骨髓内皮祖细胞和间充质干细胞对动脉粥样硬化血管内皮的修复作用
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摘要
心血管系统疾病是现代社会严重威胁人类健康,引起死亡的主要疾病。动脉粥样硬化(AS)是心血管疾病的主要病因之一,阻止AS的进展已成为基础医学和临床医学研究的热点。许多心血管疾病发生的危险因素,如老年、吸烟、糖尿病、高血压、高脂血症等都会造成以内皮细胞合成一氧化氮(NO)能力下降,细胞膜结构发生改变为主要特点的血管内皮功能失调,血管内皮功能失调是AS发生的第一步。AS治疗应包括早期阻止内皮功能失调、内皮细胞凋亡、防止AS斑块进展。如何对损伤的内皮进行修复,已成为AS研究的重点内容。
研究表明内皮祖细胞(EPC)具有修复和替代损伤血管内皮细胞的功能。将体外培养的EPC输入血管受损的大鼠体内,能促进内皮再生；生理条件下,轻微受损的血管内皮细胞可以通过邻近细胞的增殖得到修复,而强烈的氧化应激反应导致的内膜损伤则要通过循环中EPC归巢到损伤部位来修复。
研究发现,在成体外周组织,包括某些正常及损伤血管壁附近均存在一类具有骨髓间充质干细胞特征的细胞,这些细胞可能参与了血管组织更新和损伤后的修复过程。离体研究证实骨髓MSC在一定条件下可分化为ECs,分化后的MSC表达ECs特有标志,如血管内皮细胞生长因子受体2(KDR)、血管内皮细胞生长因子受体1(Flk-1)及血管细胞黏附分子(VCAM-1)等；此外间充质干细胞可能分泌多种细胞因子,为受损血管的修复和新血管的生成提供了适当的微环境。
本研究旨在建立高脂饮食导致的动脉粥样硬化模型,研究大鼠骨髓来源的EPC和MSC对动脉粥样硬化的治疗作用,比较两者的治疗效果,寻找AS细胞治疗的有效方法。
第一章骨髓FPC和MSC的分离培养、鉴定
目的
分离培养大鼠骨髓EPC和MSC,研究其生物学特性。
方法
1、无菌条件下取大鼠股骨,冲出骨髓细胞,用大鼠淋巴细胞分离液分离出单个核细胞,将其重悬在MSC培养液中培养,检测其生物学特性(流式和免疫组化分析表面标志、生长特性)及多向分化潜能(向脂肪细胞、成骨细胞分化)。
2、分离大鼠骨髓单个核细胞,将其重悬在内皮细胞培养液中培养。应用免疫细胞化学检测内皮细胞表面标志(vWF、CD31、CD144),通过UEA-1结合能力、乙酰化低密度脂蛋白(Dil-Ac-LDL)摄取能力的检测鉴定培养的内皮祖细胞,并研究细胞的生长特性和克隆形成能力。
结果
1、骨髓MSC的生物学特性：第三代贴壁细胞均表达MSC表面标志物CD29,CD90,不表达CD34、CD45造血细胞表面标志和CD31、vWF内皮细胞表面标志。第三代MSC接种后第1-2天生长较缓慢,从第3天起细胞开始增殖并进入对数生长期,细胞迅速增殖。根据公式DT=t×1g2／(1gNt-1gN0),得出MSC倍增时间49.13±0.7h。
2、培养的骨髓间充质干细胞在特定的培养条件下具有向脂肪细胞、成骨细胞分化的能力,符合骨髓间充质干细胞判断的金标准。
3、培养的EPC vWF、CD31、CD144表达阳性,具有与UEA-1结合能力和摄取DiI-AC-LDL的能力。EPC的生长特性为：接种后第1-6d为潜伏期,从第6 d起细胞开始增殖并进入对数生长期。不同传代次数之间的EPC其增值能力存在差异,P2代细胞增殖能力强于P5代,它们的增殖能力在第8天显示出差异(p<0.05),第10天出现显著差异(p<0.01),P2和P5EPC的倍增时间分别为：82.70±4.28h和90.88±5.30h。骨髓EPC具有克隆形成能力,并且克隆形成的数目与再种植细胞数量之间呈良好的线性关系,接种4000、6000、8000、10000个细胞可分别形成22.00±3.60、33.67±5.13、42.00±3.00、61.00±4.58个内皮细胞集落。
结论
从大鼠骨髓单个核细胞成功分离培养获得MSC和EPC。
