粒细胞集落刺激因子对动脉系统中慢性阻塞性病变作用的实验研究
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摘要
粒细胞集落刺激因子(G-CSF)是广泛应用于放疗和化疗患者动员干祖细胞支持造血恢复的细胞因子。近来研究表明,G-CSF可动员骨髓干细胞进入循环,促使心肌再生和血管新生而改善心脏功能。作为非侵入性治疗,因此有可能更广泛应用于心肌梗死及慢性缺血性心脏病,具备广阔的临床前景。但目前相关研究多局限于它对心脏的作用及机制,对于血管,尤其是与缺血性心脏病紧密相关的慢性动脉阻塞性病变,鲜有报道。另外,G-CSF动员后,直接效应是导致白细胞增多,可能引起高凝状态。这是否会诱发动脉阻塞病变的恶化,目前亦尚未明确。总之,G-CSF对动脉系统及慢性阻塞血管局部的影响,国内外学术界尚无定论。
新近许多研究证实,骨髓、外周血、脐血中存在着内皮祖细胞(endothelialprogenitor cells,EPCs),在损伤信号的刺激下能被招募到血管损伤局部,分化为成熟内皮细胞,参与血管新生和修复。G-CSF作为外源性干细胞动员剂,能刺激骨髓中EPCs进入外周血,加速损伤血管的再内皮化。结合上述不良反应,由此我们推断,其必然对局部血管,包括慢性动脉阻塞性病变及动脉系统本身也存在修复作用甚至副作用。
慢性动脉阻塞性病变作为一种血管损伤,是一系列常见的影响人类健康的心血管疾病,改变的不仅仅是血管的形态和结构,更重要的是其功能的变化。基于此,本研究从形态和功能两方面系统地阐述这个问题。
实验分以下四部分:
第一部分兔颈动脉慢性阻塞模型的构建及影像学评价的研究
目的:制备新型兔颈总动脉慢性阻塞动物模型,为下一步研究粒细胞集落刺激因子对动脉慢性阻塞性病变的作用提供实验基础。方法:健康实验用新西兰大白兔18只,随机分为假手术组、4周组和8周组,每组6只;利用右颈总动脉植入动脉缩窄环制备慢性动脉阻塞模型;应用颈动脉超声以及血管磁共振的影像学手段随访评价模型的构建效果;术后4周(4周组)或8周(8周组)处死动物,分离慢性阻塞缩窄环近端及远端行病理学检查并用扫描电镜、Real-time PCR检测eNOS和ET-1mRNA评估阻塞区域局部内皮结构功能的变化。结果:18只实验兔中17头成活,并成功建立颈总动脉慢性阻塞模型兔11只;植入动脉缩窄环后,4周即见颈总动脉逐步阻塞,颈总动脉超声随访显示血管内径从术前(2.00±0.1)cm下降至(1.33±0.23)cm,狭窄程度达到(48±7)%;到第8周时,血管内径更是显著下降至(0.48±0.06)cm,狭窄达到(95±3)%,与对照组及4周组相比,变化绝对值具有统计学意义(P＜0.05),同时血流速度及阻力指数亦有显著变化;兔颈总动脉磁共振显示造模前清晰可见,双侧基本对称,血管内径为(2.02±0.15)cm,但造模4周后右侧颈总动脉缩窄环部位明显狭窄(1.56±0.18)cm,8周后几乎完全阻塞,仅残留细线痕迹,与假手术组相比,差别有统计学意义(P＜0.05);相应血管段病理检查发现造模后内膜继续增厚,细胞外基质增加,以致管腔重度狭窄甚至闭塞;电镜扫描证实慢性阻塞后内皮细胞凋亡,基质表面颗粒状物增大;Real-time PCR可见慢性阻塞动脉远端血管ET-1基因的上调和eNOS基因的下调及对侧动脉的代偿性变化。结论:应用动脉收缩环4周后即可以成功制作慢性动脉阻塞性病变动物模型,8周更为显著,并出现相应血管段内皮形态及功能变化。
第二部分粒细胞集落刺激因子(G-CSF)对动脉慢性阻塞病变局部血管段的影响
目的:利用慢性动脉阻塞性病动物模型,随访观察G-CSF应用过程中血管局部功能和基质结构的变化,探索G-CSF治疗心血管疾病时对局部慢性阻塞病变动脉的影响,再评价其安全性,从而为临床实践提供新的理论依据。方法:健康实验用新西兰大白兔36只,先随机分为高脂喂养及普通喂养2组,再各自细分为3亚组分别为假手术组、慢性动脉阻塞性模型组和慢性动脉阻塞性模型加G-CSF干预组,每组6只;采用高脂喂养制备动脉粥样硬化模型;G-CSF干预为15μg/kg/d,皮下注射,连续5天,采用Giemsa染色及白细胞计数证实动员效果;造模前后做血常规及血脂生化检测,后2月取材截取慢性阻塞及对应部位局部颈总动脉做分子实验(PCR和WB)及病理学分析;造模后第3、5、7周进行双侧颈总动脉超声随访;胶原染色、MMP-9/TIMP-2免疫组化及WB研究血管基质结构变化;扫描电镜、Real-time PCRE测eNOS和ET-1mRNA评估相应血管内皮形态及功能的变化。结果:成功制备慢性阻塞性颈总动脉病变动物模型24只;高脂喂养2月后,高脂喂养3组总胆固醇、低密度脂蛋白(TC、LDL)等血脂水平明显升高,动脉粥样硬化指数(AI)由(1.21±0.16)升至(2.70±0.74),P＜0.05,有统计学差异;G-CSF动员后Giemsa染色可见粒细胞数目明显增加,同白细胞计数上升一致;造模3周后,颈动脉超声见近端颈总动脉血管内径逐步变小,血流速度变慢,阻力指数增高,应用G-CSF后变化更显著,高脂喂养兔狭窄度从(53.2±0.7)%升至(59.51±0.5)%,P＜0.05,有统计学差异;5周后,血管陆续阻塞直至完全闭合,超声可见动脉粥样硬化血管更易闭合,G-CSF可使完全阻塞时间提前2周达到统计学差异,而普通喂养兔需7周左右;病理分析HE染色可见G-CSF使内膜明显增生,内皮皱褶增多;胶原染色发现高脂喂养能使兔动脉硬化表现为肌纤维略减少而胶原、淀粉样变等物质增多,慢性阻塞后近端血管更显著,而G-CSF干预加剧类似变化;WesternBlot证实G-CSF与动脉粥样硬化一样均可使血管MMP-9和TIMP-2表达上调,加重阻塞病变;Real-time PCR发现G-CSF上调慢性阻塞动脉远端血管eNOS基因下调ET-1基因伴随对侧动脉代偿性变化,表达量经分析有统计学差异(P＜0.05);电镜扫描发现慢性阻塞动脉近端应用G-CSF促使内皮细胞增生,但在遄流下作用有限。结论:G-CSF动员具有促内皮化改善内皮功能的作用,但如高脂喂养导致动脉硬化一样,均可加重局部慢性动脉阻塞性病变,这可能与其恶化动脉粥样硬化以及诱导血管基质蛋白酶的表达,促进胶质纤维增生有关。
第三部分粒细胞集落刺激因子(G-CSF)对慢性阻塞病变动脉系统的整体作用
目的:系统地研究G-CSF在动脉粥样硬化中的作用及其对不同类型的动脉内皮的影响,结合功能与形态学观察,分析G-CSF对动脉粥样硬化等全身性动脉慢性阻塞病变的整体作用,指导临床最大效能地发挥其良好效应,防止不良作用。方法:30只健康实验用新西兰大白兔,随机分为对照组(n=6)、普通喂养+G-CSF组(n=8)、高脂喂养组(n=8)、高脂喂养+G-CSF组(n=8);+G-CSF为15ug/kgG-CSF用生理盐水稀释成0.5mL,每日皮下注射,连续5日,隔周进行,共4次;每周采血,硝酸还原酶法测定血清NO浓度,全自动血生化分析仪测量4组血脂:10周后取材,苏丹Ⅲ脂肪染色分析胸主动脉脂肪斑块;HE染色分析颈总动脉、股动脉及胸主动脉内膜厚度;胶原染色和免疫组化分析各组胸主动脉血管基质情况;逆转录多聚酶链反应(RT-PCR)测定颈总动脉、股动脉及胸主动脉MMP-9、TIMP-2、eNOS及ET-1 mRNA表达;Western-Blot检测不同动脉段血管内MMP-9、TIMP-2蛋白表达;扫描电镜观察各动脉段内皮表面形态,透射电镜观察各组胸主动脉内皮细胞超微结构线粒体的变化。结果:实验期间8只兔子死亡,最终4组分别存活有5、6、5、6只动物,达到实验完成要求;血脂动态检测发现高脂喂养能使TC、LDL等血脂水平上升,6周后高峰,与对照组相比,P＜0.05,但8周后就趋于稳定;+G-CSF使血脂高峰前移,使TC、LDL等提前2周就与对照组出现统计差异(P＜0.05),并有双峰样改变,但对TG同样无影响;NO监测提示G-CSF使血浆NO水平迅速升高,与对照组相比,2周即有统计学差异(P＜0.05);而单纯高脂喂养组则较迟缓,4周以后才逐步上升,差异达到统计学意义(P＜0.05);同时G-CSF干预组最高最快,差异有显著统计学意义(P＜0.01);苏丹Ⅲ染色发现高脂喂养组胸主动脉出现粥样斑块,+G-CSF则使斑块面积比从(57.40±1.68)%上升至(91.18±2.27)%,P＜0.05;HE染色发现+G-CSF使普通喂养组内膜厚度比值从(8.77±0.89)%上升至(11.10±1.50)%,高脂喂养分别为(14.97±1.87)%,(25.83±1.88)%,后者有统计学差异(P＜0.05);胸主动脉胶原染色提示G-CSF能使基质增生,免疫组化证实其上调MMP-9及TIMP-2的阳性率,4组依次分别为(1.08±0.06)%、(26.31±1.08)%、(27.90±1.07)%、(45.51±1.60)%,(4.07±1.01)%、(36.08±1.68)%、(71.36±3.01)%、(67.26±2.54)%;MMP-9及TIMP-2 mRNA RT-PCR的结果与以上相符,eNOS、ET-1mRNA的表达提示高脂喂养兔形成动脉粥样硬化后,反映内皮功能损害的ET-1基因表达依序为胸主动脉＞颈总动脉＞股动脉,而反映内皮功能良好的eNOS基因则反之,+G-CSF在普通喂养组出现eNOS基因的上调但高脂喂养组则下调,ET-1基因的变化趋势正好相反,此变化以胸主动脉为著,中小型动脉如颈总动脉、股动脉次之;电镜扫描发现高脂使EC凋亡增加,G-CSF能使EC数目增加,对正常动脉作用更大,但对粥样硬化动脉表面影响不明显;透射电镜则提示高脂喂养和G-CSF使细胞线粒体等超微结构病理改变严重,以两者累加为最。结论:G-CSF对血脂存在影响,以TC、LDL为主,对TG作用不大。因此它可使动脉粥样硬化恶化,具体表现为斑块面积的扩大,内膜厚度增加,可能与其对基质的影响有关;高脂喂养兔能成功制造动脉粥样硬化模型,成模后动脉内皮功能显著受损,其中以大型动脉显著,同时内皮形态出现相应变化;G-CSF对全身动脉系统都有影响,但动脉越小,影响越小;正常与粥样硬化动脉内皮形态及功能改变之间存在必然联系,其对G-CSF的反应也不同。
