【作者】 陈思；

【导师】 董念国；

【作者基本信息】 华中科技大学 ， 心血管外科学， 2010， 博士

【摘要】 目的体外分离和培养瓣膜间质细胞和内皮细胞,总结一种方便、快捷、重复性高的瓣膜细胞分离培养方法。方法采用胶原酶消化法从猪主动脉瓣中分离并体外扩增瓣膜间质细胞和内皮细胞。光学相差显微镜每日观察细胞形态。生长良好的2-3代细胞传入培养板,每日计算细胞个数绘制生长曲线。免疫荧光染色行细胞表型鉴定,激光扫描共聚焦显微镜观察并拍照。结果胶原酶消化法可成功从猪主动脉瓣中分离出间质细胞和内皮细胞,1-2周即可获得排列紧密的细胞单层。间质细胞生长排列呈放射状或漩涡状走行,并可互相重叠生长。内皮细胞紧密排列似典型鹅卵石或铺路石样分布。细胞生长曲线示间质细胞生长活跃期为传代后3-5天,内皮细胞为第4-5天。表型鉴定提示瓣膜间质细胞α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)和波形蛋白(vimentin)免疫荧光染色阳性,瓣膜内皮细胞血管性假血友病因子(Von Willebrand factor, vWF)和血小板内皮细胞粘附分子(Platelet-endothelial cell adhesion molecule, PEC AM)染色阳性。结论本研究探索的瓣膜间质细胞和内皮细胞分离培养方法简便、易行、重复性高,是一种理想的瓣膜细胞培养方法。目的建立体外瓣膜细胞钙化模型,诱导主动脉瓣间质细胞钙化,并观察钙化间质细胞表型变化。方法取传代4-8代间质细胞,随机分为2组,实验组以含p-甘油磷酸、维生素C、地塞米松的钙化培养基进行钙化诱导培养,对照组以标准培养基培养,培养2周。显微镜镜下行钙化结节计数,茜素红染色观察并半定量检测钙沉积,检测碱性磷酸酶活性,实时荧光定量逆转录聚合酶链反应(Real Time RT-PCR)检测间质细胞α-SMA及钙化相关因子(骨钙素,骨桥蛋白,核心结合因子al Cbfal)基因表达。结果间质细胞经钙化诱导培养2周可成功诱导钙化,实验组中间质细胞可自发形成钙化结节,数量显著高于对照组(156.25±17.38／孔VS 2.5±1.29／孔,P<0.05),钙沉积检测实验组显著高于对照组(17.52±2.04 VS1±0.22,P<0.05)；钙化诱导组碱性磷酸酶活性较对照组明显升高,2周时活性是对照组的2.3倍(P<0.01)。Real Time RT-PCR提示,实验组α-SMA、骨钙素、骨桥蛋白、Cbfal等mRNA表达水平均较对照组明显升高,差异有统计学意义(P<0.05)。结论瓣膜间质细胞可体外成功诱导钙化,钙化间质细胞呈现相对激活状态,表型向收缩表型和成骨表型转化,可能为瓣膜钙化的细胞学基础。目的构建瓣膜间质细胞和内皮细胞体外3D共培养钙化模型,观察瓣膜内皮细胞在间质细胞钙化过程中的调控作用。方法取4-8代间质细胞单独或与内皮细胞共种植于Ⅰ型胶原凝胶支架内,以含有p-甘油磷酸、维生素C、地塞米松的钙化培养基进行钙化诱导培养2周。测量凝胶向心性收缩评估瓣膜间质细胞收缩功能。茜素红染色观察并半定量检测钙沉积,Live/Dead试剂盒检测钙化细胞存活率,检测碱性磷酸酶活性,免疫荧光染色检测间质细胞表型激活(α-SMA), Real Time RT-PCR检测间质细胞α-SMA及钙化相关因子(骨钙素,骨桥蛋白,Cbfal) mRNA表达水平变化。结果成功构建间质细胞和内皮细胞3D共培养钙化模型。钙化诱导组间质细胞收缩能力强于对照组。茜素红钙沉积染色提示钙化诱导组基质内弥漫性钙化沉积,钙沉积明显高于对照组；凋亡检测和间质细胞表型检测提示钙化间质细胞呈现激活状态,伴随明显细胞凋亡发生以及α-SMA表达上调,碱性磷酸酶活性上升,并高表达骨钙素、Cbfal等钙化相关蛋白及转录因子;内皮细胞共培养可显著降低间质细胞钙沉积,减弱钙化间质细胞收缩能力,并下调α-SMA表达,降低碱性磷酸酶活性,抑制骨钙素、Cbfal等表达,保持骨桥蛋白相对高表达,使激活的间质细胞呈静息状态,缓解钙化。结论体外共培养瓣膜内皮细胞可缓解瓣膜间质细胞钙化的发生,其机制可能在于抑制间质细胞的激活表型变化,使间质细胞呈现相对静息状态。目的探讨并证实主动脉瓣间质细胞激活与瓣膜退行性钙化之间的相关性。方法收集换瓣手术中切除的老年钙化性退行性病变主动脉瓣作为实验组,以扩张型心肌病行心脏移植者主动脉瓣作为对照组。行HE染色观察瓣膜结构,Von Kossa染色观察瓣膜基质钙沉积,免疫组化染色观察Cbfal、骨钙素、α-SMA、波形蛋白表达及分布,逆转录PCR观察Cbfal和骨钙素mRNA表达情况。结果HE染色提示退行性钙化病变瓣膜胶原纤维增生,胶原束排列紊乱。Von Kossa染色提示钙化瓣膜间质内有明显散在点状钙沉积。免疫组织化学染色提示钙化瓣膜内Cbfal阳性表达,细胞外基质内骨钙素散在阳性表达。免疫荧光染色提示钙化瓣膜α-SMA染色阳性而无病变组为阴性或弱阳性。RT-PCR提示退行性钙化瓣膜中骨钙素和Cbfal均相对高表达,而正常瓣膜无表达,且两种mRNA表达强度成正相关。结论钙化瓣膜内间质细胞呈现激活状态,表型向成骨样表型转化。Cbfal与骨钙素表达强度成正相关,提示间质细胞的激活水平与钙化程度直接相关。更多还原

