动脉粥样硬化中几丁质酶3样蛋白1的组织学表达及干预研究
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摘要
背景目的
作为一种系统性疾病,动脉粥样硬化(atherosclerosis, AS)影响全身大中型动脉,已经成为冠状动脉疾病(coronary artery disease, CAD)的主要病理类型。研究证实AS是一种慢性炎症性疾病,主要表现为动脉管壁脂质及纤维基质堆积,管腔狭窄。根据“损伤-反应”学说,各种AS危险因素可以导致内皮细胞(endothelialcell, EC)功能障碍和(或)脱落,随后EC表达粘附分子增加,趋化血液单核细胞及T淋巴细胞粘附。单核细胞及T淋巴细胞进入内膜下启动炎性反应,分泌一系列细胞因子,如肿瘤坏死因子(tumor necrosis factor, TNF)、单核细胞趋化蛋白(monocyte chemoattractant protein, MCP)、白细胞介素(interleukin, IL)等,可进一步激活EC、血管平滑肌细胞(vascular smooth muscle cells, VSMCs)及巨噬细胞,促进AS发展。
几丁质酶3样蛋白1(chitinase3-like1, CHI3L1),又名软骨糖蛋白39、YKL-40、乳腺回归蛋白39,该蛋白质存在几丁质结合活性,但无几丁质酶活性。研究表明CHI3L1可由一系列细胞分泌,如呼吸道上皮细胞、脂肪细胞、软骨细胞、肿瘤细胞、VSMCs、巨噬细胞等。CHI3L1在急慢性炎症及细胞外基质(extracellular matrix, ECM)重构等疾病过程中起重要作用,如AS、支气管哮喘、高血压、糖尿病(diabetes mellitus, DM)等。
虽然CHI3L1与AS关系密切,但AS患者外周血CHI3L1水平与冠状动脉狭窄病变程度存在争议。一项研究认为AS患者外周血CHI3L1水平与冠状动脉狭窄病变程度成正相关,另一项调查证实AS患者外周血CHI3L1水平与冠状动脉狭窄病变程度无关。为了进一步明确CHI3L1与CAD的关系,本研究选择行主动脉-冠状动脉旁路移植术(coronary artery bypass graft, CABG)时废弃的主动脉管壁组织,应用免疫组化检测CHI3L1蛋白表达,探讨人体动脉组织CHI3L1蛋白表达与AS发病机制的关系。
材料方法
1.研究对象
(1)对象：选取2010年1月至2012年6月在山东省千佛山医院行CABG的39例患者作为研究组,其中男性患者27例,女性患者12例。对照组为11例亲属间肾脏移植手术供体者,均为男性,经入院查体均排除各种器质性疾病。
(2)标本：研究组患者行CABG时留取废弃的全层主动脉组织,39例CABG取得动脉组织81块。对照组供体者行肾脏移植手术时留取废弃的肾动脉组织,11例肾脏移植取得动脉组织55块。
2.生化分析
所有研究组患者入院后测定静脉血浆甘油三酯(triglycerides, TG)、总胆固醇(total cholesterol, TC)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol, HDL-C)、脂蛋白(a)(lipoprotein (a), Lp(a)、载脂蛋白A (apolipoprotein A, ApoA)、载脂蛋白B (apolipoprotein B, ApoB)、总胆红素(total bilirubin, TBIL)、直接胆红素(direct bilirubin, DBIL)、间接胆红素(indirect bilirubin, IBIL)、等生化指标。
3.冠状动脉造影
所有研究组患者CABG前行冠状动脉造影(coronary angiography, CAG)检查明确冠状动脉狭窄部位、范围及程度,有无钙化及迂曲,并利用Gensini积分方法定量评价患者冠状动脉病变情况。
4.组织准备及免疫组化
研究组及对照组动脉管壁组织经常规固定、脱水、石蜡包埋后切片,石蜡组织切片经常规脱蜡、抗原修复、滴加兔抗人CHI3L1多克隆抗体孵育、显色后行免疫组化,并应用图像分析系统测定CHI3L1相对表达量。
结果
1.研究组和对照组基本情况比较
研究组39例患者中合并吸烟者8例,高血压17例,DM15例。11例肾脏移植手术供体者无吸烟、高血压及DM病史。研究组患者血浆TG、TC、LDL-C及Lp(a)测定值较正常参考值显著升高,血浆HDL-C及ApoA测定值与正常参考值相比明显降低,p<0.05；血浆ApoB测定值虽然较正常参考值升高,但差异无统计学意义,p>0.05。研究组患者血浆TBIL及IBIL测定值较正常参考值显著降低,p<0.05；血浆DBIL测定值虽然较正常参考值降低,但差异无统计学意义,p>0.05。
2.CAG
研究组39例患者中合并左冠状动脉主干病变19例,三支病变14例,二支病变或合并左冠状动脉前降支近端病变6例。CAG提示冠状动脉均有不同程度的钙化、狭窄和迂曲,并且侧支循环建立不完全。采用Gensini积分系统进行定量积分以评价CAD严重程度,研究组39例患者Gensini积分平均为62.25±21.77(24-120)。
3.研究组和对照组动脉组织CHI3L1表达
研究组主动脉管壁组织经免疫组化检测均有CHI3L1表达,阳性表达主要分布于EC及VSMCs胞浆,表现为棕褐色或棕黄色,分布不均匀,呈颗粒状、片状、簇状及条纹状。对照组肾动脉管壁组织经免疫组化检测显示CHI3L1表达明显减少,着色表现为浅蓝色或无色,两者相比差异有统计学意义,p<0.05。
4.研究组动脉组织CHI3L1表达量与AS主要临床危险因素的关系
研究组患者CHI3L1相对表达量性别之间比较,差异无统计学意义,p>0.05；吸烟者与非吸烟者,高血压患者与非高血压患者,DM患者与非DM患者之间比较,差异均有统计学意义,p<0.05。研究组患者CHI3L1相对表达量与冠状动脉狭窄病变Gensini积分相关性分析表明两者呈正相关(r=0.611,P<0.05)。
结论
1.本研究利用人CABG时主动脉打孔废弃的主动脉组织为标本,检测动脉组织CHI3L1蛋白表达量较正常对照明显升高,为国内外首次报道。
2.本研究采用人活体动脉组织为样本,其研究结果提高了科学性、准确性及可信度,为研究AS发病机制增加了新的途径。
3.本研究显示CHI3L1蛋白表达量与AS主要临床危险因素、冠状动脉狭窄病变程度成正相关。AS主要临床危险因素可促进CHI3L1表达,CHI3L1可能通过调节AS主要临床危险因素在AS发病中起作用。
背景目的
动脉粥样硬化(atherosclerosis, AS)是慢性炎症性疾病,影响全身大中型动脉,已经成为冠状动脉疾病(coronary artery disease, CAD)的主要病理类型。各种AS危险因素可以导致内皮细胞(endothelial cell, EC)功能障碍和(或)脱落,趋化血液单核细胞及T淋巴细胞进入内膜下启动炎性反应,并分泌一系列细胞因子,进一步激活EC、血管平滑肌细胞(vascular smooth muscle cells, VSMCs)及巨噬细胞,促进AS发展。
几丁质酶3样蛋白1(chitinase3-like1, CHI3L1)存在几丁质结合活性,但无几丁质酶活性。研究表明CHI3L1可由一系列细胞分泌,如呼吸道上皮细胞、脂肪细胞、软骨细胞、肿瘤细胞、VSMCs,巨噬细胞等。CHI3L1在急慢性炎症及细胞外基质重构等疾病过程中起重要作用,如AS、支气管哮喘、高血压、糖尿病等。
人CHI3L1主动脉组织学研究显示,AS患者主动脉组织CHI3L1高度表达,为验证临床研究结果,本研究建立动物模型进行实验研究,以验证临床人活体主动脉组织研究的结果。为此本研究构建了有效慢病毒载体,使之能够携带针对小鼠CHI3L1的小干扰RNA (small interfering RNA, siRNA),并将慢病毒悬液局部转染ApoE-/-小鼠颈动脉斑块组织,观察CHI3L1基因沉默在延缓AS进展及稳定AS易损斑块中的作用。
材料方法
1.细胞培养
人胚肾细胞系293T细胞能够表达猿猴病毒40大T抗原,有利于慢病毒生产。选择小鼠单核巨噬细胞系RAW264.7细胞作为慢病毒体外转染的靶细胞。293T细胞及RAW264.7细胞于37℃在含有10%胎牛血清、100U/ml penicillin、100μg/ml streptomycin,5%CO2的DMEM中培养。
2.慢病毒构建及有效靶点筛选
设计4条针对小鼠CHI3L1基因的小发卡RNA (small hairpin RNA, shRNA)序列(site A、site B、site C及site D)。
site A的序列为：
5'-GCGACAACATGCTTAGCACATTTCAAGAGAATGTGCTAAGCATGTT GTCGCTT-3';
site B的序列为：
5'-GGCCATTGACACTGGCTATGATTCAAGAGATCATAGCCAGTGTCAAT GGCCTT-3';
site C的序列为：
5'-GCACTGGATTTGGATGATTTCTTCAAGAGAGAAATCATCCAAATCCA GTGCTT-3';
