CAJ | 학술논문

背景：研究已证实肝细胞生长因子基因转染可提高骨髓间充质干细胞移植效果，但其具体机制尚未完全明确。目的：探讨肝细胞生长因子基因转染对缺氧、无血清培养骨髓间充质干细胞c-Met、Bax、Bcl-2、Caspase-3表达及细胞迁移的影响。<br>　　方法：①贴壁法体外分离、扩增培养骨髓间充质干细胞。用x-gal染色法检测含肝细胞生长因子基因的重组腺病毒对骨髓间充质干细胞的感染率。②在缺氧、无血清条件下培养0，3，6，9，12 h，采用RT-PCR及Western blot测定骨髓间充质干细胞Bax、Bcl-2、Caspase-3的表达。③骨髓间充质干细胞缺氧、无血清培养6 h，采用RT-PCR及Western blot测定HGF、c-Met、Bax、Bcl-2、Caspase-3的表达。④细胞迁移划痕法观察缺氧、无血清培养6h后肝细胞生长因子转染对骨髓间充质干细胞迁移的影响。<br>　　结果与结论：①重组腺病毒对骨髓间充质干细胞的转染率与病毒感染复数具有量效关系，病毒感染复数为150时细胞的感染率达96.4%。②随着缺氧时间的延长骨髓间充质干细胞Bax、Bcl-2表达逐渐升高(P <0.05)，缺氧6 h时Bax/Bcl-2比值、Caspase-3蛋白达到最小(P<0.05)。③缺氧及无血清培养6 h后，与对照组及空载体组相比，Ad-HGF组HGF、c-Met、Bcl-2表达增高，而Bax、Caspase-3表达下降(P<0.05)，对照组与空载体组差异无显著性意义(P>0.05)。④Ad-HGF组骨髓间充质干细胞在缺氧培养6 h后，迁移率比对照组及空载体组高(P <0.05)。结果表明肝细胞生长因子基因转染上调缺氧、无血清培养骨髓间充质干细胞c-Met、Bcl-2表达，下调Bax、Caspase-3表达，增强骨髓间充质干细胞迁移能力。
배경：연구이증실간세포생장인자기인전염가제고골수간충질간세포이식효과，단기구체궤제상미완전명학。목적：탐토간세포생장인자기인전염대결양、무혈청배양골수간충질간세포c-Met、Bax、Bcl-2、Caspase-3표체급세포천이적영향。<br>　　방법：①첩벽법체외분리、확증배양골수간충질간세포。용x-gal염색법검측함간세포생장인자기인적중조선병독대골수간충질간세포적감염솔。②재결양、무혈청조건하배양0，3，6，9，12 h，채용RT-PCR급Western blot측정골수간충질간세포Bax、Bcl-2、Caspase-3적표체。③골수간충질간세포결양、무혈청배양6 h，채용RT-PCR급Western blot측정HGF、c-Met、Bax、Bcl-2、Caspase-3적표체。④세포천이화흔법관찰결양、무혈청배양6h후간세포생장인자전염대골수간충질간세포천이적영향。<br>　　결과여결론：①중조선병독대골수간충질간세포적전염솔여병독감염복수구유량효관계，병독감염복수위150시세포적감염솔체96.4%。②수착결양시간적연장골수간충질간세포Bax、Bcl-2표체축점승고(P <0.05)，결양6 h시Bax/Bcl-2비치、Caspase-3단백체도최소(P<0.05)。③결양급무혈청배양6 h후，여대조조급공재체조상비，Ad-HGF조HGF、c-Met、Bcl-2표체증고，이Bax、Caspase-3표체하강(P<0.05)，대조조여공재체조차이무현저성의의(P>0.05)。④Ad-HGF조골수간충질간세포재결양배양6 h후，천이솔비대조조급공재체조고(P <0.05)。결과표명간세포생장인자기인전염상조결양、무혈청배양골수간충질간세포c-Met、Bcl-2표체，하조Bax、Caspase-3표체，증강골수간충질간세포천이능력。
BACKGROUND:Previous studies have demonstrated that hepatocyte growth factor (HGF) gene transfection can improve the effectiveness of bone marrow mesenchymal stem cel transplantation, but the mechanism is stil unclear. OBJECTIVE:To observe the effects of HGF gene transfection on c-MET, Bax, Bcl-2, Caspase-3 of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions. METHODS:(1) Bone marrow mesenchymal stem cel s were isolated and amplified in vitro by differential adhesion method. The infection efficiency of recombinant adenovirus Ad-HGF in bone marrow mesechymal stem cel s was tested by x-gal staining. (2) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 0, 3, 6, 9, 12 hours. RT-PCR and western blot assays were used to evaluate the expression of Bax, Bcl-2, Caspase-3. (3) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 6 hours, and RT-PCR and western blot assays were adopted to detect HGF, c-Met, Bax, Bcl-2 and Caspase-3. (4) Cel scratch test was used to detect the effect of HGF transfection on the migration of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions for 6 hours. RESULTS AND CONCLUSION:(1) Transfection efficiency of bone marrow mesenchymal stem cel s was increased with multiplicity of infection in a dose-dependent manner. When the multiplicity of infection was 150, the transfection efficiency was 96.4%. (2) Expressions of Bax and Bcl-2 were gradual y increased with hypoxia time (P<0.05). The Bax/Bcl-2 ratio and Caspase-3 expression reach the minimum at 6 hours of hypoxia (P<0.05). (3) Compared with the control and Ad-LacZ groups, the expressions of HGF, c-Met, Bcl-2 increased, and the expressions of Bax and Caspase-3 decreased in the Ad-HGF group after 6 hours of culture under hypoxia and serum-free conditions (P<0.05). There was no significant difference between the control and Ad-LacZ groups. (4) The mobility of bone marrow mesenchymal stem cel s was higher in the Ad-HGF group than the control group and Ad-LacZ groups after 6 hours of culture under hypoxia (P<0.05). These findings indicate that transfection of HGF in bone marrow mesenchymal stem cel s can increase the expression of c-Met, Bcl-2 and decrease the expression of Bax, Caspase-3 under hypoxia and serum-free conditions, which also enhance the mobility of bone marrow mesenchymal stem cel s under hypoxia and serum-free conditions.