【作者】 何本鸿；

【导师】 张介眉；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的通过超声二维斑点追踪技术、组织多普勒技术、western blot和蛋白组学实验技术观察白马合剂对AS大鼠血脂水平、心功能、血流动力学及对动脉粥样硬化大鼠腹主动脉蛋白组的影响,研究白马合剂抗AS的作用及机制。方法将体重为200±20克,3-4月龄健康纯种SD雄性大白鼠60只,随机分为正常对照组、空白模型组、葱白组、马齿苋组、白马合剂组和普罗布考组6组,每组10只。参照郭延松等的方法制备动脉粥样硬化模型：适应性喂养一周后,正常对照组喂基础饲料和普通自来水,模型组及各治疗组以高脂饲料并于实验开始时于右下肢肌肉注射维生素D3针剂(3×105u/kg体重),以后每隔30天重复注射一次。于第7天除正常对照组外,其余各组行大鼠主动脉内膜球囊损伤术,各组于手术后第3天开始给药,给药体积为10ml/kg,正常对照组和空白模型组给予生理盐水；葱白提取物组按60mg·kg-1给药；马齿苋提取物组按30mg·kg-1给药；白马合剂组按10m1·kg-1给药；普罗布考组按16mg·kg-1给药。每日1次,连续给药16周。观察动物的一般状态：观察动物的饮食、精神、体重、毛发、大小便及死亡情况；于实验16周末禁食16h后,取血5ml留置2管,分别分离血清和血浆,检测血浆TC、TG、HDL-C、LDL-C。观察动物心功能的改变：GE Vivid 7 Dimension彩色超声诊断仪,10S探头,探头频率为8MHz。探头置于左胸,在取得满意的胸骨旁左室长轴和短轴二维图像后,将图像储存到光盘上,以便进一步脱机分析。获取连续3个心动周期的胸骨旁左室长轴及乳头肌水平短轴切面时应该注意内膜显示清晰,且帧频大于190。进入EchoPAC工作站,于胸骨旁左室长轴切面使用M型超声测量左心室舒张末期内径(LVIDd)和收缩末期内径(LVIDs),计算左心室缩短率(FS)、射血分数(EF)。进入二维应变软件程序二维应变界面中,在收缩末期心内膜显示最清晰时将图像冻结,沿心内膜边界手动勾画,将自动生成包含心肌内膜、中层和心外膜的感兴趣区(region of interest, ROI),获得理想的追踪节段后,可自动显示左心室乳头肌短轴6个节段的应变曲线图。记录节段心肌的收缩期峰值径向应变(RS)和收缩期峰值环向应变(CS)。计算二尖瓣舒张早期血流峰值流速(E峰)与舒张晚期血流峰值流速(A峰)的比值E/A。颈动脉斑块血流动力学：GE Vivid 7彩色超声诊断仪,1OL探头,探头频率为13.OMHz。固定大鼠后,检测大鼠左侧颈总动脉,分别于颈总动脉近、中、远段三处测量血管内径,并在二维实时状态下将取样容积置于血管中心,调整最佳取样容积,使超声束与血流方向夹角小于60。.在连续观察数个心动周期后,获得最清晰血流速度频谱曲线冻结图像。依次测量血流速度、阻力指数(RI)及脉动指数(PI)。蛋白组学研究：实验结束后,分离腹主动脉,用GE healthcare双向电泳和质谱鉴定进行差异蛋白的检测。准备好所需的仪器和试剂后,按照下列顺序进行实验检测：动物组织蛋白提取与纯化→等电聚焦(蛋白溶解→定量→上样→干胶条泡胀→等电聚焦→胶条保存)→二向SDS-PAGE(灌胶→胶条平衡→二向电泳)→染色(凝胶转移→银染→考染→凝胶保存)→图像分析(凝胶扫描→图片挑选及裁切→图谱分析)→质谱前处理(挖点→脱色→酶切→肽段浓缩除盐)→质谱检测(点样→质谱分析→数据库检索)。最后进行质谱鉴定。结果大鼠的一般状态：适应性喂养一周后,除正常对照组饮食逐渐增加外高脂饲料组饮食逐渐减少,空白模型组减少最为显著,和正常对照组比较差异显著(P<0.01)；给药一周后,模型组和各药物组大鼠精神萎靡不振,活动度明显减少,模型组精神状态最差,葱白提取物组相对精神状态较佳；和造模前比较,喂养4周后,正常对照组体重增加最多,其余各组体重增加幅度小于正常对照组；造模给药后一周,大鼠粪便稀薄,小便色黄量少,以后逐渐变干；除正常对照组毛发光亮外,造模后各组大鼠毛发干枯,没有光泽；除正常对照组外,各组大鼠均有死亡,死亡动物均表现为：体重明显减轻、皮毛干枯、精神极度萎靡、行动迟缓,死亡后体检未见明显异常。对TC和TG的影响：与正常对照组比较,模型组大鼠TC、TG明显升高(P<0.01)；与空白模型组比较,除普罗布考组可以明显降低TG外(P<0.01),其余各治疗组均有降低TC和TG的作用,但无统计学差异(P>0.05)。对HDL-C、LDL-C的影响：与正常对照组比较,模型组大鼠HDL-C水平未见明显改变,LDL-C水平明显升高(P<0.01)；与空白模型组比较,马齿苋组LDL-C明显降低(P<0.01)。对心功能的影响：与正常组比较,空白模型组LVIDd未见明显改变(P>0.05), EF、FS、Em/Am值明显降低(P<0.05,P<0.01)；与模型组比较,各组LVIDd和FS值未见明显改变(P>0.05)。各组EF值升高明显(P<0.01)。葱白提取物Em/Am值升高明显(P<0.01),而其余各组Em/Am值未见明显改变(P>0.05)；白马合剂组与葱白提取物组比较,EF没有显著差异(P>0.05)；白马合剂组与马齿苋提取物和普罗布考组比较,组间没有显著差异(P>0.05)。对颈动脉血流动力学的影响：与正常组比较,模型组血流速度减慢(P<0.01),RI升高(P<0.01),PI降低(P<0.01)；葱白提取物组和普罗布考组血流速度加快(P<0.01,P<0.05),马齿苋提取物组和白马合剂组血流速度没有统计学意义(P>0.05)；各组均可降低RI(P<0.01),升高PI(P<0.01),普罗布考组作用最好,其次是葱白提取物组,再次是马齿苋提取物组,白马合剂组最差。对腹主动脉蛋白组的影响：将白马合剂组与空白模型组腹主动脉组织蛋白进行2-DE分离各重复3次。应用ImageMaster软件对2-DE凝胶图进行分析,选取空白模型组的腹主动脉图谱为Master,白马合剂组图谱与之进行比较,发现两组的胶条有较好的重复性,测得白马合剂组肌组织和空白模型组凝胶的平均蛋白质点数分别为330±11和325±9,匹配率为89%。研究发现有66个蛋白质斑点在2组凝胶中有显著差异表达(表达量相差2倍以上者),其中蛋白点表达量上升32个,34个蛋白点表达量下降。我们用ABI4700 MALDI-TOF-TOF质谱仪进行质谱分析,将提取出来的各组腹主动脉蛋白进行质谱鉴定,然后对各鉴定的蛋白进行了数据库搜索得到鉴定蛋白点的信息,发现这些蛋白主要包括主要急性期蛋白光蛋白聚糖前体、大鼠alphal巨球蛋白受体结晶体A链、结蛋白等,表达量降低的蛋白质斑点有5个,这些蛋白主要包括肌球蛋白重链2、血清白蛋白前体等。证明差异蛋白的表达与AS密切相关。结论1.白马合剂可以降低AS大鼠TC、TG、LDL-C水平,升高HDL-C,对脂质代谢具有一定的调节作用。2.白马合剂组对AS大鼠EF、FS、E/A的影响,与葱白提取物组、普罗布考组和马齿苋提取物组未见明显差异。3.对AS大鼠血流动力学的影响,葱白提取物作用最强,白马合剂配伍后却没有明显的优势,可能与葱白提取物良好的辛温通阳的作用有关,和马齿苋配伍后并没有增效。4.大鼠腹主动脉2-DE图谱差异蛋白点的筛选与鉴定发现,白马合剂组和模型组比较有23个差异蛋白,并进行了鉴定,证明这些蛋白均与AS发生发展密切相关,白马合剂具有抗AS的作用。更多还原

