【作者】 周永红；

【导师】 陈利国；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2008， 博士

【摘要】 研究目的建立2型糖尿病血瘀证的血管内皮细胞损伤模型,为中医学病证结合细胞模型的研究提供方法学上的借鉴。实验内容包括两部分,第一部分进行糖尿病血瘀证血管内皮细胞损伤模型的研究,体现“同病异证”的特点;第二部分进行高血压病血瘀证和糖尿病血瘀证细胞损伤模型的比较研究,探讨“异病同证”的差异。第一部分2型糖尿病血瘀证血管内皮细胞损伤模型的研究实验1.2型糖尿病血瘀证患者血清对人脐静脉内皮细胞(ECV-304)的细胞活性和形态的影响目的:观察2型糖尿病血瘀证患者血清对人脐静脉内皮细胞(ECV-304)的细胞活性和细胞形态的影响。方法:ECV-304传代后取对数生长期细胞干预分组如下:N组(正常对照组)、H组(健康人血清干预)、MD组(糖尿病血瘀证患者血清干预)和MND组(糖尿病非血瘀证患者血清干预)。其中糖尿病血瘀证和非血瘀证患者血清各有3个浓度:5%,10%,20%。采用MTT法在全自动酶标仪上于570nm处测定吸光值,应用倒置相差显微镜、扫描电镜和透射电镜观察细胞形态的变化。结果:①MTT法检测细胞活性,5%浓度:MD组的OD值(0.94±0.14)较H组(1.60±0.17)明显降低,差异有统计学意义(P＜0.001);10%浓度:MD组的OD值(0.88±0.11)较H组(1.34±0.14)明显降低,差异有统计学意义(P＜0.001)。②电镜下MD组细胞和N组的细胞形态上具有明显差异。倒置相差显微镜观察发现:MD组的细胞由圆形居多变为多角形或椭圆形居多,伴间隙增宽;扫描电镜观察发现:MD组细胞收缩分离成为放射星状体,细胞表面有大小不一的圆形凹陷或突起;透射电镜观察发现:MD组胞浆内所含的吞饮泡较N组多,粗面内质网扩张伴数目减少,核糖体部分丢失。平面内质网明显扩张,线粒体肿胀,嵴消失。结论:10%浓度的2型糖尿病血瘀证患者血清严重损伤体外培养的正常人脐静脉内皮细胞(ECV-304),造成细胞活性降低和细胞形态改变。实验2.2型糖尿病血瘀证患者血清对人脐静脉内皮细胞(ECV-304)内分泌功能的影响目的:观察2型糖尿病血瘀证患者血清干预ECV-304对其内分泌功能的影响。方法:细胞干预分组方法同实验1。应用放免法测定内皮素(ET)含量,应用硝酸还原酶测定一氧化氮(NO)含量;利用双抗夹心ELISA法检测各组细胞培养上清液中内皮细胞蛋白C受体(EPCR)、血管内假性血友病因子(vWF)和血栓调节蛋白(TM)的含量。结果:①MD组的ET水平(286.03±24.03)比N组(77.88±10.07)升高,差异有统计学意义(P＜0.001);MD组的ET水平(286.03±24.03)比MND组(236.22±15.89)升高,差异有统计学意义(P＜0.001)。②MD组的NO水平(22.37±3.30)比N组(52.97±8.51)降低,差异有统计学意义(P＜0.001);MD组的NO水平(22.37±3.30)和MND组(24.55±3.79)相比差异没有统计学意义(P＞0.05)。③内皮细胞经过患者血清损伤后,MD组分泌的EPCR含量(189.66±11.76)比N组(113.94±16.04)升高,差异有统计学意义(P＜0.001);MND组分泌量(216.24±15.86)较MD组(189.66±11.76)升高,差异有统计学意义(P＜0.001)。④损伤后MD组分泌的vWF含量(79.94±8.52)比N组(51.09±9.92)升高,差异有统计学意义(P＜0.001);MND组分泌量(78.85±10.17)较MD组(79.94±8.52)减少,但差异没有统计学意义(P＞0.05)。⑤损伤后MD组分泌的TM含量(29.25±5.16)比N组的(18.47±2.59)升高,差异有统计学意义(P＜0.001);MND组分泌量(26.40±4.36)较MD组(29.25+5.16)减少,但差异没有统计学意义(P＞0.05)。结论:糖尿病血瘀证患者血清干预ECV-304后可以造成血管内皮细胞功能损伤,表现在内分泌功能的变化上,其中最为明显的是释放ET增加,而NO分泌减少,ET和NO二者之间失去平衡;反映内皮细胞内分泌功能的细胞因子如EPCR、vWF和TM等细胞损伤标志物的含量也显著上升,尤其是EPCR增加最为明显,提示糖尿病血瘀证患者血清对上述因子的表达起作用,说明糖尿病血瘀证患者血清干预正常的血管内皮细胞,可以造成内分泌功能紊乱。实验3.2型糖尿病血瘀证患者血清对人脐静脉内皮细胞(ECV-304)骨架及胞浆内游离钙的影响目的:探讨2型糖尿病血瘀证患者血清干预ECV-304对细胞骨架微丝及胞浆内游离钙浓度的影响。方法:细胞干预分组方法同实验1。应用激光扫描共聚焦显微镜,采用Fluo-3/AM作为荧光指示剂观察细胞内游离钙浓度的变化,采用荧光探针标记的鬼笔环肽染色法观察细胞肌动蛋白微丝分布的差异。结果:①胞浆内游离钙浓度以荧光强度来表示,MD组胞浆内荧光分布不均匀,呈网状或团块状,荧光强度(127.53±10.49)高于N组(92.05±6.41),差异有统计学意义(P＜0.001);MD组的荧光强度也高于MND组(108.26±7.35),差异有统计学意义(P＜0.001)。②细胞骨架微丝经过特异性抗体染色后,N组和H组细胞分布多规则清晰,彼此连接,附着在细胞内特定部位,保持着细胞的正常外形;MD组可见细胞外形发生皱缩,微丝断裂,排列紊乱,少部分区域缺失;MND组可见细胞外形发生皱缩,微丝断裂但排列较为规则,部分可见丝网状有序排列。结论:糖尿病血瘀证患者血清严重损伤体外培养的正常ECV-304,表现在胞浆内游离钙浓度的升高和细胞骨架微丝分布的变化。由于细胞内钙离子通过微丝结合蛋白来调节细胞骨架的装配与分解,高钙情况下该蛋白可使长微丝切断成片段,本研究中细胞内钙离子的升高可能介导了血瘀证血清对细胞骨架的损伤作用。实验4.