载脂蛋白E基因敲除小鼠在动脉粥样硬化研究中的应用

载脂蛋白E基因敲除小鼠是目前在动脉粥样硬化（AS）研究领域中应用最多的基因工程动物。参考国内外应用载脂蛋白E基因敲除小鼠研究AS的有关文献，就其AS病灶形成的规律及形态学改变，有氧运动和饮食对该小鼠AS病灶的影响，关于调脂药物和其它药物研究情况，骨髓移植和基因治疗对该种小鼠AS病灶的影响等方面作一综述，以期将这种基因工程小鼠动物模型更广泛地应用于中西医药研究。     

载脂蛋白E基因敲除小鼠在动脉粥样硬化研究中的应用

载脂蛋白E基因敲除小鼠是目前在动脉粥样硬化 (AS)研究领域中应用最多的基因工程动物。参考国内外应用载脂蛋白E基因敲除小鼠研究AS的有关文献 ,就其AS病灶形成的规律及形态学改变 ,有氧运动和饮食对该小鼠AS病灶的影响 ,关于调脂药物和其它药物研究情况 ,骨髓移植和基因治疗对该种小鼠AS病灶的影响等方面作一综述 ,以期将这种基因工程小鼠动物模型更广泛地应用于中西医药研究     

大鼠皮肤创伤修复过程中转化生长因子β1和β3的表达

背景：转化生长因子β在组织创伤修复中发挥核心和关键作用。 目的：观察转化生长因子β1和转化生长因子β3在大鼠皮肤瘢痕性创伤愈合过程中表达量及表达部位的变化。 方法：制备大鼠皮肤全层切伤模型,长度1.5-2.0 cm,深及筋膜层。于伤后0 h,12 h,1 d,2 d,3 d,4 d,5 d,6 d,7 d处死大鼠,取损伤部位皮肤,采用免疫组织化学染色检测各时间点转化生长因子β1和转化生长因子β3的表达,并进行定量分析。 结果与结论：免疫组织化学染色显示,在创伤愈合的早期阶段(伤后1-5 d),转化生长因子β1和转化生长因子β3免疫阳性颗粒主要出现在上皮细胞、上皮基底层细胞胞浆、巨噬细胞等免疫细胞胞浆及肉芽组织中;随着创伤修复时间的持续,免疫阳性颗粒主要出现在真皮层的成纤维细胞及细胞外基质中。其中转化生长因子β1的表达在创伤后1-5 d最强,而转化生长因子β3在创伤后六七天时开始明显表达。可见在大鼠皮肤瘢痕性创伤愈合过程中,转化生长因子β1的表达先于转化生长因子β3,提示转化生长因子β1与胶原形成及创伤修复关系密切,而转化生长因子β3在愈合后期表达量有升高趋势,其可能与创伤后期的组织改建密切相关。     

人转化生长因子β1基因重组腺病毒的构建与鉴定

背景：转化生长因子β1能促进多种细胞的生长增殖,是调控骨髓间充质干细胞向成骨方向定向分化的主要生长因子。 目的：利用AdMax系统构建人转化生长因子β1(hTGF-β1)基因的重组腺病毒表达载体。 方法：采用PCR方法克隆人转化生长因子β1基因cDNA后,插入线性化表达载体pAV-MCMV-EGFP-3FLAG中,转化E.coli DH5α感受态细胞,构建pAV-MCMV-hTGF-β1重组质粒。 结果与结论：含目的基因的重组腺病毒质粒共转染293细胞进行病毒包装、扩增后, 经PCR、Western Blot检测得到转化生长因子β1重组腺病毒,其滴度约为1.25×10¹⁰ pfu/mL。表明利用AdMax系统成功可构建人转化生长因子β1腺病毒载体,并可满足进一步的转化生长因子β1成骨作用的研究。     

转化生长因子β信号与肌肉调控

BACKGROUND: Transforming growth factor-β signaling widely existing in cells mediates cell growth, proliferation, migration, differentiation, and apoptosis. The activation of transforming growth factor-β signaling can result in muscular dystrophy. However, there have been some contradictions regarding the effects of the transforming growth factor-β signaling on muscular dystrophy. OBJECTIVE: To summarize the latest progress in the effects of the transforming growth factor-β signaling on muscle mass and function regulation to provide the solutions for the treatment of muscular dystrophy. METHODS: A computer-based online search was conducted in PubMed and Wanfang databases from 2005 to 2015 to screen the relevant literatures using Chinese and English key words “transforming growth factor-β, muscle, regulation mechanism, treatment”. A total of 102 literatures were retrieved, and 22 eligible literatures were included, summarized, and analyzed. RESULTS AND CONCLUSION: The activation of transforming growth factor-β signaling as a common cause of most muscle disorders promotes the activation of muscle satellite cells, differentiation of myocytes, myoblast infusion, the expression of muscle-specific proteins, and the inhibition of collagen synthesis, which facilitates muscular fibrosis and scar formation. Transforming growth factor-β signaling is involved in Duchenne muscular dystrophy, spinal scoliosis, type I diabetes induced skeletal muscle regenerative disorders, myocardial and cardiac remodeling. The inhibition of transforming growth factor-β signaling may result in incomplete muscle recovery. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

