ICAM-1介导的脉冲电刺激下血管内皮细胞与祖细胞粘附的实验研究
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摘要
研究背景与目的
生物电是人体的固有属性,伴随着每个生命活动的完成；细胞是机体基本组成单位,细胞膜内外某些带电离子分布和浓度不同以及细胞膜对不同离子的选择通透性,正是细胞、组织形成跨膜电位的条件,也是生物电的起源。正常情况下,细胞间紧密连接的存在可使跨膜电位均等分布于相应组织当中,组织一旦损伤,完整的细胞间紧密连接不复存在,损伤处局部跨膜电位即被削弱,健侧部位的高电势和损伤病灶的低电势随即形成一内生电场。由于内生电场激活了病变组织细胞及其周围细胞修复信号的传递,可诱导机体产生损伤修复效应,因此,如何深化认识内生电场修复作用并加以利用则显得十分重要。
基于此背景,生物电刺激在血管再生领域中的应用已经积累了一定的数据,有关策略主要围绕以下两个方面：第一,外部施加生物电刺激直接促进机体内血管再生,相当于弥补并强化内生电场诱导产生的修复效应；第二,以构建组织工程化血管为中心,通过生物电刺激的干预,从排列、粘附和抗性等方面增强种子细胞与生物材料的整合。由于血管内皮细胞覆盖所有类型血管并密切接触血流,同时也是新生血管形成的首要环节,故目前电刺激研究多集中于血管内皮细胞功能学的改变。然而,研究表明仅仅依赖于血管内皮增殖或出芽等原位修复并不能满足重建血管、改善血流的需要,内皮祖细胞(Endothelial progenitor cell, EPC)的发生发展已被证实对成体血管再生有着不可替代的作用,新生血管形成的研究方向逐渐由传统的血管形成过渡至EPC介导的血管发生。那么EPC将如何参与到电刺激促新生血管形成过程?该问题的探讨十分有意义。
随着EPC探索的深入,EPC研究已由改良培养方法和分析增殖分化能力为主的策略演变为对细胞归巢信号的重点探索。EPC归巢,指在缺血损伤的应激代偿反应中,EPC因缺血部位趋化因子的诱导而产生动员和定向迁移,然后在血管内皮层上缓慢移动,继而与血管内皮产生紧密粘附并完成跨内皮转移,最后通过直接分化和分泌等形式来完成修复反应。经证实,EPC与血管内皮之间的粘附在整个归巢过程当中起着承上启下的作用,已知多对粘附分子组合参与此调节过程。其中细胞间粘附分子-1 (Intercellular adhesion molecule, ICAM-1)属于免疫球蛋白超家族成员,其在血管内皮细胞膜上的表达不仅介导了细胞间的粘附,更重要的是通过这种结合,可将外界损伤刺激信号沿着EPC膜上配体整合素αvβ2,尤其是整合素β2,传递至EPC胞内,然后启动后续生物学信号的转导,在粘附分子调控的归巢过程当中最为关键。那么血管内皮在适宜电刺激的调节下能否增强自身与EPC的粘附?电刺激促进血管内皮捕获循环EPC的机制又是否与ICAM-1的上调有关?国内外未见同类型报道,这是开展本课题研究的主要目的。
为验证我们的设想,本课题研究分为三个部分：
(1)自主研制具有适合刺激形式的电刺激仪,获得人脐静脉内皮细胞(Human umbilical vein endothelial cell, HUVEC)并建立细胞培养电刺激模型,首先评价电刺激下培养体系的稳定性、HUVEC形态学和增殖活性的改变；
(2)诱导培养外周血EPC并进行鉴定,通过荧光标记EPC以衡量电刺激下HUVEC与EPC之间粘附强度的改变；
(3)检测电刺激下HUVEC ICAM-1的表达水平,以期探索电刺激影响HUVEC捕获EPC的可能机制。
方法
(1)电刺激仪的研制与评价
联合生物医学工程学院医学工程系,自主研制脉冲输出形式的电刺激仪；固定刺激脉宽和频率为最大值,伏安法检测不同电压幅度刺激下2h内培养液电阻,并且探测局部温度的变化；
(2)电刺激对HUVEC形态学以及增殖活性的影响
胶原酶消化法分离HUVEC,结合形态学和Ⅷ因子相关抗原对其鉴定；将HUVEC分为不同参数刺激组和常规培养组,刺激24h后观察HUVEC形态学变化,MTS法比较HUVEC增殖活性的差异；
(3)电刺激对HUVEC与EPC之间粘附强度的影响
密度梯度离心法分离外周血单个核细胞,专用培养基直接诱导培养外周血EPC,于第9天进行免疫荧光鉴定(乙酰化低密度脂蛋白和荆豆类凝集素)以及表面标志流式分析(CD34、CD133、CD31、VEGFR2和CD14)；
取第9天EPC,活性荧光染料CFSE标记后接种在单层HUVEC上,不同参数电刺激培养24h,显微镜下观察贴壁荧光标记EPC数量,荧光酶标仪读取细胞荧光强度,以荧光比率衡量粘附强度。
(4)电刺激对HUVEC ICAM-1表达的影响
24h培养结束后分别收集不同参数电刺激组和常规培养组细胞的总RNA,采用Real time PCR技术中的SYBR Green染料法,运用2-ΔΔCt法对各实验组中HUVEC ICAM-1 mRNA相对表达量进行分析。
(5)统计学方法：
数据以均数±标准差(x±s)表示,表面标志阳性率比较采用配对t检验,多组间不同时间点电阻比较采用重复测量数据的方差分析,多组间温度比较、荧光比率比较采用完全随机设计方差分析,多组间细胞增殖活性比较、基因表达量比较采用随机区组设计的方差分析：方差齐时采用Fisher法,多重比较用Bonferroni法；方差不齐时则用Welch近似法,多重比较用Dunnett T3法,P<0.05视为具有显著性差异,采用SPSS13.0统计软件计算。
结果
(1)电刺激仪的研制与评价
自主研制电刺激仪可提供幅度(0-±40V)、频率(0.01-10 Hz)、脉宽(0.1-24 ms)均可调的双相脉冲输出,配合C-Dish电极板使用共同构成细胞培养电刺激模型。固定最大脉宽和最大频率,采用伏安法测得2h内不同刺激电压强度下(0V、5V、10V、20V、和40V)培养液电阻存在显著性差异(F=174.14,P=0.000),电压强度与刺激时间存在交互效应(F=74.12,P=0.000),不同电压幅度下培养液电阻在不同刺激时间点的变化趋势不同。培养液电阻和局部温度均以10V以内电压幅度相对稳定,表示此范围内电解反应恒定,可在此基础上进行后续细胞学实验。