血府逐瘀汤及丹参酮ⅡA(TanⅡA)对抗糖尿病血瘀证患者血清致内皮功能障碍的影响目的:观察血府逐瘀汤复方及丹参酮ⅡA对抗糖尿病血瘀证患者血清致内皮功能障碍的影响。方法:N组和MD组分组方法同实验1,还有MDB组(10%2型糖尿病血瘀证患者血清和血府逐瘀汤复方药物血清共同干预24h)和MDT组(10%2型糖尿病血瘀证患者血清和TanⅡA共同干预24h)。其中血府逐瘀汤复方药物血清各有3个浓度:20%,10%和5%;TanⅡA各有3个浓度:20μg/ml,10μg/ml和5μg/ml。利用MTT法检测吸光值,利用倒置相差显微镜、扫描电镜和透射电镜观察细胞形态。根据结果对药物选取最佳作用浓度后进一步测定细胞分泌的ET和NO含量、EPCR、vWF、TM的含量。利用激光扫描共聚焦显微镜观察细胞内游离钙浓度和细胞骨架微丝的变化。结果:①倒置相差显微镜观察发现:MDB组的细胞恢复椭圆形紧密排列,边界较清楚,MDT组细胞无重叠生长现象,细胞碎片较少;扫描电镜观察发现:MDB组细胞之间有间隙,但仍然有突起连接,细胞表面有大小不一的圆形凹陷或突起;MDT组中多数细胞收缩分离呈现放射星状体;透射电镜观察发现:MDB组细胞粗面内质网数目较少,不扩张,其上附着的核糖体不丢失,线粒体数目减少,其双层膜及内嵴结构清晰,在核未见特殊改变;MDT组透射电镜结果与MDB组接近。②MDT组的OD值(1.11±0.12)较MD组(0.66±0.09)显著升高,差异有统计学意义(P＜0.001),以TanⅡA的浓度为10μg/ml时差异最为显著(P＜0.001);MDB组的OD值(0.96±0.15)较MD组(0.55±0.09)显著升高,差异有统计学意义(P＜0.001)。以血府逐瘀汤复方药物血清浓度为10%时差异最为显著(P＜0.001)。根据MTT和形态观察,选取最佳作用浓度,糖尿病血瘀证患者血清浓度为10%,TanⅡA的浓度为10μg/ml;③MDT组的ET水平(143.85±13.05)比MD组(286.03±24.03)降低,差异有统计学意义(P＜0.001);MDB组的ET水平(191.23±12.17)比MD组(286.03±24.03)降低,差异有统计学意义(P＜0.001);④MDT组的NO水平(46.86±8.12)比MD组(22.37±3.30)降低,差异有统计学意义(P＜0.001);MDB组的NO水平(29.41±4.46)比MD组(22.37±3.30)降低,但差异没有统计学意义(P＞0.05);⑤MDT组分泌的EPCR含量(132.22±13.28)与MD组(189.66±11.76)相比减少,差异有统计学意义(P＜0.001);MDB组分泌的EPCR含量(122.80±14.37)与MD组(189.66±11.76)相比也有减少,差异有统计学意义(P＜0.001)。⑥MDT组分泌的vWF含量(104.19±10.45)比MD组(79.94±8.52)升高,但差异没有统计学意义(P＞0.05);MDB组分泌的vWF含量(62.41±9.44)与MD组(79.94±8.52)相比减少,差异有统计学意义(P＜0.001)。⑦MDT组分泌的TM含量(27.14±4.61)比MD组(29.25±5.16)降低,但差异没有统计学意义(P＞0.05);MDB组分泌的TM含量(22.30±3.18)与MD组(29.25±5.16)降低,差异有统计学意义(P＜0.001)。⑧MDT组荧光强度(154.96±7.90)高于MD组(127.53±10.49),差异有统计学意义(P＜0.001);MDB组荧光强度(92.61±7.06)低于MD组(127.53±10.49),差异也有统计学意义(P＜0.001);⑨MDB组和MDT组细胞外形接近正常,骨架微丝较少但排列规则,分布清晰,和MD组有明显差异。结论:10%浓度血府逐瘀汤复方药物血清和10μg/ml中药丹参酮ⅡA可以有效对抗糖尿病血瘀证患者血清干预ECV-304造成的内皮功能障碍,减少患者血清对于细胞的损伤,对血管内皮细胞起到一定的保护作用,从而证实了在体外环境下,用于试验的中药复方和单味中药有效成分对细胞损伤模型具有的保护作用。也从药物的角度反证了细胞损伤模型的成功建立。第二部分高血压病和2型糖尿病血瘀证血管内皮细胞损伤模型的比较研究目的:通过高血压病和2型糖尿病血瘀证血管内皮细胞损伤模型的比较,为异病同证理论提供实验依据。方法:N组、H组、MD组、MDB和MDT分组方法同第一部分,还有MH组(10%高血压病血瘀证患者血清干预)、MHB组(10%高血压病血瘀证患者血清和血府逐瘀汤复方药物血清共同干预24h)和MHT组(10%高血压病血瘀证患者血清和TanⅡA共同干预24h)。实验方法同第一部分。结果:①MH组的OD值(0.98±0.12)高于MD组(0.88±0.11),但差异没有统计学意义(P＞0.05);②MH组分泌的ET含量(177.62±15.77)低于MD组(286.03±24.03),差异有统计学意义(P＜0.001);③MH组分泌的NO含量(32.61±6.76)高于MD组(22.37±3.30),差异有统计学意义(P＜0.001);④MH组分泌的EPCR含量(168.32±21.94)低于MD组(189.66±11.76),差异有统计学意义(P＜0.001);⑤MH组分泌的vWF含量(80.17±10.25)高于MD组(79.94±8.52),差异有统计学意义(P＜0.001);⑥MH组分泌的TM含量(28.36±4.94)略低于MD组(29.25±5.16),但差异没有统计学意义(P＞0.05);⑦MH组荧光强度(156.78±8.22)高于MD组(127.53±10.48),差异有统计学意义(P＜0.001);⑧MH组可见细胞间隙加大,微丝较少但排列较为规则,部分可见丝网状有序排列;与MD组骨架微丝分布差异显著。结论:高血压病患者血清和糖尿病患者血清干预正常培养的ECV-304都可以造成血管内皮细胞的损伤,在细胞形态和细胞活性、细胞内分泌功能(TM)等方面没有明显的差异,此为“证”同,可以作为“血瘀证”共同的病理生理学基础模型;而在异“病”方面,由于糖尿病和高血压病的血清也存在不同程度的差异,对细胞损伤的差异集中表现为细胞内分泌功能(EPCR和vWF、ET和NO)和细胞内钙离子的浓度变化等方面,这些差异可体现为“病”异的基础。前后结合后则明确体现了异“病”同“证”的特点,深入机制探讨将有可能对血瘀证的微观辨证提供依据。更多还原

