基因敲除动脉粥样硬化小鼠模型研究进展

基因敲除小鼠动脉粥样硬化模型是目前较为认可的探讨动脉粥样硬化发病机制及寻找新药物靶点的关键工具,也应用于潜在抗动脉粥样硬化药物的药理和毒理研究。载脂蛋白E(ApoE)、低密度脂蛋白受体(LDLR)和B类Ⅰ型清道夫受体(SR-B1)等基因的表达促进机体脂质、胆固醇和低密度脂蛋白等转运和代谢,维持血管正常功能,敲除这些基因会引起脂质转运及代谢紊乱从而诱发动脉粥样硬化及并发症的发生发展。常见的基因敲除小鼠有ApoE基因敲除、LDLR基因敲除及其改良品系,这些模型小鼠能够客观反映敲除基因对动脉粥样硬化发生的影响,且广泛应用于动脉粥样硬化的非临床研究。本文阐述了当前基因敲除小鼠动脉粥样硬化模型的机制、应用和优缺点,以期为动脉粥样硬化发病机制研究及抗动脉粥样硬化药物筛选提供参考。     

携带xylE的转基因小鼠的制备

建立了一种新的转基因小鼠致突变检测模型．选用ｘｙｌＥ基因作为诱变的靶基因,以ｐＥＳｎｘ穿梭质粒作为载体,通过显微注射法将线状ｐＥＳｎｘ导入ＩＣＲ小鼠制备Ｇ０小鼠．将显微注射后存活的３５２／５４９枚受精卵分别移入２４只受体雌鼠的输卵管中,共产生４１只仔鼠,存活３０只（Ｇ０）,经过整合检测,检测出转基因小鼠１７只,阳性率为５７％．从中筛选出２只完整整合了ｐＥＳｎｘ的转基因小鼠作为首建鼠（ｆｏｕｎｄｅｒｍｏｕｓｅ）进行繁育,目前已繁殖到第三代（Ｇ３）．经过逐代整合检测,证明转入的基因可稳定地遗传．上述结果表明已成功地制备了在基因组中整合了ｐＥＳｎｘ质粒携带ｘｙｌＥ的转基因小鼠．     

携带xylE的转基因小鼠的制备

建立了一种新的转基因小鼠致突变检测模型.选用xylE基因作为诱变的靶基因,以pESnx穿梭质粒作为载体,通过显微注射法将线状pESnx导入ICR小鼠制备G₀小鼠. 将显微注射后存活的352/549枚受精卵分别移入24只受体雌鼠的输卵管中,共产生41只仔鼠,存活30只(G₀),经过整合检测,检测出转基因小鼠17只,阳性率为57％. 从中筛选出2只完整整合了pESnx的转基因小鼠作为首建鼠(founder mouse)进行繁育,目前已繁殖到第三代(G₃). 经过逐代整合检测,证明转入的基因可稳定地遗传. 上述结果表明已成功地制备了在基因组中整合了pESnx质粒携带xylE的转基因小鼠.     

Expression of microsomal prostaglandin E synthase-1 in the spinal cord in a transgenic mouse model of amyotrophic lateral sclerosis

Prostaglandin E(2) (PGE(2)) is a key molecule involved in the neuroinflammatory processes that characterize amyotrophic lateral sclerosis (ALS). Although PGE(2) synthesis is regulated by PGE(2) synthases (PGESs), the pathological role of PGESs in ALS still remains unknown. Experiments were performed to elucidate the expression of PGESs and the localization of microsomal PGES-1 (mPGES-1) in neurons and glial cells in the spinal cord of ALS model (G93A) mice. Neurological symptom was observed in G93A mice from 14 weeks by the tail suspension test, and rotarod performances were decreased at 16 weeks and older. Western blotting revealed that the level of mPGES-1 was increased in G93A mice at 15 weeks and older. In contrast, the levels of cytosolic PGES and mPGES-2 did not change at any age. Immunohistochemical analysis demonstrated that age-dependent expression of mPGES-1 was found in motor neurons in G93A mice at 11 and 15 weeks. Immunoreactivity of mPGES-1 was also co-localized in Iba1-positive microglia in G93A mice at 15 weeks. These results suggest that mPGES-1 in motor neurons may play a role in the pathogenesis of ALS and that mPGES-1 may work sequentially in motor neurons and activated microglia to produce ALS symptoms in G93A mice.     