第二章动脉硬化大鼠动物模型的建立及动脉硬化大鼠血管内皮的衰老分析
目的
建立SD大鼠动脉硬化模型；观察大鼠血管内皮细胞衰老改变。
方法
1、SD大鼠28只,随机分为A、B、C、D 4组,每组7只。A组喂食普通饲料,B、C、D组喂食高脂饲料。大鼠高脂饲料喂养12周后测定血脂的变化,HE染色分析主要动脉血管结构的改变。
2、胶原酶灌注方法,分离胸主动脉和肾动脉血管内皮细胞,应用免疫细胞化学的方法,鉴定内皮细胞；应用细胞化学染色的方法检测衰老相关的半乳糖甘酶(SA-β-gal)活性。
结果
1、SD雄性大鼠高脂饲料喂养12周后,大鼠血液甘油三脂、总胆固醇、高密度脂蛋白胆固醇水平明显升高；高脂血症大鼠动脉血管出现明显的粥样硬化改变。
2、分离培养后的胸主动脉和肾动脉血管内皮细胞表达vWF,CD31；高脂饮食后的大鼠血管内皮细胞衰老增加,SA-β-gal活性明显增加。
结论
1、成功建立SD大鼠动脉粥样硬化模型。
2、高脂血症引起大鼠动脉结构发生病理改变,动脉血管内皮细胞衰老增加,SA-β-gal活性增加。
第三章骨髓EPC、MSC对动脉粥样硬化血管的修复作用
目的
研究大鼠骨髓来源的EPC和MSC对动脉粥样硬化治疗的效果。
方法
1、包装、纯化rAAV2-IRES-GFP,并转染EPC和MSC。
2、将转染了rAAV2-IRES-GFP的EPC和MSC经尾静脉分别移植入高脂饮食大鼠体内,病理对照组注射生理盐水。注射后两个月,检测血脂水平；HE染色观察主动脉的组织学改变；冰冻切片荧光显微镜下观察GFP标记的细胞。
3、RT-PCR检测主动脉壁一氧化氮合酶(eNOS)、细胞间黏附分子1(ICAM-1)及载脂蛋白E (apoE) mRNA的表达。
结果
与病理对照组比较,EPC和MSC细胞治疗后大鼠血脂水平呈明显下降趋势,动脉壁脂质沉积减少；主动脉冰冻切片中可以检测到GFP标记的细胞,细胞排列整齐,衬在血管内膜上；动脉璧ICAM-1的mRNA表达水平明显降低,而apoE及eNOSmRNA的表达水平与正常对照组相比有明显的增高,三种基因的表达以EPC治疗组改变最为显著,提示EPC对动脉粥样硬化的修复作用优于MSC。
结论
EPC和MSC对动脉硬化老化的大鼠血管内皮具有一定的修复作用,两者比较,EPC对动脉粥样硬化的修复作用优于MSC。
Atherosclerosis (AS) is responsible for a significant part of morbidity and mortality all over the world. The clinical manifestation of AS includes myocardial infarction, stroke, aortic aneurysm, aortic dissection as well as intermittent lameness. Not only the pathogenic factors leading to atherosclerosis are highly heterogeneous, but the causative risk factors include hyperlipidemia, hypertension, diabetes mellitus and smoking can also damage endothelial function.
Endothelial dysfunction is an early hallmark of atherosclerotic disease and may correlate to ischemic events even in the absence of arterial obstruction. Thus, repair of the injured endothelial cells is an emphasis on AS therapy.