第四部分粒细胞集落刺激因子(G-CSF)活化血小板功能参与慢性动脉阻塞
目的:探讨G-CSF在应用于干细胞动员治疗心血管疾病时是否活化血小板功能参与慢性动脉阻塞性病变。方法:16只雄性新西兰大白兔随机分为普通和高脂喂养2组,15ug/kg G-CSF用生理盐水稀释成0.5mL,每日皮下注射,连续5日,用药前后分别用血细胞计数仪测定血小板计数(PLT)、血小板比积(MPV)、血小板分布宽度(PDW)、大血小板比例(P-LCR);双抗体夹心免疫酶联试验(ELISA)测定TXB_2、P选择素等血小板相关指标;采用全血CD41-FITC/CD62P、CD63-PE荧光抗体双标流式分析法对血小板活化标记进行验证;血小板聚集仪直接测定血小板聚集能力的前后变化。结果:用药后普通喂养组除PLT计数外上述各项指标与基础状态相比均增高,但高脂喂养组除P-LCR外,其他指标无显著变化;用药前高脂喂养组的P-选择素、TXB_2高于普通喂养组,但用药前后这两项指标变化不如普通喂养组显著,无统计学意义,普通喂养组P-选择素、TXB_2前后依次分别为(16.39±2.23)ng/ml、(40.62±19.37)ng/ml,(243.09±32.7)ng/ml、(473.87±80.17)ng/ml,差异有统计学意义(P＜0.05);全血流式分析发现普通喂养组兔子血小板活化状态在G-CSF应用前后变化较大,CD62P的阳性率分别为(0.2±0.01)%、(4.57±0.24)%,CD63的阳性率分别为(1.09±0.06)%、(7.14±0.62)%,差异有统计学意义(P＜0.05);血小板聚集功能检测发现高脂喂养组只有最大聚集率(A_(max))在用药前后有统计学差异,(33.51±0.55)%vs.(38.23±0.67)%,P＜0.05;而最大聚集时间(T_(max))和第一分钟聚集率(A_1)并无统计学差异(P＞0.05);普通喂养组则三项指标在用药前后都出现统计学差异,(12.64±0.63)%vs.(36.9±0.73)%,(3.9±0.27)秒vs.(2.19±0.19)秒,(24.58±0.47)%vs.(45.58±0.96)%,P＜0.05;高脂喂养组最大聚集率(A_(max))和第一分钟聚集率(A_1)均显著大于普通喂养组,(33.51±0.55)%vs.(12.64±0.63)%,(42.05±0.81)%vs.(24.58±0.47)%,P＜0.05。结论:G-CSF在动员作用高峰能引发血小板功能的活化,其机制可能与应激反应有关;高血脂本身就是刺激因素,有激活血小板的作用,在此应激基础上G-CSF的活化作用有所钝化;P-LCR是能够反应血小板活化的早期指标,全血细胞流式分析是检测血小板活化的较灵敏方法,聚集率测定能够直接判断血小板的活化程度;结合以上研究,我们推断活化的血小板有可能参与了动脉的慢性阻塞病变。
潜在价值和创新点
1.创建了新型慢性动脉阻塞性病变模型,具有操作简便,成功率高,便于随访,能够自身对照等优点,为该系列动脉疾病研究提供了良好的实验基础;
2.实现了动脉缩窄环的国产化,效果确切可靠,填补了国内空白,已在申请国家专利;
3.将形态学观察与功能检测紧密联系,比较全面地描述了G-CSF对动脉尤其慢性阻塞局部内皮的变化;
4.结合荧光实时定量PCR及透射电镜等先进方法,从超微结构、分子水平对G-CSF对血管的作用做了客观描述;
5.从局部出发,择大中小不同类型动脉代表,对整个动脉系统在G-CSF作用下的反应做了较深刻的论述,尤其对临床常见的动脉粥样硬化具有很强的实践指导意义;
6.国内比较领先研究了G-CSF作为干细胞动员剂治疗心血管疾病时对血小板功能的影响,对慢性动脉阻塞时是否有血栓因素参与做了初步探讨。
In recent years,a large number of animal experiments and clinical studies confirmed that stem cell transplantation can improve cardiac function after myocardial infarction,and prevent ventricular remodeling,thereby improve the prognosis.Stem cells are a kind of undifferentiated cells at early stage which have multi-directional differentiation potential and self-renewal,and can differentiate into different functional cells under certain conditions to form various of tissues and organs,termed as "plasticity".Granulocyte colony-stimulating factor(G-CSF) is the cytokine that is widely used in patients with radiotherapy and chemotherapy to mobilize hematopoietic stem progenitor cells for restoration support.Recent studies have shown that G-CSF can mobilize bone marrow stem cells into circulation, migrate to the site of myocardial infarction to prompt myocardial regeneration and angiogenesis,and improve heart function after myocardial infarction.As a non-invasive treatment,there is huge potential for the application of G-CSF in the treatment of myocardial infarction.Studies in the chronic ischemic heart disease also show the same view.However,most of the studies of G-CSF focused on its effect on heart,its impact on vessels especially chronic obstructive artery diseases related to ischemic heart diseases such as thrombosis and atherosclerosis,is rarely reported. Furthermore,G-CSF motivation can influence blood system by inducing leukocytosis, resulting in hypercoagulation.It is still not clear whether G-CSF can induce the deterioration of arterial occlusive disease.In short,the impact of G-CSF on local arterial vascular system and chronic obstructive can not be determined in domestic and foreign academics.