Pharmacotherapy of systemic sclerosis

Importance of the field: Systemic sclerosis (SSc) is an uncommon autoimmune disease with variable degrees of fibroproliferation in blood vessels and certain organs of the body. There is currently no cure. The purpose of this article is to review the current literature regarding pathogenesis and treatment of complications of SSc.

Areas covered in this review: All available articles regarding research related to SSc pathogenesis and treatment listed in the PubMed database were searched; relevant articles were then reviewed and used as sources of information for this review.

What the reader will gain: This review attempts to highlight for the reader some current thought regarding mechanisms of SSc pathogenesis and how autoimmunity relates to vascular changes and fibrogenesis of the disease, as well as providing a review of results of completed clinical trials and current ongoing clinical trials that address organ-specific or global therapies for this disease. This can aid physicians who provide medical care for patients with SSc.

Take home message: SSc is a complex autoimmune disease, the pathogenesis of which, although not completely understood, is under active study; new insights into pathogenesis are continually being discovered. Although there is no effective disease-modifying treatment for patients with SSc, quality of life, morbidity and mortality can be improved by using targeted therapy directed at affecting the consequences of damage to lungs, blood vessels, kidneys and the gastrointestinal tract. Innovative approaches to treating SSc are under intense investigation.     

降植烷诱导小鼠系统性红斑狼疮模型研究

目的 建立降植烷诱导的系统性红斑狼疮（SLE）小鼠模型,并对模型进行全面验证。方法 6~8周龄雌性BALB/c小鼠随机分为两组,模型组单次ip降植烷0.5 mL,对照组单次ip生理盐水0.5 mL。注射前及注射后2、3、4、5、6个月ELISA法检测血清中抗SLE抗体（anti-SLE）、抗双链DNA抗体（anti-dsDNA）;注射前及注射后每月1次采用目测尿蛋白试纸测定小鼠尿蛋白;6个月处死动物,观察肾脏HE染色及直接免疫荧光染色后镜下变化。结果 小鼠ip降植烷2个月后,其自身抗体（anti-dsDNA、anti-SLE）浓度显著高于对照组（P<0.05、0.01）,且6个月内抗体浓度逐渐升高;6个月时,73%模型组小鼠出现++++尿蛋白;肾脏HE切片显示模型组小鼠肾脏出现肾小球肿胀、炎症细胞浸润等典型的肾病理改变,直接免疫荧光染色见模型组小鼠肾小球毛细血管存在免疫复合物沉积,对照组小鼠肾脏组织未见改变。结论 降植烷成功诱导SLE动物模型。     

Anxiety in a transgenic mouse model of cortical-limbic neuro-potentiated compulsive behavior

Anxiety and amygdalar stimulation may induce or exacerbate compulsions triggered by cortical-limbic hyperactivity, as in human obsessive-compulsive disorder (OCD). We previously created transgenic mice that exhibit OCD-like biting, movement and behavioral perseverance abnormalities. These behaviors are caused by expression of a neuro-potentiating cholera toxin (CT) transgene in dopamine D1 receptor-expressing (D1+) neurons within the amygdalar intercalated nucleus (ICN) and within cortical areas that project to orbitofrontal cortex and striatum. Here we tested whether anxiety and increased amygdalar stimulation may play a role in eliciting or exacerbating such behaviors. D1CT mice exhibited increased thigmotaxis (tendency of mice to remain along the perimeter of open areas) in the open field assay, and increased latency to first transit and reduced transit number in the light-dark assay. These studies indicate that the D1CT mice exhibit a significant increase in behavioral indicators of anxiety. Furthermore, yohimbine, a drug that induces both amygdalar stimulation and behavioral indicators of anxiety, exacerbated abnormal leaping in D1CT mice but failed to exacerbate their abnormal behavioral perseverance. These data suggest that chronic potentiation of D1+ neurons in the amygdalar ICN increases anxiety and facilitates particular compulsive behaviors.     

