丁苯酞软胶囊治疗脑小血管病的临床研究

目的探讨丁苯酞软胶囊治疗脑小血管病的临床疗效。方法选取2016年3月—2018年2月在天津中医药大学第一附属医院就诊的脑小血管病患者100例,随机分为对照组(50例)和治疗组(50例)。对照组给予常规治疗,治疗组在此基础上口服丁苯酞软胶囊,2粒/次,3次/d,持续治疗2个月。观察两组患者临床疗效,同时比较治疗前后两组患者NIHSS、Fugl-Meyer量表、MBI评分,反应性充血指数(RHI)和内皮祖细胞(EPCs)数以及细胞因子水平。结果治疗后,对照组临床有效率为60.0%,显著低于治疗组的76.0%,两组比较差异具有统计学意义(P0.01)。治疗后,两组NIHSS评分显著降低,FMA-UE、FMA-LE、MBI评分显著升高,同组治疗前后比较差异具有统计学意义(P0.05、0.01);且治疗后治疗组上述评分均显著优于对照组,两组比较差异具有统计学意义(P0.05、0.01)。两组RHI、EPCs均较治疗前明显升高,同组治疗前后比较差异具有统计学意义(P0.05);且治疗后治疗组RHI、EPCs明显高于对照组,两组比较差异具有统计学意义(P0.05)。治疗后,两组患者血清ET-1、v WF水平均显著降低,NO水平显著升高,同组治疗前后比较差异具有统计学意义(P0.05、0.01);且治疗后治疗组上述血清细胞因子水平显著优于对照组,两组比较差异具有统计学意义(P0.05、0.01)。结论丁苯酞软胶囊能显著改善脑小血管病患者的运动功能和生活自理能力,增加外周血EPCs数目、有效调节ET-1/NO平衡,具有一定的临床应用价值。     

雌激素干预的EPCs移植对糖尿病缺血性脑卒中大鼠的治疗作用

目的 探讨雌激素干预的EPCs移植对糖尿病缺血性脑卒中的治疗作用。方法 制备大鼠糖尿病缺血性脑卒中模型,24 h后通过尾静脉移植经PKH26标记的糖尿病EPCs和雌激素干预的糖尿病EPCs。移植3 d后测定各组大鼠脑缺血体积、大鼠行为学变化、缺血部位血管新生及EPCs的归巢情况。结果 与糖尿病脑缺血大鼠相比,雌激素干预的糖尿病EPCs移植能降低脑缺血体积、改善行为学评分和缺血部位的血管新生及促进EPCs归巢到损伤部位(P<0.05)。结论 雌激素干预的糖尿病EPCs移植,通过促进EPCs归巢和血管新生对糖尿病缺血性脑卒中有较好的治疗作用。     

Efficient differentiation of vascular endothelial cells from dermal-derived mesenchymal stem cells induced by endothelial cell lines conditioned medium

Objective

To directionally-differentiate dermis-derived mesenchymal stem cells (DMSCs) into vascular endothelial cells (VECs) in vitro, providing an experimental basis for studies on the pathogenesis and treatment of vascular diseases.

反式载脂蛋白A1模拟肽R-D4F对内皮祖细胞黏附及迁移功能的影响

目的探讨反式载脂蛋白A1模拟肽R-D4F对人内皮祖细胞黏附及迁移功能的影响。方法取新鲜人脐静脉血,采用密度梯法从中分离、诱导培养人内皮祖细胞,并使用Dil-ac-LDL和FITC-UEA-1染料进行双荧光染色以及流式细胞术进行鉴定。分别给于牛血清白蛋白、顺式D-4F(50μg/ml)及R-D4F(5~50μg/ml)处理后,采用黏附能力测定实验及Transwell小室观察内皮祖细胞黏附及迁移能力的变化。结果与牛血清白蛋白对照组相比,D-4F显著增强内皮祖细胞的黏附及迁移功能。相似的是,R-D4F呈剂量依赖性地增强内皮祖细胞的黏附与迁移。结论 R-D4F具有与D-4F相似的生物学作用,可促进人内皮祖细胞的黏附及迁移。     

Proteomic analysis shows differential protein expression in endothelial progenitor cells between healthy subjects and ischemic stroke patients

Abstract

Objective: An increase in the circulating concentration of endothelial progenitor cells (EPCs) is associated with a better outcome in patients with acute ischemic stroke. Likewise, EPCs are heterogeneous cells, with functional differences and different protein expressions. Our objective was to compare protein expressions of EPCs from ischemic stroke patients and healthy subjects.

