【作者】 周建良；

【导师】 董念国；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 第一部分：PEG交联去细胞瓣复合材料构建及其体外生物相容性目的：PEG交联去细胞瓣构建复合材料,并评价其体外生物相容性。方法：(1)合成功能团为丙烯酰基的枝化状PEG,同时在去细胞瓣上引入巯基,通过丙烯酰基与巯基发生迈克尔加成反应,构建PEG交联去细胞瓣复合材料。(2)生物力学测试评价PEG交联对去细胞瓣机械性能的影响；CCK-8法检测复合材料浸提液对人脐静脉内皮细胞增殖率影响,以评价其生物相容性。结果：复合材料构建条件温和、效果确切；复合材料抗拉强度明显高于去细胞瓣(P<0.05),且与天然瓣膜抗拉强度相比,差异无统计学差异(P>0.05)。复合材料与去细胞瓣毒性评级均为0级,与阴性对照组相比,差异无统计学意义(P>0.05)；人脐静脉内皮细胞在含复合材料浸提液的培养液中形态良好,增殖旺盛。结论：PEG交联去细胞瓣的方法能够构建与天然瓣膜相似抗拉强度且无细胞毒性的复合材料。第二部分：PEG交联去细胞瓣多信号复合材料构建第一节：构建RGD-复合材料目的：评价PEG交联去细胞瓣复合材料结合粘附肽(精-甘-天冬氨酸RGD肽)的可行性和有效性。方法：复合材料与1ml不同浓度(1.5mg/ml, 1mg/ml,0.5mg/ml,0.25mg/ml)GRGDSPC肽溶液在37℃反应,在不同时间点(1h,2h,4h),用分光光度法检测复合材料结合GRGDSPC肽质量,并行免疫荧光定性检测,去细胞瓣作为阴性对照。结果：GRGDSPC肽能有效地结合于复合材料,且一片复合材料与lml GRGDSPC肽(1mg/ml)在37℃条件下反应2h,结合GRGDSPC肽质量最大为(0.79±0.01)mg。结论：PEG交联去细胞瓣复合材料,能够在体外有效地结合RGD肽,构建RGD-复合材料。第二节：构建VEGF-复合材料目的：评价PEG交联去细胞瓣复合材料结合血管内皮细胞生长因子(VEGF)的可行性和有效性。方法：复合材料与1ml不同浓度(500pg/ml, 1000pg/ml,2000pg/ml) VEGF溶液在37℃反应,在不同时间点(1h,2h,4h,12h),用ELISA法检测复合材料结合VEGF质量,并行免疫荧光检测,去细胞瓣作为阴性对照。结果：VEGF能有效地结合于复合材料,且一片复合材料与1ml VEGF (1000pg/ml)在37℃反应4h,其结合VEGF质量最大为(896.87+3.27)pg。结论：PEG交联去细胞瓣复合材料,能够在体外有效地结合VEGF,构建VEGF-复合材料。第三节：构建PEG交联去细胞瓣多信号复合材料目的：评价PEG交联去细胞瓣复合材料同时结合RGD肽和VEGF的可行性和有效性。方法：复合材料与1ml反应液(包含1mg/mlGRGDSPC肽和1000pg/mlVEGF)在37℃反应4h,对反应后复合材料行免疫荧光检测,去细胞瓣作为阴性对照。结果：GRGDSPC肽和VEGF能同时有效地结合于复合材料。结论：PEG交联去细胞瓣复合材料,能够在体外有效地结合RGD肽和VEGF,构建PEG交联去细胞瓣多信号(RGD、VEGF)复合材料。第三部分：PEG交联去细胞瓣多信号复合材料生物学性能研究第一节：内皮祖细胞分离、培养与鉴定目的：分离、培养、鉴定脐带血内皮祖细胞,为其作为组织工程种子细胞提供实验依据。方法：密度梯度离心法分离新鲜脐血中单个核细胞,在含碱性成纤维细胞生长因子和血管内皮生长因子培养液中培养,通过形态学、免疫荧光和流式细胞仪等对贴壁细胞进行鉴定；并与脐静脉内皮细胞进行增殖和迁移能力比较。结果：随培养时间延长,贴壁细胞形态发生明显改变,从小圆变成梭形,逐渐形成特征性克隆和典型成熟内皮细胞鹅卵石样形态；体外培养7天后,90%以上贴壁细胞呈Dil-ac-LDL和FITC-UEA-Ⅰ双染阳性；贴壁细胞流式细胞仪分析显示：培养7 d的细胞VEGFR-2、CD34和CD133表达分别占(77.35+4.86)%、(52.42+6.64)%和(19.36+2.14)%,培养28天的细胞VEGFR-2和CD34表达分别占(81.06+7.31)%和(7.62±3.14)%,而未检测到CD133表达；人内皮祖细胞增殖和迁移能力明显高于人脐静脉内皮细胞<0.05)。结论：用密度梯度离心法和贴壁筛选法,可成功分离出脐带血内皮祖细胞,该方法简单、经济、可行；且内皮祖细胞可向内皮细胞分化,增殖和迁移能力强,有望成为理想种子细胞。第二节：PEG交联去细胞瓣多信号复合材料生物学性能测定目的：研究PEG交联去细胞瓣多信号(RGD、VEGF)复合材料对内皮祖细胞粘附和增殖的影响,为进一步构建TEHV提供依据。方法：A组去细胞瓣,B组PEG交联去细胞瓣复合材料,C组VEGF-复合材料,D组为RGD-复合材料,E组PEG交联去细胞瓣多信号(RGD、VEGF)复合材料。然后在各组材料上种植EPCs,分别在种植后2h、4h、8h用细胞计数和3H-TdR掺入法检测各组材料上细胞数和每分钟脉冲数,反映EPCs在各组材料上粘附性；分别在种植后2d、4d、8d,用细胞计数和3H-TdR掺入法检测各组瓣膜支架上细胞数和每分钟脉冲数,反映各组材料上EPCs增殖情况。在种植8d时,取各组瓣膜支架行苏木素-伊红染色和扫描电镜,以检测EPCs在各组材料上生长情况,并取E组支架材料上细胞行RT-PCR检测t-PA和eNOS的表达,以评价新内皮抗血栓形成功能。结果：(1)种植后2h、4h、8h时各组细胞数和每分钟脉冲数为：D组>E组>C组>A组>B组,其中A组与B组之间各个时间点均无统计学差异(P>0.05),8h时D组与E组之间的差异无统计学意义(P>0.05),其余时间点各组之间的差异均有统计学意义(P<0.05)。(2)种植2d、4d、8d时各组细胞数和每分钟脉冲数为：A组均大于B组(P>0.05),C组、D组和E组均大于同一时间点A组和B组(P<0.05)；种植2d时,E组>C组(P<0.05),D组>E组(P>0.05)；种植4d时,E组>D组(P<0.05),D组>C组(P<0.05)；种植8d时,E组>D组(P<0.05),D组>C组(P>0.05)。(3)苏木素-伊红染色和扫描电镜检测显示：种植8d后,与其它组相比,E组瓣膜支架上可见一致密融合细胞层,并且该新内皮层细胞t-PA和eNOS表达与人脐静脉内皮细胞相似。结论：PEG交联去细胞瓣多信号(RGD、VEGF)复合材料上RGD肽和VEGF,能够协同促进内皮祖细胞在复合材料上粘附和增殖,有利于TEHV构建。更多还原