Propylthiouracil modulates aortic vasculopathy in the oxidative stress model of systemic sclerosis

BackgroundIn systemic sclerosis (SSc) vasculopathy affects small arteries and capillaries, but recent evidences show also macrovascular alterations. Experimental data suggest that propylthiouracil (PTU) abrogates the development of cutaneous and pulmonary fibrosis during SSc. The aim of this study was to evaluate the effect of propylthiouracil on aortic lipid peroxidation, intima-media thickness and myofibroblasts differentiation in experimental SSc.MethodsSSc was induced in BALB/c mice by daily subcutaneous injections of hypochlorous acid (HOCl) for 6 weeks. Mice (n = 25) were randomized to receive daily: HOCl (n = 10), HOCl + PTU (n = 10), or vehicle (n = 5). Thoracic aorta was evaluated by histological methods to measure intima-media thickness and by immunostaining for α-smooth muscle actin (α-SMA) to assess myofibroblast differentiation. Aortic and plasma levels of malondialdehyde (MDA) were also measured.ResultsHOCl induced a significant increase in aortic intima-media thickness compared to controls (p < 0.001), while PTU administration prevented intima-media thickening (p < 0.01). Myofibroblast differentiation was also less evident in HOCl + PTU-treated animals (p < 0.05) compared to mice treated with HOCl alone. The increase in aortic and plasma MDA levels induced by HOCl, was significantly prevented by PTU administration (p < 0.05).ConclusionPTU, by reducing lipid peroxidation, prevents aortic thickening and myofibroblast differentiation induced by HOCl, reducing macrovascular alterations in experimental SSc.     

Bosentan in systemic sclerosis

Systemic sclerosis (SSc) is a relatively rare chronic connective tissue disease characterized by varying degrees of skin fibrosis and visceral organ involvement. Pulmonary compromise, including pulmonary arterial hypertension and interstitial lung disease, is currently the leading cause of death in patients with SSc. Digital ulcers are common complications which lead to substantial morbidity and functional limitation. Until recently, treatment options for these complications were quite limited. Endothelin-1 (ET-1) is a peptide that has a role in promoting both vascular injury and the fibrotic process in SSc. Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension. In patients with pulmonary hypertension secondary to SSc, bosentan therapy prevents deterioration in exercise capacity and may improve survival. No beneficial effect was found in one study in patients with interstitial lung disease and SSc. Bosentan is able to reduce the number of new digital ulcers in patients with either a history of previous ulcers or an active ulcer, without expediting the healing of existing ulcers. Bosentan therapy is contraindicated in pregnancy and causes elevated liver transaminases in up to 14% of patients. Hence, monthly pregnancy tests should be performed and hepatic function should be monitored.     

淋巴细胞活性染色质诱导系统性红斑狼疮样小鼠模型

目的建立活性染色质诱导系统性红斑狼疮(SLE)样小鼠模型。方法从ConA活化的BALB/c小鼠脾淋巴细胞中提取活性染色质,分别于d 0、d 14、d 21和d 28以染色质100μg在BALB/c小鼠尾根部及背部皮内注射免疫4次,诱导SLE样小鼠模型。目测半定量尿蛋白试纸法检测动物的尿蛋白变化,HE染色法检查动物的肾脏、脾脏病理改变,计算动物的胸腺和脾脏指数,MTT法检测ConA和LPS诱导的T、B淋巴细胞增殖反应,全自动生化分析仪检测血清中Crea和BUN水平,ELISA法检测小鼠血清中ANA、抗dsDNA、IgG1、IgG2a、IL-10、IFN-γ水平,流式细胞术检测脾脏T、B淋巴细胞亚群变化。结果诱导模型小鼠尿蛋白水平升高,出现肾小球肾炎、脾脏增生等病理改变;脾脏指数明显升高,LPS诱导的B淋巴细胞增殖反应增强;血清Crea、BUN、ANA、抗dsDNA、IgG1、IgG2a、IL-10和IFN-γ水平明显升高;脾脏CD19+、CD19+CD21+、CD19+CD23+、CD19+IgD+B淋巴细胞亚群百分比明显升高,CD4+CD25+T淋巴细胞百分比明显下降。结论 ConA活化淋巴细胞的染色质免疫同系BALB/c小鼠成功诱导了SLE样小鼠模型,其血清学、组织病理学及免疫学方面特征与人类SLE临床特征表现相似。     

