【作者】 徐鹏；

【导师】 张凯伦；

【作者基本信息】 华中科技大学 ， 外科学， 2008， 博士

【摘要】 第一部分聚乙二醇(PEG)制备猪组织工程去细胞瓣膜及其脱细胞效率的研究目的探讨PEG法在组织工程瓣膜准备中的应用价值,比较PEG不同处理时间后的去细胞效率和组织保留情况,确定适宜的PEG去细胞方案。方法猪主动脉瓣膜分为去细胞组和正常对照组。对照组取材后仅以磷酸盐缓冲液(PBS)浸泡;去细胞组用PEG和DNase I处理,按照PEG的处理时间又分为20分钟、35分钟、45分钟三组。处理后样本用苏木精—伊红染色,扫描电镜观察去细胞情况,DNA含量测定核酸去除,计算去细胞百分率;比较不同组的脱细胞效率;测定和比较不同组样本的组织厚度、组织含水量。SDS-PAGE电泳分析组织蛋白处理前后改变情况。结果组织切片观察:对照组瓣膜具有典型的三层结构,各层间均有细胞分布但以纤维层和海绵层为多;PEG处理组,瓣膜仍然具有类似对照组的三层结构,细胞外基质(ECM)结构保存完整,胶原纤维排列整齐,无明显断裂,仍呈波浪状平行排列,结构紧凑,组织无明显水肿。20分钟组与35分钟组可见细胞分布,45分钟组各层未见细胞。DNA含量测定:20分钟组、35分钟组和45分钟组去细胞百分率分别为28.17±9.58%、40.62±7.94%和95.32±3.61%。45分钟组与20分钟组和35分钟组有显著差异而后两组间无显著差异;对照组、20分钟组、35分钟组、45分钟组的组织厚度分别为:0.39±0.05mm、0.41±0.06mm、0.43±0.05mm、0.44±0.07mm;组织含水量分别为:88.37±4.27%、90.81±2.21%、91.47±2.63%、91.89±3.01%。各组组织厚度和组织含水量无显著差异。对照组和45min组蛋白提取物的浓度分别为0.46±0.07 mg/mL和0.15±0.04 mg/mL;SDS-PAGE电泳显示,在蛋白分子量25kDa~250kDa范围内,PEG组蛋白条带虽然很细微,但仍然可见。结论PEG处理45分钟法去除细胞完全,细胞外基质保存完整,与对照组比较组织厚度、组织含水量无显著差异;PEG处理能够减少蛋白数量,但对其成分改变不大。45分钟PEG处理可以有效制备猪去细胞瓣。第二部分聚乙二醇制备猪去细胞主动脉瓣膜平面双轴生物力学测定目的验证PEG法在组织工程瓣膜准备中的应用价值,观察猪主动脉瓣叶经PEG处理时间后的生物力学性质,并和天然猪主动脉瓣叶进行比较。方法猪主动脉瓣膜分为PEG组和正常对照组。瓣叶处理方案分别为:对照组,取材后仅以磷酸盐缓冲液(PBS)浸泡;PEG组,PEG浸泡振摇45分钟和DNase I处理。处理后各组部分样本用苏木精—伊红染色观察去细胞情况,DNA含量测定核酸去除,计算去细胞百分率;部分样本沿瓣叶径向和周向裁减测样,测定组织厚度后用AGS-J电子测试仪行拉伸实验获得最大应力、最大应变、弹性模量和应力—应变曲线。结果组织切片观察:对照组瓣膜具有典型的三层结构,各层间均有细胞分布但以纤维层和海绵层为多;PEG处理组,瓣膜具有三层结构,细胞外基质(ECM)结构保存完整,各层未见细胞。DNA含量测定:PEG组去细胞百分率为94.48±4.32%。正常组、PEG组的瓣叶组织厚度分别为:0.39±0.05mm、0.41±0.05mm,各组组织厚度无显著差异。力学检测,对照组、PEG组都表现出两轴向力学性能不同的各向异性特征,对照组和PEG组具有基本相同的应力—应变曲线。径向两组最大应力、最大应变、弹性模量分别为:2.831±1.036MPa、2.496±1.251MPa;0.387±0.127、0.573±0.143;17.671±0.957 MPa、16.552±1.038 MPa周向两组最大应力、最大应变、弹性模量分别为:5.103±1.078 MPa、4.897±0.989 MPa;0.207±0.059、0.219±0.214;36.854±1.566 MPa、35.741±1.207MPa。结论PEG处理45分钟法去除细胞完全,细胞外基质保存完整,脱细胞瓣叶应力—应变曲线和对照组基本相同,仍具有两轴各向异性特点;两组平面两轴向最大应力、弹性模量和周向最大应变无显著差异,径向最大应变增加。PEG制备去细胞瓣力学性能满足组织工程瓣膜要求。第三部分聚乙二醇(PEG)处理猪主动脉去细胞瓣膜支架的免疫反应性研究目的测定PEG处理后的猪主动脉瓣膜支架的免疫反应活性;并和未经过PEG处理的猪主动脉瓣膜支架进行比较。为PEG瓣膜支架的进一步运用寻找实验依据。方法采用PEG浸泡45min法处理猪主动脉瓣膜,制备去细胞瓣膜支架。选择BALB/C小鼠,采用颈部皮下包埋瓣膜。实验分为对照组和PEG组,对照组为新鲜瓣膜皮下包埋; PEG组为去细胞瓣膜皮下包埋。瓣叶组织统一剪裁为1×1cm2大小,于BALB/C小鼠颈部皮下包埋。各组分别于手术后5d、10d、20d各取小鼠六只处死后,取出皮下瓣膜和小鼠脾脏行HE染色观察组织变化情况;取全血离心后得到血清,行ELISA检测IL-2、IL-10定量分析,了解免疫活性;脾脏组织行RT-PCR检测了解IL-2、IL-10转录翻译活性并半定量分析,两组间比较。结果两组小鼠均无实验期间死亡。HE染色显示对照组免疫炎症反应较PEG组发生时间早、程度重,并且组织结构破坏较重。ELISA结果:两组比较,在不同时间点,对照组的IL-2水平均较PEG组显著增高(p<0.05);同一组内,IL-2水平成波浪样曲线,5d开始上升,在10d最高,20d较10d下降;而两组比较,在不同时间点,IL-10水平无显著差别(p>0.05);同一组内,IL-10水平呈逐渐上升的趋势。IL-2以及L-10的RT-PCR结果也呈现出类似ELISA结果的趋势。结论PEG处理后的瓣膜细胞以及细胞膜抗原成分去除完全,皮下植入后免疫炎症程度弱,时间短,组织结够破坏少,并且易于自体细胞长入。适于作为TEHV的理想支架,但其最终植入心脏瓣膜部位后的效果仍需要实验证实。第四部分聚乙二醇(PEG)处理猪主动脉去细胞瓣膜支架细胞黏附效率及不同预处理对黏附率影响的研究目的测定PEG处理后的猪主动脉瓣膜支架的细胞黏附效率;并比较其经过不同预处理后对细胞黏附率影响,为进一步完成瓣膜支架在细胞化寻找实验依据。方法选择昆明白小鼠,采用组织贴块法,原代培养主动脉成纤维细胞,选用5～8代细胞用于细胞黏附率测定。实验分为PEG组、PEG+明胶(gelatin)组、PEG+多聚赖氨酸(Poly-L-Lysine,PLL)组和PEG+胎牛血(FBS)清组。PEG组为经PEG和DNase I处理的猪主动脉瓣叶;PEG+gelatin组、PEG+PLL组和PEG+FBS组为PEG处理后瓣叶在测定细胞黏附率前,先由相应物质包被瓣叶行预处理。各组瓣叶组织大小统一为1×1cm2,置于24孔板内,每个样本按5×105数量种植细胞,种植1h后,取出玻片和瓣叶组织,培养基洗脱其上未黏附细胞。24孔板进行MTT检测未黏附细胞数量,与总细胞数相减得到黏附细胞数,计算黏附效率并比较。PEG组取出瓣叶组织放入培养瓶静态培养2周,HE染色观察细胞生长和分布情况。结果成功有效培养出小鼠主动脉成纤维细胞; PEG组、PEG+ gelatin组、PEG+ PLL组、PEG+ FBS组的细胞黏附率为:42.63%±5.19%,69.22%±6.63%,65.78%±4.45%,85.32%±3.61%。经过预处理后各组细胞黏附率均显著高于PEG组,其中以胎牛血清效果更加显著。静态培养两周后组织切片观察:瓣叶组织三层结构,细胞生长良好,各层均有细胞分布,表层细胞数量较深部多,可见有形成连续排列的细胞层。结论组织贴块法培养主动脉成纤维细胞方便有效。PEG处理瓣叶可以黏附细胞,处理瓣叶组织经预处理后可以获得更加理想的细胞黏附率。更多还原