(2)电刺激对HUVEC形态学以及增殖活性的影响
取第2或第3代HUVEC进行实验,选择10V内不同刺激参数组合,首先通过观察刺激24h后HUVEC形态学变化以进一步缩窄刺激范围,结果发现在5V/5Hz/9ms刺激下细胞主要表现为体积增大、更扁平、长梭形伸展,核周深染区牵拉细长,与细胞长轴一致,并且可随着任一刺激参数的增高而出现空泡变性、甚至脱落；随后固定脉宽、频率分别为5V和5Hz,采用MTS法检测不同脉宽(0ms、1ms、3ms、6ms、9ms)刺激下HUVEC增殖活性,以排除增殖活性差异对粘附强度变化造成的干扰,各组OD值分别为1.457、1.471、1.469、1.461和1.458,尚不能认为各实验组间HUVEC增殖活性存在显著性差异(F=1.822,P=0.218)。
(3)电刺激对HUVEC与EPC之间粘附强度的影响
外周血EPC呈长条状分布,整体密度和状态均以第9天最佳,可同时吞噬乙酰化低密度脂蛋白和结合荆豆类凝集素,Ⅷ因子相关抗原逐渐增强,表面标志CD34、CD133、CD31、VEGFR2、CD14阳性率分别为0.19±0.06%,1.67±0.52%,61.56±5.57%,70.29±7.37%,89.31±4.11%,属于正在分化的EPC,符合早期EPC范畴；活性荧光染料CFSE标记第9天收集的EPC,荧光法分析不同脉宽刺激下HUVEC与EPC粘附强度的变化。经换算,常规培养组、1ms、3ms、6ms和9ms刺激组荧光比率分别为0.441、0.470、0.489、0.557和0.370,各实验组间荧光比率存在显著性差异(F=62.165,P=0.000),与常规培养组相比,6ms刺激组增高最显著,可见适宜双相脉冲电刺激在一定程度上有利于HUVEC与EPC之间的粘附。相反,9ms刺激组荧光比率下降,显著低于常规培养组水平。
(4)电刺激对HUVEC ICAM-1表达的影响
Real time PCR检测结果显示,与常规培养组相比,lms、3ms、6ms和9ms刺激组相对表达量分别为1.23、2.42、3.79和1.90,不同脉宽刺激下HUVEC ICAM-1 mRNA相对表达量存在显著性差异(F=17.706, P=0.000),并在一定范围内随着脉宽的延长而增高,其中6ms刺激组表达量增高最显著,9ms刺激组呈现下降迹象,但仍高于正常水平。
结论
(1)成功研制出具有双相脉冲输出的电刺激仪,并且建立了相对稳定的细胞培养刺激模型,在固定5V和5Hz的前提下,HUVEC形态改变明显,呈扁平长梭形伸展,但其增殖活性并不随着刺激脉宽的延长(0、1、3、6、9ms)而改变；
(2)成功从外周血单个核细胞中直接诱导培养出EPC,经鉴定,细胞符合正在分化的早期EPC表型；
(3)双相脉冲电刺激在一定范围内有利于HUVEC与EPC之间的粘附,其中以5V/5Hz/6ms组粘附增强最为显著；
(4)双相脉冲电刺激可上调HUVEC ICAM-1 mRNA相对表达量,变化规律与细胞间粘附实验一致,同样以5V/5Hz/6ms作用最显著,提示HUVEC ICAM-1表达上调可能是电刺激促进HUVEC捕获EPC的机制之一。
创新点
(1)前期研究中多采用直流电形式对HUVEC等血管内皮细胞进行干预,有关脉冲电刺激研究报道不多,本研究在国内外较早采用正负双相脉冲形式对HUVEC进行干预,为电刺激下血管再生的体外研究提供新的实验方法。
(2)本研究首次将内皮祖细胞整合到电刺激调节血管内皮细胞功能的研究中,明确了5V/5Hz/6ms双相脉冲电刺激可增强HUVEC与EPC的粘附,为电刺激促进新生血管形成的研究提供新的理论依据,国内外尚未见同类型报道。
(3)本研究初步证实在5V/5Hz/6ms的双向脉冲电刺激下,HUVEC捕获EPC增多的机制与ICAM-1的表达上调有关,再次肯定ICAM-1粘合识别对于EPC归巢过程的重要性,也为其他防治手段下EPC的归巢信号机制提供借鉴。
Background and objective
Bioelectricity is the inherent propertiy of human body along with the completion of vital movement. Different types and concentration ion could exchange between cells and microenvironment due to the selective permeability of cell membranes. This is so called condition of membrane potential, also origin of bioelectricity. Under normal circumstance, tight junction between cells maintains an equal distribution of membrane potential. Once sufferring from wound, intact tight junction no longer exits, resulting in a weak or collapse local potential. Therefore, the potential gradient between normal region and wound will establish an endogenous electric field, which could induce cells to repair around the lesion. According to this, it is important to deepen our understanding and make good use of this electrical field.