Therapeutic effects of D-aspartate in a mouse model of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. EAE is mainly mediated by adaptive and innate immune responses that leads to an inflammatory demyelization and axonal damage. The aim of the present research was to examine the therapeutic efficacy of D-aspartic acid (D-Asp) on a mouse EAE model. EAE induction was performed in female C57BL/6 mice by myelin 40 oligodendrocyte glycoprotein (35-55) in a complete Freund's adjuvant emulsion, and D-Asp was used to test its efficiency in the reduction of EAE. During the course of study, clinical evaluation was assessed, and on Day 21, post-immunization blood samples were taken from the heart of mice for the evaluation of interleukin 6 and other chemical molecules. The mice were sacrificed, and their brain and cerebellum were removed for histological analysis. Our findings indicated that D-Asp had beneficial effects on EAE by attenuation in the severity and delay in the onset of the disease. Histological analysis showed that treatment with D-Asp can reduce inflammation. Moreover, in D-Asp-treated mice, the serum level of interleukin 6 was significantly lower than that in control animals, whereas the total antioxidant capacity was significantly higher. The data indicates that D-Asp possess neuroprotective property to prevent the onset of the multiple sclerosis.     

系统性硬化症的流行病学研究

系统性硬化症是一种罕见的自身免疫病,患者生活质量差,严重者危及生命,但其致病原因尚不明确.本文对系统性硬化症的流行病学研究有助于加深对该病的了解.     

人类p93基因转基因小鼠模型的建立及功能初探

目的 探讨心肌特异性表达的新激酶基因p93对心脏发育的影响.方法 构建携带人心肌特异启动子的α-MHC-p93转基因质粒,以显微注射法获得p93转基因首建鼠.对转基因小鼠进行形态学观察及血清水平检测.结果 成功构建了携带有人p93基因的真核表达载体α-MHC-p93,获得首建鼠7只.经繁育共获得F1、F2代转基因鼠234只,筛选出转基因阳性鼠4只,其中F1代3只,F2代1只.小鼠心脏大体形态学检测未见房室间隔缺损现象.血清学检测结果显示:p93转基因阳性鼠血Ca2+水平明显高于正常对照组[ (28±1)mg/L比(26±1)mg/L,P＜0.05],差异有统计学意义(P＜0.05);血清肌酸激酶同工酶(CK-MB)水平低于正常对照组[(624.7±268.1)U/L比(1229.8±532.4)U/L,P＜0.05];血清乳酸脱氢酶(LDH)水平及心脏质量指数(HMI)与正常对照组相比,差异无统计学意义.结论 成功建立了人p93基因的转基因小鼠模型.单一转入p93基因没有造成实验动物的房室间隔缺损及心肌肥大.     

系统性硬化症的心脏受累

系统性硬化症(SSc)患者常伴有心脏受累,尽管大多数的心脏病变都呈隐匿性进展,但是当患者出现明显的临床心血管疾病症状时,常提示预后不良.SSc可以影响心脏各个部位,最终导致心肌缺血、心脏循环障碍、心律失常,心包积液以及瓣膜损伤.在目前众多的检测方法中,超声心动图,特别是组织多普勒成像、心脏磁共振成像以及PET扫描等对早期筛选SSc相关心脏病具较高敏感性.钙通道阻断剂和血管紧张素转换酶抑制剂的使用可以改善SSc心肌灌注和心脏功能,更重要的是,对SSc心脏受累进行早期的筛查和治疗,将对其预后改善产生重要影响.     

Targeting fibrosis in systemic sclerosis

Finding effective treatments for patients with systemic sclerosis (SSc) remains one of the final frontiers in therapeutic discovery. Although remarkable progress has been made in the symptomatic treatment of various organ system manifestations, little is available that treats the underlying disease process. SSc patients do not respond to many of the medications that provide benefit in related diseases, such as systemic lupus erythematosus, polymyositis and chronic graft-versus-host disease. Current research has not even clarified whether the complex pathogenesis starts primarily in vascular, immunological or connective tissues. Herein are discussed selected emerging therapeutics and therapeutic approaches designed to target the underlying immunological and fibrotic disease processes. Distinctive fibrotic features and data from translational research consistently place transforming growth factor-beta (TGFbeta) as a central mediator in SSc. The discovery of agents targeting TGFbeta, its activation or its intracellular signaling suggest that TGFbeta pathway inhibitors efficacious for the treatment of SSc may soon be identified. IL-4 and IL-13 are other fibrotic mediators produced during immune activation that might be targeted for SSc therapy, and therapeutics targeting these interleukins are also being developed. Immune dysregulation, leading to overproduction of these or other fibrotic mediators might respond to currently available immunosuppressives: mycophenolate, cyclosporine, tacrolimus or sirolimus, alone or in combination. Nucleic acid-containing immune complexes may also contribute to toll-like receptor mediated immune dysregulation in SSc, suggesting that agents targeting the innate immune system may ameliorate SSc. Thus, the complexity of SSc pathogenesis provides a plethora of targets for urgently needed new therapies.     

