摘要目的 探讨整合素连接激酶(ILK)过表达的心肌干细胞(CSC)移植改善心肌梗死大鼠心功能的作用及其机制.方法 从新生Sprague-Dawley(SD)大鼠中分离培养CSC,构建含人ILK全长的腺病毒载体并转染CSC.将40只8周龄的清洁级SD大鼠采用随机数字表法分为假手术组、心肌梗死+生理盐水注射组(生理盐水组)、心肌梗死+转染空载体的CSC注射组(Ad-null-CSC组)和心肌梗死+转染ILK的CSC注射组(ILK-CSC组),每组10只.经冠状动脉结扎建立大鼠心肌梗死模型.建模成功15 min后,各组大鼠均在直视下分3点在心肌梗死区及梗死边缘区分别注射30μl生理盐水、Ad-null-CSC(1×105个细胞/30μl)或ILK-CSC(1×105个细胞/30μl).移植后4周,超声心动图检查各组大鼠心功能;HE染色和Masson染色观察各组大鼠左心室心肌细胞形态和纤维化情况;免疫组织化学法检测各组大鼠心肌细胞中增殖相关基因Ki-67和磷酸化组蛋白H3(p-H3)的表达;实时定量逆转录聚合酶链反应检测另外2个增殖相关基因cyclinD1和增殖细胞核抗原(PCNA)的mRNA表达.结果 (1)各组大鼠存活情况及心功能:移植后4周,40只大鼠中有3只死于术后并发症或心力衰竭,其中生理盐水组2只,Ad-null-CSC组1只,剩余37只用于实验分析.生理盐水组大鼠左心室收缩末期内径(LVESD)和左心室舒张末期内径(LVEDD)均明显高于假手术组(P分别为0.009和0.002),而左心室射血分数(LVEF)则明显低于假手术组(P =0.006).Ad-null-CSC组大鼠LVESD和LVEDD则均低于生理盐水组(P分别为0.005和0.003),但LVEF两组差异则无统计学意义(P=0.113).而ILK-CSC组大鼠LVESD和LVEDD均进一步明显低于Ad-null-CSC组(P分别为0.004和0.000 1),LVEF明显高于Ad-null-CSC组(P =0.004).(2)各组大鼠左心室心肌形态和纤维化:移植后4周,可见生理盐水组和Ad-null-CSC组大鼠心肌细胞大量死亡,细胞核固缩,纤维增生明显,蓝色胶原组织所占面积分别达(70.6±5.1)％和(57.7±3.4)％,均明显高于假手术组(8.2±2.2)％(P分别为0.002和0.001).然而,ILK-CSC组大鼠心肌细胞核固缩较少,细胞数量较多,纤维组织较少,蓝色胶原组织所占面积为(30.6±7.0)％,明显低于Ad-null-CSC组(P =0.005).(3)各组大鼠左心室心肌组织中增殖相关蛋白的表达:移植后4周,可见Ki-67和p-H3主要表达于心肌细胞细胞核中,阳性细胞细胞核内可见棕黄色颗粒.假手术组大鼠心肌细胞内未见Ki-67和p-H3阳性表达,生理盐水组和Ad-null-CSC组大鼠心肌细胞内可见少量表达,而ILK-CSC组二者表达均较高,且均明显高于Ad-null-CSC组(P分别为0.007和0.003).(4)各组大鼠左心室心肌组织中增殖相关基因mRNA的表达:移植后4周,生理盐水组和Ad-null-CSC组大鼠左心室心肌组织中cyclinD1和PCNA的mRNA表达水平均略高于假手术组,但差异均无统计学意义(P均＞0.05).而ILK-CSC组二者水平均明显高于Ad-null-CSC组(P分别为0.003和0.004).结论 过表达ILK的CSC可通过促进心肌细胞增殖、抑制左心室纤维化,进而改善心肌梗死大鼠的心功能.

abstractsObjective To investigate the effect of cardiac stem cells (CSC) overexpressing integrin-linked kinase (ILK-CSC) transplantation on cardiac function after myocardial infarction (MI) and related mechanism.Methods CSCs were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and ILK-CSC were established by transfecting recombinant adenoviral vector harboring human wild-type ILK cDNA.Forty 8-week-old rats were randomly divided into 4 groups (n =10 each group):sham group,MI plus saline injection group (saline group),MI plus CSC transfected with null vector injection group (Ad-null-CSC group),and MI plus ILK-CSC injection group (ILK-CSC group).MI was induced through coronary artery ligation,and after 15 minutes,30 μl saline,Ad-null-CSC (1 × 105 cells/30 μl) or ILK-CSC (1 × 105 cells/30 μl) were injected into the hearts of MI rats at 3 different points in infracted zone and infarct border zone.After 4 weeks,left ventricular (LV) function was examined by echocardiography,LV fibrosis was detected by HE and Masson staining,and myocardial protein expression of Ki-67 and p-H3 was evaluated by immuohistochemistry and mRNA expression of cyclinD1 and PCNA was detected by real-time RT-PCR.Results (1) Thirty-seven rats (sham group =10,saline group =8,Ad-null-CSC group =9 and ILK-CSC group =10) survived at 4 weeks after operation.Left ventricular end-systolic dimension (LVESD,P =0.009) and left ventricular end-diastolic dimension (LVEDD,P =0.002) were significantly increased,and left ventricular ejection fraction(LVEF,P=0.006) was decreased in saline group compared with those of sham group.In Ad-null-CSC group LVESD (P =0.005) and LVEDD (P =0.003) were decreased,but LVEF remained unchanged (P =0.113) compared with those of saline group.LVESD (P =0.004) and LVEDD (P =0.000 1) of ILK-CSC group were significantly decreased,and LVEF (P =0.004) was significantly increased compared with those of Ad-null-CSC group.(2) LV histology and myocardial fibrosis:there were marked myocyte loss and significant increase of myocardial fibrosis in the saline group ((70.6±5.1) ％,P=0.002) and Ad-null-CSC group((57.7 ±3.4) ％,P=0.001) compared with sham group ((8.2 ± 2.2) ％),while the fibrosis was significantly attenuated post injection of ILK-CSC ((30.6 ± 7.0) ％,P =0.005 vs.Ad-null-CSC).(3) Protein levels of mitotic genes:the results of immuohistochemistry showed that the brown positive particles were presented in the nuclei of cardiac myocytes in saline,Ad-null-CSC and ILK-CSC groups,but they were negative in sham group.The protein expression of Ki-67 (P =0.007) and phosphohistone-H3 (p-H3) (P =0.003) in ILK-CSC group was significantly higher than in Ad-null-CSC group.(4) mRNA levels of mitotic genes:the results of real-time RT-PCR showed that the mRNA levels of cyclinD1 and proliferating cell nuclear antigen (PCNA) in saline and Ad-null-CSC groups were similar as in sham group (all P ＞ 0.05).However,in ILK-CSC group they were significantly increased compared with those in Ad-null-CSC group (cyclinD1,P =0.003;PCNA,P =0.004).Conclusion Our results suggest that myocardial transplantation of CSC overexpressing ILK improves cardiac function in the post-MI rats,and this beneficial effect may be related to the enhanced proliferation of cardiac myocytes and attenuated myocardial fibrosis.