Many recent researches have shown that,as a sort of the stem cells,endothelial progenitor cells(EPCs) exist in bone marrow,peripheral blood and cord blood.The EPCs can be recruited to the local vascular injury,differentiated into mature endothelial cells,and involved in angiogenesis and repair by stimulated with injury signals.There are various methods to stimulate EPCs in peripheral blood including exercise,drugs,etc.The drugs which can increase EPCs in adult peripheral blood include stem cell factor,granulocyte colony-stimulating factor,statins,estrogen and so on confirmed by the current researches.As an exogenous stem cell mobilization agent,G-CSF can stimulate EPCs from bone marrow into the peripheral blood and accelerate re-endothelialization of the injury vascular.With the combination of well-known adverse reactions,we infer that G-CSF can be bound to have repair effects and even side effects on the local blood vessels,including chronic obstructive pulmonary arterial lesions and arterial system itself.
As a vascular injury,chronic arterial occlusive diseases are a series of common cardiovascular diseases threatening human health.The diseases are not only the altered shape and structure of blood vessels,but more importantly,the changes of functions.There is minimal study about the role of G-CSF on it especially.So,this study expounded two aspects of the problem systematically from the morphology and function.The research was divided into four parts:firstly,a new type of chronic arterial occlusive disease model was set up.Secondly,combined with the relatively mature rabbit atherosclerosis model,we analized the effects of G-CSF on the local endarterium with chronic obliteration in artery.Thirdly,we observed the effects of G-CSF on different types of arterial lesions as well as chronic obstructive arterial system was done.Finally,we studied G-CSF on platelet activated function to verify whether it is involved in chronic arterial obstruction which can make a preliminary summary of the whole study and explore a new direction for the next step.
PartⅠEstablishment and imaging evaluation of the chronic carotid artery obstructive model of rabbit
Objective:To set up a new type of rabbit chronic carotid obstructive arteries model for further studies of the effect of G-CSF on chronic obstructive arterial lesions.
Methods:Eighteen New Zealand white healthy rabbits were randomly divided into the sham-operated group,4-week group and 8-week group,each including 6 rabbits. The artery narrowing rings were implanted in the right carotid to prepare the chronic artery occlusion model.Carotid artery ultrasound and magnetic resonance vascular imaging study were used for the model evaluating.The animals were sacrificed after 4 weeks(4-weeks group) or 8 weeks(8-weeks group).The proximal and distal vessel tissues of the chronic obstructive narrowing rings were separated and performed the histopathological examination,then the structure and function changes of partial endothelial cells in obstructive region were assessed.Results:Seventeen rabbits survived and 11 were successfully set up chronic carotid obstructive artery models in a total of 18 rabbits.Carotid artery obstruction was observed gradually after implanting artery constrictor lasting for 4 weeks.Carotid artery ultrasound follow-up showed that vascular diameter from(2.00±0.1)cm preoperatively decreased to(1.33±0.23)cm,and the rate of stenosis changed to(48±7)%.Vascular diameter was significantly decreased to(0.48±0.06)cm while the rate of stenosis reached(95±3)% at 8weeks,compared with the control group and 4-weeks group(P＜0.05),while blood flow velocity and resistance index also changed significantly.Rabbit carotid artery model of magnetic resonance showed that the former is clearly visible,bilateral symmetrical,and vascular diameter was(2.02±0.15)cm,but the stenosis significantly reached(1.56±0.18)cm on the constrictor site of the right common carotid artery at 4 weeks,meanwhile the artery was almost completely blocked and had only traces of residual thin line at 8 weeks,the difference has statistical significance compared with the sham-operated group(P＜0.05).Pathological examination of related vessel model revealed that the intima continued thickening,extracellular matrix increased,resulting in severe luminal stenosis or occlusion.Scan electron microscope confirmed endothelial cell apoptosis after chronic obstruction,and matrix surface granular material increased.The ET-1 gene expression was up-regulated and the eNOS gene expression was down-regulated in distal vascular of chronic obstructive artery by Real-time PCR,meanwhile the compensatory changes in contralateral arteries were observed.Conclusions:Chronic arterial occlusive disease animal model can be successful set up 4 weeks after implanting artery constrictor.It is more significant after 8 weeks,and the endothelial morphology and function changes of corresponding paragraph of vessel were observed.
PartⅡThe effects of granulocyte colony-stimulating factor(G-CSF) on the local vascular paragraph of chronic obstructive arterial lesions
Objective:To observe the partial functions and matrix changes of blood vessel during application of G-CSF on chronic artery occlusion animal models,and to explore the impacts of G-CSF on the local vessel of the chronic obstructive arterial disease during treatment of cardiovascular diseases,then evaluate its safety,so as to provide a new theoretical basis for clinical practice.Methods:Thirty-six New Zealand white rabbits were first randomly divided into high-fat feeding group and ordinary feeding group,and then each subdivided into 3 subgroups named separately sham-operated group,chronic arterial occlusive model group and chronic arterial occlusive model add G-CSF-intervention group,each group had 6 rabbits.The atherosclerosis model was set up by using high-fat feeding;the method of G-CSF intervention was 15ug/kg/d,subcutaneous injection for 5 days,while using Giemsa staining and white blood cell count to confirm the mobilization results.The blood routin and lipid biochemical detection were done before and after model was made, then molecular(PCR and WB)experiments and pathological analysis were done based on the local carotid artery of the chronic obstructive and the corresponding parts after 2 months.Bilateral carotid artery ultrasound follow-up check was given at 3,5,7weeks after model was set up;collagen staining,MMP-9 and TIMP-2 immunohistochemical check and WB were detected to study the structure changes in vascular matrix.The form and function changes of the corresponding vascular endothelial cells were assessed by using scanning electron microscope,Real-time PCR detection of eNOS and ET-1mRNA expression.Results:Twenty-four animal models for chronic obstructive common carotid arteries were successfully established. Blood lipid items like TC and LDL were significantly elevated in the three high fat feeding groups 2 months after feeding.AI(Arteriosclerosis Index) was elevated from (1.21±0.16) to(2.70±0.74)(P＜0.05).Granulocytes were significantly increased by Giemsa staining after G-CSF motivation,correlated with the increase of WBC counting.Shrinked internal diameter,slowed bloodstream,and increased resistance index of the proximal end of common carotid artery was observed using carotid artery ultrasound 3 weeks after operation(P＜0.05).Stenosis appeared more dramatic in high fat feeding rabbits after the application of G-CSF[from(53.24±0.7)%to (59.51±0.5)%(P＜0.05)].Vessels were consecutively clogged until completely occlusion 5 weeks later,with a more dramatic change in arteriosclerosis vessels confirmed by the ultrasound.It took about 7 weeks for complete occlusion of the vessel in normal feeding rabbits,and it was 2 weeks shorter in the G-CSF group. Tunica intima proliferation and endothelium reductus growth were observed by HE staining in the G-CSF group.Arteriosclerosis in the common carotid artery of rabbits presented as decreased muscle fiber,increased collagen and amyloidosis tissues by collagen staining in the high fat feeding group,which was more obvious in the proximal end of vessel during chronic obstruction,and the change was more dramatic in the G-CSF group.Like those in arteriosclerosis,expression of MMP-9 and TIMP-2 were up regulated in the G-CSF group confirmed by Western blot,and exacerbated clogging.Increased eNOS expression and decreased ET-1 expression in the distal end of the vessel and a compensation of the opposite side were observed using Real-time PCR in the G-CSF group(P＜0.05).Endothelium hyperplasy in the proximal end of chronic obstructive vessel was observed in the G-CSF group using scanning electron microscope,the alternation was limited in turbulent flow.Conclusions:G-CSF has the ability to improve endothelial function through inducing endothelialization. However,like arteriosclerosis induced by high fat feeding,chronic obstructive artery lesions are likely to be worsened by G-CSF due to deterioration of arteriosclerosis and gelatinous fiber hyperplasia induced by increased matrix metalloproteinase in vessels.
