内皮祖细胞与雌激素联合应用防治血管内皮损伤后再狭窄的实验研究
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摘要
背景
冠心病(coronary heart disease,CHD)是现代社会严重威胁人类健康,引起死亡的主要疾病,其防治已成为基础医学和临床医学研究的热点,具体病因尚未明确。患者多伴有高血脂、高血压、糖尿病、肥胖、吸烟、体力活动减少、家族史等危险因素,男性多在40-60岁之间发病,女性多在绝经期后表现出症状。冠心病的治疗目前主要包括药物治疗、经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)和冠状动脉旁路移植术(coronary artery bypass grafting,CABG)。PCI术自上世纪七十年代应用临床以来,经过几十年的发展已成为治疗冠心病的重要手段之一,从起初的单纯球囊扩张到裸金属支架及目前的药物洗脱支架、切割球囊、支架内放射治疗等。虽然新器械、新技术不断应用于临床,但PCI术后再狭窄仍然是广大临床及科研工作者面临的一道难题。
单纯应用雌激素或内皮祖细胞(endothelial progenitor cells,EPC)防治PCI术后再狭窄已经取得了一定效果,但尚未见将两者联合应用的报道。雌激素不仅具有改善血脂代谢,促进内皮细胞(endothelial cells,EC)增生、迁移的作用,而且在EPC的动员、归巢到损伤血管局部参与损伤内皮的修复过程中发挥重要作用。
研究目的
本实验通过建立卵巢切除术后雌性大白兔腹主动脉球囊损伤模型,观察雌激素与EPC两者联合应用促进内皮修复、抑制新生内膜增生的效果是否优于单独应用。旨在探讨EPC与雌激素联合应用防治血管内皮损伤后再狭窄的效果。
材料和方法
1、雌性新西兰大白兔60只随机分为5组,每组12只。Ⅰ：假手术组；Ⅱ：对照组；Ⅲ：雌激素组；Ⅳ：EPC组；V：雌激素+EPC组。卵巢切除术后,Ⅲ组和V组给予雌激素替代治疗,其余动物生理盐水皮下注射。
2、实验动物卵巢切除前及卵巢切除后3d、2周分别采血,应用放免法检测血清雌激素水平。
3、实验动物高脂饮食6周后应用氧化酶法检测血浆总胆固醇、甘油三酯水平；用直接法检测低密度脂蛋白胆固醇及高密度脂蛋白胆固醇。
4、从兔外周血分离培养单个核细胞,制备EPC悬液以备细胞移植。
5、建立兔腹主动脉损伤模型,Ⅰ组动物仅分离暴露出股动脉而不做球囊损伤,Ⅳ组和V组动物损伤血管局部经导管给予EPC悬液2mL输注,其余动物(除Ⅰ组外)给予生理盐水2mL。
6、4周后,伊文斯蓝染色检测损伤血管段内皮修复程度,免疫组化法检测细胞增殖程度,HE染色观察管腔丢失情况。
结果
1、Ⅲ、Ⅴ组动物雌激素水平明显高于Ⅰ、Ⅱ、Ⅳ组(P<0.01)。
2、与Ⅰ、Ⅱ、Ⅳ组相比,Ⅲ、Ⅴ组动物总胆固醇、低密度脂蛋白胆固醇明显降低(P<0.01),高密度脂蛋白胆固醇明显升高(P<0.05),甘油三脂差异无统计学意义。
3、V组与Ⅲ、Ⅳ组相比内皮修复程度、细胞增殖程度及管腔丢失程度差异有统计学意义,分别为[(90.2±5.1)%vs(85.1±4.1)%、(83.9±6.5)%,P<0.05],[(15.67±2.98)%vs(26.02±4.18)%、(24.87±3.54)%,P<0.01],[(17.61±2.69)%vs(38.43±4.35)%、(41.15±4.45)%,P<0.01]。
结论
EPC与雌激素联合应用能加速内皮修复、抑制新生内膜增生,减少血管腔丢失。
Background
Coronary heart disease(CHD) is a kind of disease that is very harmful to people's health and a main reason for people's dealth in modern society.It has become the hot pot of preclinical and clinical medicine,but etiological factor has not been ascertained: Men usually suffer CHD at the age about forty to sixty years old,however,women appear symptoms almost after menopause.Patient with CHD usually get the risk factors,such as hyperlipemia,hypertension, diabetes mellitus, obesity,semoking and so on.Treating to CHD mainly include drugs, percutaneous coronary intervention(PCI), coronary artery bypass grafting (CABG),and gradually PCI has become one of the most effective tragedy.Though more and more new apparatus and technology have been applied to clinic,in-stent restenosis is a serious problem to the doctor and researcher of medicine.
People have got some effectiveness from the application of estrogen or endothelial progenitor cells(EPC) alone,but there is no report about combination of the both.Estrogen has many actions,including accommodation of blood fat,promoting the growth and migration of endothelial cells(EC) and playing a important role in EPC.
