脾气虚证模型大鼠心功能变化及其机制的研究

发布时间：2018-01-07 22:24

本文关键词：脾气虚证模型大鼠心功能变化及其机制的研究　出处：《辽宁中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:中医学基本理论认为,脾脏“为后天之本”“气血生化之源”,是提供后天生命活动所需物质最重要器官。而“脾主运化、统血”是脾脏的核心功能。现代医学已明确,维系生命活动最基本物质是三磷酸腺苷(ATP)为代表的能量物质。而ATP的来源与生成则包括了物质的消化与吸收、血液的运输、跨膜物质转运以及以线粒体为平台的氧化磷酸化等生物学过程。因此,“脾主运化、统血”功能与现代医学能量代谢的生物学过程有着密切的内在联系。但中医学理论认为,“脾主运化与统血”是脾脏象的两个不同核心功能,“脾统血”包含了“生血、行血、摄血”作用。我们根据中医学理论与现代医学能量代谢的生物学过程及其机制,认为脾生血应与血液成分的生成有关;脾摄血则与止血和凝血机制有关。而对于“行血”则提出以下科学假说:“脾统血”功能可能属于“脾主运化”功能的一部分,即物质消化与吸收后,通过血液循环系统向全身组织细胞输送的部分功能,其机制可能是脾气虚导致了BNP的变化,进而影响了c AMP-PKA通路,导致心功能发生了变化。本工作目的,运用电生理学及分子生物学技术,观察脾气虚证模型大鼠心功能变化,并探讨其可能的分子生物学机制,旨在阐明中医学“脾主运化、统血”,特别是“运化”与行血功能之间关系的科学内涵同时,为临床“从脾论治”心血管疾病提供实验依据。方法:1.实验动物与分组由本溪实验动物中心(辽宁长生生物科技有限公司)购入30只SPF级雄性SD大鼠,体重200±10g,许可证号SCXK(辽-2010-0001),进行一周时间适应性饲养后,随机分为正常组和脾气虚模型组两组,每组各15只。2.脾气虚证模型复制与评价:大鼠脾气虚证模型复制方法根据中医学病因基本理论,参照《中药新药临床研究指导原则》[1]和《中医实验动物模型方法学》[2]中制备脾虚模型的方法,运用饮食不节加劳倦内伤复合因素复制脾气虚证大鼠模型。即24小时自由饮食水,禁食不禁水48小时;同时每天进行游泳25分钟,水温在35℃~37℃之间,室温保持在23±1℃;连续造模2周后进行模型评价。脾气虚证模型成模评价方法:依据文献[3]分别对模型大鼠宏观表征主要症状,神疲、乏力、食少纳呆、身体消瘦、便软腹泻等进行了量化,剔除没有达到标准大鼠。正常组正常饮食水,没有施加刺激因素。3.核心指标检测方法3.1左心功能检测:采用小动物超声影像系统进行大鼠心功能检测;该系统由加拿大Visua Sonics公司购入,型号VEVO2100超高分辨率。于模型组造模成功后,禁食24小时,分别检测正常组及模型组大鼠左心功能,检测部位选择大鼠房室环,参照小动物超声心动图仪器内部设定的的心电图既有模式连接仪器。连接完成后,调整仪器参数:动态量程(Dynamic range),60d B;显示地图(Display map),CS;扫描速率(Sweep speed),1000Hz;对等(Contrast),50;亮度(Brightness)50。等待大鼠呼吸平稳,心电图能够清楚的展现出来后,开始记录大鼠10秒钟的超声心动图,仪器随机从10秒钟的片段中选取3段进行测量,给出平均值,从而获得各组大鼠的左室射血分数(LVEF)、左室收缩末期容积(ESV)、左室每搏输出量(SV)、左室舒张末期容积(EDV)的平均值。3.2.取材及分子生物学指标检测:取材方法:实验结束后,两组大鼠禁止饮食24小时,选用20%氨基甲酸乙酯溶液对大鼠进行麻醉,大鼠腹部备皮,解剖,5ml注射器抽取大鼠腹主动脉血;随后用医用剪刀剪开胸腔后,取大鼠心脏,医用手术刀迅速切取心脏组织,根据检测指标要求分别进行采血即组织样本制备。采用常规HE染色方法观察心脏组织形态学变化;透射电镜检测心肌组织细胞形态学改变。采用Elisa法测定血清及心肌组织BNP、c AMP的含量;采用免疫组化法测心肌细胞BNP受体蛋白的表达。采用蛋白质印迹法(Western Blot)测定心肌中BNP的表达;采用Q-PCR方法检测b FGF、PKA m RNA表达。4.统计方法:本实验所有数据统计学分析处理都由SPSS 13.0统计软件进行,并用均数±标准差(`x±s)的方式表达数据结果,采用校正t检验(方差不齐时)或独立样本t检验(方差齐时)比较计量资料,以P0.05为差异具有统计学意义。结果:1.大鼠心功能检测结果:与正常组比较,脾气虚证模型组大鼠心脏左室收缩末期容积(ESV)数值明显升高(P0.05);而左室舒张末期容积(EDV)、左室射血分数(LVEF)、左室每搏输出量(SV)则明显低于正常组(P0.05)。2.心肌组织HE染色结果:正常组大鼠心肌细胞纤维结构显示较为清楚、心肌纤维排列方式整齐、心肌细胞横向纹路清晰,并无出现炎症细胞的浸润现象,心肌细胞结构为正常;脾气虚证模型组大鼠心肌萎缩,细胞排列方式紊乱、细胞坏死,存在细胞脂肪变性及浑浊等变化。3.心肌组织透射电镜检测结果:正常组心肌细胞排列正常,肌膜结构清晰,心肌纤维排列尚规则,Z线可见。肌纤维内线粒体丰富,结构完好,线粒体无肿胀、变性及坏死;间质未见纤维组织增生及炎细胞浸润。模型组肌纤维排列疏松(水肿),偶见闰板分离,部分肌丝溶解;肌丝A带、I带结构不清,Z线仍可见;线粒体较丰富,但大小不一,线粒体基质电子密度增高。4.BNP、c AMP含量检测结果:与正常组比较,脾气虚证模型组大鼠血清及心肌组织中BNP、c AMP含量均明显升高,具有统计学意义(P0.05)。5.BNP受体表达检测结果:大鼠心肌组织中BNP受体表达,多见于细胞膜,细胞胞浆亦有表达但较少,呈棕黄色为阳性表达。正常组大鼠心肌细胞染色表现为弱阳性,而脾气虚证模型组的阳性物表达的细胞数明显多于正常组;与正常组比较,脾气虚证模型组其心肌细胞中BNP的IOD值明显升高,且差异具有统计学意义(P0.05)。6、PKA与b FGF m RNA表达检测结果:目的基因得到有效扩增,因目的基因的扩增曲线走势符合S型曲线。熔解曲线中显示没有出现杂峰,只有一个峰值,说明目的基因的定量测定可以采用该体系。对比两组间PKA及b FGF m RNA的表达情况,脾气虚证模型组比正常组具有明显的增加(P0.05)。本实验结果显示,脾气虚证模型大鼠的“脾主运化”功能损伤时,其左室收缩末期容积增大,舒张末期容积、每搏输出量等均发生变化,特别是左心射血分数发生改变,表明脾气虚“运化”功能发生障碍时,心脏射血功能也明显受到影响,即表现为“行血”功能下降。本结果支持了“脾统血”功能可能属于“脾主运化”功能一部分的假说。总结本实验结果可以认为,脾气虚证,“脾主运化”功能失职时其“统血”功能也受到影响,其可能机制之一是由于心肌组织BNP及其受体表达上调,激动了c AMP-PKA通路并通过b FGFm RNA过度表达,从而造成心肌组织形态学发生改变,最终导致其左心室射血功能下降。结论:1、脾气虚证模型大鼠左心室射血功能明显降低。其中左室收缩末期容积(ESV)升高;左室舒张末期容积(EDV)、左室射血分数(LVEF)、左室每搏输出量(SV)则明显降低。2、脾气虚证模型大鼠心肌组织萎缩,炎症细胞浸润、其肌纤维排列紊乱、肌丝与核发生溶解;线粒体大小不均。3、脾气虚证模型大鼠心肌组织与血清中BNP含量增加,其心肌细胞BNP受体表达上调。4、脾气虚证模型大鼠心肌组织c AMP含量升高、PKA及b FGFm RNA表达上调。
