CAJ | 학술논문

目的：??采用体外和体内方法研究淫羊藿苷（ICA）对人牙周膜干细胞（hPDLSCs）增殖及骨向分化的影响。方法??采用体外酶消化组织块法培养hPDLSCs，有限稀释克隆法分离纯化，检测细胞表面标志物以进行鉴定。体外实验：hPDLSCs与1×10-7?mol·L-1?ICA共同培养，用四甲基偶氮唑盐法检测其增殖情况，经碱性磷酸酶（ALP）染色后采用酶联免疫仪检测其骨向分化情况，逆转录聚合酶链反应（RT-PCR）检测其成骨基因的表达情况，茜素红染色检测其骨量表达。体内实验：将hPDLSCs和纳米羟磷灰石（nHAC）复合物经1×10-7?mol·L-1?ICA共同诱导培养后，将其植入免疫缺陷小鼠，术后30?d采用组织切片法观察成骨状况。结果??体外实验：1×10-7?mol·L-1?ICA作用于hPDLSCs后，与不含ICA的对照组相比，实验组从培养的第2天开始，hPDLSCs增殖明显；培养3、5、7?d，实验组ALP活性明显升高；培养7?d，成骨基因的相对表达量明显增高；培养14、21、28?d，实验组钙化结节的面积明显大于对照组。体内实验：实验组骨量较对照组明显增加。结论??1×10-7?mol·L-1?ICA可以促进hPDLSCs的增殖及骨向分化，提示采用ICA代替常规生长因子应用于牙周组织工程具有一定可行性。
목적：??채용체외화체내방법연구음양곽감（ICA）대인아주막간세포（hPDLSCs）증식급골향분화적영향。방법??채용체외매소화조직괴법배양hPDLSCs，유한희석극륭법분리순화，검측세포표면표지물이진행감정。체외실험：hPDLSCs여1×10-7?mol·L-1?ICA공동배양，용사갑기우담서염법검측기증식정황，경감성린산매（ALP）염색후채용매련면역의검측기골향분화정황，역전록취합매련반응（RT-PCR）검측기성골기인적표체정황，천소홍염색검측기골량표체。체내실험：장hPDLSCs화납미간린회석（nHAC）복합물경1×10-7?mol·L-1?ICA공동유도배양후，장기식입면역결함소서，술후30?d채용조직절편법관찰성골상황。결과??체외실험：1×10-7?mol·L-1?ICA작용우hPDLSCs후，여불함ICA적대조조상비，실험조종배양적제2천개시，hPDLSCs증식명현；배양3、5、7?d，실험조ALP활성명현승고；배양7?d，성골기인적상대표체량명현증고；배양14、21、28?d，실험조개화결절적면적명현대우대조조。체내실험：실험조골량교대조조명현증가。결론??1×10-7?mol·L-1?ICA가이촉진hPDLSCs적증식급골향분화，제시채용ICA대체상규생장인자응용우아주조직공정구유일정가행성。
Objective??To?evaluate?the?effects?of?Icariin?(ICA)?on?the?proliferation?and?osteogenic?differentiation?of?human?periodontal?ligament?stem?cells?(hPDLSCs)?in vitro and?in vivo. Methods??An?enzymatic?digestion?block?was?used?in vitro?to?culture?hPDLSCs,?which?were?separated?and?purified?by?limited?dilution?cloning.?The?hPDLSCs?were?identified?using?cell-surface?markers?and?cocultured?with?1×10?7 mol·L?1 ICA?solution.?The?proliferation?ability?of?these?cells?was?determined?by?thiazolyl?blue?tetrazolium?bromide?(MTT)?assay.?After?staining?with?alkaline?phosphatase?(ALP),?osteogenesis?was?detected?by?enzyme-linked?immunosorbent?assay.?Osteoblast-related?genes?were?analyzed?by?reverse?transcription-polymerase?chain?reaction.?Alizarin?red?staining?was?performed?to?measure?the?level?of?calcium?deposition.?The?hPDLSCs?were?cocultured?with?1×10?7 mol·L?1 ICA?and?nano-hydroxyapatite?scaffolds?in vivo?before?transplantation?into?subcutaneous?tissues?of?nude?mice.?Osteogenic?abilities?were?histochemically?analyzed?after?30?days?of?induction.?Results??The?hPDLSCs?were?affected?by?1×10?7 mol·L?1 ICA,?and?MTT?assay?showed?that?the?proliferation?of?the?groups?treated?with?ICA?in vitro?was?better?than?that?of?the?control?groups?on?the?second?day.?The?ALP?activity?of?the?treated?hPDLSCs?was?significantly?enhanced?after?cell?culture?for?3,?5,?and?7?days.?The?gene?expression?of?osteoblastic?markers?was?also?significantly?enhanced?after?7?days.?The?deposition?of?mineralization?after?incubation?with?1×10?7 mol·L?1?ICA?increased?compared?with?the?control?after?cell?culture?for?14,?21,?and?28?days.?Furthermore,?the?bone?expression?of?the?treatment?groups?in vivo?was?significantly?enhanced?com-pared?with?that?of?the?control?groups.?Conclusion??Treatment?with?1×10?7 mol·L?1 ICA?can?significantly?promote?proliferation?and?differentiation?of?hPDLSCs?in vitro?and?in vivo.?ICA?can?effectively?function?as?a?bioactive?growth?factor?in?periodontal?tissue?engineering?to?replace?traditional?growth?factors.