【Abstract】 Part one:Construction and in vitro biocompatibility of the composite PEG-crosslinked decellularized valveObjective:To construct a composite material by polyethylene glycol (PEG) crosslinking of decellularized valves, and evaluate the biocompatibility of the composite material in vitro.Methods:First, branched PEG with the functional group of propylene acyl was synthesized, and thiol was introduced into decellularized valves. Then the composite valve was constructed by the Michael addition reaction between propylene acyl and thiol. Second, the effects of PEG crosslinking on biomechanical properties of decellularized valve were evaluated. For assessing the biocompatibility, cell proliferation rates of the human umbilical vein endothelial cells (HUVECs) which were exposed to the extract of composite material were detected by CCK-8 assay.Results:The PEG-crosslinking reaction for the construction of composite material has a mild conditions and a high efficiency; The tensile strength of the composite materials was obviously higher than that of the decellularized ones(P<0.05), and there was no significant difference in tensile strength between composite materials and natural valves(P>0.05). The toxicity of composite material were classified Grade 0, and no significant difference of toxicity was observed between the composite material group and the negative control group; HUVECs treated with culture medium containing extracts of composite material show great cytoactivity and normal morphology.Conclusions:A noncytotoxic composite material can be obtained by PEG crosslinking with the decellularized valves, and the tensile strength of the composite material is similar to the natural one.Part two:Construction of the composite PEG-crosslinked decellularized valve with multi-signalChapter one:Construction of the composite valve conjugated with RGD peptideObjective:To evaluate the feasibility and effectiveness of conjugation of RGD (Arg-Gly-Asp) peptide on the composite PEG-crosslinked decellularized valve and optimize the reaction condition.Methods:The composite PEG-crosslinked decellularized valve was reacted with 1ml GRGDSPC peptides solution of different concentrations (1.5mg/ml, 1mg/ml, 0.5mg/ml,0.25mg/ml) at 37℃. The conjugated effect was evaluated quantitatively by spectrophotometer at different time points (1h,2h, 4h), and the qualitative tests were performed by immunofluorescence. The decellularized valves were set as negative control.Results:The GRGDSPC peptides could be conjugated effectively with the PEG-composite valve, and the quality of conjugated GRGDSPC peptides reaches a climax amount of (0.79±0.01)mg when the concentration of GRGDSPC peptides solution was lmg/ml and the reaction time was 2h.Conclusion:The construction of RGD-composite material can be achieved by grafting GRGDSPC peptide on the composite PEG-crosslinked decellularized valve in vitro.Chapter two Construction of the composite valve conjugated with VEGFObjective:To evaluate the feasibility and effectiveness of conjugation of VEGF on composite PEG-crosslinked decellularized valve and optimize the reaction condition.Methods:The composite PEG-crosslinked decellularized valve was reacted with lml VEGF solution of different concentrations (500pg/ml, 1000pg/ml,2000pg/ml) at 37℃. The quality of conjugated VEGF was measured by ELISA at different time points (1h,2h,4h,12h), and the qualitative tests were performed by immunofluorescence. The decellularized valves were set as negative