中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2012年
9期
698-702
,共5页
孟云超%姜海行%张君红%陆正峰%覃山羽%宁琳%杨文
孟雲超%薑海行%張君紅%陸正峰%覃山羽%寧琳%楊文
맹운초%강해행%장군홍%륙정봉%담산우%저림%양문
间质干细胞%骨髓%肝星状细胞%细胞凋亡%肝细胞生长因子%肝细胞生长因子激活因子
間質榦細胞%骨髓%肝星狀細胞%細胞凋亡%肝細胞生長因子%肝細胞生長因子激活因子
간질간세포%골수%간성상세포%세포조망%간세포생장인자%간세포생장인자격활인자
Mesenchymal stem cells%Bone marrow%Hepatic stellate cells%Apoptosis%Hepatocyte growth factor%Hepatocyte growth factor activator
目的 探讨大鼠骨髓间充质干细胞(BMSCs)与肝星状细胞(HSCs)共培养体系中肝细胞生长因子激活因子(HGFA)激活肝细胞生长因子(HGF)后对HSCs凋亡的影响.方法 用半透膜建立上下双层细胞共培养体系,各组培养24h、48 h及72h后,倒置相差显微镜观察细胞形态学变化；流式细胞仪检测BMSCs表面抗原及HSCs凋亡率；四甲基偶氮唑盐法检测HSCs增殖率；免疫组织化学法检测HSCs中α-平滑肌肌动蛋白表达；免疫荧光及组织化学染色法检测HGF的激活形式(即HGF-α链)；酶联免疫吸附法检测HGF及HGFA浓度.计量资料采用单因素方差分析,组间均数的比较采用q检验. 结果 不同浓度HGF在各时段对HSCs无增殖抑制作用(P＞0.05),差异无统计学意义;HGFA在各时段对HSCs有明显增殖抑制作用,且呈浓度依赖性,在24h HGFA浓度为70 ng/ml时抑制作用为(0.26±0.00),较对照组的(0.13±0.04),明显增加,差异有统计学意义(P＜0.05);72h实验组HGF-α链相对表达量37.24±1.03低于HGFA干预组的40.44±0.77,差异有统计学意义(P＜ 0.05)；两者在各时段与对照组比较,差异均有统计学意义(P＜ 0.01).实验组HSCs凋亡率在72h为40.77％±1.16％,均较HGFA干预组的33.35％±2.04高,差异有统计学意义(P＜ 0.05)；两者在各时段与对照组比较,差异有统计学意义(P＜0.05).实验组及HGFA干预组中HGF浓度的降低呈时间依赖性,较HSCs空白对照组低；实验组HGFA的浓度在各时段均较对照组高.结论 BMSCs与HSCs共培养后促进HGFA的分泌,并激活HGF使其发挥促HSCs凋亡的作用.
目的 探討大鼠骨髓間充質榦細胞(BMSCs)與肝星狀細胞(HSCs)共培養體繫中肝細胞生長因子激活因子(HGFA)激活肝細胞生長因子(HGF)後對HSCs凋亡的影響.方法 用半透膜建立上下雙層細胞共培養體繫,各組培養24h、48 h及72h後,倒置相差顯微鏡觀察細胞形態學變化；流式細胞儀檢測BMSCs錶麵抗原及HSCs凋亡率；四甲基偶氮唑鹽法檢測HSCs增殖率；免疫組織化學法檢測HSCs中α-平滑肌肌動蛋白錶達；免疫熒光及組織化學染色法檢測HGF的激活形式(即HGF-α鏈)；酶聯免疫吸附法檢測HGF及HGFA濃度.計量資料採用單因素方差分析,組間均數的比較採用q檢驗. 結果 不同濃度HGF在各時段對HSCs無增殖抑製作用(P＞0.05),差異無統計學意義;HGFA在各時段對HSCs有明顯增殖抑製作用,且呈濃度依賴性,在24h HGFA濃度為70 ng/ml時抑製作用為(0.26±0.00),較對照組的(0.13±0.04),明顯增加,差異有統計學意義(P＜0.05);72h實驗組HGF-α鏈相對錶達量37.24±1.03低于HGFA榦預組的40.44±0.77,差異有統計學意義(P＜ 0.05)；兩者在各時段與對照組比較,差異均有統計學意義(P＜ 0.01).實驗組HSCs凋亡率在72h為40.77％±1.16％,均較HGFA榦預組的33.35％±2.04高,差異有統計學意義(P＜ 0.05)；兩者在各時段與對照組比較,差異有統計學意義(P＜0.05).實驗組及HGFA榦預組中HGF濃度的降低呈時間依賴性,較HSCs空白對照組低；實驗組HGFA的濃度在各時段均較對照組高.結論 BMSCs與HSCs共培養後促進HGFA的分泌,併激活HGF使其髮揮促HSCs凋亡的作用.
