糖尿病心肌病动物模型的建立

doi:10.3969/j.issn.2095-4344.2015.27.001

中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (27): 4265-4270.doi: 10.3969/j.issn.2095-4344.2015.27.001

• 心肺移植 heart-lung transplantation • 下一篇

糖尿病心肌病动物模型的建立

刘文旗^1，2，3，戴红艳³，邢明青⁴，管军³，王晏平³

¹青岛大学医学院，山东省青岛市 266071；青岛大学医学院附属青岛市市立医院，²细胞移植实验室，³心内科，⁴临床检验科，山东省青岛市 266011

出版日期:2015-06-30 发布日期:2015-06-30
通讯作者: 管军，青岛大学医学院附属青岛市市立医院心内科，山东省青岛市 266011 王晏平，青岛大学医学院附属青岛市市立医院心内科，山东省青岛市 266011
作者简介:刘文旗，女，1989年生，山东省枣庄市人，汉族，青岛大学在读硕士，主要从事冠心病、高血压的防治及糖尿病心肌病发病机制方面的研究。
基金资助:
国家自然科学基金项目(81200175)；青岛市自然科学基金项目(13-1-4-140-jch)

Establishment of animal models of diabetic cardiomyopathy

Liu Wen-qi^{1, 2, 3}, Dai Hong-yan³, Xing Ming-qing⁴, Guan Jun³, Wang Yan-ping³

1 Qingdao University Medical College, Qingdao 266071, Shandong Province, China
2 Key Laboratory of Cellular Transplantation, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266011, Shandong Province, China
3 Department of Cardiology, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266011, Shandong Province, China
4 Department of Clinical Laboratory, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266011, Shandong Province, China

Online:2015-06-30 Published:2015-06-30
Contact: Corresponding author: Guan Jun, Department of Cardiology, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266011, Shandong Province, China Corresponding author: Wang Yan-ping, Department of Cardiology, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266011, Shandong Province, China
About author:Liu Wen-qi, Studying for master’s degree, Qingdao University Medical College, Qingdao 266071, Shandong Province, China; Key Laboratory of Cellular Transplantation, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266011, Shandong Province, China; Department of Cardiology, Qingdao Municipal Hospital Affiliated to Qingdao University Medical College, Qingdao 266011, Shandong Province, China
Supported by:
the National Natural Science Foundation of China, No. 81200175; the Natural Science Foundation of Qingdao City of China, No. 13-1-4-140-jch

摘要/Abstract

摘要：

背景：糖尿病性心肌病是导致糖尿病患者死亡率增加的重要因素，是一种严重的糖尿病并发症。因此为研究糖尿病心肌病的发病机制及诊疗提供有效的实验动物模型显得尤为重要。
目的：探索关于构建Wistar大鼠糖尿病心肌病动物模型的方法。
方法：将40只大鼠随机分为对照组(n=10)和糖尿病心肌病组(n=30)。糖尿病心肌病组应用链脲菌素60 mg/kg一次性腹腔注射法构建糖尿病心肌病大鼠模型；对照组大鼠给予等体积的枸橼酸缓冲液。两组大鼠均以非高脂高糖的普通饲料喂养。
结果与结论：与对照组相比，糖尿病心肌病组大鼠在腹腔注射链脲菌素3周后血糖显著升高，心肌细胞排列紊乱，细胞核大小不规则，心肌组织内胶原含量明显增多且胶原纤维粗大排列紊乱，同时反映糖尿病心肌病心肌纤维化的转化生长因子β1和Ⅰ型胶原的mRNA和蛋白表达水平亦明显增高。说明一次性腹腔注射大剂量的链脲菌素(60 mg/kg)并给予非高脂高糖的普通饲料喂养，可以构建稳定的1型糖尿病心肌病模型，方法安全有效，可行性高。

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, 链脲菌素, 糖尿病心肌病, 转化生长因子β1, Ⅰ型胶原, 心肌纤维化, 心肌肥厚, 国家自然科学基金

Abstract:

BACKGROUND: Diabetic cardiomyopathy, a serious complication of diabetes, is an important factor of increased mortality in patients with diabetes. Therefore, providing an effective experimental animal model is particularly important for studying the pathogenesis and treatment methods of diabetic cardiomyopathy.
OBJECTIVE: To explore the method of establishing Wistar rat models of diabetic cardiomyopathy.
METHODS: Forty rats were randomly divided into the control group (n=10) and diabetic cardiomyopathy group (n=30). The rats in the diabetic cardiomyopathy group were intraperitoneally injected with 60 mg/kg streptozotocin at a time to establish rat models of diabetic cardiomyopathy. The rats in the control group were given the same dosage of citric acid buffer by the same way. The rats in these two groups were all fed with non-fat high-sugar normal diet.
RESULTS AND CONCLUSION: Compared with the control group, after 3 weeks of injection with streptozotocinin in rat models of diabetic cardiomyopathy, blood glucose level was significantly increased, myocardial cells arranged in disorder, the nuclei were of different sizes, collagen content in the myocardial tissue was significantly increased, and collagen fibers were thick and disordered. In addition, the mRNA and protein levels of transforming growth factor β1 and type I collagen, two indices reflecting myocardial fibrosis, were markedly increased. These results indicate that intraperitoneally injecting large doses of streptozotocin (60 mg/kg) at a time and feeding with non-fat high-sugar normal diet could establish a stable rat model of type 1 diabetic cardiomyopathy. This method is safe and effective with high feasibility.