PartⅢThe impact of G-CSF on Chronic Obstructive Artery System
Objective:To systematically investigate the impact of G-CSF on various types of artery endothelial cells in arteriosclerosis and analyze its systematic impact on chronic obstructive artery systems like arteriosclerosis from functional and morphological aspects,with the hope of providing a guidance for a better application and prevention of adverse effects in clinical practice.Methods:Thirty healthy experimental New Zealand white rabbits were randomly divided into the control group(n=6),the normal feeding group(n=6),normal feeding +G-CSF group(n=8),the high fat feeding group (n=8),the high fat feeding +G-CSF group(n=8).G-CSF was injected subcutaneously at a dose of 15ug/kg diluted to 0.5mL with normal sodium for a consecutive 5 days,4 times in all once every other week.Blood sampling was collected once a week.NO concentration was assessed by nitrate reductase method.Blood lipid concentration was assessed by automatic blood biochemistry analyzer.Adipose fat plague in thoracic aorta was analyzed by SudanⅢadipose staining 10 weeks later.Intima thickness of common carotid artery,femoral artery,and thoracic aorta were analyzed using HE staining.Thoracic aorta Matrix was analyzed by collagen staining and immunohistochemical test.The mRNA expression of MMP-9,TIMP-2,eNOS and ET-1 in common carotid artery,femoral artery,and thoracic aorta were analyzed using RT-PCR.Protein expressions of MMP-9 and TIMP-2 in various arteries were analyzed using Western-Blot.Surface appearance of various artery endothelial cells was observed using scanning electron microscope.Altered ultrastructure in endothelial cells in thoracic aorta like mitochondria was analyzed using transmission electron microscope.Results:Eight rabbits died during experiment.There finally survived 5,6,5,6 animals in the four groups,respectively.Blood lipid dynamic test found an elevated blood lioid like TC and LDL in the high fat feeding group,reaching peak 6 weeks later(P＜0.05),stable 8 weeks later.The application of G-CSF shifted forward the peak of blood lipid such as TC and LDL by 2 weeks(compared with the control group,P＜0.05),presenting double hump,with no influence on TG.NO monitoring showed sharp elevation of serum NO by G-CSF(compared with the control group 2 weeks later,P＜0.05).The high fat feeding group showed a slow elevation in 4 weeks(P＜0.05),and the G-CSF intervention group showed the most significant change(P＜0.01).Adipose fat plague in thoracic aorta was observed by SudanⅢadipose staining in the high fat feeding group.G-CSF expanded the plague from(57.40±1.68)%to(91.18±2.27)%(P＜0.05).An elevation of intima thickness was found in the normal feeding group from(8.77±0.89)%to(11.10±1.50)%using HE staining,while a change from(14.97±1.87)%to(25.83±1.88)%in the high fat feeding group was found(P＜0.05).Collagen staining in thoracic aorta suggested that G-CSF induced production of matrix.Increased positive rate of MMP-9 and TIMP-2 were confirmed by immunohistochemical test[(1.08±0.06)%,(26.31±1.08)%, (27.90±1.07)%,(45.51±1.60)%,(4.07±1.01)%,(36.08±1.68)%,(71.36±3.01)%, (67.26±2.54)%,respectively in the four groups],RT-PCR confirmed a similar change of MMP-9 and TIMP-2 mRNA expression.Altered eNOS and ET-1 mRNA expression suggested the severity of ET-1,an index reflexing the impaired endothelial function,was that thoracic aorta＞common carotid artery＞femoral artery,and the expression of eNOS gene,reflexing a protective effect,changed the opposite in the model of arteriosclerosis induced by high fat feeding.G-CSF induced an up-regulation of the expression of eNOS gene in normal feeding group and a down-regulation in the high fat feeding group,meanwhile eNOS expression changed the opposite.This was more dramatic in thoracic aorta and less significant in middle and micro vessels like common carotid artery and femoral artery.The electron microscope found increased apoptosis of EC by high fat diet and increased EC by G-CSF.The latter showed the most significant in normal arteries and less in the surface of arteries in arteriosclerosis.Ultrastructure of endothelial cells like mitochondria was dramatically changed by high fat feeding and G-CSF using transmission electron microscope,which changed more significantly when they were both there.Conclusions:G-CSF deteriorated arteriosclerosis presenting as expensed plaque,thickened tunica intima mainly through impacting TC and LDL,not TG, possibly related to its impact on matrix.Model of arteriosclerosis was successfully established in the high fat feeding group,displaying impaired endothelial function especially in large arteries,and a corresponding endothelial function.G-CSF impacted the artery system.The impact was more significant in larger arteries.There were relationships in appearance and function between normal and arteriosclerosis endothelial cells,as well as in their reactions to G-CSF.
PartⅣInvolvement of G-CSF in Chronic Artery Obstruction via the Possible Mechanism of Platelet Function Activation
Objective:To investigate the involvement of G-CSF in chronic artery obstruction via the possible mechanism of activation of platelet function during its application in cardiovascular diseases as a stem cell motivator.Methods:Sixteen male New Zealand white rabbits were randomly divided into the high fat feeding group and the control group.G-CSF was injected subcutaneously at a dose of 15ug/kg diluted to 0.5mL with normal sodium for a consecutive 5 days.PLT,MPV,PDW and P-LCR were assessed using blood cell counting instrument before and after drug.Platelet related index such as TXB2 and P-selectin were evaluated using ELISA.Platelet activation markers were validated using whole blood CD41-FITC/CD62P,CD63-PE fluorescent antibody double-labeled flow cytometry analysis.The alternation in the platelet aggregation function was assessed by platelet aggregation instrument.Results:The normal feeing group showed elevated index other than PLT after drug usage compared with basal conditions.The high fat feeding group showed change only in P-LCR.The high fat feeding group showed higher P-selectin and TXB_2 than the normal feeing group.The normal feeing group also showed a more significant change in P-selectin and TXB2 before and after drug usage[(16.39±2.23)ng/ml vs.(40.62±19.37)ng/ml(P＜0.05), (243.09±32.7)ng/ml vs.(473.87±80.17)ng/ml(P＜0.05)].Whole blood cell flow cytometer analysis displayed a large variation in platelet activation before and after G-CSF usage.The positive rate of CD62P was(0.2±0.01)%and(4.57±0.24)%, respectively(P＜0.05),and that of CD63 was(1.09±0.06)%and(7.14±0.62)%, respectively(P＜0.05).Platelet aggregation function showed significant difference in A_(max) before and after drug usage in the high fat feeding group[(33.51±0.55)%vs. (38.23±0.67)%(P＜0.05)],with no change in T_(max) and A_1(P＞0.05).All the three index showed significant difference in the normal feeding group before and after drug usage[(12.64±0.63)%vs.(36.9±0.73)%,(3.9±0.27)s vs.(2.19±0.19)s,(24.58±0.47)% vs.(45.58±0.96)%,P＜0.05].A_(max) and A_1 were significantly elevated in the high fat feeding group than the normal feeding group[(33.51±0.55)%vs.(12.64±0.63)%, (42.05±0.81)%vs.(24.58±0.47)%,P＜0.05].Conclusions:G-CSF caused the activation of platelet function on its highest peak,possibly due to stress reaction. Hyperglycemia has activated platelet before,so the motivation of G-CSF was blunted. P-LCR was an early predictor for platelet activation.Whole blood cell flow cytometer analysis was a quite sensitive tool for detecting platelet activation.Platelet aggregation rate assessment helped the evaluation of its activation.Based on the above studies,we deduce that activated platelet is possibly involved in the chronic obstructive artery diseases.
Potential Applications and Innovations
1.We have established a new animal model for chronic obstructive artery diseases with the advantages of convenience,high achievement ratio,easy to follow-up,and self-controlling,providing a sound experimental foundation for further researches.
2.We realized the localization of artery constrictor made in China.The product is a domestic technology breakthrough and is under the application for national patent.
3.Combined morphology observation with function detection,we made an overall description of the impact of G-CSF on arteries,especially on the local endothelium of chronic clogged vessels.
4.Advanced methods such as Real-time PCR and scanning electron microscope were applied to make an objective description of the impact of G-CSF on vessels at the ultra structure and molecular level.
