人骨髓基质干细胞构建组织工程心脏瓣膜的研究

取心血管外科手术患者胸骨骨髓,Ficoll离心法获取单个核细胞,体外培养扩增后种植在猪去细胞主动脉瓣叶表面2周,观察细胞生长情况;免疫组化检测细胞分化结果;扫描电镜和透射电镜观察细胞种植情况.结果 经体外培养的入骨髓基质干细胞在去细胞猪主动脉瓣叶纤维网状支架表面形成一层基本连续的细胞层.认为去细胞猪主动脉瓣叶组织是良好的纤维支架,可用于构建组织工程心脏瓣膜;人骨髓基质干细胞种植在去细胞猪主动脉瓣叶上构建组织工程心脏瓣膜是可行的.     

心瓣膜组织工程中种子细胞原代联合培养的实验研究

目的：探讨体外原代联合培养、扩增,鉴定同一来源的人血管内皮细胞、平滑肌细胞、成纤维细胞的方法,为组织工程心脏瓣膜的构建提供理想的种子细胞。方法：无菌条件下取冠状动脉旁路移植术中剩余大隐静脉3cm～8cm,组织块培养法原代培养混合血管细胞,DMEM培养液培养、扩增混合血管细胞。倒置显微镜、免疫组化方法对内皮细胞、平滑肌细胞、成纤维细胞进行鉴定。结果：倒置显微镜显示原代细胞及经传代培养后的细胞,呈现内皮样细胞、成纤维样细胞混合生长的特征。免疫细胞化学检测示内皮细胞Ⅷ因子、平滑肌细胞α-肌动蛋白、成纤维细胞纤维连接蛋白相关抗原呈阳性反应。细胞经3～4次传代,细胞数量可增殖达到8×10~6～10×10~6。结论：使用组织块法原代培养可获得内皮细胞、平滑肌细胞、成纤维细胞,三种细胞经体外联合培养、传代可以达到组织工程心脏瓣膜种子细胞的足够数量。     

心瓣膜组织工程中种子细胞原代联合培养的实验研究

目的:探讨体外原代联合培养、扩增,鉴定同一来源的人血管内皮细胞、平滑肌细胞、成纤维细胞的方法,为组织工程心脏瓣膜的构建提供理想的种子细胞.方法:无菌条件下取冠状动脉旁路移植术中剩余大隐静脉3 cm～8 cm,组织块培养法原代培养混合血管细胞,DMEM培养液培养、扩增混合血管细胞.倒置显微镜、免疫组化方法对内皮细胞、平滑肌细胞、成纤维细胞进行鉴定.结果:倒置显微镜显示原代细胞及经传代培养后的细胞,呈现内皮样细胞、成纤维样细胞混合生长的特征.免疫细胞化学检测示内皮细胞Ⅷ因子、平滑肌细胞α-肌动蛋白、成纤维细胞纤维连接蛋白相关抗原呈阳性反应.细胞经3～4次传代,细胞数量可增殖达到8×106～10×106. 结论:使用组织块法原代培养可获得内皮细胞、平滑肌细胞、成纤维细胞,三种细胞经体外联合培养、传代可以达到组织工程心脏瓣膜种子细胞的足够数量.     

聚乙二醇载药缓释微球复合去细胞瓣制备组织工程心脏瓣膜支架

目的:制备聚乙二醇(PEG)载药缓释微球复合去细胞瓣组织工程心脏瓣膜(TEHV)支架。方法:制备PEG微球,电镜观察粒径。去细胞瓣与PEG微球偶联,吸附转化生长因子(TGF-β1)。行ELISA检测,分子生物学、形态学和生物力学检测。结果:PEG微球粒径(42.72+3.48)nm。酶联免疫吸附检测示缓释效果明显,7dTGF-β1释放率67.22%,PEG微球的TGF-β1包封率为82.01%。与单纯去细胞瓣膜比较,复合支架羟脯氨酸含量和DNA含量无显著变化,形态学检测显示复合支架细胞外基质联合紧密,胶原纤维结构紧凑,且瓣叶生物力学性能提高。结论:制备PEGTGF-β1缓释微球去细胞瓣复合支架可行。     

转化生长因子-β_1基因转染构建组织工程瓣膜

目的:转化生长因子(TGF)-β1基因转染细胞,研究对天然支架组织工程瓣膜(TEHV)构建作用.方法:脂质体介导pcDNA3.0/TGF-β1基因载体进入大鼠肌成纤维细胞(MFB),48 h和4周后G418筛选法培养,SABC法检测TGF-β1表达.A组将转基因细胞种植去细胞猪主动脉瓣叶,B组种植未转基因细胞,C组单纯支架,体外培养2周构建TEHV.苏木精-伊红染色、扫描电镜观察,测定羟脯氨酸、DNA含量和最大负荷力.结果:基因转染48 h和4周,MFB瞬时和稳定表达TGF-β1蛋白.A组较B组细胞生长旺盛,胶原分泌较多,力学性能增强.结论:TGF-β1基因转染MFB作为种子细胞,能较好发挥TGF-β1活性,促进TEHV构建.     

