兰州大学机构库 >基础医学院
VCAM-1+ UC-MSC修复老年性糖尿病心肌损伤的研究
Alternative TitleVCAM-1+ UC-MSC for the repair of diabetic myocardial injury in the elderly
王丽娜
Subtype硕士
Thesis Advisor魏虎来
2023-08-31
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学硕士
Degree Discipline生物化学与分子生物学
Keyword糖尿病心肌损伤,衰老,VCAM-1+ UC-MSC,氧化应激,大鼠 diabetic myocardial injury aging high-activity mesenchymal stem cells oxidative stress rats
Abstract

背景:

机体衰老过程中,心功能会逐渐减退,并发糖尿病时伴随的心肌代谢紊乱和钙紊乱、心肌间质纤维化及冠脉微血管病变等会进一步加重心功能衰弱,诱发糖尿病性心肌病变而致心功能衰弱或丧失,目前尚无有效干预策略。脐带间充质干细胞(Umbilical Cord Mesenchymal stem cells,UC-MSC)为具有自我更新和多向分化等干细胞共性能力的多能干细胞,具备低免疫源性以及较强的免疫调节能力、迁移能力等独特的生物学特性。高表达VCAM-1的脐带间充质干细胞(VCAM-1+ Umbilical Cord Mesenchymal Stem Cells,VCAM-1+ UC-MSC)相较普通UC-MSC在诸多性能方面表现出更高的活力,在心血管疾病和糖尿病的临床治疗中极具前景。

目的:

研究VCAM-1+ UC-MSC对老化糖损伤大鼠心肌细胞(H9c2)和血管内皮细胞(HUVEC)的修复保护效应,以及对老年性糖尿病心肌损伤的治疗作用,并对VCAM-1+ UC-MSC作用的机制进行初步探讨。

方法:

(1)采用特定细胞因子组合(IL-1β + IL-4 + IFN-γ)诱导UC-MSC建立高表达VCAM-1(CD106)的高活性UC-MSC(VCAM-1+ UC-MSC)。

(2)应用博来霉素和高浓度葡萄糖诱导H9c2和HUVEC细胞分别建立了老化糖损伤的H9c2和HUVEC细胞模型。

(3)VCAM-1+ UC-MSC与老化损伤模型细胞共培养,采用EdU染色、细胞周期、集落形成实验检测细胞的增殖情况;细胞划痕和transwell实验检测细胞迁移能力;Annexin V/7-AAD染色检测细胞凋亡水平;线粒体活性氧荧光探针检测线粒体活性氧水平;Western blot技术检测细胞氧化应激信号通路相关蛋白p-Nrf2,Nrf2,KEAP1的表达水平。

(4)采用高脂高糖饲料饲喂,链脲佐菌素注射自然衰老的SD大鼠,建立老年大鼠2型糖尿病及心肌损伤模型。经尾静脉注射VCAM-1+ UC-MSC,观察大鼠一般状态和体重变化;监测大鼠血糖变化及血清胰岛素、总胆固醇和甘油三酯水平;口服糖耐量实验检测大鼠葡萄糖代谢能力;HE染色观察大鼠心脏组织病理改变;ELISA法检测血清中炎性因子的分泌;qRT-PCR检测心脏组织中炎症反应相关基因的表达水平;离体微血管压力直径测定仪检测大鼠心脏主动脉血管内外径及血管收缩和舒张能力的改变情况。

结果:

(1)VCAM-1+ UC-MSC的特性:建立的高活性VCAM-1+ UC-MSC在形态学上与UC-MSC无显著差别,具有显著的成脂、成骨、成软骨分化能力。与UC-MSC比较,VCAM-1+ UC-MSC组织纤维形成能力、血管形成能力、趋化和炎性反应水平均显著增高,同时发现,与UC-MSC相比,VCAM-1+ UC-MSC旁分泌能力增强。