载脂蛋白E基因敲除小鼠冠状动脉内粥样硬化病灶的研究

目的：研究载脂蛋白E基因敲除( apoE-/-)小鼠冠状动脉内粥样硬化病灶的分布和组成；探讨病灶发生和发展的机理。方法：取60周龄和112周龄的apoE-/-小鼠心脏作连续切片，从冠状动脉在主动脉开口处连续追踪冠状动脉主干和心肌内冠状动脉小分支，行Movat特殊染色，寻找病灶。根据组织切片数计算病灶离冠状动脉开口处的距离和病灶的长度；根据病灶的长度将病灶分为大、中、小3类病灶；用图像分析仪测量血管口径；根据Movat染色结果观察病灶内组成成分。结果：在apoE-/-小鼠冠状动脉内发现从主动脉内直接延续的延伸病灶和在冠状动脉分支内形成的原位病灶；在60周龄和112周龄小鼠冠状动脉延伸病灶的发生率分别为60%和80%；有延伸病灶的冠状动脉外膜有大量炎性细胞浸润。每只apoE-/-小鼠均有原位病灶；原位病灶多发生在左室壁心肌内血管分支处和乳头肌附近；随着原位病灶增大，蛋白聚糖成分减少，细胞内外脂质成分增多；增大的原位病灶可堵塞小血管；在有原位病灶的冠状动脉外膜常可发现有增多的炎性细胞。112周龄apoE-/-小鼠大原位病灶多于60周龄小鼠 (P＜0.05)。结论：apoE-/-小鼠冠状动脉主干和心肌小分支内分别存在着延伸和原位的粥样硬化病灶，随着小鼠周龄增加，病灶增大。动脉外膜炎症和心肌收缩对血管的挤压与病灶的发生和发展有密切关系。     

高压氧预处理超长皮瓣组织血管内皮生长因子、转化生长因子β的表达

BACKGROUND: Hyperbaric oxygen (HBO) therapy can alleviate the skin flap congestion by improving the angiogenesis and increasing the oxygen content of blood in skin flaps. Although the HBO therapy ability to increase flap survival has been well described, the research on the application of HBO pretreatment in skin flap transplantation does not arouse adequate concern. OBJECTIVE: To investigate the effect of HBO pretreatment on early-stage flap congestion in the rat model of over-length dorsal random skin flaps. METHODS: Thirty-six SD rats were randomly divided into control group (n=12), HBO pretreatment group (n=12) and HBO treatment group (n=12). Rats in the HBO pretreatment group received 4 days of HBO therapy prior to transplantation, once a day. Rats in the HBO treatment group received 4 days of HBO therapy after transplantation. Rats in the control group were raised in the normal conditions after flap transplantation. At postoperative days 3 and 5, rats were sacrificed and the samples were collected. The inflammation of flap tissues was detected using hematoxylin-eosin staining. The expression of vascular endothelial growth factor and transforming growth factor-β was analyzed by immunohistochemistry staining. The flap survival rate was calculated at postoperative day 5. RESULTS AND CONCLUSION: The flap survival area of the HBO pretreatment group and HBO treatment group was larger than that of the control group (P < 0.05). At postoperative days 3 and 5, the expression levels of vascular endothelial growth factor and transforming growth factor-β in the flap tissue were higher in the HBO pretreatment group and HBO treatment group than the control group (P < 0.05). However, there was no significant difference in the flap survival area and the expression of vascular endothelial growth factor and transforming growth factor-β between HBO pretreatment and HBO treatment groups  (P > 0.05). HBO pretreatment can increase the expression of vascular endothelial growth factor and transforming growth factor-β and promotes angiogenesis in random pattern flaps, thereby improving skin flap survival.      

