【作者】 王向培；

【导师】 吴伟；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2011， 博士

【摘要】 [背景]缓慢心律失常是由窦房结或房室结自律性低下或传导系统阻滞引起,以心率减慢为特征的一类心脏传导系统疾病。主要包括窦性心动过缓、窦房或房室传导阻滞、病态窦房结综合征(SSS)等,临床常表现为头昏、黑曚、心悸、一过性意识障碍、晕厥(严重者产生阿一斯综合征)等症状,甚至可导致死亡,属于中医的“眩晕”、“昏厥”、“心悸、“怔忡”等范畴。目前现代医学对此类心律失常无满意的药物治疗,安装起搏器治疗因费用昂贵、并须有一定的设备和专业人员等原因,难以普及,转基因与干细胞移植等治疗尚处于实验阶段。近年来利用参附注射液治疗缓慢心律失常的相关研究较多,并取得不少成果,但有关其作用机制的研究相对较少。【目的】通过临床及实验研究,探讨参附注射液治疗缓慢心律失常的临床疗效、安全性及其可能的内在机制,并探讨参附注射液在诱导骨髓间质干细胞转化为心肌细胞中的作用,从而重建房室结传导功能,为生物起搏器的研制提供前提基础,同时为以后的临床应用推广提供依据。【方法】临床研究：为前瞻性、简单随机、单盲、平行对照研究,自2009年12月至2011年3月期间,从广州中医药大学第一附属医院心血管内科收取符合诊断标准的住院病人47例,随机分为治疗组26例,对照组21例,治疗组使用参附注射液20ml、对照组使用丹参注射液20ml进行治疗,疗程一周,观察住院期间两组患者的中医证候、静息心电图及实验室指标等的前后变化,治疗4天后,治疗组、对照组各随机抽取10例患者行食道调搏心电图检查,并分别静推参附注射液、阿托品,分别检测用药前、用药后10min患者的心率、窦房结恢复时间、矫正窦房结恢复时间、窦房传导时间、文氏型房室传导阻滞点等指标,观察对比其前后变化。实验研究：将18只3～4个月、体重2-2.5kg的新西兰大耳白兔,随机分为3组(n=3),每组6只,雌雄不限,先应用参附注射液按照高(参附注射液5ml)、低剂量(参附注射液1ml)、对照组(生理盐水3ml)进行兔耳缘静脉注射1周,之后经兔双侧髂后上棘抽取兔自体骨髓,并在体外对骨髓间质干细胞(MSCs)进行分离、培养、扩增。各组细胞均分为两个亚组(A组、B组),每组各3例,细胞接种后24小时,进行诱导分化,参附注射液高、低剂组中,A亚组分别在常规培养液中加入不同浓度的参附注射液(250ul/ml 50ul/ml),同时加用5-氟胞苷(5-aza),B亚组分别在常规培养液中加用5-氟胞苷(5-aza),但不加参附注射液；对照组A亚组加用5-氟胞苷(5-aza),B亚组不加任何药物。诱导24小时后,细胞换液,按照细胞培养常规,连续培养28天。通过细胞形态学、心肌细胞特异性蛋白(α肌动蛋白、肌钙蛋白-T)以鉴定诱导分化的效果,同时在动物模型建立后2周,抽血应用Elisa法检测兔血清干细胞因子(SCF)和粒细胞集落刺激因子(G-CSF)。【结果】临床研究：参附注射液治疗组中医证候积分较治疗前减少,且优于丹参注射液治疗组(P<0.05),中医证候疗效总有效率为88.5%,明显优于对照组57.1%(P<0.05)；临床症状、心电图疗效观察,与对照组比较,差异无统计学意义；食道调搏检查中参附注射液组各项指标有好转趋势,但无显著性差异,无统计学意义,与阿托品组比较,两组在窦房结恢复时间、矫正窦房结恢复时间、窦房传导时间等组间比较,差异无统计学意义,阿托品组并无明显优于参附注射液组,说明参附注射液在改善上述指标方面亦有一定的作用,但效果不显著。实验研究：培养4周后细胞爬片,经HE染色,光学显微镜下观察：培养细胞形态与心肌细胞接近程度,由好到差顺序依次为：参附注射液高剂量A组>参附注射液高剂量B组>参附注射液低剂量A组>参附注射液低剂量B组>对照组A组>对照组B组。培养细胞肌钙蛋白T检测显示：同时加入参附注射液及5—aza诱导者骨髓间质干细胞转化为心肌细胞的阳性强度高于单纯5—aza诱导者,且与剂量有一定的关系,剂量越高,转化为心肌细胞的阳性强度越高；α-肌动蛋白检测显示,同时加入参附注射液诱导者骨髓间质干细胞转化为心肌细胞的阳性强度高于单纯5—aza.诱导,与剂量有一定的关系,剂量越高,转化为心肌细胞的阳性强度越高,但相关性较差,可能与α-肌动蛋白非心肌所特有有关,也可能与样本量较少有关。兔外周血干细胞因子检测：经ELISA法检测,参附注射液高剂量组样本浓度高于对照组(P<0.05),参附注射液高剂量组与参附注射液低剂量组、参附注射液低剂量组与对照组比较,差异无统计学意义(P>0.05)：兔外周血粒细胞集落刺激因子检测：ELISA法检测,参附注射液高剂量组样本浓度高于参附注射液低剂量组及对照组(P<0.01),参附注射液低剂量组与对照组比较,差异无统计学意义(P>0.05)；结果表明：参附注射液对兔骨髓的造血因子——干细胞因子、粒细胞集落刺激因子的生成有一定的刺激作用,且药物浓度越高,其刺激作用越强；从而证明其对骨髓造血功能有一定的刺激作用,并与给药剂量有关,剂量越高,其刺激作用越强。【结论】参附注射液治疗缓慢心律失常,可明显改善患者中医证候；参附注射液可使骨髓间质干细胞向心肌细胞的转化的阳性强度显著提高；参附注射液对骨髓造血因子有一定的刺激作用；参附注射液在缓慢心律失常及骨髓间质干细胞转化为心肌细胞中的作用,值得进一步的探索研究。更多还原

【Abstract】 BackgroudBradyarrhythmia is caused by sinus node or atrioventricular node low self-regulation, or it can caused by cardiac conduction system block.This includes sinus bradycardia, sinoatrial or atrioventricular block (AVB), sick sinus syndrome (sss) and so on. It usually manifested clinically dizziness, amaurosis, heart palpitations, a transient disturbance of consciousness, syncope (seriously, have a A-Adams syndrome) and other symptoms, even death, and it belongs to Symptoms of Traditional Chinese Medicine "Vertigo " "fainting "heart palpitations" "palpitation "and other areas. Currently modern medicine has not found a satisfactory effect of the drugs to treat these arrhythmias, due to the high cost, and must have certain equipment and professional staff and other reasons; pacemaker treatment of the disease is difficult to spread, transgenic and stem cell transplantation in the treatment of this disease is still in experimental stage. In recent years have more research about Shenfu Injection in treating bradyarrhythmia disease and made a lot of achievements, however, research on its mechanism of action is relatively small.PurposeThrough clinical and experimental study, discussion the clinical efficacy, application security and the possible intrinsic mechanism of