【作者】 洪昊；

【导师】 董念国；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分组织工程瓣膜种子细胞的研究目的：采用间充质干细胞(MSCs)构建组织工程心脏瓣膜(TEHV)，并对MSCs的细胞生物学，及所构建TEHV的生物学和生物力学性能进行研究。方法：分离培养大鼠MSCs和肌成纤维细胞，进行细胞鉴定后分别种植于去细胞猪主动脉瓣叶支架上，然后在体外培养14d构建TEHV，并分别做为实验组A和B。对MSCs的细胞生物学，及两组TEHV的生物学和生物力学性能进行检测。结果：MSCs表达CD29(94．82％)和CD44(93．59％)。免疫组化和荧光染色检测显示MSCs的Vimentin染色均为阳性，部分MSCs的a-SMA染色阳性。实验组B的a-SMA、MMP-13和TIMP-1 mRNA表达均高于实验组A(P＜0．05)，而LOX蛋白含量和LOX mRNA表达均低于实验组A(P＜0．05)。两组TEHV的形态学结构相似。实验组A支架上MSCs的Vimentin染色均为阳性，部分MSCs的a-SMA染色阳性。两组TEHV的DNA和羟脯氨酸含量，最大负荷、最大应力、最大应变和弹性模量的差异无统计学意义(P＞0．05)。结论在采用MSCs体外构建TEHV过程中，其细胞生物学性能与肌成纤维细胞相比存在差异，而两者所构建TEHV的生物学和生物力学性能相当。这说明MSCs构建的TEHV并没有在性能方面体现出明显的优势。有必要通过其他方法，对MSCs构建的TEHV性能加以改善。第二部分bFGF调控组织工程瓣膜构建的研究目的：探讨碱性成纤维细胞生长因子(bFGF)对MSCs构建的TEHV的生物学和生物力学性能的影响。方法：分离培养大鼠MSCs，种植于去细胞瓣叶支架上。将支架置于含10ng／ml bFGF的培养液中，培养14d构建的TEHV做为实验组A。实验组B除培养液中不添加bFGF外，其余同实验组A。实验组C为大鼠肌成纤维细胞构建的TEHV。对MSCs的细胞生物学，及各组TEHV的生物学和生物力学性能进行检测。结果：实验组A和C的a-SMA、MMP-13和TIMP-1 mRNA表达的差异无统计学意义(P＞0．05)，但均高于实验组B(P＜0．05)。实验组A和C的LOX蛋白含量和LOX mRNA表达的差异无统计学意义(P＞0．05)，但均低于实验组B(P＜0．05)。实验组A支架的再细胞化程度明显优于实验组B和C；DNA和羟脯氨酸含量高于实验组B和C(P＜0．05)。各组TEHV生物力学性能的差异无统计学意义(P＞0．05)。结论：在采用MSCs联合bFGF体外构建TEHV过程中，通过bFGF的调控作用，可以使MSCs的表型特性、基质金属蛋白酶及其抑制物的表达增强，达到与肌成纤维细胞相当的水平；可以使MSCs的LOX蛋白和基因表达降低，达到与肌成纤维细胞相当的水平；可以提高TEHV的生物学性能。但bFGF改善TEHV生物力学性能的作用不明显。第三部分组织工程瓣膜复合支架的研究目的：采用静电纺丝技术制备复合支架，然后在支架上种植MSCs构建TEHV，并研究复合支架对TEHV的生物学和生物力学性能的影响。方法：分离培养大鼠MSCs和肌成纤维细胞。实验组A为猪主动脉瓣叶经去细胞处理后，在其表面涂铺采用静电纺丝技术制备的聚(3—羟基丁酸酯—co—4—羟基丁酸酯)(P3／4HB)纤维膜，构建复合支架。实验组B和C为去细胞猪主动脉瓣叶支架。在实验组A和B的支架上种植MSCs，在实验组C的支架上种植肌成纤维细胞，各组均在体外培养14d构建TEHV，并进行生物学和生物力学检测。结果：复合支架的整个P3／4HB纤维膜结构均一，平均直径是156±3nm，并且与去细胞瓣叶支架表面紧密结合。各组支架的再细胞化程度相当，DNA和羟脯氨酸含量差异无统计学意义(P＞0．05)。实验组A与实验组B、C相比，最大负荷、最大应力和弹性膜量明显上升(P＜0．05)，而最大应变的差异无统计学意义(P＞0．05)。结论：复合支架能明显增强TEHV的生物力学性能，而其对TEHV生物学性能的改善作用不明显。第四部分间充质干细胞联合bFGF复合支架构建组织工程瓣膜的研究目的：采用MSCs联合bFGF复合支架构建TEHV，以改善TEHV的生物学和生物力学性能。方法：分离培养大鼠MSCs。实验组A为猪主动脉瓣叶经去细胞处理后，在其表面涂铺采用缓释和静电纺丝技术制备的含有bFGF壳聚糖纳米粒的P3／4HB纤维膜，构建复合支架(bFGF)。实验组B为在去细胞瓣叶表面涂铺P3／4HB纤维膜，构建复合支架。实验组C和D为去细胞瓣叶支架。然后在各组支架上种植MSCs，实验组C在含10ng／ml bFGF的培养液中培养，其余各组均在不含bFGF的培养液中培养。各组均在体外培养14d构建TEHV。各组TEHV进行生物学和生物力学检测。结果：复合支架(bFGF)的载药量为0．00077％，包封率为88．1％，累积释放度为1d约16．3％，5d约42．4％，10d约51．1％和15d约53．9％。复合支架(bFGF)的整个bFGF壳聚糖纳米粒P3／4HB纤维膜结构均一，并且与去细胞瓣叶支架表面紧密结合。实验组A和C支架的再细胞化程度明显优于实验组B和D，DNA和羟脯氨酸含量明显高于实验组B和D(P＜0．05)。实验组A和B与实验组C和D相比，最大负荷、最大应力和弹性膜量明显上升(P＜0．05)，而最大应变的差异无统计学意义(P＞0．05)。结论：采用MSCs联合复合支架(bFGF)构建TEHV，能明显改善和增强TEHV的生物学和生物力学性能。复合支架(bFGF)的制备为构建有再生活性和良好功能人工心脏瓣膜替代物提供了新方法和新思路。更多还原

【Abstract】 Part one Construction of Tissue Engineered Heart Valve UsingDifferent Seed CellsObjective: Tissue engineered heart valve (TEHV) was constructed using mesenchymalstem cells (MSCs) as seed cells. The cell biology of MSCs, and thebiological and biomechanical properties of the TEHV were investigated.Methods: MSCs and myofibroblasts were isolated and cultured. After cell identification,cells were seeded onto decellularized aortic valve leaflet scaffold and culturedfor 14 days to constuct TEHV in group A and B. The cell biology of MSCs,and the biological and biomechanical properties of the TEHV were examed.Results: MSCs expressed CD29 (94.82%) and CD44 (93.59%). Immunocytochemistryshowed the expression of vimentin by all MSCs and the expression of a-SMAby some MSCs. The expression of a-SMA, MMP-13 and TIMP-1 mRNAwere significantly inceased in group B, compared with lower values