(2)VCAM-1+ UC-MSC对老化糖损伤H9c2和HUVEC细胞的修复保护作用:老化糖损伤H9c2细胞和HUVEC细胞周期阻滞在G0/G1期,S+G2期细胞比例显著减少;细胞增殖能力和迁移能力降低,凋亡细胞显著增加;细胞线粒体活性氧(ROS)显著积聚,氧化应激信号通路相关蛋白p-Nrf2,Nrf2水平显著降低,KEAP1水平显著增高。VCAM-1+ UC-MSC与老化糖损伤H9c2细胞或HUVEC细胞共培养处理后,受损H9c2和HUVEC细胞的细胞周期阻滞改善;增殖和迁移能力增强,细胞凋亡率降低;细胞线粒体ROS含量降低,氧化应激信号通路蛋白P-Nrf2、Nrf2水平增高,KEAP1水平显著降低。上述结果提示VCAM-1+ UC-MSC通过抑制细胞氧化应激通路改善或修复老化糖损伤的H9c2和HUVEC细胞,其作用较UC-MSC更具优势。

(3)VCAM-1+ UC-MSC对老年糖尿病心肌病大鼠的治疗效果:采用高脂高糖饲料饲喂及链脲佐菌素腹腔注射成功建立自然衰老糖尿病心肌病大鼠模型,经尾静脉注射VCAM-1+ UC-MSC进行治疗后,老年糖尿病心肌病大鼠的多饮、多食、多尿症状及体重减低现象得到改善,血糖水平降低,血清胰岛素、总胆固醇和甘油三酯水平降低;葡萄糖代谢能力增强;大鼠心肌组织病理损伤得到改善;血清中促炎因子IL-6和IL-8水平降低,抑炎因子TGF-β水平升高;心脏组织中IL-6和TGF-β基因表达水平降低;心脏组织中氧化应激信号通路相关蛋白P-Nrf2蛋白表达水平降低;大鼠心脏主动脉血管内外径及血管收缩和扩张功能均有所改善。

结论:

高活性VCAM-1+ UC-MSC能够显著保护及修复老化糖损伤H9c2和HUVEC细胞的状态及功能,减轻和改善自然衰老糖尿病心肌损伤大鼠的心脏及血管的损伤及功能。

Other Abstract

Background:

During the aging process, the heart or myocardium gradually experiences reduced function or weakness. When accompanied by diabetes mellitus, myocardial metabolic and calcium disorders, myocardial interstitial fibrosis, and coronary microvascular disease further exacerbate the weakening of myocardial function, leading to diabetic cardiomyopathy and subsequent heart failure or dysfunction. Currently, there are no effective intervention strategies available. Umbilical cord mesenchymal stem cells (UC-MSC) are multipotent stem cells with self-renewal and multidirectional differentiation capabilities. They possess low immunogenicity and unique biological characteristics such as strong immunomodulatory and migratory abilities. Previous studies have found that umbilical cord mesenchymal stem cells with high expression of VCAM-1 (VCAM-1+ UC-MSC) exhibit superior performance and vitality in various aspects, making them highly promising for clinical treatment of cardiovascular diseases and diabetes.

Objective:

The objective of this study is to investigate the reparative and protective effects of VCAM-1+ UC-MSC on myocardial cells (H9c2) and endothelial cells (HUVEC) with glucose-induced damage. Additionally, we aim to explore the therapeutic potential of VCAM-1+ UC-MSC in the treatment of myocardial damage in elderly patients with diabetes mellitus. Furthermore, we aim to preliminarily investigate the mechanisms of action of VCAM-1+ UC-MSC.

Methods:

(1) VCAM-1+ UC-MSC with high expression of VCAM-1 (CD106) were established by inducing UC-MSC with a specific combination of cytokines (IL-1β + IL-4 + IFN-γ).

(2) H9c2 and HUVEC cells were induced with bleomycin and high concentration glucose, respectively, to establish the aging glucose-induced damage models of H9c2 and HUVEC cells.

(3) VCAM-1+ UC-MSC were co-cultured with the aging damage model cells. Cell proliferation was assessed by EdU staining, cell cycle analysis, and colony formation assay. Cell migration ability was evaluated by scratch assay and transwell assay. Cell apoptosis level was measured by Annexin V/7-AAD staining. Mitochondrial reactive oxygen species (ROS) level was detected using a mitochondrial ROS fluorescent probe. The expression levels of oxidative stress signaling pathway-related proteins p-Nrf2, Nrf2, and KEAP1 were analyzed by Western blot.