[Abstract]:Objective: TCM basic theory, "the spleen is the acquired" the source of Qi and blood ", is to provide the required material acquired life activities. The most important organs and spleen transport, the blood" is the core function of spleen. Modern medicine has been clear, sustain the life of the most basic activities of substances (ATP ATP) as the representative of the energy sources and generate ATP. It includes the matter digestion and absorption of blood transport, transmembrane transport and mitochondrial oxidative phosphorylation as a platform for other biological processes. Therefore, the "spleen transport, the blood biological process function and energy metabolism in modern medicine there is a close relationship. But the theory of traditional Chinese medicine thinks," spleen transport and blood "are two different core functions as the" spleen, splenic blood "contains" blood, blood, blood ". According to our medical theory and Biological process and mechanism of modern medicine, energy metabolism, blood and spleen that should generate blood components; spleen blood intake is related to blood coagulation and hemostasis mechanism. For the "blood" is to put forward the following hypothesis: "the spleen blood" may be a part of "spleen transport". The substance of digestion and absorption, part of the function of transport to body tissues through the blood circulation system, which may be the mechanism of spleen deficiency leads to the change of BNP, thereby affecting the C AMP-PKA pathway, leading to the changes of heart function. The objective of this work, the use of electrophysiology and molecular biology technology to observe the changes of heart function model rats with spleen deficiency syndrome, and to explore its possible molecular mechanism, to learn "spleen transport clarificate, the blood", especially the scientific connotation of "the relationship between transport and blood function at the same time, for the clinical" On the treatment of cardiovascular diseases and provide experimental evidence from the spleen. Methods: 1. experimental animal and group by Benxi Experimental Animal Center (Liaoning Changsheng biotechnology limited company) bought 30 male SD SPF rats, weight 200 + 10g, license No. SCXK (Liaoning -2010-0001), a week after adaptive feeding time. Were randomly divided into normal group and spleen qi deficiency model group two groups, 15 rats in each group of.2. replication and evaluation: the model of spleen qi deficiency in spleen qi deficiency rats model according to the basic theory of the etiology of traditional Chinese medicine, preparation method for reference "method of spleen deficiency model of clinical research on new drugs of experimental animal medicine guidelines of >[1] and >[2] in model" in the use of improper diet and internal root composite factors to copy the model of spleen qi deficiency rats. 24 hours of free food and water, but the water fasting for 48 hours; at the same time every day to swim for 25 minutes, the temperature between 35 DEG ~37 DEG, keep at room temperature In 23 - 1 DEG C; was evaluated after 2 weeks of continuous modeling. The model of spleen qi deficiency model evaluation methods: Based on literature [3] were the main symptoms, rat model of macro characterization of lassitude, weakness, anorexia, body weight loss, diarrhea and other soft is quantified, excluding did not reach the standard of rats. The normal group with normal diet water, no stimulus detection factor.3. 