【Abstract】 ObjectiveThrough ultrasound 2d spot tracking technology, tissue doppler technique, western blot and proteomics experiment technology to observe the effect of white horse mixture to Lipid levels, cardiac function, blood flow dynamics on AS rats aorta proteome.Research the effect and mechanism of white horse mixture to treat AS.Methods60 pure SD male rats the body weight were 200-230 grams,3-4 months old, were randomly divided into normal control group, the blank model group, fistular onion stalk extract group, purslane group, white horse mixture group and the probucol group. Each group contains 10 rats.Prepared the model reference to Guoyan Song, etc. atherosclerosis model:After adaptive feeding a week, the normal control group fed basic diet and ordinary tap water, the model group and every treated group with high fat diet and at the beginning of the experiment in the right lower limb intramuscular injection of vitamin D3 (3×105u/kg body weight), repeated every 30 days after the injection.On the 7 day,all groups except the normal control group were given underwent balloon injury of rat aorta surgery.After 3 days of the operation,every group were given medicine 10ml/kg, volume,the normal control group and model group were given saline.fistular onion stalk extract group were 60mg.kg-1 administration, portulaca oleracea extract group were 30mg.kg-1 administration,white horse mixture group were administered by 10mg.kg-1,probucol group administered by 16mg.kg-1; give medicine 1time every day, continue administration 16 weeks.Observe the general state of animals:To observe the animal’s diet, spirit, weight, hair, urine and death;at the end of the 16 weeks,after 16h fasting,exsanguinated blood 5ml and divided into 2 tubes, serum and plasma were isolated, detected plasma TC, TG, HDL-C, LDL-C.observe the change of Cardiac function in animals:GE Vivid 7 Dimension color ultrasound diagnostic apparatus,10S probe, the probe frequency is 8MHz.Probe placed on the left chest,in obtaining a satisfactory long axis and short axis parasternal two-dimensional image, the image was saved to the disc for further off line analysis.When getting 3 consecutive cardiac cycles of the long axis and parasternal short axis view of the level of the papillary muscles,it should be noted that the endometrium showed clear, and the frame rate is greater than 190.Into EchoPAC workstations, parasternal long axis view in M-mode ultrasound using, left ventricular end diastolic diameter (LVIDd), left ventricular end systolic diameter (LVIDs), calculated left ventricular shortening (FS), ejection fraction (EF).Strain into two-dimensional two-dimensional strain software interface, in the end-systolic endocardial will show the most clear image freeze, along the endocardial border outlined manually, automatically generate included myocardial membrane, the interest in the middle and epicardialarea (region of interest, ROI), after getting a good track segments,it can automatically display the curve segments of left ventricular papillary muscle short axis 6 strain.Record peak systolic myocardial segment radial strain (RS) and peak systolic circumferential strain (CS).Calculate mitral peak early diastolic flow velocity (E peak) and peak late diastolic flow velocity (A peak) the ratio of E/A. Hemodynamics in carotid artery plaque:GE Vivid 7 color ultrasound diagnostic apparatus,10L probe, the probe frequency is 13.0MHz.Fixed the rats, the left carotid artery of rats was detected, respectively, the common