(4) Aging SD rats with type 2 diabetes and myocardial damage were established by feeding a high-fat high-sugar diet and injecting streptozotocin. VCAM-1+ UC-MSC were intravenously injected into the rats. General condition and body weight changes of the rats were observed. Blood glucose levels and serum insulin, total cholesterol, and triglyceride levels were monitored. Oral glucose tolerance test was performed to assess glucose metabolism in the rats. Histopathological changes in the heart tissue were examined by HE staining. Secretion of inflammatory factors in the serum was measured by ELISA. Expression levels of inflammation-related genes in the heart tissue were determined by qRT-PCR. Changes in the diameter and contractile and relaxant abilities of the aorta were assessed using an ex vivo microvessel pressure diameter analyzer.

Results:

(1) Characteristics of VCAM-1+ UC-MSC: The established highly active VCAM-1+ UC-MSC showed no significant differences in morphology compared to UC-MSC, but demonstrated significant adipogenic, osteogenic, and chondrogenic differentiation abilities. In comparison to UC-MSC, VCAM-1+ UC-MSC exhibited significantly increased tissue fibrogenesis, angiogenesis, chemotaxis, and inflammatory response levels. Additionally, it was observed that VCAM-1+ UC-MSC had enhanced paracrine secretion capability compared to UC-MSC.

(2) Protective effects of VCAM-1+ UC-MSC on aging glucose-damaged H9c2 and HUVEC cells: Aging glucose-damaged H9c2 cells and HUVEC cells showed cell cycle arrest at G0/G1 phase with a significant decrease in the proportion of cells in S+G2 phase. They also exhibited reduced proliferative and migratory capacities, and increased apoptosis. Accumulation of reactive ROS in cell mitochondria was observed, along with a significant decrease in the levels of oxidative stress signaling proteins p-Nrf2 and Nrf2, and a significant increase in the level of KEAP1.Co-culturing VCAM-1+ UC-MSC with aging glucose-damaged H9c2 or HUVEC cells resulted in improved cell cycle arrest in damaged H9c2 and HUVEC cells. Proliferative and migratory capacities were enhanced, and apoptosis rates were reduced. Mitochondrial ROS levels were reduced, and the levels of oxidative stress signaling proteins P-Nrf2 and Nrf2 were increased, while the level of KEAP1 was significantly decreased. These results suggest that VCAM-1+ UC-MSC exert a superior effect, compared to UC-MSC, in improving or repairing aging glucose-damaged H9c2 and HUVEC cells through inhibition of the cell oxidative stress pathway.

(3) Therapeutic effect of VCAM-1+ UC-MSC on aging diabetic cardiomyopathy rats: The natural aging diabetic cardiomyopathy rat model was successfully established by feeding with a high-fat high-sugar diet and streptozotocin. After treatment with VCAM-1+ UC-MSC via tail vein injection, the symptoms of polydipsia, polyphagia, polyuria, and weight loss in aging diabetic cardiomyopathy rats were improved. Blood glucose levels decreased, and serum insulin levels, total cholesterol levels, and triglyceride levels also decreased. Glucose metabolism capacity was enhanced. Pathological damage in cardiac tissues of the rats was improved. Serum levels of pro-inflammatory cytokines IL-6 and IL-8 decreased, while the anti-inflammatory cytokine TGF-β level increased. Gene expression levels of IL-6 and TGF-β in cardiac tissues decreased. The expression level of oxidative stress signaling pathway-related protein P-Nrf2 in cardiac tissues decreased. The inner and outer diameters of the rat's cardiac aorta, as well as vascular contraction and dilation function, were improved.

Conclusions:

Highly active VCAM-1+ UC-MSC significantly protects and repairs the damage and function of aging glucose-damaged H9c2 and HUVEC cells, improving the damage and function of the heart and blood vessels in naturally aged diabetic rats.

 

Keywords: diabetic myocardial injury, aging, high-activity mesenchymal stem cells, oxidative stress, rats

MOST Discipline Catalogue理学 - 生物学 - 生物化学与分子生物学
URL查看原文
Language中文
Other Code262010_220200924640
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/538022
Collection基础医学院
Affiliation
兰州大学基础医学院
Recommended Citation
GB/T 7714
王丽娜. VCAM-1+ UC-MSC修复老年性糖尿病心肌损伤的研究[D]. 兰州. 兰州大学,2023.
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