5.The impacts of G-CSF on the artery system were further discussed,and the representative arteries of large,middle,and small size were picked up for discussion. This is especially meaningful for atherosclerosis,which is common in clinical practice.
6.This is an early domestical study to report the impact of G-CSF on the function of platelet during the treatment of cardiovascular diseases as stem cell motivator,make an initial investigation on the possible involvement of thrombus into chronic artery obstruction.
新近许多研究证实,骨髓、外周血、脐血中存在着内皮祖细胞(endothelialprogenitor cells,EPCs),在损伤信号的刺激下能被招募到血管损伤局部,分化为成熟内皮细胞,参与血管新生和修复。G-CSF作为外源性干细胞动员剂,能刺激骨髓中EPCs进入外周血,加速损伤血管的再内皮化。结合上述不良反应,由此我们推断,其必然对局部血管,包括慢性动脉阻塞性病变及动脉系统本身也存在修复作用甚至副作用。
慢性动脉阻塞性病变作为一种血管损伤,是一系列常见的影响人类健康的心血管疾病,改变的不仅仅是血管的形态和结构,更重要的是其功能的变化。基于此,本研究从形态和功能两方面系统地阐述这个问题。
实验分以下四部分:
第一部分兔颈动脉慢性阻塞模型的构建及影像学评价的研究
目的:制备新型兔颈总动脉慢性阻塞动物模型,为下一步研究粒细胞集落刺激因子对动脉慢性阻塞性病变的作用提供实验基础。方法:健康实验用新西兰大白兔18只,随机分为假手术组、4周组和8周组,每组6只;利用右颈总动脉植入动脉缩窄环制备慢性动脉阻塞模型;应用颈动脉超声以及血管磁共振的影像学手段随访评价模型的构建效果;术后4周(4周组)或8周(8周组)处死动物,分离慢性阻塞缩窄环近端及远端行病理学检查并用扫描电镜、Real-time PCR检测eNOS和ET-1mRNA评估阻塞区域局部内皮结构功能的变化。结果:18只实验兔中17头成活,并成功建立颈总动脉慢性阻塞模型兔11只;植入动脉缩窄环后,4周即见颈总动脉逐步阻塞,颈总动脉超声随访显示血管内径从术前(2.00±0.1)cm下降至(1.33±0.23)cm,狭窄程度达到(48±7)%;到第8周时,血管内径更是显著下降至(0.48±0.06)cm,狭窄达到(95±3)%,与对照组及4周组相比,变化绝对值具有统计学意义(P＜0.05),同时血流速度及阻力指数亦有显著变化;兔颈总动脉磁共振显示造模前清晰可见,双侧基本对称,血管内径为(2.02±0.15)cm,但造模4周后右侧颈总动脉缩窄环部位明显狭窄(1.56±0.18)cm,8周后几乎完全阻塞,仅残留细线痕迹,与假手术组相比,差别有统计学意义(P＜0.05);相应血管段病理检查发现造模后内膜继续增厚,细胞外基质增加,以致管腔重度狭窄甚至闭塞;电镜扫描证实慢性阻塞后内皮细胞凋亡,基质表面颗粒状物增大;Real-time PCR可见慢性阻塞动脉远端血管ET-1基因的上调和eNOS基因的下调及对侧动脉的代偿性变化。结论:应用动脉收缩环4周后即可以成功制作慢性动脉阻塞性病变动物模型,8周更为显著,并出现相应血管段内皮形态及功能变化。
第二部分粒细胞集落刺激因子(G-CSF)对动脉慢性阻塞病变局部血管段的影响
目的:利用慢性动脉阻塞性病动物模型,随访观察G-CSF应用过程中血管局部功能和基质结构的变化,探索G-CSF治疗心血管疾病时对局部慢性阻塞病变动脉的影响,再评价其安全性,从而为临床实践提供新的理论依据。方法:健康实验用新西兰大白兔36只,先随机分为高脂喂养及普通喂养2组,再各自细分为3亚组分别为假手术组、慢性动脉阻塞性模型组和慢性动脉阻塞性模型加G-CSF干预组,每组6只;采用高脂喂养制备动脉粥样硬化模型;G-CSF干预为15μg/kg/d,皮下注射,连续5天,采用Giemsa染色及白细胞计数证实动员效果;造模前后做血常规及血脂生化检测,后2月取材截取慢性阻塞及对应部位局部颈总动脉做分子实验(PCR和WB)及病理学分析;造模后第3、5、7周进行双侧颈总动脉超声随访;胶原染色、MMP-9/TIMP-2免疫组化及WB研究血管基质结构变化;扫描电镜、Real-time PCRE测eNOS和ET-1mRNA评估相应血管内皮形态及功能的变化。结果:成功制备慢性阻塞性颈总动脉病变动物模型24只;高脂喂养2月后,高脂喂养3组总胆固醇、低密度脂蛋白(TC、LDL)等血脂水平明显升高,动脉粥样硬化指数(AI)由(1.21±0.16)升至(2.70±0.74),P＜0.05,有统计学差异;G-CSF动员后Giemsa染色可见粒细胞数目明显增加,同白细胞计数上升一致;造模3周后,颈动脉超声见近端颈总动脉血管内径逐步变小,血流速度变慢,阻力指数增高,应用G-CSF后变化更显著,高脂喂养兔狭窄度从(53.2±0.7)%升至(59.51±0.5)%,P＜0.05,有统计学差异;5周后,血管陆续阻塞直至完全闭合,超声可见动脉粥样硬化血管更易闭合,G-CSF可使完全阻塞时间提前2周达到统计学差异,而普通喂养兔需7周左右;病理分析HE染色可见G-CSF使内膜明显增生,内皮皱褶增多;胶原染色发现高脂喂养能使兔动脉硬化表现为肌纤维略减少而胶原、淀粉样变等物质增多,慢性阻塞后近端血管更显著,而G-CSF干预加剧类似变化;WesternBlot证实G-CSF与动脉粥样硬化一样均可使血管MMP-9和TIMP-2表达上调,加重阻塞病变;Real-time PCR发现G-CSF上调慢性阻塞动脉远端血管eNOS基因下调ET-1基因伴随对侧动脉代偿性变化,表达量经分析有统计学差异(P＜0.05);电镜扫描发现慢性阻塞动脉近端应用G-CSF促使内皮细胞增生,但在遄流下作用有限。结论:G-CSF动员具有促内皮化改善内皮功能的作用,但如高脂喂养导致动脉硬化一样,均可加重局部慢性动脉阻塞性病变,这可能与其恶化动脉粥样硬化以及诱导血管基质蛋白酶的表达,促进胶质纤维增生有关。
第三部分粒细胞集落刺激因子(G-CSF)对慢性阻塞病变动脉系统的整体作用
目的:系统地研究G-CSF在动脉粥样硬化中的作用及其对不同类型的动脉内皮的影响,结合功能与形态学观察,分析G-CSF对动脉粥样硬化等全身性动脉慢性阻塞病变的整体作用,指导临床最大效能地发挥其良好效应,防止不良作用。方法:30只健康实验用新西兰大白兔,随机分为对照组(n=6)、普通喂养+G-CSF组(n=8)、高脂喂养组(n=8)、高脂喂养+G-CSF组(n=8);+G-CSF为15ug/kgG-CSF用生理盐水稀释成0.5mL,每日皮下注射,连续5日,隔周进行,共4次;每周采血,硝酸还原酶法测定血清NO浓度,全自动血生化分析仪测量4组血脂:10周后取材,苏丹Ⅲ脂肪染色分析胸主动脉脂肪斑块;HE染色分析颈总动脉、股动脉及胸主动脉内膜厚度;胶原染色和免疫组化分析各组胸主动脉血管基质情况;逆转录多聚酶链反应(RT-PCR)测定颈总动脉、股动脉及胸主动脉MMP-9、TIMP-2、eNOS及ET-1 mRNA表达;Western-Blot检测不同动脉段血管内MMP-9、TIMP-2蛋白表达;扫描电镜观察各动脉段内皮表面形态,透射电镜观察各组胸主动脉内皮细胞超微结构线粒体的变化。结果:实验期间8只兔子死亡,最终4组分别存活有5、6、5、6只动物,达到实验完成要求;血脂动态检测发现高脂喂养能使TC、LDL等血脂水平上升,6周后高峰,与对照组相比,P＜0.05,但8周后就趋于稳定;+G-CSF使血脂高峰前移,使TC、LDL等提前2周就与对照组出现统计差异(P＜0.05),并有双峰样改变,但对TG同样无影响;NO监测提示G-CSF使血浆NO水平迅速升高,与对照组相比,2周即有统计学差异(P＜0.05);而单纯高脂喂养组则较迟缓,4周以后才逐步上升,差异达到统计学意义(P＜0.05);同时G-CSF干预组最高最快,差异有显著统计学意义(P＜0.01);苏丹Ⅲ染色发现高脂喂养组胸主动脉出现粥样斑块,+G-CSF则使斑块面积比从(57.40±1.68)%上升至(91.18±2.27)%,P＜0.05;HE染色发现+G-CSF使普通喂养组内膜厚度比值从(8.77±0.89)%上升至(11.10±1.50)%,高脂喂养分别为(14.97±1.87)%,(25.83±1.88)%,后者有统计学差异(P＜0.05);胸主动脉胶原染色提示G-CSF能使基质增生,免疫组化证实其上调MMP-9及TIMP-2的阳性率,4组依次分别为(1.08±0.06)%、(26.31±1.08)%、(27.90±1.07)%、(45.51±1.60)%,(4.07±1.01)%、(36.08±1.68)%、(71.36±3.01)%、(67.26±2.54)%;MMP-9及TIMP-2 mRNA RT-PCR的结果与以上相符,eNOS、ET-1mRNA的表达提示高脂喂养兔形成动脉粥样硬化后,反映内皮功能损害的ET-1基因表达依序为胸主动脉＞颈总动脉＞股动脉,而反映内皮功能良好的eNOS基因则反之,+G-CSF在普通喂养组出现eNOS基因的上调但高脂喂养组则下调,ET-1基因的变化趋势正好相反,此变化以胸主动脉为著,中小型动脉如颈总动脉、股动脉次之;电镜扫描发现高脂使EC凋亡增加,G-CSF能使EC数目增加,对正常动脉作用更大,但对粥样硬化动脉表面影响不明显;透射电镜则提示高脂喂养和G-CSF使细胞线粒体等超微结构病理改变严重,以两者累加为最。结论:G-CSF对血脂存在影响,以TC、LDL为主,对TG作用不大。因此它可使动脉粥样硬化恶化,具体表现为斑块面积的扩大,内膜厚度增加,可能与其对基质的影响有关;高脂喂养兔能成功制造动脉粥样硬化模型,成模后动脉内皮功能显著受损,其中以大型动脉显著,同时内皮形态出现相应变化;G-CSF对全身动脉系统都有影响,但动脉越小,影响越小;正常与粥样硬化动脉内皮形态及功能改变之间存在必然联系,其对G-CSF的反应也不同。