of groupA (P＜0.05). However, the content of LOX and expression of LOX mRNAwere inceased in group A, compared with lower values of group B (P＜0.05).In the scaffolds of group A, all MSCs expressed vimentin and some MSCs expressed a-SMA. TEHV in two groups showed simliar morphologcicalstructure. And the differences of DNA and hydroxyproline contents,Max-load, Max-stress, elastic modulus and Max-strain between two groupshave no statistical significance (P＞0.05).Conclusion: During the process of TEHV construction using MSCs as seed cells in vitro,the differences of cell biology between MSCs and myofibroblasts werepresent. But the biological and biomechanical properties of TEHV usingMSCs or myofibroblasts as seed cells were similar. And it is necessary toimprove the properties of TEHV using other methods.Part two Construction of Tissue Engineered Heart ValveUsing bFGFObjective: The biological and biomechanical properties of TEHV constructed by MSCsand bFGF were investigated.Methods: MSCs were isolated and cultured. Then MSCs were seeded onto decellularizedaortic valve leaflet scaffold. TEHV were cutlured with DMEM contained 10ng/ml bFGF in vitro for 14 d in group A. In group B, TEHV were cutluredwith DMEM only for 14 d. In group C, TEHV were constructed usingmyofibroblasts as seed cells. The cell biology of MSCs, and the biologicaland biomechanical properties of the TEHV were examed.Results: Group A and C showed comparable expression of a-SMA, MMP-13 andTIMP-1 mRNA (P＞0.05). However the mRNA expression weresignificantly inceased in group A and C, compared with lower values ofgroup B (P＜0.05). Group A and C showed comparable content of LOX andexpression of LOX mRNA (P＞0.05). However the content of LOX and expression of LOX mRNA were inceased in group B, compared with lowervalues of group A and C (P＜0.05). Recellularization of scaffolds in group Awas significantly improved compared to group B and C. And the DNA andhydroxyproline contents were inceased in group A, compared with lowervalues of group B and C (P＜0.05). And Max-load, Max-stress, elasticmodulus and Max-strain of TEHVs between each group have no statisticalsignificance (P＞0.05).Conclusion: During the process of TEHV construction in vitro, when regulated by bFGF,the expression of phenotype of MSCs was equal to myofibroblasts. Theexpression of matrix metalloproteinase and tissue inhibitor ofmetalloproteinase of MSCs was equal to myofibroblasts. The expression ofLOX of MSCs was equal to myofibroblasts. And the biological propertiesof TEHV can be improved. But the biomechanical properties of TEHV cannot be improved by bFGF.Part three Construction of Tissue Engineered Heart ValveUsing Hybrid ScaffoldObjective: The objective of this study was to fabricate hybrid scaffolds using anelectrospinning technique. Then TEHVs were engineered by seeding MSCsonto the scaffolds. The effects of the hybrid scaffolds on the biological andbiomechanical properties of TEHVs were investigated.Methods: MSCs and myofibroblasts were obtained from rats and cultured. In group A,porcine aortic heart valve leaflets were decellularized, coated withpoly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) using anelectrospinning technique. Group B and C were decellularized valve leafletsscaffolds. In group A and B, scaffolds