목적 탐토대서골수간충질간세포(BMSCs)여간성상세포(HSCs)공배양체계중간세포생장인자격활인자(HGFA)격활간세포생장인자(HGF)후대HSCs조망적영향.방법 용반투막건립상하쌍층세포공배양체계,각조배양24h、48 h급72h후,도치상차현미경관찰세포형태학변화；류식세포의검측BMSCs표면항원급HSCs조망솔；사갑기우담서염법검측HSCs증식솔；면역조직화학법검측HSCs중α-평활기기동단백표체；면역형광급조직화학염색법검측HGF적격활형식(즉HGF-α련)；매련면역흡부법검측HGF급HGFA농도.계량자료채용단인소방차분석,조간균수적비교채용q검험. 결과 불동농도HGF재각시단대HSCs무증식억제작용(P＞0.05),차이무통계학의의;HGFA재각시단대HSCs유명현증식억제작용,차정농도의뢰성,재24h HGFA농도위70 ng/ml시억제작용위(0.26±0.00),교대조조적(0.13±0.04),명현증가,차이유통계학의의(P＜0.05);72h실험조HGF-α련상대표체량37.24±1.03저우HGFA간예조적40.44±0.77,차이유통계학의의(P＜ 0.05)；량자재각시단여대조조비교,차이균유통계학의의(P＜ 0.01).실험조HSCs조망솔재72h위40.77％±1.16％,균교HGFA간예조적33.35％±2.04고,차이유통계학의의(P＜ 0.05)；량자재각시단여대조조비교,차이유통계학의의(P＜0.05).실험조급HGFA간예조중HGF농도적강저정시간의뢰성,교HSCs공백대조조저；실험조HGFA적농도재각시단균교대조조고.결론 BMSCs여HSCs공배양후촉진HGFA적분비,병격활HGF사기발휘촉HSCs조망적작용.
Objective To determine whether apoptosis is induced in rat hepatic stellate cells (HSCs) in response to activation of the hepatocyte growth factor (HGF) by hepatocyte growth factor activator (HGFA) by using a co-culture system of bone marrow mesenchymal stem cells (BMSCs) and HSCs.Methods In this study,cells were divided into the following five groups:HSC control group:HSCs co-cultured with fibroblast cells; HSCs blank group:HSCs cultured alone; BMSCs blank group:BMSCs cultured alone; Experimental group:BMSCs + HSCs; HGFA intervention group:HSCs treated with 70 ng/mL of HGFA.The culture systems were established in culture plates with transwell inserts,and cells were assessed at 24,48,and 72 h of growth.Dynamic changes in cell morphology were observed under an inverted phase contrast microscope.The surface markers of BMSCs and the apoptosis rate of HSCs were detected by Annexin-V-FITC/propidium iodide (PI).Expression of α-smooth muscle actin (SMA) in HSCs was evaluated by immunohistochemistry.The presence of activated HGF (HGF-α chain) was determined by immunofluorescent staining.HSC proliferation was measured by MTT assay,and the concentrations of HGF and HGFA were quantified by enzyme-linked immunosorbent assay (ELISA).Results MTT results indicated that treatment with HGF alone had no effect on HSC proliferation rate (vs.HSC blank group,P ＞ 0.05),but that 24 h treatment with HGFA significantly inhibited the proliferation rate (0.26 ± 0.00 vs.blank group:0.13 ± 0.04,P =0.02);moreover,this effect was concentration-dependent.Expression of HGF-α was lower in the experimental group than in the HGFA intervention group at 72 h (37.24 ± 1.03 vs.40.44 ± 0.77,P =0.04),and both of these groups had higher expression than the control group at all time points examined (P ＜ 0.05).The apoptosis rate was consistently higher in the experimental group than in the HGFA intervention group,but most robustly at 72 h (40.77 ± 1.16％ vs.33.35 ± 2.04％,P =0.00); moreover,the apoptosis rate was significantly higher than that in the control group at all time points examined (P ＜ 0.01).The concentration of HGF in the experimental group and the HGFA intervention group showed a time-dependent reduction,and was consistently lower than that in the HSCs control group (P ＜ 0.05).Finally,the concentration of HGFA was higher in the experimental group than in the blank group at all time points examined (P ＜ 0.05).Conclusion The BMSC-HSC co-culture system can promote secretion of HGFA from HSCs and HGF activation,thereby inducing apoptosis of HSCs.