第四部分粒细胞集落刺激因子(G-CSF)活化血小板功能参与慢性动脉阻塞
目的:探讨G-CSF在应用于干细胞动员治疗心血管疾病时是否活化血小板功能参与慢性动脉阻塞性病变。方法:16只雄性新西兰大白兔随机分为普通和高脂喂养2组,15ug/kg G-CSF用生理盐水稀释成0.5mL,每日皮下注射,连续5日,用药前后分别用血细胞计数仪测定血小板计数(PLT)、血小板比积(MPV)、血小板分布宽度(PDW)、大血小板比例(P-LCR);双抗体夹心免疫酶联试验(ELISA)测定TXB_2、P选择素等血小板相关指标;采用全血CD41-FITC/CD62P、CD63-PE荧光抗体双标流式分析法对血小板活化标记进行验证;血小板聚集仪直接测定血小板聚集能力的前后变化。结果:用药后普通喂养组除PLT计数外上述各项指标与基础状态相比均增高,但高脂喂养组除P-LCR外,其他指标无显著变化;用药前高脂喂养组的P-选择素、TXB_2高于普通喂养组,但用药前后这两项指标变化不如普通喂养组显著,无统计学意义,普通喂养组P-选择素、TXB_2前后依次分别为(16.39±2.23)ng/ml、(40.62±19.37)ng/ml,(243.09±32.7)ng/ml、(473.87±80.17)ng/ml,差异有统计学意义(P＜0.05);全血流式分析发现普通喂养组兔子血小板活化状态在G-CSF应用前后变化较大,CD62P的阳性率分别为(0.2±0.01)%、(4.57±0.24)%,CD63的阳性率分别为(1.09±0.06)%、(7.14±0.62)%,差异有统计学意义(P＜0.05);血小板聚集功能检测发现高脂喂养组只有最大聚集率(A_(max))在用药前后有统计学差异,(33.51±0.55)%vs.(38.23±0.67)%,P＜0.05;而最大聚集时间(T_(max))和第一分钟聚集率(A_1)并无统计学差异(P＞0.05);普通喂养组则三项指标在用药前后都出现统计学差异,(12.64±0.63)%vs.(36.9±0.73)%,(3.9±0.27)秒vs.(2.19±0.19)秒,(24.58±0.47)%vs.(45.58±0.96)%,P＜0.05;高脂喂养组最大聚集率(A_(max))和第一分钟聚集率(A_1)均显著大于普通喂养组,(33.51±0.55)%vs.(12.64±0.63)%,(42.05±0.81)%vs.(24.58±0.47)%,P＜0.05。结论:G-CSF在动员作用高峰能引发血小板功能的活化,其机制可能与应激反应有关;高血脂本身就是刺激因素,有激活血小板的作用,在此应激基础上G-CSF的活化作用有所钝化;P-LCR是能够反应血小板活化的早期指标,全血细胞流式分析是检测血小板活化的较灵敏方法,聚集率测定能够直接判断血小板的活化程度;结合以上研究,我们推断活化的血小板有可能参与了动脉的慢性阻塞病变。
潜在价值和创新点
1.创建了新型慢性动脉阻塞性病变模型,具有操作简便,成功率高,便于随访,能够自身对照等优点,为该系列动脉疾病研究提供了良好的实验基础;
2.实现了动脉缩窄环的国产化,效果确切可靠,填补了国内空白,已在申请国家专利;
3.将形态学观察与功能检测紧密联系,比较全面地描述了G-CSF对动脉尤其慢性阻塞局部内皮的变化;
4.结合荧光实时定量PCR及透射电镜等先进方法,从超微结构、分子水平对G-CSF对血管的作用做了客观描述;
5.从局部出发,择大中小不同类型动脉代表,对整个动脉系统在G-CSF作用下的反应做了较深刻的论述,尤其对临床常见的动脉粥样硬化具有很强的实践指导意义;
6.国内比较领先研究了G-CSF作为干细胞动员剂治疗心血管疾病时对血小板功能的影响,对慢性动脉阻塞时是否有血栓因素参与做了初步探讨。
In recent years,a large number of animal experiments and clinical studies confirmed that stem cell transplantation can improve cardiac function after myocardial infarction,and prevent ventricular remodeling,thereby improve the prognosis.Stem cells are a kind of undifferentiated cells at early stage which have multi-directional differentiation potential and self-renewal,and can differentiate into different functional cells under certain conditions to form various of tissues and organs,termed as "plasticity".Granulocyte colony-stimulating factor(G-CSF) is the cytokine that is widely used in patients with radiotherapy and chemotherapy to mobilize hematopoietic stem progenitor cells for restoration support.Recent studies have shown that G-CSF can mobilize bone marrow stem cells into circulation, migrate to the site of myocardial infarction to prompt myocardial regeneration and angiogenesis,and improve heart function after myocardial infarction.As a non-invasive treatment,there is huge potential for the application of G-CSF in the treatment of myocardial infarction.Studies in the chronic ischemic heart disease also show the same view.However,most of the studies of G-CSF focused on its effect on heart,its impact on vessels especially chronic obstructive artery diseases related to ischemic heart diseases such as thrombosis and atherosclerosis,is rarely reported. Furthermore,G-CSF motivation can influence blood system by inducing leukocytosis, resulting in hypercoagulation.It is still not clear whether G-CSF can induce the deterioration of arterial occlusive disease.In short,the impact of G-CSF on local arterial vascular system and chronic obstructive can not be determined in domestic and foreign academics.
