构建颈动脉损伤模型大鼠早期生长反应因子1及组织因子的表达

doi:10.3969/j.issn.2095-4344.2015.27.006

摘要/Abstract

摘要：

背景：脱氧核酶ED5可通过特异性抑制早期生长反应因子1的表达来阻遏下游靶基因的表达。
目的：构建颈动脉损伤模型大鼠，观察早期生长反应因子1的脱氧核酶ED5对大鼠颈动脉损伤后血浆组织因子水平的影响及防治血管内膜增生的机制。
方法：实验采用左颈总动脉内膜剥脱构建颈动脉球囊损伤模型大鼠，分别将ED5，MgCl2和FuGene6注入大鼠损伤的血管节段内。
结果与结论：病理学检查、免疫荧光染色、免疫组织化学染色、ELISA检测结果显示，建模后3，7，14，21 d，与MgCl2组和FuGene6组相比，经损伤动脉局部转染ED5后的大鼠血管组织早期生长反应因子1的表达和血浆组织因子的表达水平明显减少(P < 0.01)，且建模后7，14，21 d内膜增生程度明显减轻(P < 0.01)。结果证实，早期生长反应因子1特异脱氧核酶ED5可能通过抑制组织因子的表达，从而抑制损伤颈动脉内膜的增生。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

关键词: 实验动物, 心肺及血管损伤动物模型, 动脉损伤, 早期生长反应因子1, 组织因子, 转染, 内膜增生, 再狭窄, 介入治疗, 基因治疗, 鼠模型, 颈动脉

Abstract:

BACKGROUND: DNA enzyme targeting early growth response factor 1 (Egr-1) mRNA (ED5) can inhibit expression of downstream target genes by specifically inhibiting expression of early growth response factor 1.
OBJECTIVE: To investigate the effects of ED5 on the expression of plasma tissue factor after vascular balloon injury in rats and the mechanism of inhibiting neointimal hyperplasia.
METHODS: Intimal injury models of the left carotid artery were made in rats. Then, ED5, MgCl2 and FuGene6 were injected into the injured vascular segment.
RESULTS AND CONCLUSION: At days 3, 7, 14, 21, the expression levels of Egr-1 and tissue factor in plasma were significantly down-regulated in the ED5 transfection group compared with the MgCl2 and FuGene6 groups (P < 0.01); and neointimal hyperplasia was significantly inhibited by ED5 at days 7, 14 and 21 after modeling
(P < 0.01). ED5 may inhibit neointimal hyperplasia following balloon injury of rat common carotid artery through down-regulation of tissue factors.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Tissue Engineering, Transfection, Carotid Arteries, Genes

中图分类号:

R318

张希，苗驰. 构建颈动脉损伤模型大鼠早期生长反应因子1及组织因子的表达[J]. 中国组织工程研究, 2015, 19(27): 4293-4298.

Zhang Xi, Miao Chi. Expression of early growth response factor 1 and tissue factor in rats with carotid artery injury [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(27): 4293-4298.

图/表（结果） 6

Quantitative analysis of experimental animals
Totally 106 rats were selected, two of which were identified under immunofluorescence microscopy after transfection by ED5, and hereupon supplied. Twenty-four rats in the sham group had no loss, and another 72 rats were modeled successfully and included in result analysis, with 24 rats in each group (Figure 1).

Pathological and morphological changes in rats with carotid artery injury after ED5 transfection
In the sham group, the intima of the carotid artery was complete; at 3 days after modeling, endothelial denudation was found in the MgCl2 group and FuGene6 group, and visible thrombus formed, and moreover, there were a lot of infiltrated inflammatory cells; mild intimal hyperplasia occurred at 7 days after modeling and endometrial hyperplasia became remarkable at 21 days after modeling, and there were more vascular smooth muscle cells and collagen fibers which were disorganized and showed different forms. Compared with MgCl2 and FuGene6 groups, intimal hyperplasia group in the ED5 transfection group was significantly reduced at all time points (P < 0.01), and moreover, the number of intimal vascular smooth muscle cells and inflammatory cells was also significantly reduced and no thrombosis formed (Figure 3, Table 1).