were reseeded by MSCs and culturedover a time period of 14 days. In group C, scaffolds were reseeded by myofibroblasts and cultured over an equivalent time period. Specimens ofeach group were examined biologically and biomechanically.Results: The average diameter of the P3/4HB fibers was 156±3 nm. Electrospunmembrane of the P3/4HB firmly combined with the surface of thedecellularized valve leaflets and showed uniform fibers and microstructure.Recellularization was comparable to the specimens in each group. And thespecimens of each group revealed comparable amouts of cell mass and4-hydroxyproline (P＞0.05). However, the specimens in group A showed asignificantly increase of Max-load, Max-stress and elastic modulus, comparedto group B and C (P＜0.05). And the differences of Max-strain between threegroups were considered no statistical significance (P＞0.05).Conclusion: This study demonstrated the superiority of the hybrid scaffolds to increase thebiomechanical properties of TEHV. And compared to the decellularized valveleaflet scaffolds, the hybrid scaffolds showed similar effects on theproliferation of MSCs and formation of extracellular matrix.Part four Construction of Tissue Engineered Heart ValveUsing Mesenchymal Stem Cells and Hybrid Scaffold with bFGFObjective: TEHVs were constructed using MSCs and hybrid scaffold with bFGF. Andthe biological and biomechanical properties of the TEHV wereinvestigated.Methods: MSCs were obtained from rats and cultured, in group A, porcine aortic heartvalve leaflets were decellularized, coated with bFGF/chitosan/P3/4HBusing slow release and electrospinning technique. In group B,decellularized heart valve leaflets coated with P3/4HB using anelectrospinning technique. In group C and D, the scaffolds weredecellularized heart valve leaflets. The scaffolds in each group werereseeded by MSCs. Scaffolds in group C were cultured in DMEMsupplemented with 10 ng/ml bFGF. However, scaffolds in other groupswere cultured in DMEM without bFGF. Scaffolds in each group were cultured over a time period of 14 d to construct TEHVs. Specimens of eachgroup were examined biologically and biomechanically.Results: The protein-loading capacity of the hybrid valve scaffolds (bFGF) and theirassociation efficiency of were 0.00077% and 88.1%. In vitro release ofbFGF showed that the extent of release was 16.3% at 1d, 42.4% at 5d,51.1% at 10d and 53.9% at 15d. The electrospun membrane ofbFGF/chitosan/P3/4HB firmly combined with the surface of thedecellularized valve leaflets and showed uniform fibers and microstructure.In group A and C, recellularization of the scaffolds was significantlyimproved compared to group B and D. And biochemical analysis revealedan increase of cell mass and the content of 4-hydroxyproline compared togroup B and D (P＜0.05). In group A and B, the specimens showed asignificantly increase of Max-load, Max-stress and elastic modulus,compared to group C and D (P＜0.05), and the differences of Max-strainwere considered no statistical significance (P＞0.05).Conclusion: This study demonstrated the superiority of the hybrid scaffold with bFGFseeded by MSCs to enhance the biological and biomechanical properties ofTEHV. Hybrid scaffold with bFGF could be useful for the generation ofviable, functional heart valve prostheses.更多还原