site D的序列为：
5'-GCCAGAAGGACACTAGGTTTGTTCAAGAGACAAACCTAGTGTCCTT CTGGCTT-3'c
作为阴性对照,随机不相关序列为：
5'-GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAG AACTT-3'。
分别构建包含上述各干扰序列的穿梭质粒载体(pShuttle vectors),然后将pShuttle vectors与pGag/Pol、pRev、pVSV-G共同转染293T细胞。慢病毒体外转染RAW264.7细胞筛选有效干扰序列。收集培养72h及96h的RAW264.7细胞进行RT-PCR及western blot分析,了解各干扰序列的干扰效率。
3.动物实验
50只雄性8周龄ApoE-/-小鼠饲养于SPF级饲养室,全程高脂饮食喂饲,随机分为CHI3L1慢病毒基因沉默组及空病毒载体阴性对照组。所有ApoE-/-小鼠均行右侧颈动脉套管术,术后8周颈动脉斑块组织局部转染慢病毒悬液(滴度均为1×109TU/ml)。
4.组织准备及免疫组化
ApoE-/-小鼠经慢病毒转染干预4周后分离颈动脉斑块组织,经常规固定、脱水、包埋后切片。组织切片分别进行HE染色、CHI3L1免疫组化、油红O染色(冰冻切片)、天狼猩红染色、VSMCs α-actin免疫组化、CD68免疫组化。
5.电镜检查
ApoE-/-小鼠颈动脉斑块组织经3%戊二醛固定、漂洗、1%锇酸固定、脱水、Spon812包埋,然后经半薄切片定位,制作超薄切片。切片经柠檬酸铅和醋酸铀电子染色,应用JEOL-1011型透射电镜观察。
6. RT-PCR分析
利用RT-PCR技术分别检测RAW264.7细胞中CHI3L1mRNA表达水平及ApoE-/-小鼠颈动脉斑块组织中CHI3L1、TNF-α、MCP-1、IL、MMP-9mRNA表达水平。
7. Western blot分析
RAW264.7细胞中CHI3L1蛋白表达及ApoE-/-小鼠颈动脉斑块组织中CHI3L1、p-ERK1/2、ERK1/2、p-AKT、AKT蛋白表达水平应用western blot技术检测。
结果
1.慢病毒构建及有效靶点筛选
慢病毒构建完成后转染RAW264.7细胞,RT-PCR分析表明site A、site B、 site C及site D干扰序列基因沉默分别减少CHI3L1mRNA表达量32%、17%、65%及30%。Western blot分析表明site A、siteB、site C及site D干扰序列基因沉默分别减少CHl3L1蛋白质表达量38%、18%、64%及14%。结果提示Site C为最有效的干扰序列,最终慢病毒悬液滴度为1×109TU/ml,用于下游ApoE-/-小鼠体内实验。
2.慢病毒体内转染效应
对照组ApoE-/-小鼠颈动脉斑块组织经CHI3L1免疫组化可见阳性表达,主要分布于EC及VSMCs胞浆,表现为棕褐色或棕黄色。基因沉默组ApoE-/-小鼠颈动脉斑块组织经免疫组化提示CHI3L1表达明显减少,表现为浅蓝色或无色。RT-PCR及western blot分析结果表明基因沉默组ApoE-/-小鼠颈动脉斑块组织中CHI3L1mRNA及蛋白表达量较对照组ApoE-/-小鼠明显减少,两者相比差异有统计学意义,p<0.05。
3.电镜检查
对照组ApoE-/-小鼠颈动脉斑块组织经电镜检查提示EC及VSMCs中存在大量脂质颗粒及胆固醇结晶,EC连接中断,VSMCs以合成型为多。基因沉默组ApoE-/-小鼠颈动脉斑块组织经电镜检查表明EC及VSMCs中可见少量脂质颗粒及胆固醇结晶,钙化多见,EC连接及基底膜大多完整,VSMCs以收缩型为多。
4.慢病毒体内转染对AS斑块构成的影响
对照组ApoE-/-小鼠及基因沉默组ApoE-/-小鼠颈动脉斑块组织中脂质相对含量分别为48.8%、35.2%,两者相比后者脂质减少27.5%；胶原相对含量分别为19.5%、29.8%,两者相比后者胶原增加53.2%；VSMCs相对含量分别为14.8%、22.5%,两者相比后者VSMCs增加51.2%；巨噬细胞相对含量分别为11.9%、7.5%,两者相比后者巨噬细胞减少36%,差异均有统计学意义,p<0.05。
5.慢病毒体内转染对AS斑块炎症因子及信号转导蛋白的影响
RT-PCR分析表明基因沉默组ApoE-/-小鼠较对照组ApoE-/-小鼠颈动脉斑块组织炎症因子TNF-α、MCP-1、IL-8、MMP-9mRNA表达水平均明显减少,差异均有统计学意义,p<0.05。Western blot分析表明基因沉默组ApoE-/-小鼠较对照组ApoE-/-小鼠颈动脉斑块组织p-ERK1/2及p-AKT蛋白表达水平均明显减少,差异均有统计学意义,p<0.05。
结论
1.应用RNA干扰技术可以有效构建针对小鼠CHI3L1基因的慢病毒载体。
2.本研究发现ApoE-/-小鼠经慢病毒载体介导的CHI3L1基因沉默干预能够改善易损斑块稳定性,验证了临床人活体主动脉组织研究的结果。
3. CHI3L1基因沉默可作为抑制AS斑块进展的一个新途径,为治疗AS提供新的思路和方法。
Background and Objectives
As a systematic disease, atherosclerosis is the underlying pathology of the coronary artery disease (CAD) and is a chronic inflammatory disease. According to the "response-to-injury" hypothesis, the endothelial denudation and (or) endothelial dysfunction caused by the risk factors are the first step in the development of atherosclerosis. These activated endothelial cells facilitate monocytes infiltration into the vessel wall and the monocytes differentiate into macrophages, which accumulate lipids and remain in the vessel wall, thereby becoming foam cells. These cells mentioned above release proinflammatory molecules such as tumor necrosis factor (TNF), monocyte chemoattractant protein (MCP), and interleukin (IL), which induce further accumulation of monocytes and migration and proliferation of vascular smooth muscle cells (VSMCs).
Chitinase3-like1(CHI3L1) is a chitin-binding glycoprotein without chitinase activity. CHI3L1has been shown to act as an important regulator of acute and chronic inflammation. CHI3L1can be secreted by a variety of cells, including VSMCs and macrophages. It is also found in tissues with inflammation and extracellular tissue remodeling. Several studies have shown an important link between CHI3L1and inflammatory diseases, including asthma, hypertension, diabetes mellitus, insulin resistance, and atherosclerosis as well.
Although the relationship between CHI3L1and CAD is important, there is a controversy in the association between blood CHI3L1levels and the severity of atherosclerosis. One study showed that severity of atherosclerosis is associated with higher blood CHI3L1levels, and another paper concluded that circulating CHI3L1was not specifically related to the size of atherosclerotic stenosis. In order to elucidate the relationship between CHI3L1and CAD, we investigated the correlation between CHI3L1expression levels and pathogenesis of atherosclerosis by measuring the changes of CHI3L1in the aortic tissues of patients undergoing coronary artery bypass graft (CABG) surgery.
