【作者】 步雪峰；

【导师】 吴浩荣；

【作者基本信息】 苏州大学 ， 普外科， 2009， 博士

【摘要】 第一部分:鼠骨髓来源的内皮祖细胞的体外培养及生物学特性的研究目的将鼠骨髓来源的内皮祖细胞(endothelial progenitor cells , EPCs)诱导培养成内皮细胞(endothelial cells , ECs),鉴定并观察其在体外增殖、分化过程中相关细胞表型的变化。方法采用密度梯度离心方法获得鼠骨髓单个核细胞,分别用EGM-2培养剂进行诱导分化(实验组)和普通培养基培养(对照组),于培养4、7、10、14、21 d,用免疫荧光双标技术(CD34、VWF和FLK1等)鉴定EPCs,用MTT法分析EPCs增殖情况。于诱导分化后的1、4、7、10、14、21 d,用western blotting法检测VWF蛋白的水平,用RT-PCR和Real-time PCR法检测VEGFR 2(FLK1)、VWF的mRNA表达变化,并与未诱导培养的细胞进行比较。结果鼠骨髓单个核细胞在诱导培养的4 d细胞开始贴壁,7 d细胞大多变为梭形,排列成“铺路石”样,而对照组的细胞生长状态差,形态不如典型的“铺路石”状;MTT结果表明10 d时EPCs达增殖高峰(P<0.05);免疫染色表明,诱导培养7 d的细胞约90%呈VWF/CD34双荧光阳性,对照组的阳性率极低,随培养时间的延长阳性率无增加; western blotting结果表明,诱导培养1 d的细胞无VWF表达,培养4 d后表达逐渐增强, 10~14 d达高峰,21 d后稍减弱;Real-time PCR结果表明,培养1 d的细胞FLK1和VWF的mRNA表达低(P<0.01),4 d后表达逐渐增强,14 d达高峰,21 d后略减少,未诱导细胞无表达。结论本试验成功地从鼠骨髓单个核细胞中分离、诱导培养出EPCs,在其体外扩增分化为成熟内皮细胞的过程中,P0～P3(1～21 d) FLK1和VWF等内皮细胞标志蛋白表达逐渐增强,P3(21 d)后略有下降;而未加诱导的不能生长成内皮细胞。该细胞可作为构建血管组织工程较为理想的内皮的种子细胞。第二部分:鼠骨髓来源的平滑肌祖细胞培养、鉴定及生物学特性的研究目的将鼠骨髓来源的平滑肌祖细胞(smooth muscle progenitor cells, SPCs),诱导培养成平滑肌细胞(smooth muscle cells, SMCs),鉴定并观察其在体外增殖、分化过程中相关细胞表型的变化。方法采用密度梯度离心方法获得鼠骨髓单个核细胞,用M-199条件培养剂进行诱导其分化,于培养4、7、10、14、21 d,用免疫荧光双标技术(α-SMA、CD14)鉴定SPCs。于诱导分化后的1、4、7、10、14、21 d,用western blotting法检测α-SMA蛋白的水平,用RT-PCR和Real-time PCR法检测α-SMA的mRNA表达变化,并与未诱导培养的细胞进行比较。结果鼠骨髓单个核细胞在诱导培养的4 d细胞开始贴壁,7 d细胞大多变为梭形,14 d(第3代)融合时呈现典型的“峰”“谷”样形态;免疫荧光染色表明,培养4 d的细胞α-SMA/CD14开始出现双荧光阳性;western blotting结果表明,培养1 d的细胞无α-SMA表达,培养4 d后表达逐渐增强, 10~14 d达高峰,21 d后仍持续高水平;Real-time PCR结果表明,培养1 d的细胞α-SMA的mRNA表达低(P<0.01),4 d后表达逐渐增强,14 d达高峰(P<0.01),21 d后仍持续高水平,未诱导细胞无表达。结论本试验成功地从鼠骨髓单个核细胞中分离培养出SPCs,在其体外扩增分化为成熟平滑肌细胞的过程中,α-SMA、CD14平滑肌细胞标志蛋白及mRNA表达逐渐增强。该细胞可作为构建血管组织工程较为理想的平滑肌的种子细胞。第三部分:细胞外基质支架对内皮祖细胞生长影响的研究目的研究鼠骨髓来源内皮祖细胞(EPCs)在细胞外基质压缩和未压缩支架上的生长特性,为EPCs生长寻找更新、更适合的生物组织工程支架。方法将纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝作用下,构建毯状细胞外基质支架(extracellular matrix ECM),一组给于一定压力形成压缩支架为实验组,另一组未压缩的支架为对照组,在两种支架表面种植骨髓来源的经诱导培养的第二代内皮祖细胞(EPCs)。分别于培养1、3、7、10、14、21 d,用免疫荧光技术(VWF)鉴定EPCs,用电镜观察和分析的支架表面结构以及EPCs在支架表面的生长情况。于诱导分化后的3、7、10、14、21 d,用western blotting法检测VWF蛋白的水平,用Real-time PCR法检测VWF的mRNA表达变化。于培养10 d,用流体力学的方法测定四组不同支架的扭矩。结果1 h、3 h、5 h三个检测点的细胞贴壁率分别是:压缩组25.3%、60.4%、90.5%,未压缩组10.5%、30.4%、66.3%。分析1﹑3﹑7﹑10﹑14﹑21 d两组支架细胞的数量,结果表明随着时间的增加两组支架上细胞数明显增加,在1﹑3﹑7 d压缩组的细胞数明显高于未压缩组(p<0.001),且压缩组细胞较未压缩组细胞形态成熟,与内皮细胞相似,10 d后细胞数无明显差异,但压缩支架上形成一个较平整的细胞平面,而在未压缩支架表面细胞形态不规则、高低不平、细胞间排列松散。western blotting检测表明3﹑7﹑10 d VWF蛋白在压缩支架上表达比未压缩支架上强,14 d后两者表达无差异。real-timePCR结果显示:两组不同支架上的EPCs均表达VWF基因,两组的表达量7 d后均显著增高(p<0.05),14 d达高峰(p<0.01),21 d仍维持较高水平,而在3、7、10 d三时段中的压缩组VWF基因表达量明显高于未压缩组(p<0.05),14 d后实验组仍高于对照组,但无差异。压缩支架上种植细胞的扭矩明显低于其他三种支架(p<0.01)。结论压缩细胞外基质支架较未压缩的更能促进EPCs粘附、增殖和分化,压缩的支架可作为EPCs生长的载体,可作为一种新颖的合成人造血管的生物组织工程支架。第四部分:细胞外基质支架对平滑肌祖细胞生长影响的研究目的研究鼠骨髓来源平滑肌祖细胞(smooth muscle progenitor cells,SPCs)在细胞外基质支架上的生长特性,为SPCs生长寻找更新的生物组织工程支架。方法将纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝作用下,构建毯状细胞外基质支架(extracellular matrix ECM)。第二代SPCs种植于支架表面为实验组,种植于圆盖片上为对照组。于培养1、3、7、10、14、21 d,用免疫荧光技术(α-SMA)鉴定SPCs,用扫描电镜和透射电镜观察和分析的支架表面结构以及SPCs在支架上生长情况。分别于诱导分化后的3、7、10、14、21 d用western blotting法检测α-SMA蛋白的水平,用Real-time PCR法检测α-SMA的mRNA表达变化。结果1 h、3 h、5 h三个检测点的细胞贴壁率分别是:实验组(支架)26.40%、61.40%、91.50%,对照组(圆盖片上)11.10%、30.55%、66.90%。