丹参对慢性低氧性肺动脉高压大鼠肺组织bFGF表达的影响

目的 观察丹参对慢性低氧性肺动脉高压大鼠肺组织碱性成纤维细胞生长因子(bFGF)表达的影响,探讨丹参阻抑肺腺泡内动脉(IAPA)重建的机制。方法 Wistar大鼠随机分为3组:正常对照组、慢性低氧组、低氧丹参治疗组。低氧大鼠置于常压低氧舱3周后,测定血流动力学;观察IAPA结构变化,用免疫组化检测肺组织bFGF表达;用氯胺T比色法测定肺组织羟田氨酸含量。结果 丹参能降低平均肺动脉压,减少肺组织羟脯氨酸含量(P<0.05,P<0.01),抑制IAPA平滑肌细胞增生,降低肺组织bFGF表达强度 结论(1)丹参能抑制IAPA平滑肌细胞增生及胶原沉积而阻抑IAPA的结构重建。(2)抑制bFGF表达是丹参调节IAPA平滑肌细胞增生的重要途径之一。     

聚乙二醇去细胞瓣复合支架联合人主动脉瓣膜间质细胞体外构建组织工程心脏瓣膜

目的:运用聚乙二醇(PEG)交联猪去细胞瓣,共价接枝GRGDSPC多肽(甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-脯氨酸-半胱氨酸),并联合人主动脉瓣膜间质细胞(HAVICs)体外构建组织工程心脏瓣膜。方法:通过迈克尔加成反应,将去细胞瓣支架上引入的巯基与枝化状PEG末端丙烯酰基共价结合,使PEG交联去细胞瓣,并行生物力学检测。再利用PEG-去细胞瓣支架上未饱和的丙烯酰基与GRGDSPC多肽末端半胱氨酸的巯基发生迈克尔加成反应,对PEG-去细胞瓣支架行GRGDSPC多肽共价修饰,免疫荧光测定GRGDSPC多肽修饰效果。在RGD-PEG-去细胞瓣复合支架上种植HAVICs,缝制于生物反应器内,体外构建心脏瓣膜。接种2、4、8h后,通过细胞计数观察细胞粘附情况。体外培养10d后行形态学观察和DNA含量测定。结果:力学测试结果显示,PEG-去细胞瓣支架的最大抗张强度较单纯去细胞瓣支架明显提高[(7.53±0.32)Mpa∶(5.65±0.28)Mpa],差异有统计学意义(P<0.05),与天然主动脉瓣的差异无统计学意义(P>0.05)。免疫荧光检测显示,GRGDSPC多肽可有效地与PEG-去细胞瓣共价接枝。不同时段细胞粘附计数及形态学观察提示,RGD-PEG-去细胞瓣复合支架明显促进HAVICs的粘附,并可在瓣膜表面形成连续单细胞层。DNA含量测定提示,RGD-PEG-去细胞瓣复合支架DNA含量明显增高(P<0.05)。结论:PEG交联去细胞瓣可明显改善瓣膜支架力学性能,GRGDSPC多肽接枝的PEG-去细胞瓣复合支架,可促进种子细胞的粘附,改善瓣膜支架生物学性能。     

体外构建组织工程化同种带瓣管道的研究

利用密度梯度离心法分离出胸骨骨髓10ml间充质干细胞(HMSCs),体外培养、扩增、纯化后加入特定生长因子将其定向诱导分化为内皮细胞(ECs)并作为种子细胞;液氮保存的人同种带瓣管道表面的ECs利用SDS及EDTA等去污剂完全脱去,并作为种植支架。将已培养好的ECs高密度种植在管道支架上,静态培养2周后用扫描电镜观察管道表面再内皮化程度。结果HMSCs在特定环境下定向分化为ECs;人同种带瓣管道表面的ECs被完全脱去,而中心部分的基质细胞及弹性纤维、胶原纤维完整保留;体外构建的组织工程化带瓣管道表面重新覆盖了完整的ECs单层。证实将HMSCs定向诱导分化后的ECs种植于脱ECs的人同种带瓣管道上,构建同种组织工程化带瓣管道具有可行性。     

H2O2诱导体外培养的皮肤成纤维细胞氧化应激损伤

目的探讨H2O2对体外培养的人皮肤成纤维细胞生物学特性的影响,从细胞水平为抗皮肤衰老药物的研究提供较理想的皮肤衰老模型。方法用不同浓度的H2O2(50～300μmol/L)干预体外培养的正常皮肤成纤维细胞,MTT比色法检测细胞增殖,流式细胞仪检测细胞凋亡,羟脯氨酸比色法检测胶原含量,紫外分光光度计测定细胞中超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量。结果 H2O2浓度依赖性地抑制皮肤成纤维细胞增殖,促进细胞凋亡,降低细胞SOD的活性,减少细胞胶原含量,增加MDA含量。结论氧化应激可损伤皮肤成纤维细胞,用H2O2致皮肤成纤维细胞氧化损伤模型可用于抗皮肤衰老药物的研究。     