Many recent researches have shown that,as a sort of the stem cells,endothelial progenitor cells(EPCs) exist in bone marrow,peripheral blood and cord blood.The EPCs can be recruited to the local vascular injury,differentiated into mature endothelial cells,and involved in angiogenesis and repair by stimulated with injury signals.There are various methods to stimulate EPCs in peripheral blood including exercise,drugs,etc.The drugs which can increase EPCs in adult peripheral blood include stem cell factor,granulocyte colony-stimulating factor,statins,estrogen and so on confirmed by the current researches.As an exogenous stem cell mobilization agent,G-CSF can stimulate EPCs from bone marrow into the peripheral blood and accelerate re-endothelialization of the injury vascular.With the combination of well-known adverse reactions,we infer that G-CSF can be bound to have repair effects and even side effects on the local blood vessels,including chronic obstructive pulmonary arterial lesions and arterial system itself.
As a vascular injury,chronic arterial occlusive diseases are a series of common cardiovascular diseases threatening human health.The diseases are not only the altered shape and structure of blood vessels,but more importantly,the changes of functions.There is minimal study about the role of G-CSF on it especially.So,this study expounded two aspects of the problem systematically from the morphology and function.The research was divided into four parts:firstly,a new type of chronic arterial occlusive disease model was set up.Secondly,combined with the relatively mature rabbit atherosclerosis model,we analized the effects of G-CSF on the local endarterium with chronic obliteration in artery.Thirdly,we observed the effects of G-CSF on different types of arterial lesions as well as chronic obstructive arterial system was done.Finally,we studied G-CSF on platelet activated function to verify whether it is involved in chronic arterial obstruction which can make a preliminary summary of the whole study and explore a new direction for the next step.
PartⅠEstablishment and imaging evaluation of the chronic carotid artery obstructive model of rabbit
Objective:To set up a new type of rabbit chronic carotid obstructive arteries model for further studies of the effect of G-CSF on chronic obstructive arterial lesions.
Methods:Eighteen New Zealand white healthy rabbits were randomly divided into the sham-operated group,4-week group and 8-week group,each including 6 rabbits. The artery narrowing rings were implanted in the right carotid to prepare the chronic artery occlusion model.Carotid artery ultrasound and magnetic resonance vascular imaging study were used for the model evaluating.The animals were sacrificed after 4 weeks(4-weeks group) or 8 weeks(8-weeks group).The proximal and distal vessel tissues of the chronic obstructive narrowing rings were separated and performed the histopathological examination,then the structure and function changes of partial endothelial cells in obstructive region were assessed.Results:Seventeen rabbits survived and 11 were successfully set up chronic carotid obstructive artery models in a total of 18 rabbits.Carotid artery obstruction was observed gradually after implanting artery constrictor lasting for 4 weeks.Carotid artery ultrasound follow-up showed that vascular diameter from(2.00±0.1)cm preoperatively decreased to(1.33±0.23)cm,and the rate of stenosis changed to(48±7)%.Vascular diameter was significantly decreased to(0.48±0.06)cm while the rate of stenosis reached(95±3)% at 8weeks,compared with the control group and 4-weeks group(P＜0.05),while blood flow velocity and resistance index also changed significantly.Rabbit carotid artery model of magnetic resonance showed that the former is clearly visible,bilateral symmetrical,and vascular diameter was(2.02±0.15)cm,but the stenosis significantly reached(1.56±0.18)cm on the constrictor site of the right common carotid artery at 4 weeks,meanwhile the artery was almost completely blocked and had only traces of residual thin line at 8 weeks,the difference has statistical significance compared with the sham-operated group(P＜0.05).Pathological examination of related vessel model revealed that the intima continued thickening,extracellular matrix increased,resulting in severe luminal stenosis or occlusion.Scan electron microscope confirmed endothelial cell apoptosis after chronic obstruction,and matrix surface granular material increased.The ET-1 gene expression was up-regulated and the eNOS gene expression was down-regulated in distal vascular of chronic obstructive artery by Real-time PCR,meanwhile the compensatory changes in contralateral arteries were observed.Conclusions:Chronic arterial occlusive disease animal model can be successful set up 4 weeks after implanting artery constrictor.It is more significant after 8 weeks,and the endothelial morphology and function changes of corresponding paragraph of vessel were observed.
PartⅡThe effects of granulocyte colony-stimulating factor(G-CSF) on the local vascular paragraph of chronic obstructive arterial lesions
Objective:To observe the partial functions and matrix changes of blood vessel during application of G-CSF on chronic artery occlusion animal models,and to explore the impacts of G-CSF on the local vessel of the chronic obstructive arterial disease during treatment of cardiovascular diseases,then evaluate its safety,so as to provide a new theoretical basis for clinical practice.Methods:Thirty-six New Zealand white rabbits were first randomly divided into high-fat feeding group and ordinary feeding group,and then each subdivided into 3 subgroups named separately sham-operated group,chronic arterial occlusive model group and chronic arterial occlusive model add G-CSF-intervention group,each group had 6 rabbits.The atherosclerosis model was set up by using high-fat feeding;the method of G-CSF intervention was 15ug/kg/d,subcutaneous injection for 5 days,while using Giemsa staining and white blood cell count to confirm the mobilization results.The blood routin and lipid biochemical detection were done before and after model was made, then molecular(PCR and WB)experiments and pathological analysis were done based on the local carotid artery of the chronic obstructive and the corresponding parts after 2 months.Bilateral carotid artery ultrasound follow-up check was given at 3,5,7weeks after model was set up;collagen staining,MMP-9 and TIMP-2 immunohistochemical check and WB were detected to study the structure changes in vascular matrix.The form and function changes of the corresponding vascular endothelial cells were assessed by using scanning electron microscope,Real-time PCR detection of eNOS and ET-1mRNA expression.Results:Twenty-four animal models for chronic obstructive common carotid arteries were successfully established. Blood lipid items like TC and LDL were significantly elevated in the three high fat feeding groups 2 months after feeding.AI(Arteriosclerosis Index) was elevated from (1.21±0.16) to(2.70±0.74)(P＜0.05).Granulocytes were significantly increased by Giemsa staining after G-CSF motivation,correlated with the increase of WBC counting.Shrinked internal diameter,slowed bloodstream,and increased resistance index of the proximal end of common carotid artery was observed using carotid artery ultrasound 3 weeks after operation(P＜0.05).Stenosis appeared more dramatic in high fat feeding rabbits after the application of G-CSF[from(53.24±0.7)%to (59.51±0.5)%(P＜0.05)].Vessels were consecutively clogged until completely occlusion 5 weeks later,with a more dramatic change in arteriosclerosis vessels confirmed by the ultrasound.It took about 7 weeks for complete occlusion of the vessel in normal feeding rabbits,and it was 2 weeks shorter in the G-CSF group. Tunica intima proliferation and endothelium reductus growth were observed by HE staining in the G-CSF group.Arteriosclerosis in the common carotid artery of rabbits presented as decreased muscle fiber,increased collagen and amyloidosis tissues by collagen staining in the high fat feeding group,which was more obvious in the proximal end of vessel during chronic obstruction,and the change was more dramatic in the G-CSF group.Like those in arteriosclerosis,expression of MMP-9 and TIMP-2 were up regulated in the G-CSF group confirmed by Western blot,and exacerbated clogging.Increased eNOS expression and decreased ET-1 expression in the distal end of the vessel and a compensation of the opposite side were observed using Real-time PCR in the G-CSF group(P＜0.05).Endothelium hyperplasy in the proximal end of chronic obstructive vessel was observed in the G-CSF group using scanning electron microscope,the alternation was limited in turbulent flow.Conclusions:G-CSF has the ability to improve endothelial function through inducing endothelialization. However,like arteriosclerosis induced by high fat feeding,chronic obstructive artery lesions are likely to be worsened by G-CSF due to deterioration of arteriosclerosis and gelatinous fiber hyperplasia induced by increased matrix metalloproteinase in vessels.