carotid artery proximal, middle, distal vessel diameter measured at three and two-dimensional real-time status in the sample volume placed in the vessel center, adjusting the best samplingvolume, so that the angle between the ultrasound beam and blood flow direction is less than 60°.In continuous observation several cardiac cycle,to obtain the clearest image frozen blood flow velocity spectrum curves.Followed by measurement of blood flow velocity, resistance index (RI) and pulsatility index (PI).Proteomics research:when the experiment ended,the separated the abdominal aorta, with the GE healthcare for two-dimensional electrophoresis and mass spectrometry detection of differences protein.After preparing instruments and reagents needed, according to the following order experimental detection: animal tissue protein extraction and purification→isoelectric focusing (dissolve the protein→quantitative→sample on the protein→dry rubber strip bubble bilges→isoelectric focusing→glue to keep)→two sds-page (encapsulating compound→strip balance→2-direction electrophoresis)→Dyeing (gel transfer→silver stain transfer→and dyed→gel to keep)→test and image analysis (gel scanning→pictures selected and cutting→map analysis)→mass spectrometry pretreatment(cut→decoloring→enzyme cut→peptides enzyme concentration in addition to salt)→mass spectrometry detection (some samples→mass spectrometry analysis→searchable database). At last mass spectrum identification.ResultsThe general state of rats:adaptation after a week feeding, in addition to the normal control group,the diet of which increased gradually, the diet of high fat diet group decreased, the blank model group decreased the most significant,compared with the normal control group,it had significant difference (P<0.01); given medicine a week later, the model group and the spirit of each drug group malaise, decreased activity,model group had the worst state of mind,fistular onion stalk extract group had a better set of relative mental state;compared with modeling before,after 4 weeks of feeding, the normal control group gained the most weight, while the weight of rest group gained less than the normal control group;one week after modeling,rat feces thin, yellow urine less,then gradually become dry;addition to the normal control group,the hair of which is shiny, the making rats in each group after the model all had dry hair, not shiny; addition to the normal control group,each group had the rats dead,which showed:significant weight loss, skin dryness, mental extremely sluggish and slow,the physical examination of the dead rats did not show obvious exception.The impact on TC and TG:compared with normal control group, TC, TG of model group was significantly higher (P<0.01); compared with the blank model group, the indicators of probucol group was significantly lower (P <0.01),other treatment group can decrease TC and TQbut there was no significant difference(P> 0.05)Effects on HDL-C, LDL-C.Compared with the normal control group, there was no obvious change on HDL-C,while increase LDL-C levels obviously(P<0.01);purslane group decreased LDL-C levels obviously(P <0.01).The impact on cardiac function:compared with the normal group, Blank model group LVIDd did not show obvious change (P>0.05), while EF, FS, Em/Am reduced significantly (P<0.05, P<0.01);compared