退行性变椎间盘组织转化生长因子β1基因的表达

背景：据报道,转化生长因子β1能促进椎间盘细胞的增殖与分化,并参与其损伤修复过程。但转化生长因子β1是否参与椎间盘退变的过程？ 目的：分析在人体退行性变椎间盘组织中转化生长因子β1的表达情况,并探讨其与人体椎间盘退行性变的关系。 方法：收集正常椎间盘组织30例,退行性变人体椎间盘组织530例,采用苏木精-伊红染色、免疫印迹和RT-PCR方法进行研究,对退行性变的椎间盘组织进行病理学分型,分别检测转化生长因子β1在不同类型退变的椎间盘中表达的情况并与正常椎间盘组织进行对比分析。 结果与结论：苏木精-伊红染色病理学诊断：将退行性变的椎间盘组织根据病理学改变程度分为4型。免疫印迹法和RT-PCR法均显示：在正常和退变椎间盘组织中,转化生长因子β1均有表达,但在病变组织中随病变加重转化生长因子β1表达量随之增加,退变组织与正常组织比较差异有非常显著性意义 (P < 0.01)。说明转化生长因子β1高表达与人体椎间盘退行性变呈正相关。     

脂欣康胶囊抗载脂蛋白E基因敲除小鼠动脉粥样硬化形成作用

为观察脂欣康胶囊对载脂蛋白E(ApoE)基因敲除小鼠动脉粥样硬化 (As)的影响 ,将 6周龄ApoE基因敲除小鼠随机分为模型组与脂欣康组 ,相同遗传背景的同龄正常C57BL/6J小鼠为正常对照组。脂欣康组灌服脂欣康胶囊内之药粉 ,模型组、正常对照组均灌服生理盐水。连续灌胃 14周后 ,用 10 %福尔马林灌注后 ,取主动脉做形态学观察及图像分析。结果表明 ,正常对照组主动脉壁正常 ,无As病灶。模型组动脉粥样硬化 (As)病变显著 ,多为粥样硬化期病灶 ,可见由大量泡沫细胞形成的脂纹脂斑期病变 ,也可见由大量泡沫细胞及胆固醇结晶形成的粥样斑块期病灶。脂欣康组As病变较模型组明显减轻 ,多为早期粥样硬化 ,斑块面积显著小于模型组 ,P <0 .0 5。结果提示脂欣康有减轻ApoE基因敲除小鼠As的作用。     

环孢素干预小肠移植物中转化生长因子β1的表达

背景：小肠移植慢性排斥反应的发生是影响其长远预后的重要因素。 目的：观察环孢素对于小肠移植物中转化生长因子β1表达的影响。 方法：近交系F344(RT11vr)大鼠和Lewis(RT11)大鼠作为小肠移植的供体和受体,利用显微外科技术构建异系大鼠小肠移植模型,分别在建模后7,28,60 d进行移植物活检,常规病理学检测,应用Real time-PCR技术检测移植物内转化生长因子β1因子mRNA转录水平,并采用免疫组织化学方法对转化生长因子β1的表达进行定位。 结果与结论：在建模后7-28 d应用环孢素的过程中转化生长因子β1 mRNA转录水平呈现明显减低趋势;在28-60 d,停用环孢素后该因子表达则明显升高。说明环孢素对于异系小肠移植中转化生长因子β1的表达有一定的抑制作用。     

Characterization of transforming growth factor-beta 1 gene expression in porcine immune cells.

We have investigated the regulation of transforming growth factor beta 1 gene expression in a variety of porcine immune cell populations, including peripheral blood mononuclear cells (PBMC), peripheral blood monocytes, alveolar macrophages and lymphoid cells from various swine lymphoid tissues. Using porcine transforming growth factor beta 1 cDNA probes in Northern blot assays, messages of 2.5 and 3.5 kb TGF beta 1 mRNA were detected in the cells investigated. A variety of mitogenic and immunomodulatory substances were examined for their ability to induce TGF beta 1 mRNA expression. These include phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS), dexamethasone (Dex), tumor necrosis factor (TNF) and interleukin (IL)-1 alpha. While low level constitutive expression of TGF beta 1 mRNA was detected from all cells investigated, PMA treatment of PBMC and alveolar macrophages resulted in a more than 10-fold increase in the steady-state level of TGF beta 1 mRNA within 2 hr of PMA addition. Also, the effect of opiate drugs, methadone (Md) and morphine (Mor), on TGF beta 1 gene expression was determined. Cells treated with opiates expressed the same levels of TGF beta 1 mRNA as untreated cells. Since TGF beta 1 biological activity can be induced by opiates, the regulation of TGF beta 1 gene expression likely involves mechanisms that do not cause changes in mRNA levels.     