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: Diabetic Cardiomyopathy, Streptozotocin, Fibrosis, Myocardium

中图分类号:

R318

刘文旗，戴红艳，邢明青，管军，王晏平. 糖尿病心肌病动物模型的建立[J]. 中国组织工程研究, 2015, 19(27): 4265-4270.

Liu Wen-qi, Dai Hong-yan, Xing Ming-qing, Guan Jun, Wang Yan-ping. Establishment of animal models of diabetic cardiomyopathy[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(27): 4265-4270.

图/表（结果） 3

Quantitative analysis of experimental rats and process of diabetes induction in rats
At the end of 12 weeks, 24 rats from the DCM group and 10 rats from the control group survived, and the rest five diabetic rats from the DCM group died due to infection or diabetic ketoacidosis. Unfortunately, in the DCM group, one rat was discardded because its blood glucose was normal but did not meet the standard for successful diabetes induction. Ultimately 10 rats from the control group and 24 rats from the DCM group were included in the final analysis. The rats in the control and DCM groups were all given normal diet and were allowed free access to water. Food and water intakes were closely monitored and blood flucose level was measured weekly. Experimental procedure is shown in Figure 1.

Improvement of diabetes models
In this experiment, blood sugar level at 3 weeks after STZ injection was used as a diagnostic criterion for type 1 diabetes, which ensures the quality and stability of animal models. Male Wister rats were used to establish DCM model easily and effectively. Throughout the whole experiment, rats in the control and DCM groups were all given normal low fat diet. In addition, histopathological staining, RT-PCR, and western blot analysis were used to determine the myocardial pathological alteration of DCM clearly and credibly.

The stability of diabetes models
Injection of a large dose of STZ could damage rat pancreatic function, which ensures the efficacy of diabetes models and longer period of normal diet, leading to more severer pathological changes of type 1 diabetes. In addition, blood glucose level at 3 weeks after STZ injection as a diagnostic criterion for type 1 diabetes ensured the quality and stability of animal models.

Behavioral characteristics of rat models of diabetes
In the course of the experiment, rats from the control group were in good state of mind, whose reaction was sensitive. In addition, the body weight of rats in the control group was increased significantly and these rats had a shiny fur. The STZ-induced diabetic rats were at a diabetic state of polydipsia and polyuria with lowered body weights. Other symptoms such as hair removal, tail rot, and cataract, which are commonly associated with the diabetes complications, also appeared in the diabetic rats.

General changes of blood glucose levels
Before the STZ injection, there was no significant difference in blood glucose level in rats between the control and DCM groups (P > 0.05). Blood glucose levels were significantly increased in STZ-induced diabetic rats compared to those in rats of the control group (P < 0.05; Table 1).

Pathological variations of cardiac tissue
Through light microscopy, it was observed that the diabetic rats had increased sizes and disordered arrangement of cardiocytes compared to those in rats of the control group after 12 weeks of diabetes. Masson’s trichrome staining of rat myocardium showed that the cardiac collagen deposition in the STZ-induced diabetic
rats was dramatically increased and spread over the surrounding myocardial cells and blood vessels,
compared to that in rats of the control group (Figure 2).

Myocardial expression of TGF-β1 and type I collagen
TGF-β1 and type I collagen were regarded as a marker reflecting the activation of cardiac fibrosis[13-16]. The mRNA and protein expression levels of TGF-β1 and type I collagen , which were measured by RT-PCR (Figure 3) and western blot analysis (Figure 4) respectively, were markedly higher in the DCM group than in the control group.

参考文献

引言

材料方法

MATERIALS AND METHODS
Animals and experimental materials
Forty male healthy Wister-Albino rats (6-week-old; 180-200 g) were purchased from the Laboratory Animal Center of Lu-kang Corporation (certificate No. 20130829). Rats were maintained in an air-conditioned room at a constant temperature of 20±2 ℃ and were fed with a normal diet and water at liberty. All experimental procedures involving animals were performed in strict accordance with the guidelines approved by the Animal Ethics Committee of Qingdao Municipal Hospital, China.