Methods: Eleven ischemic stroke patients and 11 healthy subjects, matched by age and gender, were included in this study. EPC colonies were defined as early outgrowth colony forming unit-endothelial cell. Cells were lysed and proteins were purified and separated on two-dimensional gels. Gel images were analyzed using the PDQuest software and protein differences between EPCs from ischemic stroke patients and healthy subjects were identified by mass spectrometry. Results were finally validated by western blot.

Results: Proteomic analysis revealed three qualitative differences between EPCs from healthy subjects and ischemic stroke patients. Two of them, endoplasmatic reticulum protein-29 and CdC-42, were only expressed in EPCs from healthy subjects, whereas elongation factor-2 was only identified in EPCs from ischemic stroke patients. Furthermore, we identified one protein, peroxiredoxin-1, whose expression was 10 times stronger in ischemic stroke patients than in healthy subjects. Western blot analysis showed greater expression of endoplasmatic reticulum protein-29 in EPCs from healthy subjects and elongation factor-2 and peroxiredoxin-1 in EPCs from ischemic stroke patients.

Conclusion: Proteomic analysis showed differences in protein expressions of EPCs from ischemic stroke patients and healthy subjects that may be involved in mechanisms related to functional impairment.     

姜黄素对兔颈动脉斑块稳定性及EPCs活力的影响

目的:观察姜黄素对颈动脉斑块稳定性及内祖皮细胞(EPCs)的影响。方法:将30只新西兰兔随机分为空白对照组、模型组及姜黄素组,各10只,其中空白对照组予普通饲料喂养,模型组予喂养高脂饲料,姜黄素组予喂养高脂饲料+100 mg/d姜黄素。连续喂养12周后HE染色法观察3组西兰兔主动脉内膜斑块厚度、外周血总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)水平以及利用MTT法测定人纤维EPCs细胞迁徙及增殖活性。结果:1)血脂水平:与空白组比较,模型组及姜黄素组TC、TG及LDL-C均升高(P0.05),HDL-C降低(P0.05),其中姜黄素干预后上述指标有明显改善(P0.05)。2)形态学变化:空白对照组颈动脉壁厚薄均匀,结构均匀,模型组及姜黄素均出现动脉管腔狭窄,动脉内膜增厚,其中姜黄素组较模型组改善。3)EPCs增殖情况:与空白组比较,模型组及姜黄素OD值均降低(P0.05),与模型组比较,姜黄素OD有所上调(P0.05)。4)EPCs迁徙能力:空白对照组各时间点划痕愈合率均显著高于其余2组(P0.05),其中姜黄素组各时间点划痕愈合率均高于模型组(P0.05)。结论:姜黄素由明显抗动脉粥样硬化作用,其作用机制可能与促进EPCs增殖及迁徙有关。     

Kinase inhibitors for cardiovascular disease

Over the last decade, there has been substantial progress toward understanding the pathophysiology and treatment of cardiovascular diseases (CVDs). Elucidating cellular responses to the extracellular environment and signal transduction mechanisms have provided the opportunity to explore novel molecular therapeutic approaches for the treatment of CVDs. Neurohormonal stimulation has been implicated in these diseases; blockade of the renin-angiotensin and beta-adrenergic systems are examples of therapeutic effectiveness. There are multiple cell signaling cascades, some of which are beneficial or compensatory and others deleterious. The balance between these pathways, which in large part is dictated by the cellular environment, determines the outcome as a diseased or non-diseased state. Selective targeting of signaling pathways using protein kinase inhibitors, would have a potential advantage over receptor blockers. We review potential protein kinase targets and recent evidence supporting therapeutic interventional value in CVDs.     

