血管化组织工程化窦房结的构建及应用基础研究
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摘要
病态窦房结、严重房室阻滞等心律失常是影响人类健康的心血管疾病之一，随着细胞治疗、基因治疗以及组织工程技术的发展，生物起搏器已成为心脏起搏研究的热点。
目前，构建生物心脏起搏器的方法主要有三种：利用基因转染的方法调节自体心肌细胞离子电流触发起搏；向受体心脏种植起搏细胞或工程化的起搏细胞；利用组织工程技术构建组织工程化起搏组织。组织工程化窦房结是指选取适当的起搏细胞和支架材料，在体外应用组织工程的方法构建功能稳定的组织工程化起搏组织。组织工程化窦房结的形态结构更接近生理状态窦房结，细胞在三维支架上更易生长、不易分散，能够形成统一节律搏动。本实验室前期已经在体外成功构建了组织工程化窦房结，并证实该窦房结具有较好的起搏活性、体内移植出现异位起搏点。
组织工程化组织或器官的存活需要血管提供氧气、营养物质，并带走代谢产物，且血管内皮细胞对组织的成熟和分化也有一定的影响。未进行血管化构建的组织工程化组织或器官，移植体内后的存活必须依赖宿主的血管长入，但是，大多数宿主血管长入的速度都不能满足有一定体积的移植体的需求，特别是在植入的早期。因此，决定三维的组织工程化组织或器官移植成功的关键是能否有效、快速的形成功能血管。目前，组织工程组织或器官血管化的方法主要有三种：体内血管预构建；利用血管生成因子促进血管发生，和血管的体外预构。目前，组织工程化窦房结血管化的研究尚未见报道。
基于以上观点，本实验拟在我们前期组织工程化窦房结构建的研究基础上，选用同种异体胚胎源性起搏样细胞与骨髓源性内皮祖细胞联合种植，进行血管化组织工程化窦房结的体外构建和体内移植，通过形态学、免疫组织化学、Western Blot等方法探讨血管化组织工程化窦房结构建心脏生物起搏器的可行性及其可能机制，以进一步探讨组织工化窦房结移植构建心脏生物起搏器的可行性，并确定一种可行的血管化组织工程化窦房结的构建方法。
第一部分胚胎心脏祖细胞的分离、培养、
诱导及鉴定
研究目的：获取具有生理功能的大鼠胚胎源性起搏样细胞。
材料和方法：分离受孕11.5天的SD大鼠胚胎心管，采用胰酶消化的方法接种培养心脏祖细胞。培养1周，用内皮素-1诱导培养3天。观察诱导后细胞的搏动情况和细胞形态，取生长状态良好的细胞用免疫细胞荧光化学法检测起搏细胞特异性标志物HCN2、HCN4、α-actin、TnT及Cx43的表达情况，进行细胞鉴定。
结果：培养的大鼠胚胎心脏祖细胞贴壁生长，呈梭形、三角形，细胞自发搏动。内皮素-1诱导后，可见诱导后起搏样细胞生长密集、成簇生长、部分区域可出现成团搏动。免疫细胞荧光化学检测显示，起搏细胞特异性标志物HCN2、HCN4、Cx43、α-actin和TnT均为阳性表达，阳性细胞率达90%以上。
结论：内皮素-1诱导，可以获得具有生理功能的胚胎源性起搏样细胞。
第二部分骨髓源性内皮祖细胞的分离、
培养及鉴定
研究目的：获取活性好、纯度高的骨髓源性内皮祖细胞。
材料和方法：分离成年SD大鼠双侧股骨、胫骨骨髓，等量淋巴细胞分层液分离，收集单个核细胞，通过EGM-2MV条件培养基进行骨髓源性内皮祖细胞的培养。观察诱导培养后细胞的形态，取生长状态良好的细胞用免疫细胞化学的方法检测内皮祖细胞表面标志物CD31、CD34和CD133的表达，进行细胞鉴定。
结果：大鼠骨髓源性内皮祖细胞贴壁生长，多数细胞呈梭形、三角形、纺锤形或不规则形。5-7天，EPCs细胞集落出现，中间为圆形细胞群，边缘细胞为梭形、出芽，呈单层生长，类似血岛；9-11天，细胞出现“铺路石样”结构；呈现EPCs典型形态特征。免疫细胞化学法检测显示，表面标志物CD31、CD34和CD133阳性表达，阳性细胞达90%以上。
结论：等量淋巴细胞分层液分离、EGM-2MV条件培养基培养，可以获得活性好、纯度高的大鼠骨髓源性内皮祖细胞。
第三部分血管化组织工程化窦房结的
体外构建
研究目的：体外构建血管化组织工程化窦房结。
材料和方法：选择Matrigel基质胶为支架材料，将标记后的骨髓源性内皮祖细胞与胚胎源性起搏样细胞以不同的比例（2:1/1:1/1:2）与液态Matrigel基质胶混合，在37℃、5%CO2培养箱内进行体外共培养。分别在18h、3d、5d、7d、9d、11d、13d、15d、17d、19d、21d观察不同实验组细胞复合体的形态及搏动频率，免疫荧光显微镜下和苏木精-伊红染色方法观察两种细胞的分布情况。
结果：胚胎源性起搏样细胞与骨髓源性EPS细胞2:1和1:1混合组培养18h出现内皮细胞典型的“成血管现象”；1:2混合组“成血管现象”不明显。1:1和1:2混合组培养第3天，复合体大部分区域开始出现统一的搏动，且随着培养时间的延长，频率逐渐增加，培养到2周左右时频率达到最高峰；但培养至3周左右时Matrigel基质胶体积较前减小，自发搏动较前减弱。2:1混合组统一搏动不明显。免疫荧光显微镜下观察和苏木精-伊红染色结果显示，1:1混合组两种细胞密度分布相对均匀，细胞间连接广泛存在。
结论：以骨髓源性内皮祖细胞与胚胎源性起搏样细胞联合种植，可以构建血管化组织工程化窦房结；两种细胞以1:1的比例效果较好。