肥大细胞和类胰蛋白酶与心血管疾病

肥大细胞分泌的类胰蛋白酶具有多种生物学活性。这些活性与某些心血管疾病的发生发展密切相关。实验证实,在扩张型心肌病和缺血性心肌病患者的心脏组织中,类胰蛋白酶的含量要高于正常心脏组织。活化的肥大细胞可以通过多种机制降解细胞外基质,其中类胰蛋白酶促进基质金属蛋白酶的生成起到了关键的作用。此外,类胰蛋白酶能够降解纤维蛋白原,诱导内皮细胞、成纤维细胞及平滑肌细胞的增殖,降解纤维连接蛋白。它的这些活性在动脉粥样硬化、血管重构和新生以及血管内皮细胞和平滑肌细胞凋亡的过程中均起到了重要的作用。     

Absent aortic valve leaflets

A newborn with difficulty in feeding and with tachypnoea was admitted to the hospital. During the following days his condition deteriorated and he died at the age of 8 days. At autopsy we found an unusual association of hypoplastic left heart syndrome and absence of the aortic leaflets.     

Congenital absence of pulmonary valve leaflets.

Congenital absence of pulmonary valve leaflets is an uncommon condition usually associated with ventricular septal defect and an obstructive pulmonary valve ring. Twenty-one patients with these malformations are described. Twenty had an associated ventricular septal defect with ventriculoarterial concordance, and one also had transposition of the great arteries, ventricular septal defect, and obstructive pulmonary valve ring. The clinical features, cardiac catheterisation findings, and angiocardiographic results are presented. Twelve patients underwent cardiac surgery. Three patients died, one in the early, and the other two in the late postoperative period. The results, according to the surgical technique employed and postoperative cardiac catheterisation findings, showed that patients in whom the bioprostheses were implanted in the pulmonary position had a better late follow-up.     

Early in vivo experience with tissue-engineered heart valve leaflets from autologous bone marrow-derived cells

Background Currently used heart valve prostheses are associated with anticoagulation complications or limited durability. The advancement of stem cell study and tissue-engineered heart valve research may offer a relatively ideal solution to these problems. Methods Bone marrow was aspirated from sternum of lamb goats to isolate BMCs. Cells were identified by flow cytometry and its capacity of differentiation. Cellular viability was assessed with Rhdomine 123 staining. 1 × 107 cells were seeded on a patch of ...     

Endothelial cell orientation on aortic valve leaflets

Studies of the structure of blood vessels have commonly found that cells lining the vessels are lengthened in the direction of blood flow, presumably as an effect of shear stress. A study of aortic valves has, however, shown that endothelial cells covering both sides of aortic valve leaflets are arranged across the direction of flow. Aortic valve leaflets taken from seven adult mongrel dogs and fixed either open or closed with glutaraldehyde were studied with a scanning electron microscope after critical point drying. On both open and closed leaflets the endothelial cells tended to be aligned circumferentially with the free edge of the leaflet. The circumferential pattern was particularly unexpected on the leaflet surface facing the left ventricle because this surface could be expected to receive the full effect of shear stress from systolic blood flow. The finding suggested that surface cellular alignment on leaflets is determined by some force other than shear stress. The force apparently responsible for organising the collagenous layers in the leaflet, which are best adapted to bear stress, is diastolic blood pressure. Since the endothelium follows the same pattern of alignment as the layers of collagen it seems reasonable to conclude that endothelial orientation is also a response ultimately to pressure stress. The arrangement of the endothelium then serves as a readily observed indicator of those functional stresses that dominate and organise leaflet structure.     

Echocardiographic evaluation of normal and prolapsed tricuspid valve leaflets

The tricuspid valve was examined by 2-dimensional (2-D) echocardiography in 14 patients with tricuspid valve prolapse (TVP) and in 16 normal subjects. Individual leaflets were identified anatomically and for frequency of prolapse. Maximal and minimal anular sizes were measured. Multiple tomograms of the tricuspid anulus were recorded at 30 degrees intervals around the tricuspid anulus with the transducer placed at the right ventricular apex. Anuli were reconstructed from the 6 planes and corrected for body surface area. Three leaflets of the tricuspid valve could be anatomically identified in all patients. Prolapse of all 3 leaflets was observed in 6 patients, 2 leaflets in 5 and 1 in 3. Frequency of individual leaflet prolapse was 93% for the septal cusp, 86% for the anterior and 43% for the posterior. Maximal anular circumference and area in TVP were 7.9 +/- 0.6 and 8.9 +/- 1.3 cm2/m2, respectively--significantly larger than values in normal subjects (6.4 +/- 0.5 cm/m2 and 6.1 +/- 0.9 cm2/m2, respectively) (p less than 0.001). Percent reductions in circumference and area in TVP were 14 +/- 3 and 25 +/- 5%, respectively--significantly smaller values than in normal subjects (19 +/- 4 and 33 +/- 4%, respectively). Tricuspid regurgitation (TR) was detected by contrast echocardiography in 7 of 14 patients with TVP. The severity of TR appeared to be minimal in 6 of the 7 patients, and was not associated with an increase in anular size. Thus, TVP is associated with anular dilatation irrespective of associated TR, probably as a primary pathologic characteristic.