Based on the bioelectricity and the effect of endogenous electric field, electrical stimulation in vascular repair has been reported in the past decade and two main strategies will be the right direction. First, an external electrical stimulation is used to promote vascular regeneration directly, equivalent to adjust or strengthen the endogenous electrical effect. Second, taking vascular tissue engineering into consideration, integration of seed cells and the biomaterial through arrangement、adhesion and resistance is also being studied under electrical stimulation. Because vascular endothelial cell covers all of the blood vessel, intimately contacts with the blood flow and presents as the first step of angiogenesis, preliminary studies have pay more attention to the function of endothelial cell under electric field. However, recent researches show that it is difficult to meet the need of vascular reconstruction only depending on endothelial cell proliferation or sprouting in situ. Endothelial progenitor cell (EPC) has been proven to be irreplaceable in the adult neovascularization. It is indicated that research direction has been changed from traditional angiogenesis to vasculogenesis mediated by EPC. Therefore, to discuss how EPC participates in neovascularization through electrical stimulation is very meaningful.
With a rapid development of EPC study, many researchers have been focus on EPC homing instead of EPC induction、proliferation and differentiation. There are several steps in EPC homing, including cell mobilization and direct migration due to cytokines secreted from ischemia, rolling and firm adhesion with endothelium, transendothelial transfer and finally direct differentiation or emiocytosis to complete the repair process. In these steps, adhesion between EPC and endothelial cell is the most important part in the homing process and three pair adhesion molecules are involved, making a close link between EPC chemotaxis and functioning. Intercellular adhesion molecule-1 (ICAM-1 or CD54) belongs to immunoglobulin superfamily and responses for maintenance of physiological function, pathogenesis and wound healing. Its uniform ligand is integrinαvβ2, which is also called CD 11 a/CD 18 compounds. Expression of ICAM-1 in endothelial cell do mediates EPC adhesion, but more importantly, under combination with the integrinαvβ2 in EPC, especially integrinβ2, the external stimulation signal will be passed from vascular lesion to EPC, and then switch on the EPC repair process. Consequently, our research is going to find out whether electrical stimulation could do well in the adhesion between endothelial cell and EPC? And ICAM-1 up regulation could be related to the mechanism of endothelial cell capturing more EPC under electrical stimulation?
To investigate the change and mechanism of adhesion between endothelial cell and EPC under electrical stimulation, our research is divided into three parts.
(1)We are going to make the suitable electrical stimulator by ourselves and establish a stimulate culture model. In order to find out a safe and effective stimulate interval, we first conduct the evaluation including stability of culture system, morphologic and proliferation of HUVEC.
(2)We induce EPC from periphery blood and make phenotype identification. To assess the change of HUVEC-EPC adhesion, we label the EPC with fluorchrome and quantify by fluorescence intensity after co-culture with HUVEC monolayer under stimulation.
(3)To explore the possible mechanism of HUVEC capturing EPC under stimulation, we detect the expression of ICAM-1 in HUVEC which plays an important role in HUVEC-EPC adhesion.
Methods
(1)Development and evaluation of electrical stimulator
Electrical stimulator with a pulse output was made by our cooperation with school of biomedical engineering, Southern Medical University. With a fixed and maximum pulse and frequency, we assessed medium resistance using voltammetry and detected temperature of culture system under different voltage stimulation within 2h.
(2)Morphology and proliferation changes of HUVEC under different stimulation
HUVEC was digested by collagenase from human umbilical cord and identified with the morphology andⅧfactor related antigen. Then they were divided into different stimulation groups and conventional culture group. After 24h stimulation, morphology of HUVEC was observed and the proliferation activity was analyzed by MTS.
(3)egulation on HUVEC-EPC adhesion under different stimulation
Periphery blood mononuclear cells were isolated from periphery blood using density gradient centrifugation. They were seed in the special endothelial culture medium and would be induced to EPC after 9 day. Then EPC identification was determined by immunofluorescence, with a staining of Ulex europaeus lectin I and acetylated low-density lipoprotein. Phenotypic analyses of the EPC were performed by flow cytometry, including CD34, CD133, CD31, VEGFR2 and CD14.
We collected EPC at day 9 and labeled them with fluorochrome CFSE. The labeled EPC were seed in the HUVEC monolayer and stimulated for 24h under different pulse. To measure adhesion between HUVEC and EPC, CFSE-EPC adhered to monolayer was discriminated in fluorescence microscopy and quantified by fluorescence micro plate reader. An index of fluorescence ratio was used to indicate the adhesion intensity.
