光学—磁共振双模监测干细胞联合功能化自组装多肽治疗急性心肌梗死

发布时间：2018-03-30 15:08

本文选题：骨髓间充质干细胞　切入点：功能化自组装多肽纳米纤维支架　出处：《北京协和医学院》2015年博士论文

【摘要】：目的：将双重表达绿色荧光蛋白(GFP)和萤火虫荧光素酶(Fluc)的SD大鼠骨髓间充质干细胞(GFP/Fluc BMSCs),分别联合功能化自组装多肽RADA-PRG或RADA-KLT注射至Balb/c小鼠急性梗死心肌周围,利用生物发光成像(BLI)、磁共振成像(MRI)双模态示踪移植后干细胞的定位、存活、增殖和迁移并评估治疗后心功能的改善程度；结合病理HE染色及免疫组化验证体内干细胞分化、血管生成过程,从而探讨功能化自组装多肽纳米纤维支架材料提高干细胞存活率及治疗效果的可行性及可能机制。方法：构建Balb/c小鼠急性心肌梗死模型,分别在梗死心肌周围注射A组(对照组)：PBS缓冲液；B组：GFP/Fluc BMSCs; C组：GFP/Fluc BMSCs+RADA-PRG;D组:GFP/Fluc BMSCs+RADA-KLT.注射体积约40ul/只,细胞浓度约1.0×106/ml。术后第1、4、7、10、13天行BLI,术后第3、28天行MRI并计算左心室射血分数(LVEF),术后第29天取心脏组织病理行HE染色以及GFP、CD34免疫组化,并计算梗死灶内及周围血管密度。结果：自术后第1天即可观察到B、C、D三组心脏体表投射部位有明确的光学信号,第4天信号强度达到峰值,第7天开始逐渐减弱,B、D两组至第10天消失,C组至第13天消失,而A组始终未观察到光学信号。其中,C组信号强度自第4天开始均高于B组(Day 4, P=0.0008; Day 7, P=0.003; Day 10, P=10-7),而D组仅在第4天高于B组(P=0.02)。此外,三组均在第1天即可在心脏以外部位观察到光学信号,尤以头部最为明显,与心脏区域维持同样时间后消失。术后第3天四组LVEF无明显差异(P=0.59)。术后第28天,四组LVEF均较前提高,其中A、B两组仍无明显差异(P=0.21),而C、D两组均高于A(C组,P=0.01；D组,P=0.01)、B(C组,P=0.01；D组,P=0.02)两组。心脏组织HE染色及GFP免疫组化示四组梗死灶均为纤维瘢痕组织替代,B组可见GFP阳性的心肌细胞,C、D两组亦可见GFP阳性细胞,但与心肌细胞、血管结构不一致,A组未见GFP阳性细胞。CD34免疫组化示B、C、D三组梗死灶内及周围血管密度明显高于A组(B组,P=0.006；C组,P=0.0001；D组,P=0.0002),且C、D两组明显高于B组(C组,P=0.003；D组,P=0.002)。结论：将SD大鼠GFP/Fluc BMSCs注射至Balb/c小鼠急性梗死心肌周围,利用BLI、MRI双模态可以有效示踪移植后BMSCs的定位、存活、增殖和迁移,并评估治疗后心功能的改善程度。单纯用BMSCs治疗,BMSCs在体内存活时间较短,心功能未见明显改善。而联合RADA-PRG可以促进BMSCs在心肌组织的定位、增殖,加强BMSCs在体内分化并诱导血管生成的作用,从而强化治疗效果；联合RADA-KLT可以促进体内血管生成、组织修复过程,从而强化治疗效果。
[Abstract]:Objective: to inject GFP / Fluc BMSCs of SD rat bone marrow mesenchymal stem cells expressing green fluorescent protein (GFP) and fluorescein luciferase (luciferase) into the acute infarcted myocardium of Balb/c mice, respectively, in combination with functionalized self-assembly polypeptide RADA-PRG or RADA-KLT. The localization, survival, proliferation and migration of stem cells after transplantation were evaluated by bioluminescence imaging bli and magnetic resonance imaging (MRI), and the differentiation of stem cells in vivo was verified by HE staining and immunohistochemistry. To explore the feasibility and possible mechanism of functional self-assembled polypeptide nanofiber scaffolds for improving the survival rate and therapeutic effect of stem cells. Methods: the acute myocardial infarction model of Balb/c mice was established. Group A (control group: PBS buffer group B: GFP / Fluc BMSCs; group C: GFP / Fluc BMSCs RADA-PRGN D group: GFP / Fluc BMSCs RADA-KLT.Injection volume about 40ul/; The cell concentration was about 1.0 脳 10 6 / ml. Bli was performed on the 1st day, the 3rd day after operation, and the left ventricular ejection fraction (LVEFV) was calculated. On the 29th day after operation, the heart tissue was taken for HE staining and CD34 immunohistochemical staining. The density of blood vessels in and around the infarct was calculated. Results: from the first day after operation, clear optical signals could be observed in the projecting site of the heart surface in group B\ -C\ -C\ -D, and the intensity of the signal reached a peak on day 4. Group C disappeared from day 7 to day 13, group C disappeared gradually from day 7 to day 10, and group D disappeared gradually from day 7 to day 13. However, no optical signal was observed in group A. the signal intensity of group C was higher than that of group B from day 4 to day 4; Day _ 7, P _ (0.003); Day _ (10), P ~ (10 ~ (-7)), but that of group D was only higher than that of group B on day 4. Optical signals were observed on the first day in all the three groups, especially in the head, which disappeared at the same time as the heart region. There was no significant difference in LVEF between the four groups on the 3rd day after operation. The LVEF of the four groups increased on the 28th day after operation. There was still no significant difference between the two groups (P = 0.21), but the two groups were higher than those in group A (n = 0.01). He staining in cardiac tissue and GFP immunohistochemical staining showed that myocardial GFP was positive in the four groups. GFP positive cells were also found in two groups. But with cardiomyocytes, There were no GFP positive cells. CD34 immunohistochemical staining in group A showed that the density of blood vessels in and around the infarct focus in group B was significantly higher than that in group A (P = 0.006), and that in group D was significantly higher than that in group C (n = 0.0002), and that in group C was significantly higher than that in group C (n = 0.003). GFP/Fluc BMSCs was injected into the myocardium around acute infarction in Balb/c mice. The localization, survival, proliferation and migration of BMSCs after transplantation can be effectively traced by using BLII-MRI, and the degree of improvement of cardiac function after treatment can be evaluated. The survival time of BMSCs alone in vivo is shorter. The cardiac function was not obviously improved, but combined with RADA-PRG could promote the localization and proliferation of BMSCs in myocardial tissue, enhance the differentiation of BMSCs in vivo and induce angiogenesis, thus enhancing the therapeutic effect, and combined with RADA-KLT could promote angiogenesis in vivo. Tissue repair process to enhance the therapeutic effect.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R542.22

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