Shenfu Injection in treating bradyarrhythmia disease, aslo discussion the role of Shenfu Injection in the induction of bone marrow mesenchymal stem cells transform into myocardial cells, and thus restore the conduction of Atrioventricular node. This can provide the prerequisite and basis for the development of biological pacemakers, at the same time to provide the basis for clinical applicationhe in the future. MethodsClinical study:A prospective, simple random, single blind, parallel group study, from December 2009 to March 2011 period, from Guangzhou University of Traditional Chinese Medicine Department of Cardiology, First Affiliated Hospital to receive patients met the diagnostic criteria in 47 cases Were randomly divided into two group,26 in cases treatment group,21 cases in control group. Treatment group using 20ml Shenfu injection, the control group using 20ml Danshen injection for treatment, both of those treatments were continued for a week. Observed the change of traditional Chinese Syndrome, resting electrocardiogram and laboratory indicators about the two groups patients before and after the treatment,4 days after treatment, two groups were randomly selected 10 patients for esophageal pacing ECG examination, In the process of this examination, two groups of patients were intravenous inject shenfu injection and atropine injection, Before treatment, 10min after treatment were detected indicators of patients with heart rate, sinus node recovery time, corrected sinus node recovery time, sinoatrial conduction time and Venturi-type atrioventricular block point, Observed, compared to its changes before and after treatment.Animal Study:The age of 3 to 4 months, weighing 2-2.5kg of 18 New Zealand white rabbits were divided into 3 groups(n=3), each group have 6 rabbits, Without limiting the male and female. First Shenfu injection were used with the high (Shenfu 5ml) and low dose (Shenfu lml), the control group (saline 3ml) were injected throngh rabbit ear vein for 1 week, And then by rabbit bilateral posterior superior iliac spine pumping the bone marrow, and in vitro bone marrow mesenchymal stem cells (MSCs) were isolated, cultured and amplified. The cells were divided into two subgroups (A group, B group),3 cases in each group,24 hours after cell inoculation, began induced differentiation, when cells were induced to differentiate in vitro, in groups with high and low dose of Shenfu injection, A sub-group were in the regular culture medium with different concentrations of Shenfu (250ul/ml,50ul/ml), added in the 5-F cytidine (5-aza), B sub-groups were in the regular culture medium was added 5-fluoro cytidine (5-aza), but without Shenfu injection; in control group, A sub-group plus 5-FU cell Glucoside (5-aza), B sub-group without any drugs. After 24 hours, the cell medium was changed, according to conventional cell culture continuously cultured cells until 28 days。By cell morphology, cardiomyocyte specific proteins (a actin, troponin-T) to identify the effect of induced differentiation, at the same time after the animal model established for 2 weeks apply the Elisa assay to determine the serum of stem cell factor (SCF) and granulocyte colony stimulating factor (G-CSF) in rabbit blood.ResultClinical study:The Traditional Chinese Medicine syndrome score of Shenfu injection treatment group is less than before treatment, and better than Danshen injection group (P<0.05), TCM total effective rate of Shenfu injection treatment group was 88.5%, significantly better than the control group 57.1% (P<0.05); Clinical symptoms, ECG