【Abstract】 Part One Primary Study on Decelluarization Rate of Poly Ethylene Glycol in preparing porcine Tssue Egineering Hart VlvesObjective: To study the feasibility of a new decellularization method in preparing porcine tissue engineering heart valves by poly ethylene glycol, determine the decelluarity efficiency and the preservation of the ECM by the method. Methods: The porcine aortic valves were harvested from slaughterhouse and divided into control group and PEG treated group. The specimens in control group were sink in phosphate buffer saline (PBS), while that in PEG group were decellularized with poly ethylene glycol and DNAse I. The PEG group was subdivided into 20min, 35min and 45min subgroup based on the time in PEG. Histochemical studies were performed on sequential valve sections dyed with hematoxylin and eosin. Specimens also were observed by scanning electron microscope (SEM). The content of DNA was detected by photometer. And the thickness and amount of water in specimens were measured in each group. The change in tissue protein was evaluated by SDS-PAGE electrophoresis. Results: In the light microscope, the control group has typical tri-layer structure as native valves. The cells were separated in all three layers and were predominant in spongiosa and fibrosa; the PEG group also has tri-layer structure and the extra cellular matrix (ECM) was well reserved. The collagen was compact and no apparent split. Cells could be seen in 20min and 35min subgroup, but no signs in 45min subgroup. The decellularity percentage could be calculated by DNA concentration in PEG subgroups, which were 28.17±9.58%、40.62±7.94%、95.32±3.61% respectively. The difference between 45min and 20min、35min was significant, which was not between 20min and 35min. the thickness of specimens in four groups were 0.39±0.05mm、0.41±0.06mm、0.43±0.05mm、0.44±0.07mm, and the amount of water in valves were 88.37±4.27%、90.81±2.21%、91.47±2.63%、91.89±3.01%, respectively. The difference has no significance. Extracted protein of nature and PEG treated porcine aortic valves were 0.46±0.07 mg/mL and 0.15±0.04 mg/mL. However, within the examined molecular weight range, protein bands were still detectable by SDS-PAGE within the decellularized porcine aortic valve. Conclusion: Sinking in PEG for 45min could remove celluar components effectively; the extra cellular matrix (ECM) was well reserved; the thickness and amount of water in valves had no significant change. The decellularization procedure removes considerable amounts of proteins within the porcine aortic valves. The method has value in preparing porcine decelluarization heart valves.Part Two Primary Study on plane Biaxial Mechanical Properties of the PEG Treated and Natural Porcine Aortic Valve CuspObjective: To validate the reliability of the method of preparing porcine decellularization tissue engineering heart valves by poly ethylene glycol, determine and compare the biaxial mechanical properties of PEG treated and natural porcine aortic valve. Methods: The porcine aortic valves were divided into control group and PEG group. The specimens in control group were sink in phosphate buffer saline (PBS), while that in PEG group