Xiangfeng Wu, Tao Wang, Tao Liu, Fu-yu Xu, Qian Zhang
{"title":"Thbs4基因编辑BMSCs移植对糖尿病后肢缺血大鼠VEGF和Ang-1的影响及其机制","authors":"Xiangfeng Wu, Tao Wang, Tao Liu, Fu-yu Xu, Qian Zhang","doi":"10.3760/CMA.J.ISSN.1673-4181.2019.04.003","DOIUrl":null,"url":null,"abstract":"Objective \nTo investigate the effects and the mechanism of thrombospondin 4 (Thbs4) gene-edited bone marrow mesenchymal stem cells (BMSCs) transplantation on vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in diabetic rats with hind limb ischemia. \n \n \nMethods \nThirty Sprague-Dawley rats were randomly divided into the model group, BMSCs treatment group and Thbs4-BMSCs treatment group on average. After constructing the type Ⅱ diabetic rat model with hind limb ischemia, 100 μl normal saline, BMSCs suspension and Thbs4-BMSCs suspension (cell number: 2×106) were locally injected into the ischemic injury area of rats for the model group, BMSCs group and Thbs4-BMSCs group, respectively. The rats were sacrificed on the 14th day after stem cell transplantation, and the muscle tissues near the ischemic area were collected. The relative expression of VEGF and p-Smad2/3 protein was detected by Western Blot. The Ang-1 protein expression was detected by immunofluorescence staining. The levels of related genes were detected by qRT-PCR, and the von Willebrand Factor (vWF) protein expression was detected by immunohistochemistry staining. \n \n \nResults \nThe relative expression levels of VEGF, Ang-1 and vWF protein in the Thbs4-BMSCs group were significantly higher than those in the model group and BMSCs group (VEGF protein: P<0.01 and P<0.05). The mRNA expression of VEGF and Ang-1 were significantly up-regulated, the differences were statistically significant(VEGF mRNA: all P<0.01; Ang-1: P<0.01 and P<0.05). The expression of p-Smad2/3 protein in the Thbs4-BMSCs group was significantly higher than that in the model group and the BMSCs treatment group (all P<0.01). The expression of p-Smad2/3 protein was significantly decreased after the addition of p-Smad2/3 pathway inhibitor, the differences were statistically significant (P<0.05). \n \n \nConclusions \nThbs4-BMSCs transplantation can effectively promote angiogenesis in diabetic rats with hind limb ischemia, and the effect of angiogenesis may be related to the activation of Smad2/3 signaling pathway. \n \n \nKey words: \nDiabetes mellitus; Hind limb ischemia; Mesenchymal stem cell transplantation; Thrombospondin 4; Angiogenesis","PeriodicalId":61751,"journal":{"name":"国际生物医学工程杂志","volume":"42 1","pages":"294-300"},"PeriodicalIF":0.0000,"publicationDate":"2019-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Effect of Thbs4 gene editing BMSCs transplantation on VEGF and Ang-1 in diabetic hind limb ischemia rats and related mechanism\",\"authors\":\"Xiangfeng Wu, Tao Wang, Tao Liu, Fu-yu Xu, Qian Zhang\",\"doi\":\"10.3760/CMA.J.ISSN.1673-4181.2019.04.003\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Objective \\nTo investigate the effects and the mechanism of thrombospondin 4 (Thbs4) gene-edited bone marrow mesenchymal stem cells (BMSCs) transplantation on vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in diabetic rats with hind limb ischemia. \\n \\n \\nMethods \\nThirty Sprague-Dawley rats were randomly divided into the model group, BMSCs treatment group and Thbs4-BMSCs treatment group on average. After constructing the type Ⅱ diabetic rat model with hind limb ischemia, 100 μl normal saline, BMSCs suspension and Thbs4-BMSCs suspension (cell number: 2×106) were locally injected into the ischemic injury area of rats for the model group, BMSCs group and Thbs4-BMSCs group, respectively. The rats were sacrificed on the 14th day after stem cell transplantation, and the muscle tissues near the ischemic area were collected. The relative expression of VEGF and p-Smad2/3 protein was detected by Western Blot. The Ang-1 protein expression was detected by immunofluorescence staining. The levels of related genes were detected by qRT-PCR, and the von Willebrand Factor (vWF) protein expression was detected by immunohistochemistry staining. \\n \\n \\nResults \\nThe relative expression levels of VEGF, Ang-1 and vWF protein in the Thbs4-BMSCs group were significantly higher than those in the model group and BMSCs group (VEGF protein: P<0.01 and P<0.05). The mRNA expression of VEGF and Ang-1 were significantly up-regulated, the differences were statistically significant(VEGF mRNA: all P<0.01; Ang-1: P<0.01 and P<0.05). The expression of p-Smad2/3 protein in the Thbs4-BMSCs group was significantly higher than that in the model group and the BMSCs treatment group (all P<0.01). The expression of p-Smad2/3 protein was significantly decreased after the addition of p-Smad2/3 pathway inhibitor, the differences were statistically significant (P<0.05). \\n \\n \\nConclusions \\nThbs4-BMSCs transplantation can effectively promote angiogenesis in diabetic rats with hind limb ischemia, and the effect of angiogenesis may be related to the activation of Smad2/3 signaling pathway. \\n \\n \\nKey words: \\nDiabetes mellitus; Hind limb ischemia; Mesenchymal stem cell transplantation; Thrombospondin 4; Angiogenesis\",\"PeriodicalId\":61751,\"journal\":{\"name\":\"国际生物医学工程杂志\",\"volume\":\"42 1\",\"pages\":\"294-300\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-08-28\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"国际生物医学工程杂志\",\"FirstCategoryId\":\"1087\",\"ListUrlMain\":\"https://doi.org/10.3760/CMA.J.ISSN.1673-4181.2019.04.003\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"国际生物医学工程杂志","FirstCategoryId":"1087","ListUrlMain":"https://doi.org/10.3760/CMA.J.ISSN.1673-4181.2019.04.003","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Effect of Thbs4 gene editing BMSCs transplantation on VEGF and Ang-1 in diabetic hind limb ischemia rats and related mechanism
Objective
To investigate the effects and the mechanism of thrombospondin 4 (Thbs4) gene-edited bone marrow mesenchymal stem cells (BMSCs) transplantation on vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in diabetic rats with hind limb ischemia.
Methods
Thirty Sprague-Dawley rats were randomly divided into the model group, BMSCs treatment group and Thbs4-BMSCs treatment group on average. After constructing the type Ⅱ diabetic rat model with hind limb ischemia, 100 μl normal saline, BMSCs suspension and Thbs4-BMSCs suspension (cell number: 2×106) were locally injected into the ischemic injury area of rats for the model group, BMSCs group and Thbs4-BMSCs group, respectively. The rats were sacrificed on the 14th day after stem cell transplantation, and the muscle tissues near the ischemic area were collected. The relative expression of VEGF and p-Smad2/3 protein was detected by Western Blot. The Ang-1 protein expression was detected by immunofluorescence staining. The levels of related genes were detected by qRT-PCR, and the von Willebrand Factor (vWF) protein expression was detected by immunohistochemistry staining.
Results
The relative expression levels of VEGF, Ang-1 and vWF protein in the Thbs4-BMSCs group were significantly higher than those in the model group and BMSCs group (VEGF protein: P<0.01 and P<0.05). The mRNA expression of VEGF and Ang-1 were significantly up-regulated, the differences were statistically significant(VEGF mRNA: all P<0.01; Ang-1: P<0.01 and P<0.05). The expression of p-Smad2/3 protein in the Thbs4-BMSCs group was significantly higher than that in the model group and the BMSCs treatment group (all P<0.01). The expression of p-Smad2/3 protein was significantly decreased after the addition of p-Smad2/3 pathway inhibitor, the differences were statistically significant (P<0.05).
Conclusions
Thbs4-BMSCs transplantation can effectively promote angiogenesis in diabetic rats with hind limb ischemia, and the effect of angiogenesis may be related to the activation of Smad2/3 signaling pathway.
Key words:
Diabetes mellitus; Hind limb ischemia; Mesenchymal stem cell transplantation; Thrombospondin 4; Angiogenesis
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