【Abstract】 ObjectiveTo establish the vascular endothelial cell（VEC） injury model of syndrome（BSS） associated with diabetes（DB）,to observe the model cells’ change of cytoactive,shape,ultrastructure and function,and to provide reference in the methodology for the establishment of traditional Chinese medicine（TCM） cell model based on integration of disease and syndrome.PartⅠ.Research on the injury model by the serum of DB patients with BSS1.Effects of ECV-304 incubated by the serum of patients with BSS associated with diabetes[Objective]To observe the effects of ECV-304 incubated by the serum of patients with BSS associated with diabetes.[Methods]ECV-304 were obtained by cultured conventionally.It was divided into 4 groups,ECV-304 cultured with DMEM（Group N）,with the serum of healthy person（Group H）,with the serum of patients with BSS associated with diabetes（Group MD） and the serum of diabetes patients with no-BSS（Group MND）.The cytoactive of ECV-304 were assayed by MTT chromatometry,and morpHological changes were identified by light microscope and electron microscopy.All groups were measured.[Results]①There was a statistically significant difference（P＜0.001）regarding the OD in the MD Group and H Group.②By the inverse phase contrast microscope,we discovered a significant difference between the Group MD and Group MND.[Conclusion]The serum of patients with BSS associated with diabetes could injury ECV-304 of the cytoactive and shape.2.Preliminary research on the mechanisms of injury ECV-304 by the serum of patients with BSS associated with diabetes on endothelial dysfunction[Objective]To explore the mechanisms of ECV-304 incubated by the serum of patients with BSS associated with diabetes.[Methods]ECV-304 was divided into 4 groups the same as the 1^st experiment.The nitric oxide（NO） excreted by model cells was assayed by nitric acid deoxidizing enzyme method,and the endothelin（ET） was assayed by non-balance method.The injury symbols of VEC:vWF,TM and EPCR excreted by model cells were assayed by enzyme-linked immunosorbent assay （ELISA）.All groups were measured.[Results]①There was a significant difference（P＜0.001） regarding the ET excreted by ECV-304 between the Group MD and Group H,Group N,Group MND.②There was a significant difference（P＜0.001） regarding the NO excreted by ECV-304 between the Group MD and Group N,Group H.③There was a significant difference（P＜0.001） regarding the EPCR excreted by ECV-304 between the Group MD and Group N,Group H,Group MND.