In the context of regeneration, published data from animal studies have unveiled that endothelial progenitor cells(EPC) effectively contribute in restoring endothelial function.The mechanisms involved in regeneration of damaged endothelial cells have been suggested in some studies, showing that regeneration of endothelial cells were promoted when EPC were injected into rats of vascular injury. Animal studies have demonstrated that tissue ischemia upregulates many growth factors and cytokines, such as VEGF and SDF-1, which stimulated the release of EPC through eNOS and MMP-dependent pathways in the bone marrow. In addition, specific homing of EPC at the damaged site is directed by the interaction between chemokines produced locally and their specific receptors.
Mesenchymal stem cells(MSC) first identified in the marrow stroma are a group of multipotent progenitor cells which can differentiate along multiple lineages giving rise to cartilage, bone, fat, muscle and vascular tissue. Some studies indicate that MSC play important roles in the progress of atherosclerosis. Specifically,MSC can differentiate into ECs that regenerate the damaged endothelial layer. Moreover some cytokine could be secreted by MSC,which provides a proper microenvironment for repairing the vascular injury and eovascularization.
In this study, we establish an experimental animal model of atherosclerosis using rats by high-fat diet, and investigate the therapeutic effect of bone marrow-derived EPC and MSC on AS.
PartⅠ
Isolation and culture of rat bone marrow endothelial progenitor cells and mesenchymal sten cells
objective
In this study, we established the method of isolation and culture bone marrow-derived endothelial progenitor cells and mesenchymal stem cells, and investigated their biological characteristics and multipotential capacity.
Methods
1.Rat bone marrow-derived MSC were isolated by density gradient centrifugation, and cultured in MSC culture medium. Biological characteristics of MSC were detected by immunofluorescence staining, flow cytometry and growth curve, the differentiation potential of cells were tested using standard differentiation conditions for osteoblasts and adipocytes.
2.Rat bone marrow-derived EPC were isolated by density gradient centrifugation, and cultured in EPC culture medium. immunophenotypic characterizations were analyzed using cytoimmunostaining, the ability of uptaking of acetylated low-density lipoprotein (acetylated LDL) and binding to UEA-1 was detected, and the growth characteristics and clonogenic potential were observed.
Results
1.MSC revealed a surface antigen profile that is coincidence with that reported for BM-MSC, that is, CD29+,CD90+, and do not express surface markers of hematopoetic cells and endothelial cells, namely CD34-，CD45-, CD31-,vWF-. Moreover, these cells could differentiate into osteoblasts and adipocytes under specified culture condition. The growth curve of the third passage MSC showed that the cells grew slowly in the first two days and begin to speed up from the third day after plating. The doubling time was 49.13±0.7 hours.
2.The endothelial specific markers vWF、CD31 and CD 144 were expressed by the cultured cells and these cells were capable of incorporating Dil-Ac-LDL and binding to lectin UEA-1. EPC showed different proliferative potential in different passages. The proliferative potency of the second passage cells was higher than fifth passage, their doubling time was 82.70±4.28 and 90.88±5.30 hours, respectively. The formation of endothelial cell colonies was found when the cells were plated at a low density. There was a linear relationship between the number of plated cell and the number of coloniesConclusion
EPC and MSC can be isolated and cultured succesfully from the rat bone marrow.
Part II
The establishment of AS rat model and analysis of endothelial senescence after high-fat diet challenge
Objective
The aim of this study was to establish an experimental rat model of atherosclerosis, and investigate the changes of rat arterial endothelial senescence.
Methods
1. Twenty eight male healthy SD rats were randomly divided into four groups. In the control group (A) rats were fed with basic food, while in other groups rats were fed with high fat-diet. After three months the levels of plasma total cholesterol (TC), triglyceride (TG) and high density lipoprotein-cholesterol (HDL-C) were measured with biochemical techniques. The pathological changes of arterial vessels were examined by HE staining.
2. The endothelial cells from arterial vessels of high-fat and normal diet rat were isolated and identified by immunocytochemistry after a 12-wk high-fat dietary treatment, Senescence-associated (3-galactosidase (SA-β-gal) activity was examind using the method of cytochemistry.