Objective
To investigate the effect of application of endothelial progenitor cells combined with estrogen on the recovery of endothelium,inhibition of Neonitima proliferation and restenosis after injury of vascular endothelium.
Materials and Methods
1、Sixty New Zealand white rabbits were divided into five groups randomly, which were sham operation group(group-Ⅰ), control group(group-Ⅱ), estrogen treated group(group-Ⅲ), EPC treated group(group-Ⅳ), estrogen combined with EPC treated group(group-Ⅴ), twelve rabbits in each group.Three days after ovariectomy, animals in groupⅢand groupⅤwere treated with estrogen, and the other rabbits were given physiological saline instead.
2、Level of estrogen was detected at different time, including before ovariectomy and 3 days and 2 weeks after ovariectomy.
3、The level of blood lipids(including total cholesterol, triglyceride, high density lipoprotein choesterol and low density lipoprotein cholesterol) was also detected after hyperlipemia feed for 6 weeks.
4、Mononuclear cells were isolated from peripheral blood of rabbits and cultured for implatation.
5、We established the animal model of injured abdominal aorta. Animals in sham-operation group were exposed the femoral artery without injury. At the same time, we gave 2ml suspension of EPC to the rabbits in groupⅣandⅤthrough catheter and the others (exceptⅠgroup) were given physiological saline instead.
6、Effectiveness of endothelium recovery, degree of cell proliferation and lumen loss were evaluated after 4 weeks.
Results
1、The level of estrogen in groupⅢandⅤwere obviously higher than in groupⅠ,ⅡandⅣ(P<0.01)
2、The level of blood lipids,includimg total cholesterol(P<0.01), low density lipoprotein cholesterol(P<0.01) and high density lipoprotein choesterol(P<0.05) are statistically different between groupⅢ,Ⅴand groupⅠ,ⅡⅣ.
3、There are also statistical significance in the degree of endothelium recovery, cell proliferation and lumen loss between groupⅤand groupⅢ,Ⅳ[(90.2±5.1) %vs(85.1±4.1)% and (83.9±6.5)%, P<0.05], [(15.67±2.98)%vs(26.02±4.18)% and (24.87±3.54)%, P<0.01],[(17.61±2.69)%vs(38.43±4.35)% and (41.15±4.45)%, P<0.01].
Conclusion
Applicatin of EPC combined with estrogen after injury of vascular endothelium can speed up the process of endothelial recovery, inhibit neonitima proliferation and reduce lumen loss significantly.
冠心病(coronary heart disease,CHD)是现代社会严重威胁人类健康,引起死亡的主要疾病,其防治已成为基础医学和临床医学研究的热点,具体病因尚未明确。患者多伴有高血脂、高血压、糖尿病、肥胖、吸烟、体力活动减少、家族史等危险因素,男性多在40-60岁之间发病,女性多在绝经期后表现出症状。冠心病的治疗目前主要包括药物治疗、经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)和冠状动脉旁路移植术(coronary artery bypass grafting,CABG)。PCI术自上世纪七十年代应用临床以来,经过几十年的发展已成为治疗冠心病的重要手段之一,从起初的单纯球囊扩张到裸金属支架及目前的药物洗脱支架、切割球囊、支架内放射治疗等。虽然新器械、新技术不断应用于临床,但PCI术后再狭窄仍然是广大临床及科研工作者面临的一道难题。
单纯应用雌激素或内皮祖细胞(endothelial progenitor cells,EPC)防治PCI术后再狭窄已经取得了一定效果,但尚未见将两者联合应用的报道。雌激素不仅具有改善血脂代谢,促进内皮细胞(endothelial cells,EC)增生、迁移的作用,而且在EPC的动员、归巢到损伤血管局部参与损伤内皮的修复过程中发挥重要作用。
研究目的
本实验通过建立卵巢切除术后雌性大白兔腹主动脉球囊损伤模型,观察雌激素与EPC两者联合应用促进内皮修复、抑制新生内膜增生的效果是否优于单独应用。旨在探讨EPC与雌激素联合应用防治血管内皮损伤后再狭窄的效果。
材料和方法
1、雌性新西兰大白兔60只随机分为5组,每组12只。Ⅰ：假手术组；Ⅱ：对照组；Ⅲ：雌激素组；Ⅳ：EPC组；V：雌激素+EPC组。卵巢切除术后,Ⅲ组和V组给予雌激素替代治疗,其余动物生理盐水皮下注射。
2、实验动物卵巢切除前及卵巢切除后3d、2周分别采血,应用放免法检测血清雌激素水平。
3、实验动物高脂饮食6周后应用氧化酶法检测血浆总胆固醇、甘油三酯水平；用直接法检测低密度脂蛋白胆固醇及高密度脂蛋白胆固醇。
4、从兔外周血分离培养单个核细胞,制备EPC悬液以备细胞移植。
5、建立兔腹主动脉损伤模型,Ⅰ组动物仅分离暴露出股动脉而不做球囊损伤,Ⅳ组和V组动物损伤血管局部经导管给予EPC悬液2mL输注,其余动物(除Ⅰ组外)给予生理盐水2mL。
6、4周后,伊文斯蓝染色检测损伤血管段内皮修复程度,免疫组化法检测细胞增殖程度,HE染色观察管腔丢失情况。
结果
1、Ⅲ、Ⅴ组动物雌激素水平明显高于Ⅰ、Ⅱ、Ⅳ组(P<0.01)。
2、与Ⅰ、Ⅱ、Ⅳ组相比,Ⅲ、Ⅴ组动物总胆固醇、低密度脂蛋白胆固醇明显降低(P<0.01),高密度脂蛋白胆固醇明显升高(P<0.05),甘油三脂差异无统计学意义。
3、V组与Ⅲ、Ⅳ组相比内皮修复程度、细胞增殖程度及管腔丢失程度差异有统计学意义,分别为[(90.2±5.1)%vs(85.1±4.1)%、(83.9±6.5)%,P<0.05],[(15.67±2.98)%vs(26.02±4.18)%、(24.87±3.54)%,P<0.01],[(17.61±2.69)%vs(38.43±4.35)%、(41.15±4.45)%,P<0.01]。