were decellularized with poly ethylene glycol and DNAse I. Histochemical studies were performed on sequential valve sections dyed with hematoxylin and eosin. The content of DNA was detected by photometer. Rectangle specimens were dissected from the central belly region of the leaflet in either radial or circumferential directions. After the thickness of the specimens was measured in each group, the specimens were mounted onto the AGS-J device and got tensile test. The stress-strain curve, max-stress, max-load, max-strain and Elastic modulus were got from the experimental data. Results: in the light microscope, the control group has typical tri-layer structure as native valves. The cells were separated in all three layers and were predominant in spongiosa and fibrosa; the PEG group also has tri-layer structure and the extra cellular matrix (ECM) was well reserved, no cellular signs could be seen. The decellularizaty percentage was 94.48±4.32%. The thickness of specimens in two groups was 0.39±0.05mm and 0.41±0.05mm. The two groups had similar stress-strain curve, and there was difference in mechanical properties between radial and circumferential directions. The max max-stress, max-strain and elastic modulus of the two groups were 2.831±1.036MPa、2.496±1.251MPa;0.387±0.127、0.573±0.143;17.671±0.957 MPa、16.552±1.038 MPa in radial direction and 5.103±1.078 MPa、4.897±0.989 MPa ; 0.207±0.059、0.219±0.214 ; 36.854±1.566 MPa、35.741±1.207MPa in circumferential direction, respectively. Conclusion: Sinking in PEG for 45min could remove celluar components effectively; the thickness of valves had no significant change. The two groups had character of mechanical anisotropy. There was no significant difference in max-stress and elastic modulus in radial or circumferential direction between the two groups. The PEG trated porcine arotic valve has biaxial mechanical properties and could be an alternative for tissue engineering heart valve scaffold.Part Three Evaluation on the immunogenic property of PEG treated porcine tissue engineering valvesObjective: To compare and evaluate the immunogenic property of nature porcine aortic valve and PEG treated porcine decellularated tissue engineering heart valves. Methods: The nature porcine aortic valves were treated with PEG as the method we used previously. The nature valves and PEG treated valves were cut into 1×1cm2. The BALB/C mice were divided into two groups: control and PEG groups, which were then subcutaneous embedded with nature and PEG treated valves in nape, respectively. On 5d, 10d, 20d after valves were subcutaneous embedded, six mice in each group were executed and the valves and spleen were explanted and dyed with hematoxylin and eosin. The serum was got from the whole blood and then the content of IL-2, IL-10 in serum was measured by ELISA assay. The transcription activities of IL-2, IL-10 in mice spleen were self-quantity measured by RT-PCR. The result of them was compared between the two groups. Results: There were no deaths between the two groups in the time of experiment. The histology examination showed that the rejection happened in control group was earlier and severer than PEG group. And the tissue construction was destroyed badly in control group