control.Results:The VEGF could be conjugated effectively on the composite valve by PEG, and the maximum quality of conjugated VEGF was (896.87±3.27)pg when the concentration of VEGF solution was 1000pg/ml and reaction time was 4h.Conclusion:The construction of VEGF-composite material can be achieved by grafting VEGF on the composite PEG-crosslinked decellularized valve in vitro.Chapter three:Construction of the composite PEG-crosslinked decellularized valve with multi-signalObjective:To assess the probability and effectiveness of conjugation of RGD peptide and VEGF on composite PEG-crosslinked decellularized valve simultaneously.Methods:The composite PEG-crosslinked decellularized valve were reacted with lml reaction mixture solution (including lmg/ml GRGDSPC peptide and 1000pg/ml VEGF) at 37℃for 4h. Then the immunofluorescence was taken to make a qualitative evaluation of the conjugation effects. The decellularized valves were set as negative control.Results:The GRGDSPC peptides and VEGF could be conjugated effectively onto composite material by PEG.Conclusion:The construction of composite PEG-crosslinked decellularized valve with multi-signal (RGD and VEGF) can be achieved by conjugating the RGD peptide and VEGF on the composite PEG-crosslinked decellularized valve in vitro. Part three:Study on biological properties of the composite PEG-crosslinked decellularized valve with multi-signalChapter one:The isolation, Culture and identification of EPCsObjective:To provide human endothelial progenitor cells (EPCs) for the further study by isolation, culture and identification of EPCs from human umbilical cord blood.Methods:Fresh umbilical blood was collected and density gradient centrifugated to isolate mononuclear cells (MNCs). The cells then were cultured in the medium supplemented with VEGF and bFGF. Morphology, immunofluorescence and flow cytometry were applied to confirm that the attached cells were EPCs. The proliferation and migration capability of EPCs were compared with HUVECs.Results:As the time went on, the shape of the attached cells changed from small round to spindle. the typical colonies were gradually formed and the cobblestone-like morphology as a specific characteristic of mature endothelial cells was observed; after been cultured for 7 days, more than 90% of the attached cells express both Dil-acLDL and FITC-UEA-Ⅰ. Flow cytometric analysis showed that the positive staining rate of the attached cells for VEGFR2、CD34 and CD133 were (77.35±4.86)%, (52.42±6.64)% and (19.36±2.14)% respectively, and at the 28th day, the positive staining rate of attached cells for VEGFR2 and CD34 were (81.06±7.31)% and (7.62±3.14)% respectively while CD133 can not been stained. Compared with HUVECs, EPCs have more potent potential of proliferation and migration (P<0.05).Conclusion:EPCs can be isolated successfully from human umbilical cord blood by a simple, economical and feasible method composed of density gradient centrifugation and adherent filtration. EPCs can be introduced to endothelial cells and have great capacity of proliferation and migration, which makes it an ideal seed cells. Chapter two:Measurement