CAJ | 학술논문

目的探讨与骨髓间充质干细胞(BMSCs)共培养的肝细胞可耐受慢加急性肝功能衰竭患者血清的细胞毒性,以及共培养细胞对肝功能衰竭血清的肝功能支持作用.方法收集慢性乙型肝炎慢加急性肝功能衰竭患者血清(n=18)及正常志愿者血清(n=18).建立猪肝细胞与BMSCs最适共培养体系.实验分为4组:肝功能衰竭血清单纯肝细胞培养组(Hep-F)、肝功能衰竭血清共培养组(Co-F)、正常人血清单纯肝细胞培养组(Hep-N)和正常人血清共培养组(Co-N).血清浓度依次为10％、20％、40％、60％、80％和100％.培养24 h后收集上清液检测白蛋白水平和肝细胞周期确定最合适的肝功能衰竭血清浓度.观察细胞生长状态、超微结构及细胞活力.分别检测60％浓度血清培养组超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)、谷氨酰胺(GLN)和胆碱酯酶(CHE)水平.更换为含10％胎牛血清的完全培养基继续培养24 h,收集Hep-FB、Co-FB、Hep-NB和Co-NB组上清液,重复检测SOD、LDH、GLN和CHE水平.结果共培养组肝细胞在60％浓度的肝功能衰竭血清中贴壁数量最多,呈岛状聚集生长.肝细胞表达白蛋白水平最佳,G2～S期细胞比例升至32.08％,细胞内超微结构接近正常,细胞活力较对照组明显改善.Co-F组SOD、GLN和LDH水平与Co-N组之间差异无统计学意义(P＞0.05),仅CHE水平有所下降,但与Hep-F组比较显著提高.Co-FB组LDH、GLN、CHE活性较Hep-F组有显著改善.结论与BMSCs共培养的肝细胞可耐受中高浓度慢加急性肝功能衰竭血清的细胞毒性,有效维持肝细胞功能.
목적 탐토여골수간충질간세포(BMSCs)공배양적간세포가내수만가급성간공능쇠갈환자혈청적세포독성,이급공배양세포대간공능쇠갈혈청적간공능지지작용.방법 수집만성을형간염만가급성간공능쇠갈환자혈청(n=18)급정상지원자혈청(n=18).건립저간세포여BMSCs최괄공배양체계.실험분위4조:간공능쇠갈혈청단순간세포배양조(Hep-F)、간공능쇠갈혈청공배양조(Co-F)、정상인혈청단순간세포배양조(Hep-N)화정상인혈청공배양조(Co-N).혈청농도의차위10％、20％、40％、60％、80％화100％.배양24 h후수집상청액검측백단백수평화간세포주기학정최합괄적간공능쇠갈혈청농도.관찰세포생장상태、초미결구급세포활력.분별검측60％농도혈청배양조초양화물기화매(SOD)、유산탈경매(LDH)、곡안선알(GLN)화담감지매(CHE)수평.경환위함10％태우혈청적완전배양기계속배양24 h,수집Hep-FB、Co-FB、Hep-NB화Co-NB조상청액,중복검측SOD、LDH、GLN화CHE수평.결과 공배양조간세포재60％농도적간공능쇠갈혈청중첩벽수량최다,정도상취집생장.간세포표체백단백수평최가,G2～S기세포비례승지32.08％,세포내초미결구접근정상,세포활력교대조조명현개선.Co-F조SOD、GLN화LDH수평여Co-N조지간차이무통계학의의(P＞0.05),부CHE수평유소하강,단여Hep-F조비교현저제고.Co-FB조LDH、GLN、CHE활성교Hep-F조유현저개선.결론 여BMSCs공배양적간세포가내수중고농도만가급성간공능쇠갈혈청적세포독성,유효유지간세포공능.
Objective To clarify the tolerance of hepatocytes co-cultured with bone marrow mesenchymal stem cells (BMSCs) to the cytotoxicity of sera from the patients with liver failure induced,and the supporting effects on liver failure sera by co-culture.Methods Serum samples from 18 patients with acute-on-chronic liver failure due to hepatitis B were collected with informed consent.Normal serum was obtained from 18 normal volunteers.The optimal co-cultivation of BMSCs and primary hepatocytes was generated.Four groups were creased as follows:hepatocyte alone with liver failure serum (Hep-F),co-culture with liver failure serum (Co-F),hepatocyte alone with normal serum (Hrp-N),and co-culture with normal serum (Co-N).The additional serum concentration was set as 10％,20％,40％,60％,80％ and 100％,respectively.The albumin expression and cell cycle of hepatocytes after 24 h culture were examined to determine the optimal liver failure serum concentration.The morphology and viability were also monitored.The indices of hepatic functions in every group were tested.Subsequently,the cells were inoculated in complete medium containing 10％ fetal bovine serum for another 24 h,and hepatocyte specific functions of Hep-FB,Co-FB,Hep-NB,and Co-NB were examined,respectively.Results A well attachment and self-organization of islet-like hepatocyte aggregates were encouraged in Co-F group with 60％ liver failure serum.It also achieved the best albumin secretion and largest population of hepatocytes in G2-S phase.The cell ultrastructure remained similar to that of hepatocytes in vivo.Meanwhile,hepatocyte viability assay showed a metabolically active,viable cell population in Co-F group instead of quite a few dead cells in Hep-F group.The activities of superoxide dismutase (SOD),lactate dehydrogenase (LDH),glutamine (GLN) and CHE in Co-F group were all significantly increased as compared with those in Hep-F group (P ＜ 0.05).The similar trends were observed between Co-FB group and Hep-FB group except SOD.Conclusion The co-cultured hepatocytes with bone marrow MSCSs could counteract the cytotoxicity of midhigh concentration serum with acute-on-chronic liver failure,which would manifest a well hepatic supporting function.