第四部分血管化组织工程化窦房结的在体研究
研究目的：血管化组织工程化窦房结体内移植，观察移植体功能发挥、局部血流变化，和VEGFR-2、PI3K、AKT等因子的表达情况。
材料和方法：健康成年SD大鼠60只，雌雄不限，体重200-250g，随机分为3组，每组20只。正常对照组大鼠不做任何处理，非血管化组织工程化窦房结组将体外构建的组织工程化窦房结移植入大鼠心脏的左室壁心外膜下，血管化组织工程化窦房结组将体外构建的血管化组织工程化窦房结移植入大鼠心脏的左室壁心外膜下。定期监测各组大鼠心电图情况；术后4周、8周，激光多普勒血流仪在体测定左室壁血流量；HE染色观测移植组织血管形成； Western Blot方法检测移植区域VEGFR-2、PI3K、AKT等因子的表达情况。
结果：非血管化组织工程化窦房结组和血管化组织工程化窦房结组出现异位起搏的室性早搏。术后4周，血管化组织工程化窦房结组移植局部血流量高于正常对照组，非血管化组织工程化窦房结组低于正常对照组；术后8周，三组心室壁血流量无差异。术后4周，血管化组织工程化窦房结中有大量新生血管形成，非血管化组织工程化窦房结组新生血管较少；术后8周，血管化组织工程化窦房结组和非血管化组织工程化窦房结组中新生血管情况无差别。术后4周血管化组织工程化窦房结组中VEGFR-2、PI3K、AKT等因子在移植区域的表达水平高于非血管化组织工程化窦房结组和正常对照组；术后8周，三种因子的表达在3个实验组中无差异。
结论：用本实验方法构建的血管化组织工程化窦房结植入体内可长期存活，产生异位起搏点；血管化组织工程化窦房结可较早期建立血液供应；发生血管化的机制可能与PI3K/Akt信号通路有关。
Bradyarrhythmia such as Sick sinus, severe atrial-ventricular block etc is a commoncardiovascular diseases that endanger human health.With the rapid development of genetherapy, cell therapy, as well as bioengineering technology, biological pacemaker hasbecome a research hotspot of cardiac pacing.
At present, there are three main methods of constructing biological pacemaker:by usingthe method of gene regulation of autologous cardiomyocytes ion current trigger pacing;planting pacemaker cells or engineering to receptor cardiac pacemaker cells;structuringtissue engineering pacemaker tissue by tissue engineering.Tissue engineering sinus node isto select appropriate pacemaker cells and support material, for tissue engineering in vitrotissue engineering method to build functional stability pacing organizations. Themorphology of the tissue engineering sinus node are closer to the physiological state of thesinus node, cells in the three dimensional support are more easily to grow, not easy todisperse, able to form a unified rhythm pulsation. In addition, the tissue transplants to theheart can establish a new pacemaker, adapt to the neurohumoral regulation and with thedevelopment of body to growth. Currently,our experiment have successfully constructed invitro tissue engineering sinus node, the organization can maintain good sinus nodepacemaker activity,via in vivo transplantation experiments found that the tissue intransplantation area can appear ectopic pacemaker.