PartⅢThe impact of G-CSF on Chronic Obstructive Artery System
Objective:To systematically investigate the impact of G-CSF on various types of artery endothelial cells in arteriosclerosis and analyze its systematic impact on chronic obstructive artery systems like arteriosclerosis from functional and morphological aspects,with the hope of providing a guidance for a better application and prevention of adverse effects in clinical practice.Methods:Thirty healthy experimental New Zealand white rabbits were randomly divided into the control group(n=6),the normal feeding group(n=6),normal feeding +G-CSF group(n=8),the high fat feeding group (n=8),the high fat feeding +G-CSF group(n=8).G-CSF was injected subcutaneously at a dose of 15ug/kg diluted to 0.5mL with normal sodium for a consecutive 5 days,4 times in all once every other week.Blood sampling was collected once a week.NO concentration was assessed by nitrate reductase method.Blood lipid concentration was assessed by automatic blood biochemistry analyzer.Adipose fat plague in thoracic aorta was analyzed by SudanⅢadipose staining 10 weeks later.Intima thickness of common carotid artery,femoral artery,and thoracic aorta were analyzed using HE staining.Thoracic aorta Matrix was analyzed by collagen staining and immunohistochemical test.The mRNA expression of MMP-9,TIMP-2,eNOS and ET-1 in common carotid artery,femoral artery,and thoracic aorta were analyzed using RT-PCR.Protein expressions of MMP-9 and TIMP-2 in various arteries were analyzed using Western-Blot.Surface appearance of various artery endothelial cells was observed using scanning electron microscope.Altered ultrastructure in endothelial cells in thoracic aorta like mitochondria was analyzed using transmission electron microscope.Results:Eight rabbits died during experiment.There finally survived 5,6,5,6 animals in the four groups,respectively.Blood lipid dynamic test found an elevated blood lioid like TC and LDL in the high fat feeding group,reaching peak 6 weeks later(P＜0.05),stable 8 weeks later.The application of G-CSF shifted forward the peak of blood lipid such as TC and LDL by 2 weeks(compared with the control group,P＜0.05),presenting double hump,with no influence on TG.NO monitoring showed sharp elevation of serum NO by G-CSF(compared with the control group 2 weeks later,P＜0.05).The high fat feeding group showed a slow elevation in 4 weeks(P＜0.05),and the G-CSF intervention group showed the most significant change(P＜0.01).Adipose fat plague in thoracic aorta was observed by SudanⅢadipose staining in the high fat feeding group.G-CSF expanded the plague from(57.40±1.68)%to(91.18±2.27)%(P＜0.05).An elevation of intima thickness was found in the normal feeding group from(8.77±0.89)%to(11.10±1.50)%using HE staining,while a change from(14.97±1.87)%to(25.83±1.88)%in the high fat feeding group was found(P＜0.05).Collagen staining in thoracic aorta suggested that G-CSF induced production of matrix.Increased positive rate of MMP-9 and TIMP-2 were confirmed by immunohistochemical test[(1.08±0.06)%,(26.31±1.08)%, (27.90±1.07)%,(45.51±1.60)%,(4.07±1.01)%,(36.08±1.68)%,(71.36±3.01)%, (67.26±2.54)%,respectively in the four groups],RT-PCR confirmed a similar change of MMP-9 and TIMP-2 mRNA expression.Altered eNOS and ET-1 mRNA expression suggested the severity of ET-1,an index reflexing the impaired endothelial function,was that thoracic aorta＞common carotid artery＞femoral artery,and the expression of eNOS gene,reflexing a protective effect,changed the opposite in the model of arteriosclerosis induced by high fat feeding.G-CSF induced an up-regulation of the expression of eNOS gene in normal feeding group and a down-regulation in the high fat feeding group,meanwhile eNOS expression changed the opposite.This was more dramatic in thoracic aorta and less significant in middle and micro vessels like common carotid artery and femoral artery.The electron microscope found increased apoptosis of EC by high fat diet and increased EC by G-CSF.The latter showed the most significant in normal arteries and less in the surface of arteries in arteriosclerosis.Ultrastructure of endothelial cells like mitochondria was dramatically changed by high fat feeding and G-CSF using transmission electron microscope,which changed more significantly when they were both there.Conclusions:G-CSF deteriorated arteriosclerosis presenting as expensed plaque,thickened tunica intima mainly through impacting TC and LDL,not TG, possibly related to its impact on matrix.Model of arteriosclerosis was successfully established in the high fat feeding group,displaying impaired endothelial function especially in large arteries,and a corresponding endothelial function.G-CSF impacted the artery system.The impact was more significant in larger arteries.There were relationships in appearance and function between normal and arteriosclerosis endothelial cells,as well as in their reactions to G-CSF.
PartⅣInvolvement of G-CSF in Chronic Artery Obstruction via the Possible Mechanism of Platelet Function Activation
Objective:To investigate the involvement of G-CSF in chronic artery obstruction via the possible mechanism of activation of platelet function during its application in cardiovascular diseases as a stem cell motivator.Methods:Sixteen male New Zealand white rabbits were randomly divided into the high fat feeding group and the control group.G-CSF was injected subcutaneously at a dose of 15ug/kg diluted to 0.5mL with normal sodium for a consecutive 5 days.PLT,MPV,PDW and P-LCR were assessed using blood cell counting instrument before and after drug.Platelet related index such as TXB2 and P-selectin were evaluated using ELISA.Platelet activation markers were validated using whole blood CD41-FITC/CD62P,CD63-PE fluorescent antibody double-labeled flow cytometry analysis.The alternation in the platelet aggregation function was assessed by platelet aggregation instrument.Results:The normal feeing group showed elevated index other than PLT after drug usage compared with basal conditions.The high fat feeding group showed change only in P-LCR.The high fat feeding group showed higher P-selectin and TXB_2 than the normal feeing group.The normal feeing group also showed a more significant change in P-selectin and TXB2 before and after drug usage[(16.39±2.23)ng/ml vs.(40.62±19.37)ng/ml(P＜0.05), (243.09±32.7)ng/ml vs.(473.87±80.17)ng/ml(P＜0.05)].Whole blood cell flow cytometer analysis displayed a large variation in platelet activation before and after G-CSF usage.The positive rate of CD62P was(0.2±0.01)%and(4.57±0.24)%, respectively(P＜0.05),and that of CD63 was(1.09±0.06)%and(7.14±0.62)%, respectively(P＜0.05).Platelet aggregation function showed significant difference in A_(max) before and after drug usage in the high fat feeding group[(33.51±0.55)%vs. (38.23±0.67)%(P＜0.05)],with no change in T_(max) and A_1(P＞0.05).All the three index showed significant difference in the normal feeding group before and after drug usage[(12.64±0.63)%vs.(36.9±0.73)%,(3.9±0.27)s vs.(2.19±0.19)s,(24.58±0.47)% vs.(45.58±0.96)%,P＜0.05].A_(max) and A_1 were significantly elevated in the high fat feeding group than the normal feeding group[(33.51±0.55)%vs.(12.64±0.63)%, (42.05±0.81)%vs.(24.58±0.47)%,P＜0.05].Conclusions:G-CSF caused the activation of platelet function on its highest peak,possibly due to stress reaction. Hyperglycemia has activated platelet before,so the motivation of G-CSF was blunted. P-LCR was an early predictor for platelet activation.Whole blood cell flow cytometer analysis was a quite sensitive tool for detecting platelet activation.Platelet aggregation rate assessment helped the evaluation of its activation.Based on the above studies,we deduce that activated platelet is possibly involved in the chronic obstructive artery diseases.
Potential Applications and Innovations
1.We have established a new animal model for chronic obstructive artery diseases with the advantages of convenience,high achievement ratio,easy to follow-up,and self-controlling,providing a sound experimental foundation for further researches.
2.We realized the localization of artery constrictor made in China.The product is a domestic technology breakthrough and is under the application for national patent.
3.Combined morphology observation with function detection,we made an overall description of the impact of G-CSF on arteries,especially on the local endothelium of chronic clogged vessels.
4.Advanced methods such as Real-time PCR and scanning electron microscope were applied to make an objective description of the impact of G-CSF on vessels at the ultra structure and molecular level.
5.The impacts of G-CSF on the artery system were further discussed,and the representative arteries of large,middle,and small size were picked up for discussion. This is especially meaningful for atherosclerosis,which is common in clinical practice.
6.This is an early domestical study to report the impact of G-CSF on the function of platelet during the treatment of cardiovascular diseases as stem cell motivator,make an initial investigation on the possible involvement of thrombus into chronic artery obstruction.
引文
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