内皮祖细胞对骨髓间充质干细胞－种植体复合物成骨能力的影响

目的：研究内皮祖细胞（EPCs）对骨髓间充质干细胞（BMSCs）膜片－种植体复合体成骨能力的影响。方法：分离培养大鼠 BMSCs 并采用细胞膜片技术培养细胞膜片;分离培养大鼠骨髓来源 EPCs;将 EPCs 加载于 BMSCs 细胞膜片上并检测相关基因;制作细胞膜片－种植体复合体并植入裸鼠皮下,8周取材,行 Micro-CT 检查及组织学检测。结果：体外实验显示EPCs 加载于 BMSCs 细胞膜片后成骨相关 Runx-2、ALP、BMP2和成血管相关 VEGF 的基因表达提高;裸鼠体内实验取材 Mi-cro-CT 检查显示加载 EPCs 组成骨量较高,HE 切片显示加载 EPCs 组成骨面积较多,免疫组化检测显示加载 EPCs 组 Runx2、BMP2表达较高。结论：加载 EPCs 能提高 BMSCs-种植体复合物的成骨能力。     

流体剪切应力对晚期内皮祖细胞生物学功能的影响

目的 研究流体剪切应力处理对晚期内皮祖细胞（endothelial progenitor cells,EPCs）体外及体内生物学功能的影响。 方法 密度梯度离心法分离大鼠骨髓单核细胞,应用EGM-2MV进行体外培养。以3~4代的EPCs,即晚期EPCs为靶细胞,对其施以1.2 Pa剪切应力处理。采用EdU标记技术、黏附能力测定实验、改良的Boyden小室、Annexin V/PI、β 半乳糖苷酶检测法、Matrigel法、荧光定量RT PCR等方法分别检测剪切应力对晚期EPCs增殖、黏附、迁移、凋亡、衰老、体外成血管及VEGF mRNA表达等生物学功能的影响。应用大鼠颈动脉损伤模型及细胞原位移植等实验手段检测剪切应力预处理对晚期EPCs修复受损内皮的影响。结果1.2 Pa剪切应力处理可不同程度提高晚期EPCs的增殖、黏附、迁移及成血管能力（P＜0.01）,上调VEGF的基因表达,抑制晚期EPCs的衰老及凋亡（P＜0.01）;移植经剪切应力预处理的晚期EPCs可加速损伤内皮的修复,减缓内膜的增生。结论 流体剪切应力可改善晚期EPCs的功能活性,提高晚期EPCs修复损伤血管内皮的能力, 这为EPCs的临床应用及剪切应力介导的细胞疗法提供了实验依据。     

Impaired development and dysfunction of endothelial progenitor cells in type 2 diabetic mice

AimDysfunction of circulating endothelial progenitor cells (EPCs) has been shown to affect the development of microvascular diseases in diabetes patients. The aim of this study was to elucidate the development and mechanical dysfunction of EPCs in type 2 diabetes (T2D).MethodsThe colony-forming capacity of EPCs and differentiation potential of bone marrow (BM) c-Kit(+)/Sca-I(+) lineage-negative mononuclear cells (KSL) were examined in T2D mice, db/db mice and KKA^y mice, using EPC colony-forming assay (EPC-CFA).ResultsT2D mice had fewer BM stem/progenitor cells, and proliferation of KSL was lowest in the BM of db/db mice. In T2D mice, the frequency of large colony-forming units (CFUs) derived from BM-KSL was highly reduced, indicating dysfunction of differentiation into mature EPCs. Only a small number of BM-derived progenitors [CD34(+) KSL cells], which contribute to the supply of EPCs for postnatal neovascularization, was also found. Furthermore, in terms of their plasticity to transdifferentiate into various cell types, BM-KSL exhibited a greater potential to differentiate into granulocyte macrophages (GMs) than into other cell types.ConclusionT2D affected EPC colony formation and differentiation of stem cells to mature EPCs or haematopoietic cells. These data suggest opposing regulatory mechanisms for differentiation into mature EPCs and GMs in T2D mice.