than that in PEG group. The result of ELISA assay showed that at each sample time point, the content of IL-2 in control group was higher than PEG group; and the curve of IL-2 was wave like, up from 5d, max at 10d, down but still high at 20d. There no significant difference between the two groups in the content of IL-10 at each time point. the curve of IL-10 was escalated. The result of RT-PCR of IL-2 and IL-10 got the same trend as that of ELISA. Conclusion: PEG treated porcine heart valves get rid of the immunogenicity thoroughly. The rejection happened after subcutaneous embedded is moderate and transitory. There have little tissue construction destroyed, and the fibroblast could adhere and live in easily. The decelluarationed valve is suit for TEHV scsffold.Part Four Evaluation on the cell adhesion efficiency of PEG treated porcine tissue engineering valves and the impact of surface modificationObjective: To get in sight of the cell adhesion efficiency of PEG treated porcine decellularated tissue engineering heart valves, and evaluate the impact of surface modification on cell adhesion efficiency of PEG treated valves. Methods: The thoracic aorta of kunming mouse was separated carefully and cut into small tissue pieces. The explants were seeded onto culture flasks. Cell was observed through phase contrast microscope and fibroblasts from 5-8 generations are applied to cell adhesion experiment. All the valve specimens were cut into 1cm×1cm and divided into PEG group, PEG + gelatin group, PEG + Poly-L-Lysine (PLL) group, PEG + fetal bovine serum (FBS)group. The specimens in PEG group were PEG treated valves with no surface modification, while that in PEG + gelatin group, PEG + PLL group, PEG + FBS group were PEG treated valves pre-coated with gelatin, poly-l-lysine and fetal bovine serum before experiment, respectively. These specimens were put into 24 cell culture medium. 5×105 fibroblasts were added into every cell and culture for 1 h. After that the valves were took out and washed out carefully to get the fibroblasts that did not adhere to the valve could stay in the culture medium. The number of cells in culture medium was got by MTT assay, so the number of cells adhere on valve can be calculated. Some of the valves in PEG group were transferred into culture flasks and static culture for two weeks, and then the valves were checked by histology. Results: The fibroblast can be culture and harvest from the method of tissue pieces culture effectively. The rate of cell adhere to the valves in PEG group, PEG + gelatin group, PEG + PLL group and PEG + FBS group was 42.63%±5.19%,69.22%±6.63%,65.78%±4.45%,85.32%±3.61%, respectively. The valves in surface modification groups has significant higher cell adhere rate than that in PEG group. After two weeks static culture, the histology examination showed that the valve preserved tri-layer structure as native valves. The cells were separated in all three layer and the number of them in superficial was more than that in deeper. The continued cell line could be seen in the surface. Conclusion: PEG treated porcine heart valves have excellent in vitro biocompatibility; the valve scaffold can obviously improve cell adhere efficiency after surface modification.更多还原