分析在1﹑3﹑7﹑10﹑14﹑21 d两组细胞的数量,结果表明随着时间的增加两组细胞数明显增加,在1﹑3﹑7 d实验组的细胞数明显高于对照组(p<0.001);扫描电镜显示在支架表面形成一个完整的较平整的细胞平面,细胞间排列紧密,细胞有较多的突起,如典型“平滑肌”形状;而对照组细胞形态不规则、高低不平、细胞间排列松散。透射电镜发现对照组的细胞为单层,而实验组平滑肌祖细胞为多层,呈三维立体结构,更接近天然血管的结构。western blotting检测表明3﹑7﹑10 dα-SMA蛋白在实验组表达比对照组强,14 d后两者表达无差异。real-time PCR结果显示:两组的SPCs均表达α-SMA基因,两组的表达量7 d后均显著增高(p<0.05),14 d达高峰(p<0.01),21 d仍维持较高水平;而在3、7、10 d三时段中的实验组α-SMA基因表达量明显高于对照组(p<0.05),14 d后两组表达相当,无差异。21 d后实验组仍维持高水平,对照组明显下降(p<0.01)。结论细胞外基质支架能显著促进SPCs粘附、增殖和分化,可作为SPCs生长的载体,可作为一种新颖的合成人造血管的生物组织工程支架。第五部分:内皮祖细胞和平滑肌祖细胞共同构建细胞化生物组织工程血管支架的研究目的研究鼠骨髓来源平滑肌祖细胞(smooth muscle progenitor cells, SPCs)和内皮祖细胞(endothelial progenitor cells , EPCs)在细胞外基质支架上的生长特性,为生物组织工程血管寻找更新的种子细胞和支架。方法将鼠骨髓来源单个核细胞( bone mononuclear cells, BMCs)分别用不同的培养基进行诱导分化,培养作为种子细胞。将纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝作用下,构建毯状细胞外基质(extracellular matrix, ECM)支架,先在支架表面种植诱导培养骨髓来源的第二代SPCs,4 d后再种植上第二代的EPCs(实验组);而在圆玻片上(对照组)。用扫描电镜和透射电镜观察和分析支架表面结构以及细胞在两种不同载体上生长情况。结果诱导培养的第二代SPCs,种植在支架上,4 d时倒置显微镜示SPCs在支架表面细胞融合呈现典型的“峰”“谷”样形态,当EPCs被种植10 d时扫描电镜显示在支架表面形成一个完整的较平整的细胞平面。透射电镜发现在多层平滑肌祖细胞表面有单层内皮祖细胞,呈三维立体结构,具有天然血管的内膜和中膜,更接近天然血管的结构。而对照组的细胞生长状态差,且为单层细胞结构。结论BMCs将是组织工程血管最理想的细胞来源,细胞外基质支架能显著促进SPCs和EPCs粘附、增殖和分化,可作为SPCs和EPCs生长的载体,可作为一种新颖的合成人造血管的生物组织工程支架。更多还原

【Abstract】 Part I Study the Culture Condition and Biological Characteristics of Endothelial Progenitor Cells from Rat Marrow in VitroObjective To elucidate a simple method of isolating endothelial progenitor cells ( EPCs) from rat marrow monocytes (MNCS) and observe the endothelial cell specific expression profile during proliferation and differentiation in vitro.Methods MNCS were isolated from rat marrow using ficoll density gradient centrifugation, induced, proliferated and differentiated in EGM-2 culture medium(the experiment group) and common medium (the control group) respectively. The EPCs were identified by immunofluorescent staining (CD34、VWF dipl-mark and FLK1) at 4,7,10,14,21 d, respectively. The proliferation of EPCs was evaluated by MTT, and endothelial cell specific markers (VWF、FLK1) were determined with western blotting and Real-time PCR at 1、4、7、10、14、21 d, respectively, and compare with the control group. Results During culturing process, the cells became spindle shaped and displayed endothelium-like cobble-stone morphology with outgrowth at 4 d and 7 d, but cells of the control group were worse in the condition and the morphous than the experiment group .Result of MTT indicated that the number of EPCs increased to peak at 10 d (P<0.05). Percentage of the cells that were positive for CD34/VWF was 90% at 7 d, and that of the control group extremely low, Percentage of positive was no increase. Expression for VWF at 1 d was not found with western blotting, however,gradually increased at 4 d and reached to the top during 10 d to 14 d. The results with Real-time PCR indicated that the expression of VWF and FLK1 mRNA within non-induced cells was not found, gradually increased at 4 d and reached to top at 14 d, the low-expression at 1 d was significantly different from the other ones (P<0.01).Conclusion These data demonstrated that EPCs could be isolated from rat marrow monocytes (MNCS) and cultured in vitro. The EPCs could be used as endothelial seeding cells