with model group,LVIDd and FS didn’t show obvious change(P>0.05).EF of every group significantly increased (P<0.01).Em/Am of fistular onion stalk extract group was significantly increased (P<0.01),while other group didn’t show obvious change (P>0.05);There wasn’t significant difference of EF between white horse mixture group and fistular onion stalk extract group(P>0.05),so was the result among white horse mixture group,portulaca extracts group and probucol group.the impact of Carotid artery blood flow velocity:compared with the normal group,blood flow of model group decreased significantly (P<0.01), RI rised significantly (P<0.01), PI was significantly lower (P<0.05);blood flow of portulaca extracts group and white horse mixture group had no significance (P>0.05).Each group can decrease RI (P<0.01) and increase PI (P<0.01),probucol group had the best effect,then fistular onion stalk extract group,portulaca extracts group,at last was white horse mixture group. the impact ofabdominal aortic protein group.Separate abdominal aortic tissue protein of white horse mixture and blank model group by 2-DE,then repeated 3 times each group.Used ImageMaster software to analyse 2-DE Gel figure,Select the abdominal aortic map of blank model group as Master, compared white mixture group with it,atlas found two group of strip have better repeatability, white mixture group measured the muscle tissue and blank model group gel respectively the average protein points 330+11 and 325+9, matching rate for 89%.Study found 66 protein in two groups gel spots,about which there were significant differences in expressing (expression differs twice the amount above),among them protein point express increased 32,34 protein point express content decreased.We used ABI4700 MALDI-TOF-TOF Mass spectrometer to mass spectrometry analysis,every group was extracted abdominal aortic proteins in mass spectrometry identification.Then the proteins of each appraisal on the database search identified protein point of information.Found these proteins included mainly the acute phase protein chitosan precursor, light protein alphal gigantic globulin receptors in rats, and protein crystals A chain,etc,there were 5 protein spot,the expression of which decreased,these protein mainly includes myosin heavy chain 2, serum albumin precursor,etc.They proofed the expression of differences protein was closely related with AS.Conclusion1,White horse mixture can reduce the TC, TG, LDL-C level of AS rats, increase HDL-C level,it plays a certain regulatory role on lipid metabolism.2,There was no significant difference among fistular onion stalk extract group,probucol group and portulaca extracts group on the effect of EF、FS、E/A as to AS rats.3,As to the effect of blood flow dynamics on rats,fistular onion stalk had the best effect,white horse mixture didn’t show obvious superiority,it may be relevant to the Xinwentongyang effect of fistular onion stalk extract,while used with portulaca,there wasn’t better effect.4,Selection and identification of differences protein points on 2-DE atlas about rats’ aortic showed,there were 23 differences proteins identified between white mixture group and blank model group,it was proved that these proteins were closely correlated with the happen of AS, white horse mixture had the effect of resistance to AS.更多还原