Reagents and instruments are listed as follows:
Reagent and instrument	Source
Streptozotocin	Sigma, USA
Glucometer, blood glucose test strip	Roche, Germany
Type I collagen antibody, polyvinylidene fluoride membranes, enhanced chemiluminescence kit	Millipore, USA
TGF-β1 antibody	Abcam, USA
second antibody labeled horseradish peroxidase	Jinqiao Zhongshan, China
Radioimmunoprecipitation assay buffer, enhanced bicinchoninic acid protein assay kit	Beyotime, China
Trizol reagent	Invitrogen, USA
LightCycler^® FastStart DNA Master SYBR Green I, Sensiscript Reverse Transcription Kit	TaKaRa Biotechnology, China

Preparation of rat models of diabetes
Following 1 week of acclimatization, rats were assigned randomly to a control group (n=10) and a DCM group (n=30). Blood glucose levels were measured initially before the experiment and then once a week throughout the experiment. Diabetes was induced by a single intraperitoneal injection with streptozotocin (STZ)
(60 mg/kg body weight) dissolved in 0.1 mol/L sodium citrate buffer (pH adjusted to 4.2), whereas rats in the control group were simply injected with 0.1 mol/L citrate buffer. Three weeks after injection with STZ, blood was collected from tail vein and samples were analyzed for blood glucose using a glucometer^[5-9].

Standards for the success in diabetes inducation
Rats with fasting blood glucose levels higher than 16.7 at the end of 3 weeks after STZ injection were considered as developing diabetes^[5-9].

Tissue collection and histopathological staining
At 12 weeks of the experiment, all rats were euthanized under deep anesthesia using pentobarbital and the whole hearts were harvested and weighed. Then, the left ventricle weight/ body weight) was calculated to observe the degree of left ventricular hypertrophy^[10-12]. The left ventricles were fixed in 4% paraformaldehyde for 48 hours, dehydrated and embedded with paraffin. Thereafter, the tissues were cut into 6-µm slices, heated at 60 ℃ for 3 hours, dewaxed, and stained with hematoxylin and eosin. The degree of ?brosis was determined by measuring the content of collagen in the heart slices using Masson Trichrome method. Each slice was analyzed under a microscope at 400× magnification. The collagen volume fraction was determined as the ratio of interstitial collagen area to myocardial area.

Western blot analysis
Protein fractions were isolated in ice-cold radioimmunoprecipitation assay buffer and protein concentrations were determined using the enhanced bicinchoninic acid protein assay kit. Protein fractions were denatured in a loading buffer, and 100 µg of each sample was loaded into alternating lane of 10% sodium dodecyl sulfate-polyacrylamide gel. Protein blots were transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk, the blots were washed with a mixture of Tris-buffered saline and Tween 20 and incubated overnight at 4 ℃ with an appropriate primary antibody (anti-β-actin, anti-transforming growth factor β1 (TGF-β1), and anti-collagen I antibodies were used at 1:1 000, 1:400 and 1:2 000 dilutions, respectively) Membranes were washed with Tris-buffered saline and Tween 20 and then incubated with the appropriate secondary horseradish peroxidase-conjugated antibodies at a 1:1 000 dilution and then washed again. Finally, the protein blots were visualized using an enhanced chemiluminescence kit. Relative intensities of protein bands were measured using Gel-Pro analyzer. β-Actin was used as a comparison.

Real-time (RT)-PCR analysis
The total ribonucleic acid (RNA) was extracted from the frozen myocardial tissues using Trizol reagent, according to the manufacturer’s instructions. The concentration of extracted total RNA was quanti?ed using spectrophotometry. The reverse transcription of RNA to complementary deoxyribonucleic acid (cDNA) was performed using Sensiscript Reverse Transcription Kit. Total cDNA was amplified with LightCycler® FastStart DNA Master SYBR Green I. Primers for rats TGF-β1, type I collagen, and β-actin were designed by TaKaRa Biotechnology Corporation: TGF-β1 sense: 5’-AGG TAA CGC CAG GAA TTG TTG CTA-3’, antisense: 5’-CAT TGC TGT CCC GTG CAG A-3’; collagen I sense: 5’-AGG GAC CCT TAG GCC ATT GTG TA-3’, antisence: 5’-GAC ATG TTC AGC TTT GTG GAC CTC-3’; β-actin sense: 5’-AGA CCT TCA ACA CCC CAG-3’, antisense: 5’-CAC GAT TTC CCT CTC AGC-3’. Amplification process included an initial denaturation step of 30 seconds at 95 ℃ followed by 40 cycles of three-step procedure (denaturation at 95 ℃ for 5 seconds, annealing at 60 ℃ for 30 seconds, and extension at 60 ℃ for 30 seconds) and then a metting-curve procedure at 60 ℃ for 1 minute. At the end of each cycle, fluorescence emitted by the SYBR Green I dye was measured. For each PCR product, a single narrow peak was obtained through melting curve analysis at a specific metting-curve temperature, indicating specific amplifications. The amount of TGF-β1, type I collagens, and β-actin transcripts was calculated from their respective standard curves using the LightCycler software. Samples were tested for three times, and the average values were used for quantification. Finally, the relative expression levels of each target gene were normalized to the messenger RNA (mRNA) of the internal standard gene β-actin.