in vascular tissue engineering.Part II Study the culture and bionomics of smooth muscle progenitorcells from rat marrow in VitroObjective To elucidate a simple method of isolating smooth muscle progenitor cells ( SPCs) from rat marrow monocytes (MNCS) and observe the smooth muscle progenitor cells specific expression profile during proliferation and differentiation in vitro.Methods MNCS isolated by ficoll density gradient centrifugation from rat marrow,induced and proliferated and differentiated then identified by immunofluorescent staining (α-SMA,CD14) at 4, 7, 10, 14, 21 d, respectively. and smooth muscle cells specific markers (α-SMA, CD14) were determined with western blotting and Real-time PCR at 1, 4, 7,10, 14, 21 d, respectively.Results During culturing, cells start adherence and became spindle shaped with outgrowth at 4 d and 7 d , and present typical“peak”“valley”at 14 d (3 generation). dipl- positive forα-SMA、CD14 at 4 d. Expression forα-SMA at 1 d not found with western blotting ,but it gradually enhanced at 4 d and reached to the top from 10 d to 14 d ,and still to remain high-level after 21 d .The results with Real-time PCR indicated that the expression ofα-SMA mRNA within noninduced cells was not found ,but after being induced it gradually enhanced at 4 d and got to top at 14 d, and still to remain high-level after 21 d , low-expression at 1 d was significantly different from the other ones(P<0.01).Conclusion These data demonstrated that SPC could be isolated and cultured from rat marrow monocytes (MNCS), and during culturing Smooth muscle cell-specific marker expressions, such asα-SMA and CD14 potently enhanced from 1 d to 21 d. SPCs can be used as smooth muscle seeding cells for constructing vascular tissue engineering.Part III Study the growth effect on endothelial progenitor cells with extracellular matrix scaffoldObjective To explore the characteristics of endothelial progenitor cells (EPCs) on pressed and unpressed extracellular matrix (ECM) scaffolds and to find a novel bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels.Methods EPCs were induced from mesenchymal stem cells isolated from rat bone marrow and cultured in EGM-2 culture medium. The scaffold was comprised from three major ECM proteins (i.e. fibrinogen, fibronectin and laminin) which can be easily molded into mat-like form and gelled by the addition of fresh plasma. Then, pressure was applied to make a pressed scaffold. EPCs were seeded on both pressed and unpressed scaffolds. the surface structure of the scaffold and growth state of EPCs on the scaffold were observed and analysed by electron microscope. The characteristics of those EPCs on different kinds of scaffolds were studied with EPC-specific VWF by immunofluorescent, western blotting and a real-time PCR technique at DIV3 (days in vitro), DIV7, DIV10, DIV14, DIV21,respectively. In addition, the hydromechanical characteristics of four kinds of scaffolds were detected by a rheometer.Results The ratios of cell adhesion at 1 h,3 h,5 h after seeded on pressed scaffold and unpressed scaffolds were 25.3%,60.4%,90.5% and 10.5%, 30.4%,66.3% respectively. Cell numbers on the two scaffold types at