Expression of Egr-1 in rats with carotid artery injury after ED5 transfection
Immunohistochemical staining results showed the Egr-1 expressed weakly in the sham group, but exhibited a high expression at 3, 7, 14, 21 days after modeling. ED5 could significantly reduce the expression of Egr-1, which was significantly different from that in the MgCl2 and FuGene6 groups at the same time (P < 0.01; Figure 4 and Table 2).

Changes in plasma tissue factor levels in rats with carotid artery injury at different time after ED5 transfection
ELISA results showed that only a small amount of vascular tissue factors expressed in the sham group, and the expression of tissue factor was gradually increased in the MgCl2 and FuGene6 groups after balloon dilation, which was significantly higher than that in the sham group (P < 0.01). However, ED5 could significantly reduce the expression of tissue factor compared with the MgCl2 and FuGene6 (P < 0.01; Table 3). It indicated that ED5 can certainly inhibit expression of tissue factor induced by endothelial injury.

参考文献

引言

Coronary heart disease has an increased incidence year by year in China. Although percutaneous coronary intervention (PCI) can realize vascular reconstruction in coronary heart disease patient, a higher incidence of post-PCI restenosis seriously impacts the long-term effects. Drug-eluting stents, which have been widely used, significantly reduce the incidence of restenosis[1-3], but recent clinical studies have shown that drug-eluting stents after implantation cannot completely avoid short-term thrombosis and long-term restenosis[4-6]. Therefore, pathological features and control of restenosis after treatment has become an important issue for cardiovascular disease research. With the rapid development of gene transfer technology, gene therapy will become a new and promising treatment for restenosis[7]. Early growth response factor 1 (Egr-1) is a transcription factor with zinc finger, which regulates the expression of many downstream genes[8-9]. Exogenous stimuli increase Egr-1 expression to promote cell proliferation and intimal hyperplasia, leading to restenosis and other pathologic vascular repair responses. Therefore, to inhibit the expression of Egr-1 can effectively prevent the abnormal proliferation of vascular smooth muscle cells. Post-PCI thrombogenesis is closely associated with proliferation, migration and transformation of vascular smooth muscle cells[10-11]. Deoxyribozyme is a deoxyribose molecule with enzymatic activity after the discovery of ribozymes. It has phosphatase activity and can catalyze and specifically shear RNA[12], which can act as a meaningful tool in different disease states. ED5 can inhibit the expression of downstream target genes by specific down-regulation of Egr-1.

Our experiments aimed to construct a rat model of carotid artery injury to observe the effects of ED5 on tissue factor expression and intimal hyperplasia after balloon injury of the common carotid artery and to explore the possible mechanism underlying ED5 against intimal hyperplasia due to vascular balloon injury.

材料方法

Design
Randomized controlled animal experiments.

Time and setting
The experiments were completed at the Animal
Laboratory of China Medical University from October 2010 to March 2012.

Materials
Totally 106 male Wistar rats, weighing 350-400 g, were provided by the Experimental Animal Center of China Medical University (license No. SCXK(Liao)2008-0005).

Experimental instruments and reagents for detection of Egr-1 and tissue factor expression in rats with carotid artery injury:
Instruments and reagents	Source
2F Fogarty catheter	Baxter Healthcare Corporation, USA
FuGene6	Roche, USA
Egr-1 rabbit anti-rat monoclonal antibody	Santa Cruz, USA
ELISA kit for tissue factor	R&D systems
ED5	Shanghai Boya Biotechnology Co., Ltd., China

Methods
Establishing rat models of carotid artery injury
As reported previously[13-14], 10% chloral hydrate was intraperitoneally injected into rats (30 mg/kg), and then, the left carotid artery of rats was bluntly dissected via the middle path of the rat neck and blocked temporarily by vascular chips following by insertion of 2F catheter from the external carotid artery to the left common carotid artery. After that, 0.2 mL normal saline was injected into the airbag, and the catheter was withdrawn fully along the common carotid artery followed by deflating the balloon. The above- mentioned process was repeated three times, resulting in intimal injury of the left carotid artery. After modeling, the external carotid artery was ligated to restore the blood flow and the wound was stitched. Intramuscular injection of 8×104 U penicillin was done to prevent post-modeling infection, and the rats were fed normally and allowed free access to water.