(4)Regulation on ICAM-1 expression of HUVEC under different stimulation
Total RNA was collected from different groups where HUVEC had been stimulated for 24h and then ICAM-1 mRNA was detected by SYBR Green staining, one of the methods about real time PCR. Finally, ICAM-1 mRNA relative expression was analyzed using 2-ΔΔCt.
Statistical analysis
Data was showed as mean±standard deviation, surface markers between two group were compared using paired Sample T test, resistance at different time points between groups were compared using repeated measurement of variance analysis, temperature and fluorescence ratio between different groups were compared using one-way ANOVA, proliferation index and relative gene expression were compared using two-way ANOVA. Once there was significant difference between groups, two methods of multiple comparison were selectively used, Bonferroni for equal varicances assumed while Dunnett T3 for not assmed. P<0.05 was consider to be significant difference and all the data was analyzed by SPSS13.0 software.
Results
(1)Development and evaluation of electrical stimulator
Our self-made stimulator could provide an biphasic pulse, including adjustable voltage(0-40V)、frequency(0.01～10Hz) and pulse width (0.1-24ms). The simulated model was set up with the combination of C-Dish electrode. With a fixed and maximum pulse width and frequency, there was a significant difference in medium resistance detected by voltammetry among different voltage groups(0V,5V,10V, 20V and 40V)(F=174.14, P=0.000). Moreover, there was a significant interaction between voltage and stimulation time, suggesting a different trend of different time under different voltage. According to the analysis, both the resistance and temperature in 10V group was relative stable, indicating a stable electrolysis reaction during our stimulation process indirectly.
(2)Morphology and proliferation changes of HUVEC under different stimulation
To further select the suitable parameters, we first chose the passage 2 or 3 of HUVEC for stimulation within 10V. After various selection, typical morphological changes of HUVEC was present in 5V/5Hz/9ms, including flat and fusiform shape, deeply stained peri-nuclei zone stretched to the cell long axis. Then we fixed the frequency and voltage at 5V and 5Hz, utilized MTS to detect the proliferation of HUVEC under different pulse width. The OD values in 0ms,1ms,3ms,6ms,9ms group were 1.457,1.471,1.469,1.461 and 1.458 respectively, showing that no significant difference was among them (F=1.822, P=0.218).
(3)Regulation on HUVEC-EPC adhesion under different stimulation
EPC induced from periphery blood was present as spindle shape. They could uptake acLDL and exhibit UEA lectin binding capability simultaneously at day 9, also with an enhanced synthesis ofⅧfactor related antigen. The expression profile of EPC at day 9 were CD34(0.19%±0.06%), CD133(1.67%±0.52%), CD14(89.31%±4.11%), CD31(61.56%±5.57%) and VEGFR2(70.29%±7.37%). All these showed that adhesion cells presented as differentiating early EPC.
EPC from day 9 was collected for CFSE labeling. Fluorometric method was used to detect the fluorescence intensity of adhesion CFSE-EPC in different pulse width. Results showed that fluorescence ratio were 0.441,0.470,0.489,0.557 and 0.370 in 0ms,1ms,3ms,6ms and 9ms group respectively. There was a significant difference among these groups and 6ms group was the best of all, indicating that suitable electrical stimulation could benefit for the HUVEC-EPC adhesion to some extent. In contrast, the ratio of 9ms group decreased significantly, even lower than the control group.
(4)Regulation on ICAM-1 expression of HUVEC under different stimulation
Compared with control group by real time PCR, ICAM-1 mRNA expression of HUVEC in different groups, including lms,3ms,6ms and 9ms were 1.23,2.42,3.79 and 1.90 respectively. Significant difference was present among them (F=17.706, P =0.000), indicating that up regulation expression was follow with an extension of pulse width. Taking 6ms group in particular, its expression was the significantly highest while there was a decreasing trend in 9ms group.
Conclusion
(1)It was successful to make an electrical stimulator with a biphasic pulse and set up a safe stimulated model. There is no change in proliferation of HUVEC but a typical change of morphology from 0ms to 9ms. HUVEC presented as more flat and spindle.
(2)EPC was induced from PBMC successfully and it belonged to differentiating early EPC according to the phenotype.
(3)Biphasic electrical stimulation was benefit for the HUVEC-EPC adhesion and 5V/5Hz/6ms would be the best.
(4)Electrical stimulation could up regulate the mRNA expression of ICAM-1. Its change was consistent with the trend of HUVEC-EPC adhesion. It is suggest that electrical stimulation contributing to HUVEC-EPC adhesion should be related to the up regulation of ICAM-1 expression.
Point of innovation
(1)Regulation on the endothelial cell using direct current have been reported in previous studies and there were few literatures about pulse stimulation. Our research is successful to regulate HUVEC under biphasic pulse stimulation. It will provide a novel method to the future studies about vascular regeneration through electrical stimulation.
(2)We are the first to make integration between EPC and endothelial cell under 5V/5Hz/6ms electrical stimulation. The stimulated strengthen HUVEC-EPC adhesion is probably a new theory about electrical stimulation contributing to neovascularization. So far, there is no report about this concept.
(3)Our research has suggested that up regulation of ICAM-1 may be responsible for the mechanism of endothelial cell capturing more EPC under electrical stimulation. These will help us to reaffirm the importance of ICAM-1 for EPC homing and provide a good reference for EPC homing under other stimulation methods.