3.1 core indicators of left ventricular function detected by small animal imaging system of rat cardiac function detection; the system by the Canadian Visua Sonics company purchased VEVO2100 type ultra high resolution. In the model group after modeling, fasting for 24 hours, respectively, left heart the function of the normal group and the model group rats were detected, rat atrioventricular ring detection site, according to small animal echocardiography instrument set the ECG model as connecting instrument. The connection is completed, the adjustment of the instrument parameters: dynamic range (Dynamic range), 60d B (Display map); map display, CS; scanning rate (Sweep speed), 1000Hz (Contrast, 50); equivalence; brightness (Brightness) 50. rats for respiration, ECG can be clearly demonstrated after the start of rats was recorded 10 seconds of ultrasonic cardiogram. The instrument from 10 randomly selected 3 seconds of footage segments were measured, gives the average value, so as to obtain the rats left ventricular ejection fraction (LVEF), left ventricular end systolic volume (ESV), left ventricular stroke volume (SV), left ventricular end diastolic volume (EDV) of the average detection.3.2. material and molecular biology index: sampling method: after the end of the experiment, two groups of rats to prohibit eating for 24 hours, with 20% urethane solution on rats were anesthetized rat abdominal skin, anatomy, 5ml syringe from rat abdominal aortic blood; followed by medical scissors after thoracic, from rat heart medical, surgical Rapid knife cut heart tissue, according to the detection requirements for blood or tissue samples were prepared by conventional HE. Observe the morphological changes of heart tissue staining; cell morphology change detection of myocardial tissue by transmission electron microscopy. The determination of serum and myocardial tissue BNP by Elisa method, the content of C AMP; the expression of BNP in myocardial cell receptor test protein by Western blot method. (Western Blot) expression of myocardial BNP were determined by Q-PCR method; detection of B FGF PKA, m RNA expression of.4. statistical methods: the statistical analysis of all data processing by the statistical software SPSS 13, and the mean and standard deviation (`x + S). Expression of the results, the correction of t test (variance missing) or independent samples t test (Fang Chaqi) measurement data are compared with P0.05, the difference was statistically significant. Results: the cardiac function of 1. rats respectively. Results: compared with normal group, spleen qi deficiency model rats, left ventricular end systolic volume (ESV) were significantly increased (P0.05); left ventricular end diastolic volume (EDV), left ventricular ejection fraction (LVEF), left ventricular stroke volume (SV) was significantly lower than that of normal group (P0.05).2. myocardial tissue HE staining results: normal rat myocardial cell fiber structure shows more clearly, the arrangement of myocardial fiber neat, myocardial cells transverse lines clear, there is no infiltration of inflammatory cells, myocardial cell structure is normal; spleen qi deficiency model group rat myocardial atrophy, cell arrangement disorder, cell necrosis. The cellular fatty degeneration and turbidity changes of.3. in myocardial tissue by electron microscopy results: normal myocardial cells were normal, muscle membrane structure clear, myocardial fibers still rules, the Z line is visible. In skeletal muscle fibers rich in mitochondria and mitochondrial structure intact, no The swelling, degeneration and necrosis; no interstitial fibrous tissue hyperplasia and inflammatory cell infiltration. The model group loosely arranged muscle fibers (edema), occasionally intercalated plate separation, part of myofilament dissolved; filaments A band, I band structure is not clear, the Z line is still visible; mitochondria were abundant, but not the size of a line. Mitochondrial matrix increased electron density.4.BNP, c test results of AMP content: compared with the normal group, spleen qi deficiency model rats serum and myocardial tissue BNP, C content of AMP were increased significantly, with statistical significance (P0.05).5.BNP receptor expression results: expression of BNP receptor in rat myocardium, found in the cell membrane the cytoplasm was also expressed, but less, brownish yellow positive expression. Normal rats myocardial cells were weakly positive, the number of cells and expression of spleen qi deficiency model group were significantly higher than the normal group; comparing with normal group, spleen qi deficiency model group, the cardiac muscle fine Cell BNP of IOD increased obviously, and the difference was statistically significant (P0.05).6, PKA B and FGF m test results: the expression of RNA gene was effectively amplified, because the amplification curve of target gene in line with the trend of the S curve. The melting curve shows no impurity peaks, only one peak, indicating the quantitative determination the target gene can be used in the system. The expression of PKA and B were compared between two groups of FGF m of RNA, spleen qi deficiency model group significantly increased than normal group (P0.05). The experimental results show that the model of spleen qi deficiency rats "spleen transport function injury, the left ventricular end systolic increased volume, end diastolic volume, stroke volume and change, especially the left ventricular ejection fraction changes showed that spleen deficiency" transport "dysfunction, cardiac ejection function was significantly affected by the performance of the" blood "function Drop. The results support the "splenic blood" function may belong to the spleen transport function part of the hypothesis. The experimental results can be summarized that "spleen deficiency syndrome, spleen transport function of dereliction of duty on the part of its" the blood "function is also affected, one of the mechanisms may be due to the upregulation of BNP expression and its receptors in cardiac muscle tissue, excited C AMP-PKA pathway by B FGFm overexpression of RNA, resulting in myocardial tissue morphological changes, resulting in the left ventricular ejection function decreased. Conclusion: 1. The left ventricular ejection function of spleen qi deficiency model rats decreased obviously. The left ventricular end systolic volume (ESV) increased; left ventricular end diastolic volume (EDV), left ventricular ejection fraction (LVEF), left ventricular stroke volume (SV) decreased.2, myocardial tissue in the rat model of spleen qi deficiency atrophy, inflammatory cell infiltration, the muscle fibers arranged in disorder, myofilament and nuclear dissolution; The size of mitochondria was not equal to.3. In the spleen qi deficiency syndrome rats, the BNP content in myocardium and serum increased, the expression of BNP receptor in cardiac myocytes increased by.4, and the C AMP content in myocardial tissue increased, the expression of PKA and B FGFm RNA increased in rats with spleen qi deficiency syndrome.

【学位授予单位】：辽宁中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R228

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