④There was a significant difference（P＜0.001） regarding the vWF excreted by ECV-304 between the Group MD and Group N.⑤And the same condition of TM happened between the Group MDand the Group N significantly（P＜0.001）.[Conclusion]The serum of patients with BSS associated with diabetes could injury ECV-304 and caused endothelial dysfunction.It was possible that BSS influnenced the expression of molecules in vascular endothelial cells.3.Preliminary research on the mechanisms of injury ECV-304 by the serum of patients with BSS associated with diabetes on intracellular free calcium([Ca²⁺]i) and cytoskeleton[Objective]To explore the mechanisms of ECV-304 incubated by the serum of patients with BSS associated with diabetes.[Methods]ECV-304 was divided into 4 groups the same as the 1^st experiment.Model cells’ intracellular free calcium([Ca²⁺]i) and cytoskeleton were assayed by laser scanning confocal microscope（LSCM）.All groups were measured.[Results]①There was a significant difference（P＜0.001） regarding the intracellular calcium concentration([Ca²⁺]i) of ECV-304 between the Group MD and Group H,Group N,Group MND.②by the cytoskeleton,the Group MD was distinctly different with the Group MND.[Conclusion]The serum of patients with BSS associated with diabetes could injury ECV-304 and caused Model cells’ intracellular free calcium([Ca²⁺]i) and cytoskeleton change.4.Protective effects of Xuefn Zhuyu decoction and TanⅡA on the injury model by the serum of patients with BSS associated with diabetes on endothelial dysfunction [Objective]To observe the protective effectes of Xuefu Zhuyu decoction and TanⅡA on the cellar injury model by the serum of patients with BSS associated with diabetes on endothelia dysfunction.[Methods]ECV-304 were obtained by cultured conventionally.It was divided into 4 groups,Group N and Group MD is the same as 1^st experiment,the group incubated with the serum of patients with BSS associated with diabetes and the mice serum of Xuefu Zhuyu decoction（Group MDB）,the group with the serum of diabetes patients with BSS and TanⅡA（Group MDT）.The activeness of ECV-304 were assayed by MTT chromatometry,and morpHological changes were identified by light microscope and electron microscopy.The nitric oxide（NO） excreted by model cells was assayed by nitric acid deoxidizing enzyme method,and the endothelin（ET） was assayed by non-balance method.The injury symbols of VEC:vWF,TM and EPCR excreted by model cells were assayed by enzyme-linked immunosorbent assay（ELISA）.Model cells’ intracellular free calcium ([Ca²⁺]i) and cytoskeleton were assayed by laser scanning confocal microscope（LSCM）.All groups were measured.