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Title: [Transplantation of cardiac stem cells overexpressing integrin-linked kinase improves cardiac function in a rat model of acute myocardial infarction].
Author: Li JJ, Liu Y, Zhao J, Li AY, Wang DM.
Journal: Zhonghua Xin Xue Guan Bing Za Zhi; 2017 Oct 24; 45(10):880-886. PubMed ID: 29081179.
Abstract:
Objective: 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 (1×10(5) cells/30 μl) or ILK-CSC (1×10(5) 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. 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30 μl生理盐水、Ad-null-CSC(1×10(5) 个细胞/30 μl)或ILK-CSC(1×10(5) 个细胞/30 μl)。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。.

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