was observed.The fourth generation of OPCs was analyzed for expression of OPCs specific markers O4,Soxl0 and platelet derived growth factor alpla receptors(PDGFαR) and the capacity to differentiate into oligodendrocytes by immunofluorescence staining.At the same time,the effects of B27 and N1 on OPCs growth state were inspected as well.Results For a short-time culture,OPCs had typical bipolar or tripolar morphology and proliferated in good condition.For a long-time culture,all 4 generations OPCs had typical bipolar or tripolar morphology;the fourth generation OPCs highly(＞ 90％) expressed 04,Sox10 and PDGFαR,after induction,OPCs could be differentiated into oligodendrocytes.After 4 generations of long-time culture,OPCs already maintained the original sharp,high purity and had the capacity to differentiate into oligodendrocytes.It was indicated that this culture system was suitable for human OPCs for a long-time culture.Conclusions Overall,using this culture system,isolated human OPCs not only can be stably cultured and proliferated in vitro,but also have the capacity to differentiate into oligodendrocytes.From this reproducible method,a large number of human OPCs can be stably obtained in vitro as convenient as possible.And this will provide a new option for clinical applications.This method uses fewer cytokines.Therefore,this method will provide stable,efficient and economical OPCs for cell therapy of myelination disorders or myelin damage diseases.