Materials and Methods
1. Study population
From2010to2012,39patients with CAD scheduled for CABG surgery were investigated and defined as a research group. Eleven normal subjects who donated kidneys were investigated as the control group. Aortic specimens were obtained from the aorta that was routinely removed during the CABG surgery and the discarded renal arterial tissues were collected from the11subjects.
2. Biochemical analysis
Venous blood samples were obtained for the measurement of serum triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), lipoprotein (a)(Lp (a)), apolipoprotein A (ApoA), apolipoprotein B (ApoB), total bilirubin (TBIL), direct bilirubin (DBIL) and indirect bilirubin (IBIL) as well.
3. Coronary angiography
All CAD patients were confirmed by the coronary angiography (CAG). Gensini score system was used to assess the CAD severity.
4. Tissue preparation and histological analysis
The arterial tissues obtained from the CABG surgery and kidney donators were fixed in4%buffered formalin, embedded in paraffin and sectioned. After deparaffinage, hydration and antigen retrieve, the sections were incubated with the rabbit antihuman CHI3L1antibody.
Results
1. Baseline characteristics
The serum TG, TC, LDL-C, and Lp (a) levels in the research group were elevated, whereas the serum HDL-C, ApoA, TBIL, and IBIL levels were decreased, compared with the normal reference values (p<0.05).
2. Coronary atherosclerotic lesions in the research group
CAG in the research group showed that19patients were with left main lesions,6patients were with two coronary arteries lesions, and14patients were with three coronary arteries lesions. The average Gensini score was62.25±21.77.
3. Presence of CHI3L1in the human arterial tissues
In the arterial tissues obtained from the healthy donors little CHI3L1expression could be demonstrated according to the immunohistochemical staining. CHI3L1was overexpressed in the arterial specimens of CAD patients.
4. Correlation between the arterial CHI3L1expression and the clinical risk factors of,, atherosclerosis
The expression levels of CHI3L1did not differ between males and females, differ between smokers and non-smokers, hypertensives and non-hypertensives, diabetics and non-diabetics. The relative expression levels of CHI3L1was elevated in smokers, patients with hypertension, or diabetes mellitus, and gender had no significant effect. The linear correlation analysis revealed that arterial CHI3L1expression levels were correlated with the Gensini scores (r=0.61l,p<0.05).
Conclusions
1. In the present study, we found that CHI3L1was overexpressed in aorta of patients with coronary atherosclerosis and it might provide a new approach to the research of atherosclerosis.
2. The relative expression levels were correlated with the atherosclerotic risk factors and the severity of CAD and CHI3L1could be a predictor of the coronary arterial stenosis.
Background and Objectives
Atherosclerosis is a chronic inflammatory disease and is the underlying pathology of the coronary artery disease (CAD). The endothelial denudation and (or) endothelial dysfunction are the first step in the development of atherosclerosis. The activated endothelial cells facilitate monocytes infiltration into the vessel wall. These monocytes differentiate into macrophages, which accumulate lipids and remain in the vessel wall, thereby becoming foam cells. These cells synthesize and release proinflammatory molecules, which induce further accumulation of monocytes and migration and proliferation of vascular smooth muscle cells (VSMCs).