载脂蛋白E基因多态性与疾病的相关性研究

载脂蛋白E（ApoE）是一种重要的血浆脂蛋白,由3种等位基因构成：E2、E3和E4。ApoE作为一种载脂蛋白,在脂质运输和代谢过程中发挥重要作用,而目前越来越多的研究表明：ApoE在免疫调节方面发挥重要作用,从而参与到多种疾病的发生发展中。近年来发现,ApoE及其基因多态性与高脂血症、动脉粥样硬化、Alzheimer病、神经系统病变及脓毒血症等人类疾患的发生发展有着密切关系。     

The cross-talk between transforming growth factor-beta1 and ultrasound stimulation during mechanotransduction of rat tenocytes

Ultrasound is an effective noninvasive treatment for various tendinopathies. However, how tenocytes convert ultrasound stimulation into cascades of cellular and molecular events is not well understood. The purpose of this study is to elucidate the signaling pathways of tenocytes during ultrasound stimulation. Primary cultures of tenocytes were harvested from Achilles tendons of Sprague-Dawley rats. The viability and proliferation of tenocytes, their genes expression, and the signaling pathways after ultrasound treatment with or without specific inhibitors were evaluated and analyzed. The results showed that ultrasound treatment (100 mW/cm(2) for 20 min) significantly enhanced matrix metalloproteinase 13 (MMP-13), c-Fos, and c-Jun gene expression, increased JNK and p38, but not extracellular signal-regulated kinase-1/2 (ERK1/2), phosphorylation at 5 min, and sustained up to 60 min. JNK inhibitor and p38 inhibitor, but not ERK1/2 inhibitor, attenuated ultrasound-dependent induction of MMP-13 expression, indicating that the JNK and p38 pathways are required for ultrasound-induced MMP-13 expression in tenocytes. We also found that SB431542 (transforming growth factor-beta (TGF-β) receptor kinases inhibitor) suppressed ultrasound-induced MMP?13 and c-Fos gene expression, and p38 phosphorylation. This study revealed that ultrasound treatment stimulates tenocytes proliferation and regulates their matrix metabolism through the cross-talk between TGF-β and ultrasound-induced mitogen-activated protein kinases (MAPKs) signaling pathways.     

The cross-talk between transforming growth factor-beta1 and ultrasound stimulation during mechanotransduction of rat tenocytes

Ultrasound is an effective noninvasive treatment for various tendinopathies. However, how tenocytes convert ultrasound stimulation into cascades of cellular and molecular events is not well understood. The purpose of this study is to elucidate the signaling pathways of tenocytes during ultrasound stimulation.

Primary cultures of tenocytes were harvested from Achilles tendons of Sprague–Dawley rats. The viability and proliferation of tenocytes, their genes expression, and the signaling pathways after ultrasound treatment with or without specific inhibitors were evaluated and analyzed.

The results showed that ultrasound treatment (100 mW/cm² for 20 min) significantly enhanced matrix metalloproteinase 13 (MMP-13), c-Fos, and c-Jun gene expression, increased JNK and p38, but not extracellular signal-regulated kinase-1/2 (ERK1/2), phosphorylation at 5 min, and sustained up to 60 min. JNK inhibitor and p38 inhibitor, but not ERK1/2 inhibitor, attenuated ultrasound-dependent induction of MMP-13 expression, indicating that the JNK and p38 pathways are required for ultrasound-induced MMP-13 expression in tenocytes. We also found that SB431542 (transforming growth factor-beta (TGF-β) receptor kinases inhibitor) suppressed ultrasound-induced MMP‐13 and c-Fos gene expression, and p38 phosphorylation.

This study revealed that ultrasound treatment stimulates tenocytes proliferation and regulates their matrix metabolism through the cross-talk between TGF-β and ultrasound-induced mitogen-activated protein kinases (MAPKs) signaling pathways.     

No evidence for cholinergic problems in apolipoprotein E knockout and apolipoprotein E4 transgenic mice

The varepsilon4 allele of the apolipoprotein E gene constitutes the major genetic risk factor to develop Alzheimer's disease. If and how this protein contributes to the pathological cascade of Alzheimer's disease is not known. The varepsilon4 allele particularly affects the cholinergic defect, which is one of the most consistent neurotransmitter problems in an Alzheimer's disease brain.We have analysed several parameters of the cholinergic system in brain of apolipoprotein E knockout mice as well as in transgenic mice overexpressing human apolipoprotein E4. We analysed the distribution of cholinergic fibers, the number and morphology of cholinergic neurons and the enzymatic activity of acetylcholinesterase and choline acetyltransferase in different brain regions. Finally, we analysed the distribution and the binding parameters of [3H]hemicholinium-3, a specific marker for the high affinity choline transporter in different brain sections and regions.This extensive effort failed to show any consistent difference in the cholinergic parameters studied, in either the apolipoprotein E4 transgenic mice or in the apolipoprotein E knockout mice, compared to age-matched non-transgenic mice. We conclude that the apolipoprotein E4 is not deleterious per se for the cholinergic system in mouse brain.     