on biological properties of the composite PEG-crosslinked decellularized valve with multi-signalObjective:To investigate the effect of composite valve with multi-signal on the adhesion and proliferation of EPCs, and provide the basis for the further construction of TEHVMethods:The experiment were assigned into 5 groups:Group A were decellularized valves, Group B were composite PEG-crosslinked decellularized valves, Group C were VEGF-composite valve, Group D were RGD-composite valve and Group E were multi-signal composite valve(n=8). EPCs were seeded onto the valve of different groups, the cells number and counts per minute(cpm) were detected by cell counting and thymidine(3H-TdR) up-take method at 2h、4h、8h respectively after seeding to estimate the adhesion amount of EPCs in different groups. the cells number and cpm of each group were detected using cell counting and thymidine(3H-TdR) up-take method at 2d、4d、8d respectively after seeding to evaluate the proliferation of EPCs. Hematoxylin and eosin (HE) stain and scanning electron microscopy (SEM) were performed to give a morphology evidence of the growth of EPCs at 8d after seeding. Finally the expression of t-PA and eNOS of group E were measured by RT-PCR to assess the antithrombotic function of the multi-signal composite valve.Results:(1) After been seeded for 2h、4h and 8h, the results of cell count and cpm in each group were as the following:Group D>Group E>Group C>Group A>Group B, but there was no significant difference between Group A and Group B(P> 0.05), there was also no significant difference between Group D and Group E at the 8h after the seeding(P>0.05), but the differences in the other groups were significant at the other time points(P<0.05), (2) At the 2d,4d and 8d after been seeded, the results of cell count and cpm in each group were as the following: Group A was always higher than Group B(P>0.05), the cell number and cpm in group C、Group D and Group E were significantly higher than those in Group A and Group B (P<0.05) at the time point. At the 2d after the seeding, the cell number and cpm in Group E was higher than those in Group C (P<0.05), the cell number and cpm in Group D was higher than those in Group E(P>0.05); at the 4d after the seeding, the cell number and cpm in Group E higher those in group D(P<0.05), the cell number and cpm in Group D was higher than those in Group C(P<0.05); at the 8d after the seeding, the cell number and cpm in Group E was higher than those in Group D(P<0.05), the cell number and cpm in Group D was higher than those in Group C(P>0.05). (3) the results of HE and SEM indicated that the confluent and compact monolayer could be formed on the surface of Group E compared with other groups at 8d after the seeding, and the expression level of t-PA and eNOS gene in this neo-endothelium were very similar to those in HUVECs.Conclusion:The RGD peptides and VEGF conjugated on the composite PEG-crosslinked decellularized valve can achieve a synergistic promotion on the adhesion and proliferation of EPCs seeded on the composite valve, which is useful for the construction of tissue engineering heart valves.更多还原