CAJ | 학술논문

目的观察人血小板裂解液(HPL)对脐带间充质干细胞(UC-MSCs)的形态、增殖、表面标志、诱导分化能力的影响.方法取正常足月妊娠产妇脐带血,离心后收集血沉棕黄层及血清按4∶1混合后-20℃冻存,反复冻融2次后制成HPL,按10％比例加入培养基.分离获得UC-MSCs,在含10％ HPL或10％胎牛血清(FBS)的培养基中培养,比较培养30 d所获得的累积倍增级、最大传代代数所获得的细胞数及第3代细胞6孔板中培养6d所获得的细胞量;通过流式细胞仪检测HPL对培养细胞的表面标志的影响;观察HPL对培养细胞的成骨、成脂及成软骨诱导分化能力的影响.结果 HPL或FBS培养的UC-MSCs形态相同,均为长梭形,传至第3代形态无明显改变.在HPL或FBS中培养30 d所获得的累积倍增级比较差异无统计学意义(P＞0.05);HPL培养的细胞在第10代的细胞量[(7.5±0.4)×106个]明显多于10％ FBS组[(4.3±0.2)×106个];第3代UC-MSCs在6孔板培养6d后,HPL组所得细胞数明显多于FBS组(P＜0.05).流式结果显示,UC-MSCs表面高表达CD44、CD73、CD90、CD105,不表达或低表达CD11b、CD19、CD31、CD34、CD45及人类白细胞抗原DR基因(HLA-DR).与10％ FBS培养的UC-MSCs比较,10％ HPL培养并未影响其表面标记的表达.HPL或FBS培养的UC-MSCs均能进行三系诱导分化,其分化能力无明显差异.结论 HPL可以代替FBS来进行培养和扩增UC-MSCs.在保持其形态、表面标志、分化能力不变的情况下,UC-MSCs在HPL环境下增殖更快.
목적 관찰인혈소판렬해액(HPL)대제대간충질간세포(UC-MSCs)적형태、증식、표면표지、유도분화능력적영향.방법 취정상족월임신산부제대혈,리심후수집혈침종황층급혈청안4∶1혼합후-20℃동존,반복동융2차후제성HPL,안10％비례가입배양기.분리획득UC-MSCs,재함10％ HPL혹10％태우혈청(FBS)적배양기중배양,비교배양30 d소획득적루적배증급、최대전대대수소획득적세포수급제3대세포6공판중배양6d소획득적세포량;통과류식세포의검측HPL대배양세포적표면표지적영향;관찰HPL대배양세포적성골、성지급성연골유도분화능력적영향.결과 HPL혹FBS배양적UC-MSCs형태상동,균위장사형,전지제3대형태무명현개변.재HPL혹FBS중배양30 d소획득적루적배증급비교차이무통계학의의(P＞0.05);HPL배양적세포재제10대적세포량[(7.5±0.4)×106개]명현다우10％ FBS조[(4.3±0.2)×106개];제3대UC-MSCs재6공판배양6d후,HPL조소득세포수명현다우FBS조(P＜0.05).류식결과현시,UC-MSCs표면고표체CD44、CD73、CD90、CD105,불표체혹저표체CD11b、CD19、CD31、CD34、CD45급인류백세포항원DR기인(HLA-DR).여10％ FBS배양적UC-MSCs비교,10％ HPL배양병미영향기표면표기적표체.HPL혹FBS배양적UC-MSCs균능진행삼계유도분화,기분화능력무명현차이.결론 HPL가이대체FBS래진행배양화확증UC-MSCs.재보지기형태、표면표지、분화능력불변적정황하,UC-MSCs재HPL배경하증식경쾌.
Objective To investigate the impact of human platelet lysates (HPL) on the morphology,proliferation,cell surface markers,and differentiation capacity of umbilical cord-derived mesenchymal stem cells (UC-MSCs).Methods Cord blood was collected from umbilical cord after the delivery of placenta.Four buffy coat units and one serum unit were pooled and frozen at-20 ℃.After two freeze-thaw cycles,at least 10 units of freeze-thaw lysed human platelets were further pooled to yield HPL.UC-MSCs were isolated and cultured in medium containing either 10％ HPL or fetal bovine serum (FBS).To compare the effect of HPL on MSCs expansion,the 30 d cumulative population doublings were determined,UC-MSCs were maximally expanded,and the number of cells cultured in six-well plate for 6 d was counted.Cell surface antigen phenotyping was performed with UC-MSCs cultured in either 10％ HPL or 10％ FBS at passage 3.The effect of HPL on the differentiation capacity of UC-MSCs was also examined.Resuits HPL has no effect on the morphology of UC-MSCs ; as shown by spindle-like cells at passage 3.The 30 d cumulative population doublings were statistical comparable between HPL and FBS.However,the number of cells maximally expanded at passage ten in HPL [(7.5 ±0.4) × 106] cells was much greater than in FBS [(4.3 ±0.2) × 106] cells.After culturing for 6 d,cell number/well in the HPL group was greater than the FBS group (P ＜ 0.05).UC-MSCs were strongly positive against CD44,CD73,CD90,and CD105; whereas they were negative for CD11b,CD19,CD 31,CD34,CD45,or Human leukocyte antigen-DR (HLA-DR).No significant differences between HPL and FBS were detected.UC-MSCs derived from both HPL and FBS demonstrated differentiation toward the osteogenic,adipogenic,and chondrogenic lineages.Conclusion HPL may be considered as an alternative to FBS for the culture and expansion of UC-MSCs in vitro.HPL favors very rapid expansion while maintaining the morphology,cell surface markers,and differentiation capacity.