生物电是人体的固有属性,伴随着每个生命活动的完成；细胞是机体基本组成单位,细胞膜内外某些带电离子分布和浓度不同以及细胞膜对不同离子的选择通透性,正是细胞、组织形成跨膜电位的条件,也是生物电的起源。正常情况下,细胞间紧密连接的存在可使跨膜电位均等分布于相应组织当中,组织一旦损伤,完整的细胞间紧密连接不复存在,损伤处局部跨膜电位即被削弱,健侧部位的高电势和损伤病灶的低电势随即形成一内生电场。由于内生电场激活了病变组织细胞及其周围细胞修复信号的传递,可诱导机体产生损伤修复效应,因此,如何深化认识内生电场修复作用并加以利用则显得十分重要。
基于此背景,生物电刺激在血管再生领域中的应用已经积累了一定的数据,有关策略主要围绕以下两个方面：第一,外部施加生物电刺激直接促进机体内血管再生,相当于弥补并强化内生电场诱导产生的修复效应；第二,以构建组织工程化血管为中心,通过生物电刺激的干预,从排列、粘附和抗性等方面增强种子细胞与生物材料的整合。由于血管内皮细胞覆盖所有类型血管并密切接触血流,同时也是新生血管形成的首要环节,故目前电刺激研究多集中于血管内皮细胞功能学的改变。然而,研究表明仅仅依赖于血管内皮增殖或出芽等原位修复并不能满足重建血管、改善血流的需要,内皮祖细胞(Endothelial progenitor cell, EPC)的发生发展已被证实对成体血管再生有着不可替代的作用,新生血管形成的研究方向逐渐由传统的血管形成过渡至EPC介导的血管发生。那么EPC将如何参与到电刺激促新生血管形成过程?该问题的探讨十分有意义。
随着EPC探索的深入,EPC研究已由改良培养方法和分析增殖分化能力为主的策略演变为对细胞归巢信号的重点探索。EPC归巢,指在缺血损伤的应激代偿反应中,EPC因缺血部位趋化因子的诱导而产生动员和定向迁移,然后在血管内皮层上缓慢移动,继而与血管内皮产生紧密粘附并完成跨内皮转移,最后通过直接分化和分泌等形式来完成修复反应。经证实,EPC与血管内皮之间的粘附在整个归巢过程当中起着承上启下的作用,已知多对粘附分子组合参与此调节过程。其中细胞间粘附分子-1 (Intercellular adhesion molecule, ICAM-1)属于免疫球蛋白超家族成员,其在血管内皮细胞膜上的表达不仅介导了细胞间的粘附,更重要的是通过这种结合,可将外界损伤刺激信号沿着EPC膜上配体整合素αvβ2,尤其是整合素β2,传递至EPC胞内,然后启动后续生物学信号的转导,在粘附分子调控的归巢过程当中最为关键。那么血管内皮在适宜电刺激的调节下能否增强自身与EPC的粘附?电刺激促进血管内皮捕获循环EPC的机制又是否与ICAM-1的上调有关?国内外未见同类型报道,这是开展本课题研究的主要目的。
为验证我们的设想,本课题研究分为三个部分：
(1)自主研制具有适合刺激形式的电刺激仪,获得人脐静脉内皮细胞(Human umbilical vein endothelial cell, HUVEC)并建立细胞培养电刺激模型,首先评价电刺激下培养体系的稳定性、HUVEC形态学和增殖活性的改变；
(2)诱导培养外周血EPC并进行鉴定,通过荧光标记EPC以衡量电刺激下HUVEC与EPC之间粘附强度的改变；
(3)检测电刺激下HUVEC ICAM-1的表达水平,以期探索电刺激影响HUVEC捕获EPC的可能机制。
方法
(1)电刺激仪的研制与评价
联合生物医学工程学院医学工程系,自主研制脉冲输出形式的电刺激仪；固定刺激脉宽和频率为最大值,伏安法检测不同电压幅度刺激下2h内培养液电阻,并且探测局部温度的变化；
(2)电刺激对HUVEC形态学以及增殖活性的影响
胶原酶消化法分离HUVEC,结合形态学和Ⅷ因子相关抗原对其鉴定；将HUVEC分为不同参数刺激组和常规培养组,刺激24h后观察HUVEC形态学变化,MTS法比较HUVEC增殖活性的差异；
(3)电刺激对HUVEC与EPC之间粘附强度的影响
密度梯度离心法分离外周血单个核细胞,专用培养基直接诱导培养外周血EPC,于第9天进行免疫荧光鉴定(乙酰化低密度脂蛋白和荆豆类凝集素)以及表面标志流式分析(CD34、CD133、CD31、VEGFR2和CD14)；
取第9天EPC,活性荧光染料CFSE标记后接种在单层HUVEC上,不同参数电刺激培养24h,显微镜下观察贴壁荧光标记EPC数量,荧光酶标仪读取细胞荧光强度,以荧光比率衡量粘附强度。
(4)电刺激对HUVEC ICAM-1表达的影响
24h培养结束后分别收集不同参数电刺激组和常规培养组细胞的总RNA,采用Real time PCR技术中的SYBR Green染料法,运用2-ΔΔCt法对各实验组中HUVEC ICAM-1 mRNA相对表达量进行分析。
(5)统计学方法：
数据以均数±标准差(x±s)表示,表面标志阳性率比较采用配对t检验,多组间不同时间点电阻比较采用重复测量数据的方差分析,多组间温度比较、荧光比率比较采用完全随机设计方差分析,多组间细胞增殖活性比较、基因表达量比较采用随机区组设计的方差分析：方差齐时采用Fisher法,多重比较用Bonferroni法；方差不齐时则用Welch近似法,多重比较用Dunnett T3法,P<0.05视为具有显著性差异,采用SPSS13.0统计软件计算。
结果
(1)电刺激仪的研制与评价
自主研制电刺激仪可提供幅度(0-±40V)、频率(0.01-10 Hz)、脉宽(0.1-24 ms)均可调的双相脉冲输出,配合C-Dish电极板使用共同构成细胞培养电刺激模型。固定最大脉宽和最大频率,采用伏安法测得2h内不同刺激电压强度下(0V、5V、10V、20V、和40V)培养液电阻存在显著性差异(F=174.14,P=0.000),电压强度与刺激时间存在交互效应(F=74.12,P=0.000),不同电压幅度下培养液电阻在不同刺激时间点的变化趋势不同。培养液电阻和局部温度均以10V以内电压幅度相对稳定,表示此范围内电解反应恒定,可在此基础上进行后续细胞学实验。