Chitinase3-like1(CHI3L1) is a chitin-binding glycoprotein without chitinase activity. CHI3L1has been shown to act as an important regulator of acute and chronic inflammation. CHI3L1is secreted by a variety of cells, including VSMCs and macrophages. It is also found in tissues with inflammation and extracellular tissue remodeling. Several studies have shown an important link between CHI3L1and inflammatory diseases, including asthma, hypertension, diabetes mellitus, insulin resistance, and atherosclerosis as well.
The relationship between CHI3L1and atherosclerosis is important, in order to verify the therapeutic value of CHI3L1, we constructed lentiviral vectors, which could efficiently deliver small interfering RNAs (siRNAs), and aimed at knocking down CHI3L1to explore the mechanisms of CHI3L1in atherosclerosis in apolipoprotein E-knockout (ApoE-/-) mice.
Materials and Methods
1. Cell culture
The293T human embryonic kidney cell line and the RAW264.7mouse macrophage cell line were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with10%fetal bovine serum (FBS),100U/ml penicillin,100μg/ml streptomycin, in a humidified incubator with5%CO2.
2. Lentivirus construction and target screening for RNAi
Four different sequences (site A, site B, site C, and site D) of mice CHI3L1gene were designed as the target for RNA interference (RNAi).
The sequence of site A was:
5'-GCGACAACATGCTTAGCACATTTCAAGAGAATGTGCTAAGCATGTT GTCGCTT-3';
the sequence of site B was:
5'-GGCCATTGACACTGGCTATGATTCAAGAGATCATAGCCAGTGTCAAT GGCCTT-3';
the sequence of site C was:
5'-GCACTGGATTTGGATGATTTCTTCAAGAGAGAAATCATCCAAATCCA GTGCTT-3';
the sequence of site D was:
5'-GCCAGAAGGACACTAGGTTTGTTCAAGAGACAAACCTAGTGTCCTT CTGGCTT-3'.
As a negative control, the scrambled sequence (mock siRNA) was:
5'-GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAG AACTT-3'.
The pShuttle vectors containing the different mouse CHI3L1RNAi sequences were constructed. A lentivirus was produced by cotransfection of the pShuttle vector, pGag/Pol, pRev, and pVSV-G into the293T cells. The lentiviruses were used to transfect the RAW264.7cells. The RAW264.7cells were collected for RT-PCR and western blot experiment at72h and96h after transfection.
3. Animal experiment
We obtained50male ApoE-/-mice,8weeks old. All the mice were fed a high-fat diet. The mice were divided into2groups:empty lentivirus control group and CHI3L1lentivirus silenced group. A constrictive silica collar was placed on the right common carotid artery of mice. Eight weeks after surgery, the lentiviral suspension at1×109TU/ml was instilled into the right common carotid artery.
4. Tissue preparation and histological analysis
The right common carotid artery obtained from the mice was immersed in4%formaldehyde. Six crosssections in each mouse were used for a particular type of staining. One section was stained with hematoxylin and eosin. Another section was immunostained with rabbit antimouse CHI3L1antibody. Collagen and lipids were identified by Sirius red staining and oil red O staining. VSMCs and macrophages were immunostained with a-actin antibody and CD68antibody.
5. Transmission electron microscopy
The fresh mice arterial tissues were used to undergo electron microscope examination. The arterial tissues were placed in2.5%glutaraldehyde, then the tissues were postfixed in1%osmium tetroxide followed by staining with2%uranyl acetate. Then the tissues were dehydrated through ethanol and were embedded in Spon812. Finally, the sections were stained with uranyl acetate followed by lead citrate and examined in a JEM-1010electron telescope.
6. RT-PCR analysis
The mRNA expression levels of CHI3L1in RAW264.7cells, CHI3L1, TNF-a, MCP-1, IL-8, and MMP-9in mice were analysed using the RT-PCR.
7. Western blot analysis
The protein expression levels of CHI3L1, p-ERK1/2, ERK1/2, p-AKT, and AKT in RAW264.7cells and in mice were assayed by western blot.
Results
1. Effects of lentiviral transfection in vitro
The RAW264.7cell line was transfected with four different CHI3L1siRNAs. Site A, site B, site C, and site D exhibited32%,17%,65%, and30%reduction in mRNA expression. Site A, site B, site C, and site D exhibited38%,18%,64%, and14%reduction in protein expression. Then the site C was the most effective vector in blocking CHI3L1expression. The site C lentivirus and mock lentivirus were produced at a viral titer of1×109TU/ml for further in vivo studies.
2. Effects of lentiviral transfection on CHI3L1expression in plaques
In the control group, CHI3L1expression could be demonstrated according to the immunohistochemical staining. However, little CHI3L1was expressed in the silenced group. Compared with the control group, the silenced group showed reduced CHI3L1protein expression by50%. In addition, there were significant differences of CHI3L1mRNA expression levels between control group and silenced group.
3. Electron microscopy analysis
For electron microscopy, in control group most of the endothelial cells denudated and there were a large number of lipid granules under the basement membrane in the vessel wall. The atherosclerotic plaques were occupied with necrotic particles, calcification crystals and cellular debrises. However, in silenced group the number of lipid granules was relatively decreased. Collagen bundles and elastic fibers were seen on the vessel side of the endothelium.
4. Effects of lentiviral transfection on plaque composition
The relative content of lipids in plaque tissues of the control group and the silenced group was48.8%and35.2%, the relative reduction of lipids content in the silenced group was27.5%. The relative content of collagen was19.5%and29.8%, the relative increase of collagen content was53.2%. The relative VSMCs content was14.8%and22.5%, the relative increase of VSMCs content was51.2%. The relative macrophages content was11.9%and7.5%, the relative reduction of macrophages content was36%.
5. Effects of CHI3L1gene silencing on inflammatory mediators and signal transduction protein in the lesions
The CHI3L1silenced group showed lower mRNA expression levels of TNF-a, MCP-1, IL-8, MMP-9, compared with the control group (p<0.05). The silenced group showed lower protein expression levels of p-ERK1/2and p-AKT, compared with the control group (p<0.05).
Conclusions
1. In the present study, we applied lentivirus mediated shRNA to knock down the mice CHI3L1gene.
2. The silence of CHI3L1diminished the atherosclerotic burden and increased plaque stability in ApoE-/-mice, which might provide a new therapeutic approach to the treatment of atherosclerosis.
