[1]陈文明,蹇明辉,仇治梅,等.TLR4基因修饰BMSCs移植对大鼠急性心肌梗死后心功能的影响[J].第三军医大学学报,2019,41(15):1438-1444.
 CHEN Wenming,JIAN Minghui,QIU Zhimei,et al.Effect of bone marrow mesenchymal stem cells modified with Toll-like receptor 4 on cardiac function in rats after acute myocardial infarction[J].J Third Mil Med Univ,2019,41(15):1438-1444.
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TLR4基因修饰BMSCs移植对大鼠急性心肌梗死后心功能的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第15期
页码:
1438-1444
栏目:
基础医学
出版日期:
2019-08-15

文章信息/Info

Title:
Effect of bone marrow mesenchymal stem cells modified with Toll-like receptor 4 on cardiac function in rats after acute myocardial infarction
作者:
遵义医科大学附属医院心血管内科
Author(s):
CHEN Wenming JIAN Minghui QIU Zhimei SHI Bei

Department of Cardiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province, 563099, China

关键词:
Toll样受体-4骨髓间充质干细胞急性心肌梗死心功能
Keywords:
Toll-like receptor 4 bone marrow mesenchymal stem cells acute myocardial infarction cardiac function
分类号:
R329-33; R331.31; R542.22
文献标志码:
A
摘要:

目的 探讨Toll样受体-4(Toll-like receptor 4,TLR4)修饰的骨髓间充质干细胞(bone marrow mesenchymal stem Cells,BMSCs)对心肌梗死后心脏心功能的影响。方法 取大鼠骨髓体外分离扩增培养BMSCs,将携带目的基因重组慢病毒载体(Lv-EGFP-TLR4)及空病毒载体(Lv-EGFP)分别转染至BMSCs;用ELISA检测Lv-EGFP-TLR4转染后BMSCs细胞上清液TLR4的表达。选用成年SD大鼠,结扎左冠状动脉前降支建立急性心肌梗死模型,随机分为3组(n=18):对照组、Lv-EGFP-BMSCs组、Lv-EGFP-TLR4-BMSCs组。分别将Lv-EGFP-BMSCs和Lv-EGFP-TLR4-BMSCs多点注射到大鼠心肌梗死区周边,对照组注射等量PBS;细胞移植第7天,用荧光显微镜追踪EGFP阳性细胞表达,Western blot检测梗死区周边TLR4的蛋白表达;细胞移植第28天,用超声心动图检测大鼠心功能,HE和MASSON染色观察心肌胶原纤维化程度,用ELISA检测血清Ⅰ型前胶原羧基端肽(PⅠCP)和Ⅲ型前胶原氨基端肽(PⅢNT)表达水平。结果 Lv-EGFP-TLR4转染BMSCs后,细胞上清液中TLR4的表达明显增多,浓度达到(31.55 pg/mL)。细胞移植第7d,心肌梗死区域有 EGFP 标记的 BMSCs,Lv-EGFP-TLR4-BMSCs组的绿色荧光强度明显优于Lv-EGFP-BMSCs组,对照组无 EGFP 表达,TLR4蛋白表达也明显高于Lv-EGFP-BMSCs组(P<0.01)。细胞移植第28 d,Lv-EGFP-TLR4-BMSCs组左室射血分数[LVEF(44.36±5.62)%]和短轴缩短率[LVFS(22.88±9.89)%]较Lv-EGFP-BMSCs组[LVEF(18.41±5.66)%,LVFS(18.41±5.66)%]均明显增加(P<0.01),左心室收缩末径[LVDs(5.41±0.71)]和左心室舒张末径[LVDd(7.11±0.79)]值较Lv-EGFP-BMSCs组[LVDs(6.66±0.91),LVDd(8.06±0.96)]均明显缩小(P<0.01);血清中Lv-EGFP-TLR4-BMSCs组PⅠCP(72.88 μg/L)和PⅢNP(7.33 μg/L)蛋白浓度较Lv-EGFP-BMSCs组[PⅠCP(83.79 μg/L)和PⅢNP(9.51 μg/L)]减少(P<0.05);Lv-EGFP-TLR4-BMSCs组心肌纤维化程度也明显低于Lv-EGFP-BMSCs组。结论 TLR4基因修饰BMSCs移植治疗心肌梗死,进一步减轻心肌纤维化程度,从而改善心肌梗死大鼠的心功能。

Abstract:

ObjectiveTo investigate the effect of bone marrow mesenchymal stem cells (BMSCs) modified with Toll-like receptor 4 (TLR4) on cardiac function in rats after acute myocardial infarction. MethodsBMSCs were isolated from rat bone marrow and then cultured in vitro. The recombinant lentivirus vector carrying the target gene (Lv-EGFP-TLR4) and empty virus vector (Lv-EGFP) were transfected into BMSCs respectively. The expression of TLR4 in the supernatant of BMSCs cells transfected with Lv-EGFP-TLR4 was detected by ELISA. Adult SD rats were randomly divided into 3 groups (n=18): control group, Lv-EGFP-BMSCs group and Lv-EGFP-TLR4-BMSCs group. Lv-EGFP-BMSCs and Lv-EGFP-TLR4-BMSCs were injected into the periphery of myocardial infarction in rats, respectively. The control group was injected with the same amount of PBS. On the 7th day of transplantation, the expression of EGFP positive cells was tracked by fluorescence microscopy, and the expression of TLR4 was detected by Western blotting. Cardiac function was measured by echocardiography on 28 d after transplantation. Myocardial collagen fibrosis was observed by HE and MASSON staining. Serum levels of procollagen type I carboxyl terminal peptide (PICP) and procollagen type Ⅲ amino terminal peptide (PⅢNT) were measured by ELISA. ResultsAfter transfection of BMSCs with Lv-EGFP-TLR4, the expression of TLR4 was increased significantly in the supernatant of BMSCs, and reached 31.55 pg/mL in 48 h after transfection. On the 7th day of cell transplantation, the green fluorescence intensity of BMSCs labeled with EGFP in infarct area was significantly higher in Lv-EGFP-TLR4-BMSCs group than in Lv-EGFP-BMSCs group, and no EGFP expression was seen in control group, and the expression of TLR4 protein was also significantly higher than that in Lv-EGFP-BMSCs group (P<0.01). On the 28th day of cell transplantation, the Lv-EGFP-TLR4-BMSCs group showed significantly higher left ventricular ejection fraction [LVEF, (44.36±5.62)%] and short-axis shortening rate [LVFS, (22.88±9.89)%] when compared with the Lv-EGFP-BMSCs group [(18.41±5.66)%, (18.41±5.66)%, P<0.01], but left ventricular end-systolic diameter (LVDs, 5.41±0.71) and left ventricular end-diastolic diameter (LVDd, 7.11±0.79) were statistically decreased than the other group (6.66±0.91, 8.06±0.9, P<0.01) The Lv-EGFP-TLR4-BMSCs group also had significantly lower serum levels of PICP (72.88 vs 83.79 μg/L) and PⅢNT (7.33 vs 9.51 μg/L) than those in Lv-EGFP-BMSCs group (P<0.05). The severity of myocardial fibrosis in Lv-EGFP-TLR4-BMSCs group was also significantly lower than that in Lv-EGFP-BMSCs group. ConclusionTransplantation of TLR4 modified BMSCs can treat myocardial infarction, attenuate the severity of myocardial fibrosis, and thereby improve the cardiac function of myocardial infarction rats. 

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更新日期/Last Update: 2019-08-13