结论
EPC与雌激素联合应用能加速内皮修复、抑制新生内膜增生,减少血管腔丢失。
Background
Coronary heart disease(CHD) is a kind of disease that is very harmful to people's health and a main reason for people's dealth in modern society.It has become the hot pot of preclinical and clinical medicine,but etiological factor has not been ascertained: Men usually suffer CHD at the age about forty to sixty years old,however,women appear symptoms almost after menopause.Patient with CHD usually get the risk factors,such as hyperlipemia,hypertension, diabetes mellitus, obesity,semoking and so on.Treating to CHD mainly include drugs, percutaneous coronary intervention(PCI), coronary artery bypass grafting (CABG),and gradually PCI has become one of the most effective tragedy.Though more and more new apparatus and technology have been applied to clinic,in-stent restenosis is a serious problem to the doctor and researcher of medicine.
People have got some effectiveness from the application of estrogen or endothelial progenitor cells(EPC) alone,but there is no report about combination of the both.Estrogen has many actions,including accommodation of blood fat,promoting the growth and migration of endothelial cells(EC) and playing a important role in EPC.
Objective
To investigate the effect of application of endothelial progenitor cells combined with estrogen on the recovery of endothelium,inhibition of Neonitima proliferation and restenosis after injury of vascular endothelium.
Materials and Methods
1、Sixty New Zealand white rabbits were divided into five groups randomly, which were sham operation group(group-Ⅰ), control group(group-Ⅱ), estrogen treated group(group-Ⅲ), EPC treated group(group-Ⅳ), estrogen combined with EPC treated group(group-Ⅴ), twelve rabbits in each group.Three days after ovariectomy, animals in groupⅢand groupⅤwere treated with estrogen, and the other rabbits were given physiological saline instead.
2、Level of estrogen was detected at different time, including before ovariectomy and 3 days and 2 weeks after ovariectomy.
3、The level of blood lipids(including total cholesterol, triglyceride, high density lipoprotein choesterol and low density lipoprotein cholesterol) was also detected after hyperlipemia feed for 6 weeks.
4、Mononuclear cells were isolated from peripheral blood of rabbits and cultured for implatation.
5、We established the animal model of injured abdominal aorta. Animals in sham-operation group were exposed the femoral artery without injury. At the same time, we gave 2ml suspension of EPC to the rabbits in groupⅣandⅤthrough catheter and the others (exceptⅠgroup) were given physiological saline instead.
6、Effectiveness of endothelium recovery, degree of cell proliferation and lumen loss were evaluated after 4 weeks.
Results
1、The level of estrogen in groupⅢandⅤwere obviously higher than in groupⅠ,ⅡandⅣ(P<0.01)
2、The level of blood lipids,includimg total cholesterol(P<0.01), low density lipoprotein cholesterol(P<0.01) and high density lipoprotein choesterol(P<0.05) are statistically different between groupⅢ,Ⅴand groupⅠ,ⅡⅣ.
3、There are also statistical significance in the degree of endothelium recovery, cell proliferation and lumen loss between groupⅤand groupⅢ,Ⅳ[(90.2±5.1) %vs(85.1±4.1)% and (83.9±6.5)%, P<0.05], [(15.67±2.98)%vs(26.02±4.18)% and (24.87±3.54)%, P<0.01],[(17.61±2.69)%vs(38.43±4.35)% and (41.15±4.45)%, P<0.01].
Conclusion
Applicatin of EPC combined with estrogen after injury of vascular endothelium can speed up the process of endothelial recovery, inhibit neonitima proliferation and reduce lumen loss significantly.
引文
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