efficacy, compared with the control group, no significant difference; Esophageal pacing ECG Shenfu injection group in a better trend in the indicators, but no significant difference or no statistically significant; Compared with the atropine injection group, the the difference of the two groups in the sinus node recovery time, corrected sinus node recovery time, sinoatrial conduction time were not statistically significant; The atropine injection group not significantly better than Shenfu injection group, indicating that the Shenfu injection can also improve the indicators described above, but the effect was not significant.Animal Study:After 4 weeks of cell culture, preparation climbing film, by HE staining were observed under light microscope:cells close to the form and extent of myocardial cells, from good to bad in the order:Shen Fu-pin high-dose A group> Shen Fu-pin high-dose B group> A reference group of low-dose needle attached to> participate in group B with low-dose needle> control group A group> control group B group. Troponin T test showed that cultured cells: added both Shenfu injection and 5-aza at the same time for inducing bone marrow mesenchymal stem cells transformed into myocardial cells positive intensity higher than that only induced by 5-aza, and have a certain relationship with the dose, the higher the dose, the higher the positive strength of transformed Into myocardial cells; a-actin analysis showed that, added both Shenfu injection and 5-aza at the same time for inducing bone marrow mesenchymal stem cells transformed into myocardial cells positive intensity higher than that only induced by 5-aza, and have a certain relationship with the dose, the higher the dose, the higher the positive strength of transformed Into myocardial cells, however, somewhat less relevant, this may be related to a-actin-specific non-cardiac related, also may be relevanted to small sample size. Rabbit peripheral blood stem cell factor test:Detected by ELISA, the sample concentrations of high dose Shenfu injection group was higher than the control group(P<0.05), that of high dose Shenfu injection group compared with low dose group and Shenfu injection low dose group compared with control group were no Significance statistical difference (P> 0.05);Rabbit peripheral blood of granulocyte colony stimulating factor test:Detected by ELISA, the sample concentrations of high dose Shenfu injection group was higher than the low dose Shenfu injection and the control group(P<0.05), that of low dose Shenfu injection group compared with control group was no Significance statistical difference (P> 0.05);The results showed that:Shenfu injection have a certain stimulus in the production of rabbit bone marrow hematopoietic growth factors-stem cell factor, Granulocyte colony stimulating factor, and the higher drug concentration, the stronger the effect of its stimulus; Those proved that Shenfu injection have a certain stimulating effect for bone marrow hematopoietic, and associated with with the drug dose, the higher the dose, the stronger the effect of its stimulation.ConclusionUseing Shenfu injection in treating bradyarrhythmia disease can significantly improve patient TCM syndrome; Shenfu injection enable increase the rate of conversion of bone marrow mesenchymal stem cells transfor into myocardial cells Significantly; Shenfu injection have a certain stimulus for bone marrow stem cells; the role of Shenfu injection in bradyarrhythmia and bone marrow mesenchymal stem cells transfor into myocardial cells worth further exploration and research.更多还原