作为一种系统性疾病,动脉粥样硬化(atherosclerosis, AS)影响全身大中型动脉,已经成为冠状动脉疾病(coronary artery disease, CAD)的主要病理类型。研究证实AS是一种慢性炎症性疾病,主要表现为动脉管壁脂质及纤维基质堆积,管腔狭窄。根据“损伤-反应”学说,各种AS危险因素可以导致内皮细胞(endothelialcell, EC)功能障碍和(或)脱落,随后EC表达粘附分子增加,趋化血液单核细胞及T淋巴细胞粘附。单核细胞及T淋巴细胞进入内膜下启动炎性反应,分泌一系列细胞因子,如肿瘤坏死因子(tumor necrosis factor, TNF)、单核细胞趋化蛋白(monocyte chemoattractant protein, MCP)、白细胞介素(interleukin, IL)等,可进一步激活EC、血管平滑肌细胞(vascular smooth muscle cells, VSMCs)及巨噬细胞,促进AS发展。
几丁质酶3样蛋白1(chitinase3-like1, CHI3L1),又名软骨糖蛋白39、YKL-40、乳腺回归蛋白39,该蛋白质存在几丁质结合活性,但无几丁质酶活性。研究表明CHI3L1可由一系列细胞分泌,如呼吸道上皮细胞、脂肪细胞、软骨细胞、肿瘤细胞、VSMCs、巨噬细胞等。CHI3L1在急慢性炎症及细胞外基质(extracellular matrix, ECM)重构等疾病过程中起重要作用,如AS、支气管哮喘、高血压、糖尿病(diabetes mellitus, DM)等。
虽然CHI3L1与AS关系密切,但AS患者外周血CHI3L1水平与冠状动脉狭窄病变程度存在争议。一项研究认为AS患者外周血CHI3L1水平与冠状动脉狭窄病变程度成正相关,另一项调查证实AS患者外周血CHI3L1水平与冠状动脉狭窄病变程度无关。为了进一步明确CHI3L1与CAD的关系,本研究选择行主动脉-冠状动脉旁路移植术(coronary artery bypass graft, CABG)时废弃的主动脉管壁组织,应用免疫组化检测CHI3L1蛋白表达,探讨人体动脉组织CHI3L1蛋白表达与AS发病机制的关系。
材料方法
1.研究对象
(1)对象：选取2010年1月至2012年6月在山东省千佛山医院行CABG的39例患者作为研究组,其中男性患者27例,女性患者12例。对照组为11例亲属间肾脏移植手术供体者,均为男性,经入院查体均排除各种器质性疾病。
(2)标本：研究组患者行CABG时留取废弃的全层主动脉组织,39例CABG取得动脉组织81块。对照组供体者行肾脏移植手术时留取废弃的肾动脉组织,11例肾脏移植取得动脉组织55块。
2.生化分析
所有研究组患者入院后测定静脉血浆甘油三酯(triglycerides, TG)、总胆固醇(total cholesterol, TC)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol, LDL-C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol, HDL-C)、脂蛋白(a)(lipoprotein (a), Lp(a)、载脂蛋白A (apolipoprotein A, ApoA)、载脂蛋白B (apolipoprotein B, ApoB)、总胆红素(total bilirubin, TBIL)、直接胆红素(direct bilirubin, DBIL)、间接胆红素(indirect bilirubin, IBIL)、等生化指标。
3.冠状动脉造影
所有研究组患者CABG前行冠状动脉造影(coronary angiography, CAG)检查明确冠状动脉狭窄部位、范围及程度,有无钙化及迂曲,并利用Gensini积分方法定量评价患者冠状动脉病变情况。
4.组织准备及免疫组化
研究组及对照组动脉管壁组织经常规固定、脱水、石蜡包埋后切片,石蜡组织切片经常规脱蜡、抗原修复、滴加兔抗人CHI3L1多克隆抗体孵育、显色后行免疫组化,并应用图像分析系统测定CHI3L1相对表达量。
结果
1.研究组和对照组基本情况比较
研究组39例患者中合并吸烟者8例,高血压17例,DM15例。11例肾脏移植手术供体者无吸烟、高血压及DM病史。研究组患者血浆TG、TC、LDL-C及Lp(a)测定值较正常参考值显著升高,血浆HDL-C及ApoA测定值与正常参考值相比明显降低,p<0.05；血浆ApoB测定值虽然较正常参考值升高,但差异无统计学意义,p>0.05。研究组患者血浆TBIL及IBIL测定值较正常参考值显著降低,p<0.05；血浆DBIL测定值虽然较正常参考值降低,但差异无统计学意义,p>0.05。
2.CAG
研究组39例患者中合并左冠状动脉主干病变19例,三支病变14例,二支病变或合并左冠状动脉前降支近端病变6例。CAG提示冠状动脉均有不同程度的钙化、狭窄和迂曲,并且侧支循环建立不完全。采用Gensini积分系统进行定量积分以评价CAD严重程度,研究组39例患者Gensini积分平均为62.25±21.77(24-120)。
3.研究组和对照组动脉组织CHI3L1表达
研究组主动脉管壁组织经免疫组化检测均有CHI3L1表达,阳性表达主要分布于EC及VSMCs胞浆,表现为棕褐色或棕黄色,分布不均匀,呈颗粒状、片状、簇状及条纹状。对照组肾动脉管壁组织经免疫组化检测显示CHI3L1表达明显减少,着色表现为浅蓝色或无色,两者相比差异有统计学意义,p<0.05。
4.研究组动脉组织CHI3L1表达量与AS主要临床危险因素的关系
研究组患者CHI3L1相对表达量性别之间比较,差异无统计学意义,p>0.05；吸烟者与非吸烟者,高血压患者与非高血压患者,DM患者与非DM患者之间比较,差异均有统计学意义,p<0.05。研究组患者CHI3L1相对表达量与冠状动脉狭窄病变Gensini积分相关性分析表明两者呈正相关(r=0.611,P<0.05)。
结论
1.本研究利用人CABG时主动脉打孔废弃的主动脉组织为标本,检测动脉组织CHI3L1蛋白表达量较正常对照明显升高,为国内外首次报道。
2.本研究采用人活体动脉组织为样本,其研究结果提高了科学性、准确性及可信度,为研究AS发病机制增加了新的途径。
3.本研究显示CHI3L1蛋白表达量与AS主要临床危险因素、冠状动脉狭窄病变程度成正相关。AS主要临床危险因素可促进CHI3L1表达,CHI3L1可能通过调节AS主要临床危险因素在AS发病中起作用。
背景目的
动脉粥样硬化(atherosclerosis, AS)是慢性炎症性疾病,影响全身大中型动脉,已经成为冠状动脉疾病(coronary artery disease, CAD)的主要病理类型。各种AS危险因素可以导致内皮细胞(endothelial cell, EC)功能障碍和(或)脱落,趋化血液单核细胞及T淋巴细胞进入内膜下启动炎性反应,并分泌一系列细胞因子,进一步激活EC、血管平滑肌细胞(vascular smooth muscle cells, VSMCs)及巨噬细胞,促进AS发展。
几丁质酶3样蛋白1(chitinase3-like1, CHI3L1)存在几丁质结合活性,但无几丁质酶活性。研究表明CHI3L1可由一系列细胞分泌,如呼吸道上皮细胞、脂肪细胞、软骨细胞、肿瘤细胞、VSMCs,巨噬细胞等。CHI3L1在急慢性炎症及细胞外基质重构等疾病过程中起重要作用,如AS、支气管哮喘、高血压、糖尿病等。
人CHI3L1主动脉组织学研究显示,AS患者主动脉组织CHI3L1高度表达,为验证临床研究结果,本研究建立动物模型进行实验研究,以验证临床人活体主动脉组织研究的结果。为此本研究构建了有效慢病毒载体,使之能够携带针对小鼠CHI3L1的小干扰RNA (small interfering RNA, siRNA),并将慢病毒悬液局部转染ApoE-/-小鼠颈动脉斑块组织,观察CHI3L1基因沉默在延缓AS进展及稳定AS易损斑块中的作用。
材料方法
1.细胞培养
人胚肾细胞系293T细胞能够表达猿猴病毒40大T抗原,有利于慢病毒生产。选择小鼠单核巨噬细胞系RAW264.7细胞作为慢病毒体外转染的靶细胞。293T细胞及RAW264.7细胞于37℃在含有10%胎牛血清、100U/ml penicillin、100μg/ml streptomycin,5%CO2的DMEM中培养。
2.慢病毒构建及有效靶点筛选
设计4条针对小鼠CHI3L1基因的小发卡RNA (small hairpin RNA, shRNA)序列(site A、site B、site C及site D)。
site A的序列为：
5'-GCGACAACATGCTTAGCACATTTCAAGAGAATGTGCTAAGCATGTT GTCGCTT-3';
site B的序列为：
5'-GGCCATTGACACTGGCTATGATTCAAGAGATCATAGCCAGTGTCAAT GGCCTT-3';
site C的序列为：
5'-GCACTGGATTTGGATGATTTCTTCAAGAGAGAAATCATCCAAATCCA GTGCTT-3';
site D的序列为：