DIV1, DIV3, DIV7, DIV10, DIV14, DIV21 increased in evidence. pressed scaffolds has got a larger cell number(p<0.001)at DIV1, DIV3, DIV7,but no significant difference was found after DIV10. Furthermore, cell shapes of EPCs on pressed scaffolds were more mature and more similar to endothelial cell. A level cell surface on pressed scaffolds was achieved. However, on unpressed scaffolds, cell shapes were irregular and gaps between cells at the top of the scaffold were rather distant. Western blotting assays revealed: EPCs on pressed scaffolds expressed more protein VWF at DIV3,vDIV7, vDIV10, but no significant difference after DIV14. Real-time PCR results showed: EPCs on the two different groups of scaffolds all expressed VWF gene, The quantity of their expression in the two groups were all enhanced at DIV7(p<0.05), peaked at DIV14(p<0.01)and still maintained at a high level at DIV21. The quantity of VWF gene expression in the pressed group was much higher than that in the unpressed group at DIV3, DIV7, DIV10, but there was no significant difference after DIV14. Hydromechanical result show torque of pressed and growthed-cell scaffold was obviously lower than other scaffold (p<0.01).Conclusions Pressed ECM scaffolds can promote adhesion, proliferation and differentiation on EPCs. Pressed scaffolds can be used as the matrix for EPC and fabricated into a novel synthetic tissue bio-engineered vascular scaffold. Part IV Study the growth effect on smooth muscle progenitor cells with extracellular matrix scaffoldObjective To explore the characteristics of smooth muscle progenitor cells (SPCs) on the extracellular matrix (ECM) scaffolds and to find a novel bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels.Methods second generation SPCs from mesenchymal stem cells isolated and cultured and induced from rat bone marrow were growthed on the scaffold(the experiment group), that comprises three major ECM proteins (i.e. fibrinogen, fibronectin and laminin) which can be easily molded into mat-like form and gelled by the addition of fresh plasma.and cells were the control group on the cover glass. the surface structure of the scaffold and growth state of SPCs on the scaffold were observed and analysed by scanning electron microscope (SEM)and transmission electron microscope (TEM);The characteristics of those SPCs on the experiment group and the control group were studied with SPC-specificα-SMA by immunofluorescent, ,western blotting and by an real-timePCR technique at DIV3 (days in vitro), DIV7, DIV10, DIV14, DIV21.Results The ratios of cell adhesion at 1 h, 3 h, 5 h after seeded on scaffold and control group were 26.40%,61.40%,91.50% and 11.10%,30.55%,66.90% respectively. Cell numbers on the experiment group and the control group at DIV1, DIV3, DIV7, DIV10, DIV14, DIV21 increased in evidence. the experiment group has got a larger cell number(p<0.001)at DIV1, DIV3, DIV7,but no significant difference after DIV10. Furthermore, cell shapes of SPCs on scaffolds were more level, more compact and have major ecphymas as typical smooth muscle cell by SEM. Detected by TEM , SPCs of the control group were monolayer ,on the other hand ,ones of the experiment