Results
1. Adult SD male rat were fed with high-fat diet to induce to heperlipemia. After 12 weeks of feeding, the levels of serum triacetylglycerid, total cholesterol and high density lipoprotein cholesterol were elevated in high-fat fed mice compared with normal diet fed mice.
2. No pathological changes were observed in the aorta in control group by HE staining, but the arterial vessels showed pathology changes of atherosclerosis.
3. High-fat diet increased the prevalence of endothelial senescence, SA-β-gal acitivies increased significantly in postdietary challenge.
Conclusion
1. The AS rat model was established succesfully.
2. High-fat diet damaged arterail vessels and caused arterial endothelial cell senescence, and increased senescence-associated SA-β-gal activities in rat.
PartⅢ
The repairing effect of bone marrow-derived EPC and MSC on AS
Objective
In this study, the repairing effect of bone marrow-derived EPC and MSC on AS was investigated.
Methods
1. rAAV2-IRES-GFP was packed and purified, and was used to transduce EPC and MSC.1、packing and purifing rAAV2-IRES-GFP,and EPC and MSC were transferred by rAAV2-IRES-GFP
2. Group A (normal control) was injected with saline; Group B (untreated model control) was injected with saline; Group C EPC transduced by rAAV2-IRES-GFP were transplanted into model rat; Group D MSC transduced by rAAV2-IRES-GFP were transplanted into model rat. After 2 months, the level of blood lipid was tested. The change of aorta histology was observed by HE staining and GFP-labeled cells by frozen section.
3.Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression level of eNOS, ICAM-1 and APOE in artery vessel.
Results
The level of blood lipid in all treated rats decreased substantialy compared with the model group. The EPC and MSC transferred with rAAV2-IRES-GFP were found in the endothelial monolayer of artery vessel of EPC-treated group and MSC-treated group, respectively. The result of HE staining suggested that, the lipid deposits in aortic endothelium in both cell transferred groups were less than those in the untreated group.
RT-PCR suggested that eNOS, apoE expression level of arterial vascular wall in MSC treatment group and EPC treatment group was both significantly higher than untreated control group, but still lower than the normal diet controls. The expression levels of ICAM-1 in treatment group was significantly lower than the pathological control group (p<0.05).
These results showed that the therapeutical effect of EPC on AS was more obvious than that of MSC.
Conclusion
Both bone marrow-derived EPC and MSC had the repairing effect on arterial endothelium of AS, but the therapeutical effect of EPC was more obvious than that of MSC on the damaged arterial endothelium.
研究表明内皮祖细胞(EPC)具有修复和替代损伤血管内皮细胞的功能。将体外培养的EPC输入血管受损的大鼠体内,能促进内皮再生；生理条件下,轻微受损的血管内皮细胞可以通过邻近细胞的增殖得到修复,而强烈的氧化应激反应导致的内膜损伤则要通过循环中EPC归巢到损伤部位来修复。