5'-GCCAGAAGGACACTAGGTTTGTTCAAGAGACAAACCTAGTGTCCTT CTGGCTT-3'c
作为阴性对照,随机不相关序列为：
5'-GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAG AACTT-3'。
分别构建包含上述各干扰序列的穿梭质粒载体(pShuttle vectors),然后将pShuttle vectors与pGag/Pol、pRev、pVSV-G共同转染293T细胞。慢病毒体外转染RAW264.7细胞筛选有效干扰序列。收集培养72h及96h的RAW264.7细胞进行RT-PCR及western blot分析,了解各干扰序列的干扰效率。
3.动物实验
50只雄性8周龄ApoE-/-小鼠饲养于SPF级饲养室,全程高脂饮食喂饲,随机分为CHI3L1慢病毒基因沉默组及空病毒载体阴性对照组。所有ApoE-/-小鼠均行右侧颈动脉套管术,术后8周颈动脉斑块组织局部转染慢病毒悬液(滴度均为1×109TU/ml)。
4.组织准备及免疫组化
ApoE-/-小鼠经慢病毒转染干预4周后分离颈动脉斑块组织,经常规固定、脱水、包埋后切片。组织切片分别进行HE染色、CHI3L1免疫组化、油红O染色(冰冻切片)、天狼猩红染色、VSMCs α-actin免疫组化、CD68免疫组化。
5.电镜检查
ApoE-/-小鼠颈动脉斑块组织经3%戊二醛固定、漂洗、1%锇酸固定、脱水、Spon812包埋,然后经半薄切片定位,制作超薄切片。切片经柠檬酸铅和醋酸铀电子染色,应用JEOL-1011型透射电镜观察。
6. RT-PCR分析
利用RT-PCR技术分别检测RAW264.7细胞中CHI3L1mRNA表达水平及ApoE-/-小鼠颈动脉斑块组织中CHI3L1、TNF-α、MCP-1、IL、MMP-9mRNA表达水平。
7. Western blot分析
RAW264.7细胞中CHI3L1蛋白表达及ApoE-/-小鼠颈动脉斑块组织中CHI3L1、p-ERK1/2、ERK1/2、p-AKT、AKT蛋白表达水平应用western blot技术检测。
结果
1.慢病毒构建及有效靶点筛选
慢病毒构建完成后转染RAW264.7细胞,RT-PCR分析表明site A、site B、 site C及site D干扰序列基因沉默分别减少CHI3L1mRNA表达量32%、17%、65%及30%。Western blot分析表明site A、siteB、site C及site D干扰序列基因沉默分别减少CHl3L1蛋白质表达量38%、18%、64%及14%。结果提示Site C为最有效的干扰序列,最终慢病毒悬液滴度为1×109TU/ml,用于下游ApoE-/-小鼠体内实验。
2.慢病毒体内转染效应
对照组ApoE-/-小鼠颈动脉斑块组织经CHI3L1免疫组化可见阳性表达,主要分布于EC及VSMCs胞浆,表现为棕褐色或棕黄色。基因沉默组ApoE-/-小鼠颈动脉斑块组织经免疫组化提示CHI3L1表达明显减少,表现为浅蓝色或无色。RT-PCR及western blot分析结果表明基因沉默组ApoE-/-小鼠颈动脉斑块组织中CHI3L1mRNA及蛋白表达量较对照组ApoE-/-小鼠明显减少,两者相比差异有统计学意义,p<0.05。
3.电镜检查
对照组ApoE-/-小鼠颈动脉斑块组织经电镜检查提示EC及VSMCs中存在大量脂质颗粒及胆固醇结晶,EC连接中断,VSMCs以合成型为多。基因沉默组ApoE-/-小鼠颈动脉斑块组织经电镜检查表明EC及VSMCs中可见少量脂质颗粒及胆固醇结晶,钙化多见,EC连接及基底膜大多完整,VSMCs以收缩型为多。
4.慢病毒体内转染对AS斑块构成的影响
对照组ApoE-/-小鼠及基因沉默组ApoE-/-小鼠颈动脉斑块组织中脂质相对含量分别为48.8%、35.2%,两者相比后者脂质减少27.5%；胶原相对含量分别为19.5%、29.8%,两者相比后者胶原增加53.2%；VSMCs相对含量分别为14.8%、22.5%,两者相比后者VSMCs增加51.2%；巨噬细胞相对含量分别为11.9%、7.5%,两者相比后者巨噬细胞减少36%,差异均有统计学意义,p<0.05。
5.慢病毒体内转染对AS斑块炎症因子及信号转导蛋白的影响
RT-PCR分析表明基因沉默组ApoE-/-小鼠较对照组ApoE-/-小鼠颈动脉斑块组织炎症因子TNF-α、MCP-1、IL-8、MMP-9mRNA表达水平均明显减少,差异均有统计学意义,p<0.05。Western blot分析表明基因沉默组ApoE-/-小鼠较对照组ApoE-/-小鼠颈动脉斑块组织p-ERK1/2及p-AKT蛋白表达水平均明显减少,差异均有统计学意义,p<0.05。
结论
1.应用RNA干扰技术可以有效构建针对小鼠CHI3L1基因的慢病毒载体。
2.本研究发现ApoE-/-小鼠经慢病毒载体介导的CHI3L1基因沉默干预能够改善易损斑块稳定性,验证了临床人活体主动脉组织研究的结果。
3. CHI3L1基因沉默可作为抑制AS斑块进展的一个新途径,为治疗AS提供新的思路和方法。
Background and Objectives
As a systematic disease, atherosclerosis is the underlying pathology of the coronary artery disease (CAD) and is a chronic inflammatory disease. According to the "response-to-injury" hypothesis, the endothelial denudation and (or) endothelial dysfunction caused by the risk factors are the first step in the development of atherosclerosis. These activated endothelial cells facilitate monocytes infiltration into the vessel wall and the monocytes differentiate into macrophages, which accumulate lipids and remain in the vessel wall, thereby becoming foam cells. These cells mentioned above release proinflammatory molecules such as tumor necrosis factor (TNF), monocyte chemoattractant protein (MCP), and interleukin (IL), which induce further accumulation of monocytes and migration and proliferation of vascular smooth muscle cells (VSMCs).
Chitinase3-like1(CHI3L1) is a chitin-binding glycoprotein without chitinase activity. CHI3L1has been shown to act as an important regulator of acute and chronic inflammation. CHI3L1can be secreted by a variety of cells, including VSMCs and macrophages. It is also found in tissues with inflammation and extracellular tissue remodeling. Several studies have shown an important link between CHI3L1and inflammatory diseases, including asthma, hypertension, diabetes mellitus, insulin resistance, and atherosclerosis as well.
Although the relationship between CHI3L1and CAD is important, there is a controversy in the association between blood CHI3L1levels and the severity of atherosclerosis. One study showed that severity of atherosclerosis is associated with higher blood CHI3L1levels, and another paper concluded that circulating CHI3L1was not specifically related to the size of atherosclerotic stenosis. In order to elucidate the relationship between CHI3L1and CAD, we investigated the correlation between CHI3L1expression levels and pathogenesis of atherosclerosis by measuring the changes of CHI3L1in the aortic tissues of patients undergoing coronary artery bypass graft (CABG) surgery.