Grouping and intervention
Ten rats were excluded because of failure in model establishment. Another 96 rats which were modeled successfully were equally randomized into four groups and treated with different surgeries[15]. Sham group: only the left carotid artery was isolated with no balloon injury; MgCl2 group: after balloon injury of the left common -carotid artery, rats were locally given 200 μL MgCl2 (1 mmol/L); FuGene6 group: after balloon injury of the left common carotid artery, rats were given 200 μL MgCl2 (1 mmol/L) containing 30 μL Fugene6 (203.4 g MgCl2 • H2O was dissolved in 800 mL water and diluted into 1 L, and then divided into aliquots undergoing autoclave); ED5 transfection group: after balloon injury of the left common carotid artery, rats were given 200 μL MgCl2 (1 mmol/L) containing 30 μL Fugene6 and 500 μg ED5. After that, six rats from each group were killed at days 3, 7, 14 and 21 after modeling.

In vivo ED5 transfection
500 μg FITC-labeled ED5 and 30 μL FuGene6 were thoroughly mixed into the compound, dissolved in 170 μL of 1 mmol/L MgCl2 solution. During the preparation of balloon injury models, the compound was injected via a micro-syringe into the external carotid artery. Twenty-four hours after transfection, two rats were sacrificed, and local vascular specimens were taken immediately, placed into liquid nitrogen and made into frozen sections (slice thickness of 5 μm) followed by observed under a fluorescent microscope in the darkness. If there was a lot of green fluorescence in the remained tunica intima and tunica media under the stimulation at wavelength of 460-490 nm, it meant a successful transfection.

Sample collection and vascular specimens
After modeling 7, 14, 21 days, the rats in each group were fixed in supine position under anesthesia, an incision was made along the original one under sterile conditions to separate the left common carotid artery. The proximal and distal ends of the carotid artery were ligated surgically, and the balloon-dilated segment, about 1 cm, was cut sharply. The vessels were rinsed with cold heparin saline quickly, and fixed with 40 g/L paraformaldehyde for 24 hours followed by pathological examination. The vascular specimens were then subject to 60%, 70%, 80%, 90%, 100% gradient ethanol dehydration, transparency with xylene, waxdip, paraffin-embedding to prepare paraffin blocks that were cut into serial sections (0.4 μm in thickness). Each specimen was cut into six tissue sections for hematoxylin-eosin staining. Based on the pathological image analysis system, changes in intima and media thickness and luminal stenosis rate were observed, determined and analyzed.

Stenosis rate (%)=(1-existing lumen area/area below the internal elastic lamina)×100%

Immunohistochemistry staining
Paraffin sections were deparaffinized and hydrated, incubated 3% hydrogen peroxide with for 15 minutes at room temperature, immersed in 0.01 mol PL citrate buffer (pH 6.0), heated to boiling in a microwave oven, repeated twice with an interval of 5 minutes, incubated in 10% goat serum at 37 ℃ for 15 minutes. Then, rabbit anti-Egr-1 polyclonal antibody was added, overnight at 4 ℃. Biotinylated goat anti-rabbit IgG antibody was added at room temperature for 20 minutes, and then peroxidase solution was added at room temperature for 20 minutes followed by 3,3'-diaminobenzidine developing. Positive plate served as a positive control and PBS in place of hybridization solution as a negative control. Egr-1 positive cells mainly presented with nuclear staining and brownish yellow. Four sections from each specimen were selected to count the absorbance value at six randomized visual fields under 400-fold light microscope. Then, the mean value was calculated.

Determination of plasma tissue factor levels
The levels of tissue factors in plasma were detected using ELISA. Blood samples (2 mL) were extracted from the heart of all animals at 3, 7, 14, 21 days after modeling. The samples were placed into a tube containing 60 μL of 10% EDTA disodium and 1000 U (0.1 mL) of aprotinin, mixed uniformly at 4 ℃, centrifuged at 3 000 r/min for 10 minutes followed by plasma separation, and then, the samples were saved at -80 ℃ under test. Plasma tissue factor levels were determined in strict accordance with the instructions.

Main outcome measures
Egr-1 and tissue factor levels in different groups.

Statistical analysis
All data were analyzed statistically using SPSS 13.0 software. Measurement data were expressed as mean±SD. One-way analysis of variance (least significant difference test) was used for intergroup comparison, and a value of P < 0.01 was considered significant.