研究发现,在成体外周组织,包括某些正常及损伤血管壁附近均存在一类具有骨髓间充质干细胞特征的细胞,这些细胞可能参与了血管组织更新和损伤后的修复过程。离体研究证实骨髓MSC在一定条件下可分化为ECs,分化后的MSC表达ECs特有标志,如血管内皮细胞生长因子受体2(KDR)、血管内皮细胞生长因子受体1(Flk-1)及血管细胞黏附分子(VCAM-1)等；此外间充质干细胞可能分泌多种细胞因子,为受损血管的修复和新血管的生成提供了适当的微环境。
本研究旨在建立高脂饮食导致的动脉粥样硬化模型,研究大鼠骨髓来源的EPC和MSC对动脉粥样硬化的治疗作用,比较两者的治疗效果,寻找AS细胞治疗的有效方法。
第一章骨髓FPC和MSC的分离培养、鉴定
目的
分离培养大鼠骨髓EPC和MSC,研究其生物学特性。
方法
1、无菌条件下取大鼠股骨,冲出骨髓细胞,用大鼠淋巴细胞分离液分离出单个核细胞,将其重悬在MSC培养液中培养,检测其生物学特性(流式和免疫组化分析表面标志、生长特性)及多向分化潜能(向脂肪细胞、成骨细胞分化)。
2、分离大鼠骨髓单个核细胞,将其重悬在内皮细胞培养液中培养。应用免疫细胞化学检测内皮细胞表面标志(vWF、CD31、CD144),通过UEA-1结合能力、乙酰化低密度脂蛋白(Dil-Ac-LDL)摄取能力的检测鉴定培养的内皮祖细胞,并研究细胞的生长特性和克隆形成能力。
结果
1、骨髓MSC的生物学特性：第三代贴壁细胞均表达MSC表面标志物CD29,CD90,不表达CD34、CD45造血细胞表面标志和CD31、vWF内皮细胞表面标志。第三代MSC接种后第1-2天生长较缓慢,从第3天起细胞开始增殖并进入对数生长期,细胞迅速增殖。根据公式DT=t×1g2／(1gNt-1gN0),得出MSC倍增时间49.13±0.7h。
2、培养的骨髓间充质干细胞在特定的培养条件下具有向脂肪细胞、成骨细胞分化的能力,符合骨髓间充质干细胞判断的金标准。
3、培养的EPC vWF、CD31、CD144表达阳性,具有与UEA-1结合能力和摄取DiI-AC-LDL的能力。EPC的生长特性为：接种后第1-6d为潜伏期,从第6 d起细胞开始增殖并进入对数生长期。不同传代次数之间的EPC其增值能力存在差异,P2代细胞增殖能力强于P5代,它们的增殖能力在第8天显示出差异(p<0.05),第10天出现显著差异(p<0.01),P2和P5EPC的倍增时间分别为：82.70±4.28h和90.88±5.30h。骨髓EPC具有克隆形成能力,并且克隆形成的数目与再种植细胞数量之间呈良好的线性关系,接种4000、6000、8000、10000个细胞可分别形成22.00±3.60、33.67±5.13、42.00±3.00、61.00±4.58个内皮细胞集落。
结论
从大鼠骨髓单个核细胞成功分离培养获得MSC和EPC。
第二章动脉硬化大鼠动物模型的建立及动脉硬化大鼠血管内皮的衰老分析
目的
建立SD大鼠动脉硬化模型；观察大鼠血管内皮细胞衰老改变。
方法
1、SD大鼠28只,随机分为A、B、C、D 4组,每组7只。A组喂食普通饲料,B、C、D组喂食高脂饲料。大鼠高脂饲料喂养12周后测定血脂的变化,HE染色分析主要动脉血管结构的改变。
2、胶原酶灌注方法,分离胸主动脉和肾动脉血管内皮细胞,应用免疫细胞化学的方法,鉴定内皮细胞；应用细胞化学染色的方法检测衰老相关的半乳糖甘酶(SA-β-gal)活性。
结果
1、SD雄性大鼠高脂饲料喂养12周后,大鼠血液甘油三脂、总胆固醇、高密度脂蛋白胆固醇水平明显升高；高脂血症大鼠动脉血管出现明显的粥样硬化改变。
2、分离培养后的胸主动脉和肾动脉血管内皮细胞表达vWF,CD31；高脂饮食后的大鼠血管内皮细胞衰老增加,SA-β-gal活性明显增加。
结论
1、成功建立SD大鼠动脉粥样硬化模型。
2、高脂血症引起大鼠动脉结构发生病理改变,动脉血管内皮细胞衰老增加,SA-β-gal活性增加。
第三章骨髓EPC、MSC对动脉粥样硬化血管的修复作用
目的
研究大鼠骨髓来源的EPC和MSC对动脉粥样硬化治疗的效果。
方法
1、包装、纯化rAAV2-IRES-GFP,并转染EPC和MSC。
2、将转染了rAAV2-IRES-GFP的EPC和MSC经尾静脉分别移植入高脂饮食大鼠体内,病理对照组注射生理盐水。注射后两个月,检测血脂水平；HE染色观察主动脉的组织学改变；冰冻切片荧光显微镜下观察GFP标记的细胞。
3、RT-PCR检测主动脉壁一氧化氮合酶(eNOS)、细胞间黏附分子1(ICAM-1)及载脂蛋白E (apoE) mRNA的表达。
结果
与病理对照组比较,EPC和MSC细胞治疗后大鼠血脂水平呈明显下降趋势,动脉壁脂质沉积减少；主动脉冰冻切片中可以检测到GFP标记的细胞,细胞排列整齐,衬在血管内膜上；动脉璧ICAM-1的mRNA表达水平明显降低,而apoE及eNOSmRNA的表达水平与正常对照组相比有明显的增高,三种基因的表达以EPC治疗组改变最为显著,提示EPC对动脉粥样硬化的修复作用优于MSC。
结论
EPC和MSC对动脉硬化老化的大鼠血管内皮具有一定的修复作用,两者比较,EPC对动脉粥样硬化的修复作用优于MSC。
Atherosclerosis (AS) is responsible for a significant part of morbidity and mortality all over the world. The clinical manifestation of AS includes myocardial infarction, stroke, aortic aneurysm, aortic dissection as well as intermittent lameness. Not only the pathogenic factors leading to atherosclerosis are highly heterogeneous, but the causative risk factors include hyperlipidemia, hypertension, diabetes mellitus and smoking can also damage endothelial function.
Endothelial dysfunction is an early hallmark of atherosclerotic disease and may correlate to ischemic events even in the absence of arterial obstruction. Thus, repair of the injured endothelial cells is an emphasis on AS therapy.