[Results]①By the inverse pHase contrast microscope,we discovered that the Group MDB and Group MDT is the same shape as the Group N.②There was a significant difference（P＜0.001）of the secretion of OD,NO,ET,EPCR,vWF,TM and intracellular calcium concentration between the Group MDB and the Group MD.③There was a significant difference（P＜0.001） of the secretion of OD,NO and ET between the Group MDT and the Group MD,but no differences in the intracellular calcium concentration and the injury symbols.④by the cytoskeleton,the Group MDB and Group MDT were distinctly different with the Group MD.[Conclusion]The Xuefu Zhuyu decoction and TanⅡA had the protective effectes on the cellar injury model by the serum of patients with BSS associated with diabetes on endothelia dysfunction and from the point of medicine,they could recover endothedial dysfunction induced by the cellar injury model the medicine.PartⅡ.Differences between injury models by the serum of EH patients with BSS and by the serum of DB patients with BSS [Objective]To investigate the differences between injury models by the serum of EH patients with BSS and by the serum of DB patients with BSS.[Methods] ECV-304 were obtained by cultured conventionally.It was divided into 4 groups. Group N,Group H and Group MD is the same as 1^st experiment,the group incubated with the serum of patients with BSS associated with EH（Group MH）.The activeness of ECV-304 were assayed by MTT chromatometry,and morpHological changes were identified by light microscope and electron microscopy.The nitric oxide（NO） excreted by model cells was assayed by nitric acid deoxidizing enzyme method,and the endothelin（ET） was assayed by non-balance method.The injury symbols of VEC: vWF,TM and EPCR excreted by model cells were assayed by enzyme-linked immunosorbent assay（ELISA）.Model cells’ intracellular free calcium([Ca²⁺]i) and cytoskeleton were assayed by laser scanning confocal microscope（LSCM）.All groups were measured.[Results]There was a significant difference of the secretion of OD, NO,ET,EPCR,vWF,intracellular calcium concentration and cytoskeleton between the Group MD and the Group MH.[Conclusion]The results clearly show that the changes between the Group MD and the Group MH is partly related to the basis of BSS.Furthermore,by the example of BSS,it can partially explain the mechanism of "different disease and same syndrome" in Traditional Chinese Medicine.更多还原