(2)电刺激对HUVEC形态学以及增殖活性的影响
取第2或第3代HUVEC进行实验,选择10V内不同刺激参数组合,首先通过观察刺激24h后HUVEC形态学变化以进一步缩窄刺激范围,结果发现在5V/5Hz/9ms刺激下细胞主要表现为体积增大、更扁平、长梭形伸展,核周深染区牵拉细长,与细胞长轴一致,并且可随着任一刺激参数的增高而出现空泡变性、甚至脱落；随后固定脉宽、频率分别为5V和5Hz,采用MTS法检测不同脉宽(0ms、1ms、3ms、6ms、9ms)刺激下HUVEC增殖活性,以排除增殖活性差异对粘附强度变化造成的干扰,各组OD值分别为1.457、1.471、1.469、1.461和1.458,尚不能认为各实验组间HUVEC增殖活性存在显著性差异(F=1.822,P=0.218)。
(3)电刺激对HUVEC与EPC之间粘附强度的影响
外周血EPC呈长条状分布,整体密度和状态均以第9天最佳,可同时吞噬乙酰化低密度脂蛋白和结合荆豆类凝集素,Ⅷ因子相关抗原逐渐增强,表面标志CD34、CD133、CD31、VEGFR2、CD14阳性率分别为0.19±0.06%,1.67±0.52%,61.56±5.57%,70.29±7.37%,89.31±4.11%,属于正在分化的EPC,符合早期EPC范畴；活性荧光染料CFSE标记第9天收集的EPC,荧光法分析不同脉宽刺激下HUVEC与EPC粘附强度的变化。经换算,常规培养组、1ms、3ms、6ms和9ms刺激组荧光比率分别为0.441、0.470、0.489、0.557和0.370,各实验组间荧光比率存在显著性差异(F=62.165,P=0.000),与常规培养组相比,6ms刺激组增高最显著,可见适宜双相脉冲电刺激在一定程度上有利于HUVEC与EPC之间的粘附。相反,9ms刺激组荧光比率下降,显著低于常规培养组水平。
(4)电刺激对HUVEC ICAM-1表达的影响
Real time PCR检测结果显示,与常规培养组相比,lms、3ms、6ms和9ms刺激组相对表达量分别为1.23、2.42、3.79和1.90,不同脉宽刺激下HUVEC ICAM-1 mRNA相对表达量存在显著性差异(F=17.706, P=0.000),并在一定范围内随着脉宽的延长而增高,其中6ms刺激组表达量增高最显著,9ms刺激组呈现下降迹象,但仍高于正常水平。
结论
(1)成功研制出具有双相脉冲输出的电刺激仪,并且建立了相对稳定的细胞培养刺激模型,在固定5V和5Hz的前提下,HUVEC形态改变明显,呈扁平长梭形伸展,但其增殖活性并不随着刺激脉宽的延长(0、1、3、6、9ms)而改变；
(2)成功从外周血单个核细胞中直接诱导培养出EPC,经鉴定,细胞符合正在分化的早期EPC表型；
(3)双相脉冲电刺激在一定范围内有利于HUVEC与EPC之间的粘附,其中以5V/5Hz/6ms组粘附增强最为显著；
(4)双相脉冲电刺激可上调HUVEC ICAM-1 mRNA相对表达量,变化规律与细胞间粘附实验一致,同样以5V/5Hz/6ms作用最显著,提示HUVEC ICAM-1表达上调可能是电刺激促进HUVEC捕获EPC的机制之一。
创新点
(1)前期研究中多采用直流电形式对HUVEC等血管内皮细胞进行干预,有关脉冲电刺激研究报道不多,本研究在国内外较早采用正负双相脉冲形式对HUVEC进行干预,为电刺激下血管再生的体外研究提供新的实验方法。
(2)本研究首次将内皮祖细胞整合到电刺激调节血管内皮细胞功能的研究中,明确了5V/5Hz/6ms双相脉冲电刺激可增强HUVEC与EPC的粘附,为电刺激促进新生血管形成的研究提供新的理论依据,国内外尚未见同类型报道。
(3)本研究初步证实在5V/5Hz/6ms的双向脉冲电刺激下,HUVEC捕获EPC增多的机制与ICAM-1的表达上调有关,再次肯定ICAM-1粘合识别对于EPC归巢过程的重要性,也为其他防治手段下EPC的归巢信号机制提供借鉴。
Background and objective
Bioelectricity is the inherent propertiy of human body along with the completion of vital movement. Different types and concentration ion could exchange between cells and microenvironment due to the selective permeability of cell membranes. This is so called condition of membrane potential, also origin of bioelectricity. Under normal circumstance, tight junction between cells maintains an equal distribution of membrane potential. Once sufferring from wound, intact tight junction no longer exits, resulting in a weak or collapse local potential. Therefore, the potential gradient between normal region and wound will establish an endogenous electric field, which could induce cells to repair around the lesion. According to this, it is important to deepen our understanding and make good use of this electrical field.