In the context of regeneration, published data from animal studies have unveiled that endothelial progenitor cells(EPC) effectively contribute in restoring endothelial function.The mechanisms involved in regeneration of damaged endothelial cells have been suggested in some studies, showing that regeneration of endothelial cells were promoted when EPC were injected into rats of vascular injury. Animal studies have demonstrated that tissue ischemia upregulates many growth factors and cytokines, such as VEGF and SDF-1, which stimulated the release of EPC through eNOS and MMP-dependent pathways in the bone marrow. In addition, specific homing of EPC at the damaged site is directed by the interaction between chemokines produced locally and their specific receptors.
Mesenchymal stem cells(MSC) first identified in the marrow stroma are a group of multipotent progenitor cells which can differentiate along multiple lineages giving rise to cartilage, bone, fat, muscle and vascular tissue. Some studies indicate that MSC play important roles in the progress of atherosclerosis. Specifically,MSC can differentiate into ECs that regenerate the damaged endothelial layer. Moreover some cytokine could be secreted by MSC,which provides a proper microenvironment for repairing the vascular injury and eovascularization.
In this study, we establish an experimental animal model of atherosclerosis using rats by high-fat diet, and investigate the therapeutic effect of bone marrow-derived EPC and MSC on AS.
PartⅠ
Isolation and culture of rat bone marrow endothelial progenitor cells and mesenchymal sten cells
objective
In this study, we established the method of isolation and culture bone marrow-derived endothelial progenitor cells and mesenchymal stem cells, and investigated their biological characteristics and multipotential capacity.
Methods
1.Rat bone marrow-derived MSC were isolated by density gradient centrifugation, and cultured in MSC culture medium. Biological characteristics of MSC were detected by immunofluorescence staining, flow cytometry and growth curve, the differentiation potential of cells were tested using standard differentiation conditions for osteoblasts and adipocytes.
2.Rat bone marrow-derived EPC were isolated by density gradient centrifugation, and cultured in EPC culture medium. immunophenotypic characterizations were analyzed using cytoimmunostaining, the ability of uptaking of acetylated low-density lipoprotein (acetylated LDL) and binding to UEA-1 was detected, and the growth characteristics and clonogenic potential were observed.
Results
1.MSC revealed a surface antigen profile that is coincidence with that reported for BM-MSC, that is, CD29+,CD90+, and do not express surface markers of hematopoetic cells and endothelial cells, namely CD34-，CD45-, CD31-,vWF-. Moreover, these cells could differentiate into osteoblasts and adipocytes under specified culture condition. The growth curve of the third passage MSC showed that the cells grew slowly in the first two days and begin to speed up from the third day after plating. The doubling time was 49.13±0.7 hours.