【Abstract】 Objective:To improve the techniques of isolating and culturing porcine aortic valvular interstitial cells and endothelial cells in vitro.Methods:Interstitial cells and endothelial cells were isolated from fresh harvested porcine aortic valves by collagenase digestion. Cellular morphologic change was observed under phase contrast microscope. Cell proliferation was detected by direct count and shown as growth curve. Immunocytochemical staining was used to identify the purities of cell populations.Results:Enzymatic digestion methods yielded aortic interstitial and endothelial cells in vitro successfully. Cellular monolayer was formed after 2 weeks of culture. Cultured interstitial cells were spindle-shaped and overlapped layers of parallel, whereas endothelial cells displayed a cobblestone morphology. The growth curve showed that the active growth phase was during 3rd-5th day after passage for interstitial cells, and 4th-5th day for endothelial cells. Interstitial cells were positive stained forα-SMA and vimentin, and endothelial cells positive stained for vWF and PECAM.Conclusion:The results of these studies provided recommendations for the improved protocol for isolating and culturing interstitial cells and endothelial cells from porcine aortic valve. Objective:to construct an induced calcification model of aortic valves by inducting calcification in porcine aortic valvular interstitial cells (VICs) in vitro.Methods:VICs were used for experiments after 4-8 passages. Cells were cultured in osteogenic media or in normal media. After 2 weeks, calcified nodules were quantified by direct counting under microscope. Calcium deposit was stained and measured by Alizarin Red S staining and assay. Activity of alkaline phosphatase was measured. mRNA expression of alpha smooth muscle actin (a-SMA) and calcification related factors such as osteocalcin, osteopontin and Core-binding factor a/Runx2 (Cbfal/Runx2) were measured by real time reverse transcription PCR.Results:VICs could calcify after 2 weeks of osteogenic induction with calcified nodules formed. Quantification of calcified nodules and calcification deposit were higher in experimental group than in control group (156.25±17.38 VS 2.5±1.29,17.52±2.04 VS 1±0.22). Activity of ALP was higher in experimental group than that of control group (5.286U/mg vs 2.298U/mg). Real Time RT-PCR indicated that expression of a-SMA, as well as calcification related markers like osteocalcin, osteopontin and Cbfal/Runx-2 was much higher in experimental group that those of control group (P＜0.05).Conclusion:VICs were activated during progress of calcification with phenotype shifting to contraction and ossification, which might be the pathological basis of valvular calcification. Objective:To observe the regulation of valvular endothelial cells on interstitial cells during calcification in a 3D co-culture model.Methods:Porcine aortic valvular interstitial cells (VIC) and endothelial cells (VEC) were isolated and expanded by collagenase methods. VIC at 4-8 passages were cultured with or without VEC in collagen I gel scaffolds, and osteogenic media was used to induce calcification of interstitial cells for up to 2 weeks. Calcium deposit was stained and measured by Alizarin Red S staining and assay, viability through Live/Dead assay, interstitial cell phenotype through alpha smooth muscle actin (α-SMA) immunohistochemistry and Real Time RT-PCR, activity of alkaline phosphatase. calcification related factors (osteocalcin and Cbfal) through Real Time RT-PCRResults:VICs could calcify after 2 weeks of osteogenic induction in collagen I gel scaffolds. Calcification was associated with increased apoptosis, stronger compaction of gels, higher activity of alkaline phosphatase, and upregulation of a-SMA expression and as well as calcification related markers like osteocalcin and Cbfal. The presence of VECs reduced the amount of calcification with a significant decrease in activated interstitial phenotype, indicated by lower calcium deposit, decreased gel compaction, reduced activity of alkaline phosphatase, and downregulated expression of a-SMA, osteocalcin and Cbfal mRNA.Conclusion:Co-culture with valvular endothelial cells in vitro could relieve calcification of interstitial cells, indicating that VEC may maintain tissue homeostasis by limiting VIC activation. Objective:To study the correlation between the expression of calcification related factors and degenerative calcified aortic valves.Methods:Degenerative calcified aortic valves were collected during aortic replacement surgeries as experimental group samples, whereas aortic valves from heart transplantation as control. HE staining was performed to indicate the structure of valves. Von kossa staining was used to show calcium deposit in diseased valves. Cbfal, osteocalcin, a-SMA and vimentin were stained by immunofluorescence. Expression of Cbfal and osteocalcin mRNA were measured via reverse transcription PCR.Results:HE staining showed fibroplasias and mess collagen array. Von Kossa staining confirmed the presence of calcification deposit in calcified valves. Immunofluorescence showed distribution of Cbfal and osteocalcin in calcified valves, and positive expression of a-SMA in calcified valves while negative in control group samples. RT-PCR revealed the increased mRNA levels of osteocaclin and Runx2 in degenerative calcified valves, and there was positive correlation between them.Conclusion:Interstitial cells in calcified valves were activated, showing a osteoblast differentiated phenotype. There is positive correlation between the expression of Cbfal and osteocalcin, indicating that the interstitial cell activation level is directly related to the valvular calcification level.更多还原