讨论

In this experiment, the rat model of carotid artery injury model was used to simulate the post-PCI restenosis process in human in order to investigate the correlation between vascular endothelial injury and plasma tissue factor expression and the interventional effects of ED5 in the occurrence and development of restenosis. Pathological processes of in vivo thrombus formation is very complex, but its fundamental step is to initiate coagulation process, and tissue factor is an important promoter of in vivo physiological coagulation[16], which can be released from vascular endothelial cells and platelet surface activated by vascular endothelial injury[17]. Thrombosis formation can be mediated by generating serine protease after vascular modeling[18]. These proteases induce the migration and proliferation of smooth muscle cells by mitogen and chemotactic effects of vascular endothelial smooth muscle cells, and thereby result in vascular stenosis through proinflammatory reactions. After vascular modeling, the coagulation system in the artery is activated by exposure of tissue factors. Besides procoagulant effect, tissue factors can mediate smooth muscle cell migration and proliferation, and promote the development of restenosis. Tissue factor plays an important role as a bridge in coagulation and inflammatory responses. Tissue factor can accelerate and aggravate the inflammatory response[19]. Research on regulator mechanism of tissue factor expression will not only clarify the new molecular mechanisms of intimal hyperplasia, but also contribute to the development of new ideal drugs that inhibit intimal hyperplasia. Tissue factor may become a new target for drug prevention and treatment of cardiovascular disease.

DNAzyme is a DNA molecule with enzymatic activity, 10-23 DNAzyme as one of which has phosphatase activity. Bilateral substrate recognition sequences of 10-23 DNAzyme can be specifically combined with RNA substrate, and in the RNA strand sequence, with respect to the catalytic sequences, unpaired purine and paired pyrimidine residues constitute specific phosphodiesterase cleavage sites, thereby to cut off the RNA substrate[20]. ED5 can inhibit Erg-1 expression by cutting off Erg-1 mRNA, and thereby deter intimal hyperplasia after vascular modeling[14].

Studies have shown that after arterial modeling, Erg-1 may be a practical and desirable target to prevent post-PCI restenosis[21-24]. This is because Egr-1 as an important nuclear transcription factor can couple extracellular stimuli for intracellular signal transduction to mediate the expression of over 30 kinds of downstream target genes, such as inducing proliferation and migration of vascular smooth muscle cells, platelet-derived growth factor, insulin-like growth factor and basic fibroblast growth factor[23-25]. ED5 is discovered to inhibit the expression of downstream target genes by specific regulation of Egr-1 expression. Now, two GC-rich Egr-1 binding sites are confirmed definitely in Egr-1 promoters, and activated Egr-1 expression can be controlled by directly binding to these sites[7, 24]. Therefore, ED5 as a new potent inactivation factor on RNA level can also suppress tissue factor transcription and expression to some extent, and accordingly, reduce short-term formation of thrombosis and neointimal hyperplasia following arterial modeling. FuGene6 as a carrier can improve the transfection efficiency. Liposomes can be used for a variety of tissues and cells, and characterized to have less restriction on gene fragment size, have no harm to transduced cells and have good reproducibility, which have been considered to be effective in vivo transfection reagents[24].

Experimental findings from the present study showed that there were different degrees of intimal hyperplasia and luminal stenosis after modeling, but these phenomena were alleviated by ED5 intervention. The Egr-1 and tissue factor expressed weakly in the sham group, but exhibited a high expression at 3, 7, 14, 21 days after modeling. ED5 could significantly reduce the mRNA expression of Egr-1 and tissue factor, which was significantly different from that in the MgCl2 and FuGene6 groups at the same time. As described above, ED5 may inhibit expression of downstream genes by cutting off Egr-1 mRNA expression, so as to inhibit short-term thrombosis and neointimal hyperplasia following balloon injury of common carotid artery. Many factors can lead to vascular stenosis. In this experiment, local ED5 transfection of injured vessels exerted obvious effects to prevent early thrombosis and intimal hyperplasis, but could not completely stop the occurrence and development of restenosis after modeling. Consequently, in-depth studies on relevant factors from different angles and aspects are necessary.

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