Main outcome measures
Myocardial pathological alteration of DCM was determined. The differences of the amount of mRNA and protein expression of TGF-1β and type I collagen detected by RT-PCR and western blot assay between the control and DCM groups were statistically analyzed.

Statistical analysis
SPSS 17.0 statistical software was applied for data analysis. Data were presented as the mean ± SEM. Comparisons between the control and DCM groups were performed using unpaired student’s t test. A value of P < 0.05 was considered statistically significant.

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

讨论

The main pathogenic factors of DCM include myocardial interstitial fibrosis, a large accumulation of collagen and endproducts from glycosylation, and energy metabolism dysfunction. The pathological changes increase the stiffness of ventricular wall and decrease the compliance of the ventricle, which leads to ventricular systolic and diastolic dysfunction[17-19]. Previous animal studies have revealed that the pathological changes of DCM that features myocardial hypertrophy and myocardial fibrosis can develop 3 weeks after a single intraperitoneal dose of STZ[3, 20]. Clinical studies have reported that at the early stage of DCM, collagen accumulates, among which type I and III collagen fibers significantly increase, and cross-linking of glycated collagen forms. Ventricular wall stiffens and diastolic function is impaired, which is similar to the changes of restrictive cardiomyopathy[21]. Left ventricular hypertrophy appears and systolic function is also impaired at the advanced stage. In addition, experiments in vitro have shown that the stimulation of a high glucose concentration enhances extracellular matrix accumulation and induces the formation of oxygen radicals, which impairs the diastolic function of the heart and promotes occurrence and development of myocardial interstitial fibrosis in patients with DCM[22-24]. The concrete pathogenesis of DCM is complex and there has been no specific drug to treat DCM. This study is aimed to investigate the method of constructing a model of DCM to provide reliable and vertical theoretical basis for the research of its pathogenesis.

Studies have indicated that STZ can cause diabetes without ketonemia and have no evident cardiac toxicity and low mortality, which accords with the requirement of the reagent itself for model construction which has no adverse effect on the heart[25]. Intraperitoneal injection is chosen for its simplicity, safety and minimal injury. Several reports have recommended that for preparation of diabetic rats, STZ is intraperitoneally injected within the range of 40-65 mg/kg[26] as well as following the classic experimental methods to establish models of type I diabetes[5-9]. The model rats manifest persistent high blood glucose levels, polydipsia, polyphagia, polyuria, weight loss, and low spirits, which was consistent with the clinical characteristics of type 1 diabetes. Hematoxylin-eosin staining and Masson’s trichrome staining of the tissue sections showed that hypertrophic myocardial cells were arranged in disorder, and bulky collagen fibers were interconnected into a web, poorly arranged and widely distributed in myocardial interstitial tissue, which conformed to the reports[27-28]. In addition, the left ventricular coefficient of STZ-induced rats in the DCM group was higher than that in rats of the control group. These indicate that rat models of type 1 DCM, which was characterized by the pathological changes of ventricular remodeling, including myocardial hypertrophy and myocardial interstitial fibrosis, was successfully constructed in this study.

Previous studies have shown that the stimulation of high glucose concentration can accelerate cardiac fibroblasts to produce collagen, fibronectin and TGF-β1[29]. In addition, long-term stimulation of high glucose level can also activate renin-angiotensin system and increase the expression of Ang II and its receptors[30], which facilitates formation of myocardial fibrosis and large accumulation of collagen and stimulates TGF-β1, an important expression of fibrogenic cytokines, to result in myocardial hypertrophy[31]. A large number of studies have found that TGF-β1 contents ascend in diabetic myocardial tissues. The over-expression of TGF-β1 can lead to accumulation of extracellular matrix, which is the main cause of myocardial interstitial remodeling in DCM[32]. The results in this study showed that disordered arrangement of cardiomyocytes and myocardial interstitial fibrosis were observed in the pathological sections of the STZ-induced rat models of DCM after long-term stimulation of high glucose level. The protein and mRNA expression levels of TGF-β1 and type I collagen in rats, which were measured by western blot and RT-PCR analysis respectively, were significantly higher in the DCM group than in the control group, which indicates that they might be involved in the pathological course of ventricular remodeling in DCM and the efficacy of animal model of DCM constructed in this study should be further validated.

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

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糖尿病心肌病动物模型的建立

Establishment of animal models of diabetic cardiomyopathy

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