Materials and Methods
1. Study population
From2010to2012,39patients with CAD scheduled for CABG surgery were investigated and defined as a research group. Eleven normal subjects who donated kidneys were investigated as the control group. Aortic specimens were obtained from the aorta that was routinely removed during the CABG surgery and the discarded renal arterial tissues were collected from the11subjects.
2. Biochemical analysis
Venous blood samples were obtained for the measurement of serum triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), lipoprotein (a)(Lp (a)), apolipoprotein A (ApoA), apolipoprotein B (ApoB), total bilirubin (TBIL), direct bilirubin (DBIL) and indirect bilirubin (IBIL) as well.
3. Coronary angiography
All CAD patients were confirmed by the coronary angiography (CAG). Gensini score system was used to assess the CAD severity.
4. Tissue preparation and histological analysis
The arterial tissues obtained from the CABG surgery and kidney donators were fixed in4%buffered formalin, embedded in paraffin and sectioned. After deparaffinage, hydration and antigen retrieve, the sections were incubated with the rabbit antihuman CHI3L1antibody.
Results
1. Baseline characteristics
The serum TG, TC, LDL-C, and Lp (a) levels in the research group were elevated, whereas the serum HDL-C, ApoA, TBIL, and IBIL levels were decreased, compared with the normal reference values (p<0.05).
2. Coronary atherosclerotic lesions in the research group
CAG in the research group showed that19patients were with left main lesions,6patients were with two coronary arteries lesions, and14patients were with three coronary arteries lesions. The average Gensini score was62.25±21.77.
3. Presence of CHI3L1in the human arterial tissues
In the arterial tissues obtained from the healthy donors little CHI3L1expression could be demonstrated according to the immunohistochemical staining. CHI3L1was overexpressed in the arterial specimens of CAD patients.
4. Correlation between the arterial CHI3L1expression and the clinical risk factors of,, atherosclerosis
The expression levels of CHI3L1did not differ between males and females, differ between smokers and non-smokers, hypertensives and non-hypertensives, diabetics and non-diabetics. The relative expression levels of CHI3L1was elevated in smokers, patients with hypertension, or diabetes mellitus, and gender had no significant effect. The linear correlation analysis revealed that arterial CHI3L1expression levels were correlated with the Gensini scores (r=0.61l,p<0.05).
Conclusions
1. In the present study, we found that CHI3L1was overexpressed in aorta of patients with coronary atherosclerosis and it might provide a new approach to the research of atherosclerosis.
2. The relative expression levels were correlated with the atherosclerotic risk factors and the severity of CAD and CHI3L1could be a predictor of the coronary arterial stenosis.
Background and Objectives
Atherosclerosis is a chronic inflammatory disease and is the underlying pathology of the coronary artery disease (CAD). The endothelial denudation and (or) endothelial dysfunction are the first step in the development of atherosclerosis. The activated endothelial cells facilitate monocytes infiltration into the vessel wall. These monocytes differentiate into macrophages, which accumulate lipids and remain in the vessel wall, thereby becoming foam cells. These cells synthesize and release proinflammatory molecules, which induce further accumulation of monocytes and migration and proliferation of vascular smooth muscle cells (VSMCs).
