摘要
第一部分：壳聚糖/肝素层层自主装涂层的生物相容性的实验研究
目的：研究壳聚糖/肝素层层自组装涂层对CD133+内皮祖细胞的黏附、增殖相关基因表达的影响,并从分子生物学角度探讨其作为促内皮修复功能药物洗脱支架涂层材料的可行性。
方法：1.分离人脐血单个核细胞,免疫磁珠分选CD133+内皮祖细胞；2.制备壳聚糖/肝素层层自组装涂层,1%明胶涂层以及玻璃对照皿,接种并培养分选所得内皮祖细胞；3.免疫荧光鉴定内皮祖细胞；4.抽提总RNA, RT-PCR法比较不同培养介质中细胞的eNOS、VE-cadherin、KDR、PECAM-1、Sirtuin-1、Thrombomodulin等基因的表达差异。
结果：1.重力梯度离心及磁珠分选法所得CD133+内皮祖细胞纯度较高(92.88%±0.51%(n=4)),在VEGF诱导下能较好的分化为内皮细胞；2. eNOS、VE-cadherin、KDR、PECAM-1和Sirtuin-1在壳聚糖/肝素层层自组装涂层组表达丰度较玻璃对照组显著增高(P<0.05),与明胶组差异不明显,Thrombomodulin在壳聚糖涂层表达丰度较明胶组、玻璃组表达均增高(P<0.05)。
结论：1.重力梯度离心及磁珠分选法所得CD133+内皮祖细胞纯度较高(92.88%±0.51%(n=4)),在VEGF诱导下能较好的分化为内皮细胞；2. eNOS、VE-cadherin、KDR、PECAM-1和Sirtuin-1在壳聚糖/肝素层层自组装涂层组表达丰度较玻璃对照组显著增高(P<0.05),与明胶组差异不明显,Thrombomodulin在壳聚糖涂层表达丰度较明胶组、玻璃组表达均增高(P<0.05)；3.壳聚糖涂层能促进内皮祖细胞的黏附、增殖,生物相容性好,为理想的生物材料。
第二部分：壳聚糖/肝素层层自组装(LBL)支架、LBL-Drug支架的制备及相关特性研究
目的：制备壳聚糖/肝素层层自组装涂层支架(LBL), LBL-Drug支架,并对其机械性能、坚韧性、亲水性、药物释放特点、细胞黏附能力等理化性能做检测。
方法：1.通过浸泡法制备LBL支架,喷涂法制备雷帕霉素药物洗脱支架(Drug),非对称涂层处理方法制备LBL-Drug支架；2.电镜观察支架压握及扩张后变化,支架坚韧性实验,测定所制备支架的接触角,LBL-Drug支架的药物体外释放测试,以及体外模拟内皮细胞黏附实验,观察支架的性能。
结果：1.成功制备了壳聚糖／肝素层层自组装支架(LBL)、LBL-Drug支架,对其物理性能及药物释放等特点进行检测,其表征特点符合支架制作的工艺要求,压握及扩张后涂层覆盖均匀无开裂脱落,在干性湿性条件下扩张均无明显差异,支架接触角的检测表明经过层层自组装技术处理后支架亲水性增强,具有良好的生物相容性；2. LBL-Drug洗脱支架雷帕霉素释放曲线呈爆发性释放的特点,与目前上市的雷帕霉素药物洗脱支架的释放特点一致；3.体外模拟细胞黏附试验可以得出经LBL处理的支架细胞黏附能力增强,支架涂层材料具有良好的生物相容性。
结论：1.上述支架制备工艺符合植入性冠脉支架的基本要求；2.经LBL处理的支架其亲水性增强,生物相容性增加;3.经LBL处理的支架能促进细胞黏附的能力,可以促进早期内皮修复。
第三部分：动物实验评价促内皮修复药物洗脱支架的有效性和安全性
目的：小型猪动物模型中观察促内皮修复药物洗脱支架(LBL-Drug支架)的有效性和安全性。
方法：1.将BMS及第二部分所制备的LBL、Drug、LBL-Drug支架随机植入小型猪的前降支、回旋支和右冠状动脉,通过过度扩张形成冠脉损伤模型。术后1周,1月,3月,6月时,观察支架内皮覆盖情况、内膜增生情况、植入部位炎症；2.6月时血管内超声评价内膜增生情况；3.1、3、6月随访冠脉造影时经冠脉注入乙酰胆碱,观察内皮依赖的血管收缩反应,了解内皮功能。
结果：1.各组支架内皮覆盖速度具有差异,其中LBL和LBL-Drug组覆盖速度较快,在植入1周后即开始有部分内皮覆盖,其速度快于BMS组,而单纯Drug组支架的内皮覆盖率最慢,1月时四组内皮均覆盖完整,至6月时,四组内皮覆盖完好,其中LBL和LBL-Drug组较BMS组及Drug组比较,内皮相对光滑,连接紧密,BMS组出现内膜增殖导致管腔狭窄；2.通过病理切片观察及计算机辅助测量,Drug-LBL组和Drug组管腔面积在1月、3月、6月时与其他两组低,差异有统计学意义(P<0.05),而BMS组和LBL组间差异无统计学意义(P>0.05); Drug-LBL组和Drug组的新生内膜面积和新生内膜厚度在1月、3月、6月时比其他两组低,其差异有统计学意义(P<0.05),BMS组和LBL组间差异不明显(P>0.05); LBL-Drug组和Drug组狭窄率在1月、3月、6月时比其他两组低,差异有统计学意义(P<0.05),BMS组最高。3.从炎症细胞浸润上看,1月时Drug支架炎症浸润最严重,炎症积分高,各组1月时的炎症积分分别为：BMS0.71±0.69, LBL0.5±0.5, Drug 1.71±0.69, LBL-Drug 0.71±0.69。Drug炎症积分较其他三组高,其差别有统计学意义(P<0.05),BMS,LBL及LBL-Drug组间差别无统计学意义(P>0.05),3月及6月时炎症细胞浸润较1月时减轻,各支架组的炎症积分差别无统计学意义(P<0.05)；4.6月时IVUS检查见BMS组支架内明显内膜增生,管腔狭窄率60%左右,LBL组支架内仅轻度内膜增生,Drug支架组和LBL-Drug支架组,未见明显内膜增生；5.乙酰胆碱观察内皮依赖的血管收缩试验可以得出,3个月时受乙酰胆碱最明显,而6个月时则开始恢复。Drug组支架对于内皮功能的影响最大且持续时间最长,而带有LBL涂层的支架则受内皮功能的影响相对小,且在3个月时开始恢复,6个月时基本恢复到安装支架时的水平。LBL支架对内皮功能的影响较Drug支架小,且内皮功能恢复早。