The plasminogen activator system in fibroblasts from systemic sclerosis

Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are two major subsets of SSc, diffuse cutaneous Systemic sclerosis (dSSc) and limited cutaneous Systemic sclerosis (lSSc). Fibroblasts play a key role in SSc. The expression and function of the urokinase (uPA)-mediated plasminogen activation (PA) system, a well-characterized system of serine-proteases involved in several pathological processes, has been investigated in SSc fibroblasts. The expression of the components of the PA system, including uPA, its type-1 and type-2 inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), was examined by Western blot in fibroblasts from patients affected by limited and diffuse forms of SSc. uPA and PAI-1 secretion increased only in fibroblasts from lSSc lesions compared to normal fibroblasts. PAI-2 levels were decreased in fibroblasts from both SSc forms. Interestingly, fibroblasts from areas not adjacent to the lesions (not-affected) of the diffuse form showed reduced levels of PAI-1 and increased uPAR expression. Adhesion experiments showed reduced adherence to VN of fibroblasts from lSSc lesions and from non-affected areas of the diffuse form, as compared to normal controls. These results suggest a role for uPA and PAI-1 in the lSSc form, likely related to the activation of latent forms of cytokines and to the accumulation of ECM components, whereas a role for uPAR can be hypothesized in the evolvement of the diffuse form, based on its up-regulation in the non-affected areas.     

Reduction of the cerebrovascular volume in a transgenic mouse model of Alzheimer's disease

Combined evidence from neuroimaging and neuropathological studies shows that signs of vascular pathology and brain hypoperfusion develop early in Alzheimer's disease (AD). To investigate the functional implication of these abnormalities, we have studied the cerebrovascular volume and selected markers of blood-brain barrier (BBB) integrity in 11-month-old 3×Tg-AD mice, using the in situ brain perfusion technique. The cerebrovascular volume of distribution of two vascular space markers, [³H]-inulin and [¹⁴C]-sucrose, was significantly lower (−26% and −27%, respectively; p < 0.01) in the brain of 3×Tg-AD mice compared to non-transgenic littermates. The vascular volume reduction was significant in the hippocampus (p < 0.01), but not in the frontal cortex and cerebellum. However, the brain transport coefficient (Clup) of [¹⁴C]-d-glucose (1 μM) and [³H]-diazepam was similar between 3×Tg-AD mice and controls, suggesting no difference in the functional integrity of the BBB. We also report a 32% increase (p < 0.001) in the thickness of basement membranes surrounding cortical microvessels along with a 20% increase (p < 0.05) of brain collagen content in 3×Tg-AD mice compared to controls. The present data indicate that the cerebrovascular space is reduced in a mouse model of Aβ and tau accumulation, an observation consistent with the presence of cerebrovascular pathology in AD.     

DNA-based immunization breaks tolerance in a hepatitis C virus transgenic mouse model

Chronic hepatitis C infection (HCV) is associated with high morbidity and mortality due to limited treatment options. HCV transgenic mice can be used as surrogate model of chronic HCV infection and may therefore be a small animal model for the evaluation of therapeutic vaccination strategies. We immunized transgenic mice expressing HCV structural proteins with an HCV core and a mouse IL-2 expression plasmid to study the cellular and humoral immune responses. DNA-based immunization induced a significant CD4+ T cell proliferative response and a CD8+ cytotoxic T cell response. Only low amounts of anti-HCV core antibodies were detected after genetic immunization. No liver damage was observed in the liver of immunized mice despite low level HCV core transgene expression and the presence of peripheral cellular immunity. These results demonstrate that DNA-based immunization may result in activation of previously tolerant T cells and is therefore a promising approach for treatment of chronic HCV infection.