Chitinase3-like1(CHI3L1) is a chitin-binding glycoprotein without chitinase activity. CHI3L1has been shown to act as an important regulator of acute and chronic inflammation. CHI3L1is secreted by a variety of cells, including VSMCs and macrophages. It is also found in tissues with inflammation and extracellular tissue remodeling. Several studies have shown an important link between CHI3L1and inflammatory diseases, including asthma, hypertension, diabetes mellitus, insulin resistance, and atherosclerosis as well.
The relationship between CHI3L1and atherosclerosis is important, in order to verify the therapeutic value of CHI3L1, we constructed lentiviral vectors, which could efficiently deliver small interfering RNAs (siRNAs), and aimed at knocking down CHI3L1to explore the mechanisms of CHI3L1in atherosclerosis in apolipoprotein E-knockout (ApoE-/-) mice.
Materials and Methods
1. Cell culture
The293T human embryonic kidney cell line and the RAW264.7mouse macrophage cell line were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with10%fetal bovine serum (FBS),100U/ml penicillin,100μg/ml streptomycin, in a humidified incubator with5%CO2.
2. Lentivirus construction and target screening for RNAi
Four different sequences (site A, site B, site C, and site D) of mice CHI3L1gene were designed as the target for RNA interference (RNAi).
The sequence of site A was:
5'-GCGACAACATGCTTAGCACATTTCAAGAGAATGTGCTAAGCATGTT GTCGCTT-3';
the sequence of site B was:
5'-GGCCATTGACACTGGCTATGATTCAAGAGATCATAGCCAGTGTCAAT GGCCTT-3';
the sequence of site C was:
5'-GCACTGGATTTGGATGATTTCTTCAAGAGAGAAATCATCCAAATCCA GTGCTT-3';
the sequence of site D was:
5'-GCCAGAAGGACACTAGGTTTGTTCAAGAGACAAACCTAGTGTCCTT CTGGCTT-3'.
As a negative control, the scrambled sequence (mock siRNA) was:
5'-GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAG AACTT-3'.
The pShuttle vectors containing the different mouse CHI3L1RNAi sequences were constructed. A lentivirus was produced by cotransfection of the pShuttle vector, pGag/Pol, pRev, and pVSV-G into the293T cells. The lentiviruses were used to transfect the RAW264.7cells. The RAW264.7cells were collected for RT-PCR and western blot experiment at72h and96h after transfection.
3. Animal experiment
We obtained50male ApoE-/-mice,8weeks old. All the mice were fed a high-fat diet. The mice were divided into2groups:empty lentivirus control group and CHI3L1lentivirus silenced group. A constrictive silica collar was placed on the right common carotid artery of mice. Eight weeks after surgery, the lentiviral suspension at1×109TU/ml was instilled into the right common carotid artery.
4. Tissue preparation and histological analysis
The right common carotid artery obtained from the mice was immersed in4%formaldehyde. Six crosssections in each mouse were used for a particular type of staining. One section was stained with hematoxylin and eosin. Another section was immunostained with rabbit antimouse CHI3L1antibody. Collagen and lipids were identified by Sirius red staining and oil red O staining. VSMCs and macrophages were immunostained with a-actin antibody and CD68antibody.
5. Transmission electron microscopy
The fresh mice arterial tissues were used to undergo electron microscope examination. The arterial tissues were placed in2.5%glutaraldehyde, then the tissues were postfixed in1%osmium tetroxide followed by staining with2%uranyl acetate. Then the tissues were dehydrated through ethanol and were embedded in Spon812. Finally, the sections were stained with uranyl acetate followed by lead citrate and examined in a JEM-1010electron telescope.
6. RT-PCR analysis
The mRNA expression levels of CHI3L1in RAW264.7cells, CHI3L1, TNF-a, MCP-1, IL-8, and MMP-9in mice were analysed using the RT-PCR.
7. Western blot analysis
The protein expression levels of CHI3L1, p-ERK1/2, ERK1/2, p-AKT, and AKT in RAW264.7cells and in mice were assayed by western blot.
Results
1. Effects of lentiviral transfection in vitro
The RAW264.7cell line was transfected with four different CHI3L1siRNAs. Site A, site B, site C, and site D exhibited32%,17%,65%, and30%reduction in mRNA expression. Site A, site B, site C, and site D exhibited38%,18%,64%, and14%reduction in protein expression. Then the site C was the most effective vector in blocking CHI3L1expression. The site C lentivirus and mock lentivirus were produced at a viral titer of1×109TU/ml for further in vivo studies.
2. Effects of lentiviral transfection on CHI3L1expression in plaques
In the control group, CHI3L1expression could be demonstrated according to the immunohistochemical staining. However, little CHI3L1was expressed in the silenced group. Compared with the control group, the silenced group showed reduced CHI3L1protein expression by50%. In addition, there were significant differences of CHI3L1mRNA expression levels between control group and silenced group.
3. Electron microscopy analysis
For electron microscopy, in control group most of the endothelial cells denudated and there were a large number of lipid granules under the basement membrane in the vessel wall. The atherosclerotic plaques were occupied with necrotic particles, calcification crystals and cellular debrises. However, in silenced group the number of lipid granules was relatively decreased. Collagen bundles and elastic fibers were seen on the vessel side of the endothelium.
4. Effects of lentiviral transfection on plaque composition
The relative content of lipids in plaque tissues of the control group and the silenced group was48.8%and35.2%, the relative reduction of lipids content in the silenced group was27.5%. The relative content of collagen was19.5%and29.8%, the relative increase of collagen content was53.2%. The relative VSMCs content was14.8%and22.5%, the relative increase of VSMCs content was51.2%. The relative macrophages content was11.9%and7.5%, the relative reduction of macrophages content was36%.
5. Effects of CHI3L1gene silencing on inflammatory mediators and signal transduction protein in the lesions
The CHI3L1silenced group showed lower mRNA expression levels of TNF-a, MCP-1, IL-8, MMP-9, compared with the control group (p<0.05). The silenced group showed lower protein expression levels of p-ERK1/2and p-AKT, compared with the control group (p<0.05).
Conclusions
1. In the present study, we applied lentivirus mediated shRNA to knock down the mice CHI3L1gene.
2. The silence of CHI3L1diminished the atherosclerotic burden and increased plaque stability in ApoE-/-mice, which might provide a new therapeutic approach to the treatment of atherosclerosis.
引文
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