Based on the bioelectricity and the effect of endogenous electric field, electrical stimulation in vascular repair has been reported in the past decade and two main strategies will be the right direction. First, an external electrical stimulation is used to promote vascular regeneration directly, equivalent to adjust or strengthen the endogenous electrical effect. Second, taking vascular tissue engineering into consideration, integration of seed cells and the biomaterial through arrangement、adhesion and resistance is also being studied under electrical stimulation. Because vascular endothelial cell covers all of the blood vessel, intimately contacts with the blood flow and presents as the first step of angiogenesis, preliminary studies have pay more attention to the function of endothelial cell under electric field. However, recent researches show that it is difficult to meet the need of vascular reconstruction only depending on endothelial cell proliferation or sprouting in situ. Endothelial progenitor cell (EPC) has been proven to be irreplaceable in the adult neovascularization. It is indicated that research direction has been changed from traditional angiogenesis to vasculogenesis mediated by EPC. Therefore, to discuss how EPC participates in neovascularization through electrical stimulation is very meaningful.
With a rapid development of EPC study, many researchers have been focus on EPC homing instead of EPC induction、proliferation and differentiation. There are several steps in EPC homing, including cell mobilization and direct migration due to cytokines secreted from ischemia, rolling and firm adhesion with endothelium, transendothelial transfer and finally direct differentiation or emiocytosis to complete the repair process. In these steps, adhesion between EPC and endothelial cell is the most important part in the homing process and three pair adhesion molecules are involved, making a close link between EPC chemotaxis and functioning. Intercellular adhesion molecule-1 (ICAM-1 or CD54) belongs to immunoglobulin superfamily and responses for maintenance of physiological function, pathogenesis and wound healing. Its uniform ligand is integrinαvβ2, which is also called CD 11 a/CD 18 compounds. Expression of ICAM-1 in endothelial cell do mediates EPC adhesion, but more importantly, under combination with the integrinαvβ2 in EPC, especially integrinβ2, the external stimulation signal will be passed from vascular lesion to EPC, and then switch on the EPC repair process. Consequently, our research is going to find out whether electrical stimulation could do well in the adhesion between endothelial cell and EPC? And ICAM-1 up regulation could be related to the mechanism of endothelial cell capturing more EPC under electrical stimulation?
To investigate the change and mechanism of adhesion between endothelial cell and EPC under electrical stimulation, our research is divided into three parts.
(1)We are going to make the suitable electrical stimulator by ourselves and establish a stimulate culture model. In order to find out a safe and effective stimulate interval, we first conduct the evaluation including stability of culture system, morphologic and proliferation of HUVEC.
(2)We induce EPC from periphery blood and make phenotype identification. To assess the change of HUVEC-EPC adhesion, we label the EPC with fluorchrome and quantify by fluorescence intensity after co-culture with HUVEC monolayer under stimulation.
(3)To explore the possible mechanism of HUVEC capturing EPC under stimulation, we detect the expression of ICAM-1 in HUVEC which plays an important role in HUVEC-EPC adhesion.
Methods
(1)Development and evaluation of electrical stimulator
Electrical stimulator with a pulse output was made by our cooperation with school of biomedical engineering, Southern Medical University. With a fixed and maximum pulse and frequency, we assessed medium resistance using voltammetry and detected temperature of culture system under different voltage stimulation within 2h.
(2)Morphology and proliferation changes of HUVEC under different stimulation
HUVEC was digested by collagenase from human umbilical cord and identified with the morphology andⅧfactor related antigen. Then they were divided into different stimulation groups and conventional culture group. After 24h stimulation, morphology of HUVEC was observed and the proliferation activity was analyzed by MTS.
(3)egulation on HUVEC-EPC adhesion under different stimulation
Periphery blood mononuclear cells were isolated from periphery blood using density gradient centrifugation. They were seed in the special endothelial culture medium and would be induced to EPC after 9 day. Then EPC identification was determined by immunofluorescence, with a staining of Ulex europaeus lectin I and acetylated low-density lipoprotein. Phenotypic analyses of the EPC were performed by flow cytometry, including CD34, CD133, CD31, VEGFR2 and CD14.
We collected EPC at day 9 and labeled them with fluorochrome CFSE. The labeled EPC were seed in the HUVEC monolayer and stimulated for 24h under different pulse. To measure adhesion between HUVEC and EPC, CFSE-EPC adhered to monolayer was discriminated in fluorescence microscopy and quantified by fluorescence micro plate reader. An index of fluorescence ratio was used to indicate the adhesion intensity.
