CAJ | 학술논문

目的体外培养并转染了产甲酸草酸杆菌(Ox.F)草酸分解基因frc和oxc的成人骨髓间充质干细胞(hMSCs-frc-oxc),并诱导其向肝细胞分化.方法体外培养转染了草酸分解基因的P4代hMSCs,并通过含40 μg/L肝细胞生长因子(HGF)± 10 μg/L胰岛素样生长因子(FGF)-4所组成的培养液诱导其向肝细胞分化.在分化过程中通过显微镜观察分化细胞形态变化.以实时定量聚合酶链反应(Real-time PCR)、Western blot技术和免疫细胞化学技术检测细胞内肝细胞特异标志物甲胎蛋白(AFP)、白蛋白(ALB)、细胞角蛋白-18(CK-18)和目的基因frc、oxc及其产物融合蛋白myc-FCOAT、flag-OCOAD的表达.成熟肝细胞L-02和转染了空病毒载体的hMSCs (hMSCs-vector)分别作为阳性对照组和阴性对照组.结果可分解草酸的hMSCs-frc-oxc在体外可成功培养,细胞经上述培养液诱导后,细胞形态逐渐变短,变成多角形.Real-time PCR可检测在诱导组细胞中AFP、ALB、CK-18和草酸分解基因frc、oxc表达,而未经诱导细胞组AFP、ALB、CK-18则未表达,未转染目的基因的细胞组frc、oxc未见表达.免疫细胞化学检测诱导28 d后细胞可表达ALB,未经诱导细胞组ALB表达呈阴性.Western blot技术检测诱导第14、28天细胞中AFP、CK-18表达较未诱导组明显增加(P＜0.05),与成熟肝细胞相似,并表达myc-FCOAT、flag-OCOAD,而未转染目的基因细胞组表达呈阴性.结论含有草酸分解基凶的hMSCs-frc-oxc体外通过一定条件诱导可以向肝细胞方向分化,经诱导分化后细胞仍可成功表达frc和oxc基因及其产物.
목적 체외배양병전염료산갑산초산간균(Ox.F)초산분해기인frc화oxc적성인골수간충질간세포(hMSCs-frc-oxc),병유도기향간세포분화.방법 체외배양전염료초산분해기인적P4대hMSCs,병통과함40 μg/L간세포생장인자(HGF)± 10 μg/L이도소양생장인자(FGF)-4소조성적배양액유도기향간세포분화.재분화과정중통과현미경관찰분화세포형태변화.이실시정량취합매련반응(Real-time PCR)、Western blot기술화면역세포화학기술검측세포내간세포특이표지물갑태단백(AFP)、백단백(ALB)、세포각단백-18(CK-18)화목적기인frc、oxc급기산물융합단백myc-FCOAT、flag-OCOAD적표체.성숙간세포L-02화전염료공병독재체적hMSCs (hMSCs-vector)분별작위양성대조조화음성대조조.결과 가분해초산적hMSCs-frc-oxc재체외가성공배양,세포경상술배양액유도후,세포형태축점변단,변성다각형.Real-time PCR가검측재유도조세포중AFP、ALB、CK-18화초산분해기인frc、oxc표체,이미경유도세포조AFP、ALB、CK-18칙미표체,미전염목적기인적세포조frc、oxc미견표체.면역세포화학검측유도28 d후세포가표체ALB,미경유도세포조ALB표체정음성.Western blot기술검측유도제14、28천세포중AFP、CK-18표체교미유도조명현증가(P＜0.05),여성숙간세포상사,병표체myc-FCOAT、flag-OCOAD,이미전염목적기인세포조표체정음성.결론 함유초산분해기흉적hMSCs-frc-oxc체외통과일정조건유도가이향간세포방향분화,경유도분화후세포잉가성공표체frc화oxc기인급기산물.
Objective To induce the differentiation of human bone mesenchmal stem cells with oxalate-degrading genes (hMSCs-frc-oxc) into hepatocyte-like cells in vitro.Methods The hMSCs-frc-oxc in passage 4 were cultured and induced to differentiate into hepatocyte-like cells in differentiation medium supplemented with 40 μg/L hepatocyte growth factor (HGF) and 10 μg/L fibroblast growth factors-4 (FGF-4).The morphological changes of cells were observed microscopically.The RNA and protein levels of hepatic markers [alpha fetal protein (AFP),cytokeratin-18 (CK-18) and albumin (ALB)] and oxalate-degrading genes (frc and oxc) were detected by using immunocytochemistry,Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR).L-02 cells and transfected mock-vehicle hMSCs (hMSCsvector) were used as positive and negative controls,respectively.Results The hMSCs-frc-oxc were cultured successfully in vitro.After induction,hMSCs-frc-oxc gradually transformed into polygonal cells.Real-time PCR revealed the expression of hepatic markers (AFP,ALB and CK-18) and oxalate-degrading genes (frc and oxc) in hMSCs-frc-oxc after inducting for 14 days and 28 days,whereas hMSCs cultured alone did not show liver specific gene expression.Immunocytochemistry revealed positive ALB expression in cells differentiating for 28 days.Western blotting revealed that the expression levels of AFP and CK-18 in differentiated cells were higher than in undifferentiated cells (P ＜ 0.05).The differentiated hMSCs-frc-oxc also expressed the oxalate-degrading genes products (myc-FCOAT and flag-OCOAD).Conclusion The hMSCs with oxalate-degrading genes could be induced to differentiate into hepatocyte-like cell by HGF and FGF-4 at certain concentrations and the hepatic differentiated hMSCs-frc-oxc could express the oxalate-degrading genes and their products.