结论：1.所制备的四组支架在小型猪冠状动脉植入的模型是可行的,通过1-6个月的随访可以评价其安全性及有效性。2. LBL-Drug组和Drug组在1月,3月,6月时其管腔面积大于BMS组和LBL组,狭窄率低于BMS组和LBL组,新生内膜面积和新生内膜厚度低于BMS和LBL组。Drug组在植入1月后炎症反应最大。LBL-Drug支架在植入6月后内膜覆盖完整规则,无支架内狭窄,支架周围炎症反应低,结果理想。3.采用非对称涂层技术(LBL-Drug支架),支架内层单纯涂层LBL促进内皮细胞修复,支架外层采用可降解材料及抗增殖药物,在抑制平滑肌细胞增殖同时促进内皮快速覆盖,使支架在有效性和安全性见达到平衡。该新型支架的动物实验数据将有助于提供内皮修复药物洗脱支架的进一步研制的关键参数,为下一步深入研究和应用打下理论和实验基础。
Part I Studies on Biocompatibility between Chitosan Coat and CD133+ Endothelial Progenitor Cells
Objective To investigate the effect of chitosan/heperin layer-by-layer self assembly coat film to the adhesion,proliferation and differentiation of CD133+ endothelial progenitor cells(EPCs), and to use molecular biologial technique to investigate its feasibility as coating material of drug eluting stent.
Methods (1) Use gravity gradient centrifugation to isolate mononuclear cells from human umbilical cord blood, and use magnetic activated cell sorting to sort CD133+ cells. (2) To fabricate chitosan/heperin layer-by-layer self assembly coat film,1% gelatin coat and bare glass cell culture dish as control group, then culture the cells. (3) Immunofluorescen assessment of the cultured cells.(4) To extract RNA from the cells, and to use RT-PCR method to test mRNA expression levers of eNOS、VE-cadhern、KDR、PECAM-1、Sirtuin-1、Thrombomodulin in different culture mediums.
Results (1) Gravity gradient centrifugation and magnetic activated cell sorting methods can obtain pure CD133+ EPCs, and can well different to endothelial cells induced by VEGF; (2) Compared with bare glass, Chitosan coating film can significantly promote the adhesion,proliferation and differentiation of EPCs.
Conclusion Chitosan coating film can promote the adhesion, proliferation and differentiation of EPCs, with good biocompatibility, and can be a promising biomaterial.
Part II The Preparation and Related Properties Study of Chitosan /Heparin Layer-by-layer Self Assembly (LBL) and LBL-Drug Stents
Objective To prepare chitosan / heparin layer-by-layer self assembly stent (LBL), LBL-Drug stent, and to test its physical and chemical properties as mechanical properties, toughness, hydrophilic, drug release characteristics, and cell adhesion abilities.
Methods (1) To prepare LBL stent by soaking method, and prepare Rapamycin eluting stent by spraying method, and prepare LBL-Drug stent by asymmetric coating method; (2) To observe the performance of stents by observing the stents after pression and expansion pressure via electron microscope, performing stent tough experiment, determining the stents contact angle, performing drug release test of LBL-Drug stents in vitro, and endothelial cell adhesion in vitro experiments.