(4)Regulation on ICAM-1 expression of HUVEC under different stimulation
Total RNA was collected from different groups where HUVEC had been stimulated for 24h and then ICAM-1 mRNA was detected by SYBR Green staining, one of the methods about real time PCR. Finally, ICAM-1 mRNA relative expression was analyzed using 2-ΔΔCt.
Statistical analysis
Data was showed as mean±standard deviation, surface markers between two group were compared using paired Sample T test, resistance at different time points between groups were compared using repeated measurement of variance analysis, temperature and fluorescence ratio between different groups were compared using one-way ANOVA, proliferation index and relative gene expression were compared using two-way ANOVA. Once there was significant difference between groups, two methods of multiple comparison were selectively used, Bonferroni for equal varicances assumed while Dunnett T3 for not assmed. P<0.05 was consider to be significant difference and all the data was analyzed by SPSS13.0 software.
Results
(1)Development and evaluation of electrical stimulator
Our self-made stimulator could provide an biphasic pulse, including adjustable voltage(0-40V)、frequency(0.01～10Hz) and pulse width (0.1-24ms). The simulated model was set up with the combination of C-Dish electrode. With a fixed and maximum pulse width and frequency, there was a significant difference in medium resistance detected by voltammetry among different voltage groups(0V,5V,10V, 20V and 40V)(F=174.14, P=0.000). Moreover, there was a significant interaction between voltage and stimulation time, suggesting a different trend of different time under different voltage. According to the analysis, both the resistance and temperature in 10V group was relative stable, indicating a stable electrolysis reaction during our stimulation process indirectly.
(2)Morphology and proliferation changes of HUVEC under different stimulation
To further select the suitable parameters, we first chose the passage 2 or 3 of HUVEC for stimulation within 10V. After various selection, typical morphological changes of HUVEC was present in 5V/5Hz/9ms, including flat and fusiform shape, deeply stained peri-nuclei zone stretched to the cell long axis. Then we fixed the frequency and voltage at 5V and 5Hz, utilized MTS to detect the proliferation of HUVEC under different pulse width. The OD values in 0ms,1ms,3ms,6ms,9ms group were 1.457,1.471,1.469,1.461 and 1.458 respectively, showing that no significant difference was among them (F=1.822, P=0.218).
(3)Regulation on HUVEC-EPC adhesion under different stimulation
EPC induced from periphery blood was present as spindle shape. They could uptake acLDL and exhibit UEA lectin binding capability simultaneously at day 9, also with an enhanced synthesis ofⅧfactor related antigen. The expression profile of EPC at day 9 were CD34(0.19%±0.06%), CD133(1.67%±0.52%), CD14(89.31%±4.11%), CD31(61.56%±5.57%) and VEGFR2(70.29%±7.37%). All these showed that adhesion cells presented as differentiating early EPC.
EPC from day 9 was collected for CFSE labeling. Fluorometric method was used to detect the fluorescence intensity of adhesion CFSE-EPC in different pulse width. Results showed that fluorescence ratio were 0.441,0.470,0.489,0.557 and 0.370 in 0ms,1ms,3ms,6ms and 9ms group respectively. There was a significant difference among these groups and 6ms group was the best of all, indicating that suitable electrical stimulation could benefit for the HUVEC-EPC adhesion to some extent. In contrast, the ratio of 9ms group decreased significantly, even lower than the control group.
(4)Regulation on ICAM-1 expression of HUVEC under different stimulation
Compared with control group by real time PCR, ICAM-1 mRNA expression of HUVEC in different groups, including lms,3ms,6ms and 9ms were 1.23,2.42,3.79 and 1.90 respectively. Significant difference was present among them (F=17.706, P =0.000), indicating that up regulation expression was follow with an extension of pulse width. Taking 6ms group in particular, its expression was the significantly highest while there was a decreasing trend in 9ms group.
Conclusion
(1)It was successful to make an electrical stimulator with a biphasic pulse and set up a safe stimulated model. There is no change in proliferation of HUVEC but a typical change of morphology from 0ms to 9ms. HUVEC presented as more flat and spindle.
(2)EPC was induced from PBMC successfully and it belonged to differentiating early EPC according to the phenotype.
(3)Biphasic electrical stimulation was benefit for the HUVEC-EPC adhesion and 5V/5Hz/6ms would be the best.
(4)Electrical stimulation could up regulate the mRNA expression of ICAM-1. Its change was consistent with the trend of HUVEC-EPC adhesion. It is suggest that electrical stimulation contributing to HUVEC-EPC adhesion should be related to the up regulation of ICAM-1 expression.
Point of innovation
(1)Regulation on the endothelial cell using direct current have been reported in previous studies and there were few literatures about pulse stimulation. Our research is successful to regulate HUVEC under biphasic pulse stimulation. It will provide a novel method to the future studies about vascular regeneration through electrical stimulation.
(2)We are the first to make integration between EPC and endothelial cell under 5V/5Hz/6ms electrical stimulation. The stimulated strengthen HUVEC-EPC adhesion is probably a new theory about electrical stimulation contributing to neovascularization. So far, there is no report about this concept.
(3)Our research has suggested that up regulation of ICAM-1 may be responsible for the mechanism of endothelial cell capturing more EPC under electrical stimulation. These will help us to reaffirm the importance of ICAM-1 for EPC homing and provide a good reference for EPC homing under other stimulation methods.
引文
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