2.The endothelial specific markers vWF、CD31 and CD 144 were expressed by the cultured cells and these cells were capable of incorporating Dil-Ac-LDL and binding to lectin UEA-1. EPC showed different proliferative potential in different passages. The proliferative potency of the second passage cells was higher than fifth passage, their doubling time was 82.70±4.28 and 90.88±5.30 hours, respectively. The formation of endothelial cell colonies was found when the cells were plated at a low density. There was a linear relationship between the number of plated cell and the number of coloniesConclusion
EPC and MSC can be isolated and cultured succesfully from the rat bone marrow.
Part II
The establishment of AS rat model and analysis of endothelial senescence after high-fat diet challenge
Objective
The aim of this study was to establish an experimental rat model of atherosclerosis, and investigate the changes of rat arterial endothelial senescence.
Methods
1. Twenty eight male healthy SD rats were randomly divided into four groups. In the control group (A) rats were fed with basic food, while in other groups rats were fed with high fat-diet. After three months the levels of plasma total cholesterol (TC), triglyceride (TG) and high density lipoprotein-cholesterol (HDL-C) were measured with biochemical techniques. The pathological changes of arterial vessels were examined by HE staining.
2. The endothelial cells from arterial vessels of high-fat and normal diet rat were isolated and identified by immunocytochemistry after a 12-wk high-fat dietary treatment, Senescence-associated (3-galactosidase (SA-β-gal) activity was examind using the method of cytochemistry.
Results
1. Adult SD male rat were fed with high-fat diet to induce to heperlipemia. After 12 weeks of feeding, the levels of serum triacetylglycerid, total cholesterol and high density lipoprotein cholesterol were elevated in high-fat fed mice compared with normal diet fed mice.
2. No pathological changes were observed in the aorta in control group by HE staining, but the arterial vessels showed pathology changes of atherosclerosis.
3. High-fat diet increased the prevalence of endothelial senescence, SA-β-gal acitivies increased significantly in postdietary challenge.
Conclusion
1. The AS rat model was established succesfully.
2. High-fat diet damaged arterail vessels and caused arterial endothelial cell senescence, and increased senescence-associated SA-β-gal activities in rat.
PartⅢ
The repairing effect of bone marrow-derived EPC and MSC on AS
Objective
In this study, the repairing effect of bone marrow-derived EPC and MSC on AS was investigated.
Methods
1. rAAV2-IRES-GFP was packed and purified, and was used to transduce EPC and MSC.1、packing and purifing rAAV2-IRES-GFP,and EPC and MSC were transferred by rAAV2-IRES-GFP
2. Group A (normal control) was injected with saline; Group B (untreated model control) was injected with saline; Group C EPC transduced by rAAV2-IRES-GFP were transplanted into model rat; Group D MSC transduced by rAAV2-IRES-GFP were transplanted into model rat. After 2 months, the level of blood lipid was tested. The change of aorta histology was observed by HE staining and GFP-labeled cells by frozen section.
3.Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression level of eNOS, ICAM-1 and APOE in artery vessel.
Results
The level of blood lipid in all treated rats decreased substantialy compared with the model group. The EPC and MSC transferred with rAAV2-IRES-GFP were found in the endothelial monolayer of artery vessel of EPC-treated group and MSC-treated group, respectively. The result of HE staining suggested that, the lipid deposits in aortic endothelium in both cell transferred groups were less than those in the untreated group.
RT-PCR suggested that eNOS, apoE expression level of arterial vascular wall in MSC treatment group and EPC treatment group was both significantly higher than untreated control group, but still lower than the normal diet controls. The expression levels of ICAM-1 in treatment group was significantly lower than the pathological control group (p<0.05).
These results showed that the therapeutical effect of EPC on AS was more obvious than that of MSC.
Conclusion
Both bone marrow-derived EPC and MSC had the repairing effect on arterial endothelium of AS, but the therapeutical effect of EPC was more obvious than that of MSC on the damaged arterial endothelium.
引文
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