Results (1) Successfully prepare chitosan / heparin layer-by-layer self-assembly (LBL) stent, LBL-Drug stent, and test its physical properties and of drug release characteristics. The properties of our prepared stents meet the requirements of stent production process. The coating of the stent is smooth and and well-distrubuted without cracking after pression and expansion. No significant difference in the expansion between the dry and wet conditions. The contact angle analysis shows that the hydrophilicity of stents enhenced after self-assembly technique treatment, with good biocompatibility. (2) The sirolimus releasing characteristics of LBL-Drug eluting stent is explosive release curve characteristics, and conform releasing characteristics of the current on sale rapamycin drug-eluting stent. (3) In vitro cell adhesion test shows thar the cell adhesion capacity of the stent is enhanced by LBL processing treatment, and stent coating material has good biocompatibility.
Conclusion (1) The coronary stent technology meets the basic requirements of scaffold preparing technology; (2) Both the hydrophilicity and biocompatibility of stent enchanced by the LBL processing handling; (3) By the LBL processing handling, the stent gain the ability of promoting cell adhesion, and can promote early endothelial repairment.
Part III Animal experimental study on efficacy and safety of promoting endothelial repair drug-eluting stent
Objective To observe the efficacy and safety of promoting endothelial repair drug-eluting stent in the minipigs model.
Methods (1) The BMS、LBL、Drug and LBL-Drug stents were implanted in pig left anterior descending, circumflex and right coronary artery, formed by over-expansion model of coronary injury. stent endothelial coverage, intimal hyperplasia, implant site inflammation were observed after 1 week,1 month,3 months and 6 months of operaion; (2) To evaluate endometrial hyperplasia 6 month by intravascular ultrasound after 6 month of operation; (3) At the time of 1、3 and 6 month follow-up coronary angiography after coronary injection of acetylcholine, endothelial function was evaluated by observe endothelium-dependent vasoconstriction.
Results 1. Each group has a different rate of stent endothelial coverage. LBL and LBL-Drug group have the fastest coverage speed, and endothelial begin to cever after 1 week which is faster than BMS group. And Drug group has the lowest endothelial cover rate. At 1 month, all group have complete endothelial cover, of which, LBL group and LBL-Drug group have relative smooth and regular endothelial compared with BMS group and Drug group. And endothelial hyperplasia leads to some extent of stenosis in BMS group. 2. By pathological observation and computer-aided measurement, Drug-LBL group and the Drug group have wider lumen area at 1 month, 3 months and 6 months, when compared with BMS group and LBL group,the difference is statistic significance(P<0.05), and no difference between BMS group and LBL group; compared with BMS group and LBL group, LBL-Drug group and Drug group have lower neointima area and neointima thickness at 1,3 and 6 months, and the difference is significance (P<0.05), while the BMS group and LBL group are no significant difference (P> 0.05). Stenosis rate at 1 month,3 months and 6 months of LBL-Drug group and Drug group are lower than the other two groups, the difference is statistically significant (P<0.05), BMS group was the highest of 4 groups.3. Judging from the infiltration of inflammatory cells,1 month Drug stents has the most severe inflammatory infiltration, with a highest inflammation score. Inflammation score of each group at 1 month are:BMS 0.71±0.69, LBL 0.5±0.5, Drug 1.71±0.69, LBL-Drug 0.71±0.69. Drug group has the highest inflammation score, the difference is statistically significant (P <0.05), other groups have no significant difference (P> 0.05), At 3 months and 6 months, the infiltration of inflammatory cells reduced when compared with 1 month, the inflammation score in each group is no significant difference (P<0.05); 4. At 6 month, IVUS examination revealed BMS group of significant in-stent neointimal hyperplasia and stenosis rate of 60%, LBL Group only mild in-stent neointimal hyperplasia, no significant intimal hyperplasia in Drug stent group and LBL-Drug stent group; 5. Acetylcholine endothelium-dependent vasoconstriction observation test suggests that, each stent group is affected most notable by acetylcholine at 3 months. Therefore, the response begins to recover at 6 month. The endothelial function effect in Drug group is of the largest and longestm of 4 groups, while the LBL coating stents with endothelial function is affected relatively small, and at 3 months, begin to recover, and recovered at 6 months. The endothelial function effect in LBL stent is little and the recovery is earlier compared with other groups.
Conclusion (1) The implantation of prepared coronary stent of the four groups of in the minipig model is feasible. evaluate the safety and efficacy of the stents by 1-6 months of follow-up. (2) At the time of 1 month,3 months and 6 months, LBL-Drug group and Drug group have a wider lumen area and lower stenosis rate compared with BMS group and LBL group, and their neointimal area and neointimal thickness are less than that of the BMS group and LBL group. At 1 month after implantation, Drug group has the greatest inflammatory response. Compared with BMS、LBL and Drug stent, LBL-Drug Stent has a relatively complete and regular intimal coverage with no in-stent restenosis, and inflammatory reaction around the stent is slight,which can be thought to a satisfactory results. (3) Using asymmetric coating technology (LBL-Drug stents), inner layer of stent can promote endothelial cells repairment, and outer layer of stent apply of biodegradable scaffold and anti-proliferation of materials, which can inhibit smooth muscle cells proliferation and reduce the inflammatory response, with good biocompatibility. These animal datas will provide the development of prohealing endothelial stent with core parameters, and will make a theoretic and experimental basis for further research and application.
引文
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