group were multiplayer which was 3D stereochemical structure and got close to natural vascular. However, cell shapes of the control group were irregular and gaps between cells at the top of the scaffold were rather distant. Western blotting assays revealed: SPCs on ECM scaffolds expressed more proteinα-SMA at DIV3, DIV7, DIV10 ,but no significant difference after DIV14. Real-time PCR results showed: SPCs on the two different groups all expressedα-SMA gene, The quantity of their expression in the two groups were all enhanced at DIV7(p<0.05 ), peaked at DIV14(p<0.01) and still maintained at a high level at DIV21. The quantity ofα-SMA gene expression in the experiment group was much higher than that in the control group at DIV3,DIV7,DIV10(p<0.05), but there was no significant difference after DIV14. that of the control group significant decrease after DIV21.conclusions scaffolds can promote adhesion, proliferation and differentiation on SPCs. ECM scaffolds can be used as the matrix for SPCs and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.Part V Experimental Research on Construction of Tissue engineering Blood Vessel scaffold with cells from smooth muscl progenitor cells and endothelial progenitor cellsObjective To explore the characteristics of smooth muscle progenitor cells (SPCs) and endothelial progenitor cells ( EPCs) on the extracellular matrix (ECM) scaffolds and to find a novel seeds cells and bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels.Methods induced, proliferated and differentiated in different culture medium from rat bone mononuclear cells (BMCs). SPCs and EPCs of the second generation from mononuclear cells isolated and cultured and induced from rat bone marrow were growthed on the scaffold(the experiment group), that comprises three major ECM proteins (i.e. fibrinogen, fibronectin and laminin) which can be easily molded into mat-like form and gelled by the addition of fresh plasma. and cells were the control group on the cover glass. the surface structure of the scaffold and growth state of SPCs and EPCs on the scaffold were observed and analysed by scanning electron microscope (SEM)and transmission electron microscope (TEM).Results BMCs were induced in EGM-2 culture medium, and BMCs were induced in M-199 culture medium with bFGF. second filial generation SPCs that induced were planted on the ECM-scaffold, SPCs under inverted/ fluorescent microscope present typical“peak”“valley”at 4 d , and second filial generation EPCs were planted on the scaffold with the SPCs after 4 d, A level cell surface on scaffolds was achieved under SEM After10 d. And multiplayer SMCs were found on the surface of monolayer EPCs under TEM, which had three diamensions stereochemical structure with endomembrane and tunica media and got close to natural blood vessel. The growth of the control group cells with monolayer construction was weaker .Conclusions BMCs will be most ideal cells source for Tissue engineering Blood Vessel. ECM scaffolds can promote adhesion, proliferation and differentiation on SPCs and EPCs.ECM scaffolds can be used as the matrix for SPCs and EPCs and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.更多还原