Reduced cardiac functional reserve in apolipoprotein E knockout mice

Several years ago, the authors reported that aortic flow velocity under resting conditions was significantly higher in apolipoprotein E knockout (apoE-KO) mice than in age-matched C57Black/6J wildtype (WT) controls. The goal of this study was to examine whether the cardiac functional reserve is impacted in response to a pharmacological stress agent in apoE-KO mice. Cardiac function was measured noninvasively by the Doppler ultrasound method at baseline and at 1 min, 5 min, 10 min, and 20 min after intraperitoneal injection of dobutamine at the doses of 1 microg/g, 3 microg/g, or 10 microg/g in 16-month-old male apoE-KO (n = 9) and WT (n = 10) mice under light anesthesia with 1.5% isoflurane via inhalation. The baseline peak and mean aortic flow velocities were 39% to 48% higher, and left ventricular contractility measured by peak acceleration rate of aortic flow velocity was 24% higher in apoE-KO compared with WT mice (P < 0.01). Dobutamine stress dose-dependently increased cardiac function, which, however, was significantly smaller with a right shift of the dose-response curve in apoE-KO mice compared with WT controls. The hypotensive response to dobutamine was not significantly different between the 2 groups. Thus, despite an elevated resting aortic flow velocity and left ventricular contractility, cardiac functional reserve in response to dobutamine stress was significantly reduced in apoE-KO mice, which could be the consequence of coronary atherosclerosis and endothelial dysfunction that limits blood supply to the heart.     

Expression of transforming growth factor-beta1 and transforming growth factor-beta receptors in hepatocellular carcinoma and dysplastic nodules.

In this study we analyzed by immunohistochemistry the expression of TGF-beta1 protein and TGF-beta receptors I and II in 4 low-grade dysplastic nodules, 2 high-grade dysplastic nodules, 6 early, 22 small, and 62 advanced hepatocellular carcinomas. The expression of TGF-beta1 protein by hepatocytes was decreased in advanced hepatocellular carcinoma compared with small or early hepatocellular carcinoma(P < .05). Frequent and intense staining of TGF-beta1 protein was noted in the sinusoidal endothelium of advanced hepatocellular carcinomas despite of its decreased staining in hepatocellular carcinoma cells. Reduced expression of TGF-beta receptors I and II compared with surrounding nontumorous tissue were noted from the early hepatocellular carcinoma stage suggesting that down-regulation of TGF-beta receptors is correlated with progression from premalignant to malignant phenotype. Reduced expression of both TGF-beta1 and TGF-beta receptor II in neoplastic hepatocytes were also significantly correlated with increased tumor size and increased proliferative activity(P < .05). These findings suggest that during hepatocarcinogenesis, the inhibitory effects of TGF-beta1 protein on hepatocellular carcinoma cells is outweighed by its effects on stromal elements, which, overall, contributes indirectly to a tumor growth stimulatory environment. Also, the growth-inhibitory effects of TGF-beta1 may have been further negated by reduced TGF-beta receptors on hepatocellular carcinoma cells.     

载脂蛋白E基因多态性与散发性老年性痴呆病的关系

目的:探讨载脂蛋白E(apoE)外显子4和增强子元件基因多态性与散发性Alzheimer病(AD)的关系。方法:应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术分别检测apoE外显子4和内含子1内增强子元件(IE1)基因型。结果:(1)ApoE外显子4基因多态性:AD组ε3/4基因型频率(0.381)和ε4等位基因频率(0.226)显著高于对照组(P＜0.05)。(2)ApoEIE1基因多态性AD组G/G基因型频率(0.595)显著高于对照组(P＜0.05)。(3)有apoEε4个体患AD风险为无ε4个体的3倍,比值比为2.932,95%可信区间1.379~6.226;G/G基因型个体患AD风险为G/C、C/C个体的2倍,比值比为2.223,95%可信区间1.075~4.599;经统计分析发现apoEε4与IE1G/G呈非常显著性正相关(P＜0.01);排除apoEε4后发现IE1G/G与AD发病风险无关。结论:ApoEε4等位基因是个体发生AD的危险因素,IE1G/G增加AD发病风险是因其与ε4相关所致。