Tissue engineered tissue or organ survival need blood vessels to provide oxygen andnutrients and take away the metabolites, and vascular endothelial cells also have a certainimpact on maturation and differentiation of the organizations. Before vascularizationconstruction of tissue-engineered tissues or organs transplantation in vivo survival mustrely on the host's vascular ingrowth. However, most of the speed of the host vascularingrowth can not meet the demand of a certain thickness transplant body, especially in theearly days of implantable. Therefore, it was decided that the key to the success of thethree-dimensional tissue engineered tissue or organ is effective, rapid formation offunctional vascular. Currently, tissue engineering of tissue or organ vascularization methodmainly have three: pre-built of the blood vessels; angiogenic factors to promoteangiogenesis and vascular in vitro prefabricated. At present, the sinus node vascularizationof tissue engineering has not been reported.
Based on the above point of view, the present study proposed in previous tissueengineered sinus node built as base, choose allogeneic embryonic origin of pacemaker cells and bone marrow-derived endothelial progenitor cells combined planting, vasculartissue engineering sinus node constructed in vitro and in vivo transplantation,byimmunohistochemistry, Western Blot and other methods to explore the feasibility ofvascularized tissue engineering sinus build cardiac biological pacemaker and its possiblemechanism, in order to develop a viable vascularized tissue engineering sinus nodeconstruction method.
Part one: The isolation, culture, induction and appraisal ofEmbryonic cardiac progenitor cells
Objective: To obtain embryonic endogenous pacemaker cells, which had physiologicfunction.
Materials and Methods: SD rat embryo heart tube conception11.5days was separated bytrypsin digestion method of inoculation culture of cardiac progenitor cells. After one weekof culture, endothelin-1-induced cultured for3days. Observed the beating and cellmorphology of cells after induction in good condition to take the growth of cells withimmunofluorescence chemical method to detect specific markers of HCN2pacemaker cells,HCN4, α-actin, TnT and Cx43expression, cells were identified.
Results: The embryonic endogenous pacemaker cells were adherent cells, which werein fusiform and triangular shape, the cells also had spontaneous cells beating. Application ofendothelin-1-induced embryonic cardiac progenitor cells, it can be seen the growth ofpacemaker cells in dense clusters growth after the induction, some part of the regions canappear lumps pulsation. Immune cell fluorescent chemical testing results showed that thepacemaker cells’ specific markers: HCN2, HCN4, Cx43, α-actin and TnT were all positiveexpression, and the masculine cell rate was above90%.Conclusion: Through the induce of endothelin-1, embryonic origin of pacemaker cells canbe successfully obtained.
Part two: The isolation, culture and appraisal of Bonemarrow-derived endothelial progenitor cells
Objective: To obtain good activity and pure bone marrow-derived endothelial progenitorcells.
Materials and methods: Separation adult SD rats bilateral femur, tibia bone marrow, theequivalent lymphocytes layered liquid separation, collection of mononuclear cells, bonemarrow-derived endothelial progenitor cells were cultured in the EGM-2MV conditionsmedium. Observe cellular morphology which are after induced cultured, and take the cellswhich growth in good condition to detect the expression of endothelial progenitor cellsurface marker VIII factor, CD31, CD34, and CD133by immunocytochemistry, thenidentified the cells.
Results: Most of the cells were showed in fusiform, triangular, spindle-shaped or irregularshape. About5th-7th days, the colony of EPCs appeared, which intermediate was circularcell populations and border cells send forth as spindle budding and appeared monolayergrowth, this structure was similar to blood islands. At9th-11th days, the cells showed atypical "cobblestone" structure, which was the typical morphological characteristics ofEPCs. The immunocytochemistry assay results showed that the surface markers factorCD31, CD34, and CD133were all in positive expression, and the masculine cell rate wasabove90%.
Conclusion: Through the induction culture of conditioned medium, good activity and purebone marrow-derived endothelial progenitor cells can be successfully obtained.
Part three: In vitro construct of vascularized tissueengineering sinus node
Objective: To construct a vascularized tissue engineering sinus node in vitro.
Materials and Methods: Select Matrigel as support material, bone marrow-derivedendothelial progenitor cells and embryo-derived pacemaker cells in different proportions(2:1/1:1/1:2) with liquid Matrigel matrix glue mixed in a5%CO2incubator at37℃forin vitro co-culture. Respectively in18h,3d,5d,7d,9d,11d,13d,15d,17d,19d,21dinverted phase contrast microscope to observe the morphology and the beating rate of thedifferent experimental group cells complex, immunofluorescence microscopy observationand hematoxylin-eosin staining method to observe the distribution of two kinds of cells.
Results: Inverted phase contrast microscope observation shows that co-culture differentproportions of cells mixed with Matrigel, the2:1and1:1mixed group appeared typical“vascular phenomenon” of endothelial cells18hafter cultured, while the “vascularphenomenon” was not obvious in the1:2mixed group. The1:1and1:2mixed group appeared the phenomenon of unified beating in most of the area of the complex after thecultivate, the unified pulsation of2:1mixed group was not obvious. In the third days, inmost area of the complex began to appear unified beating, frequency gradually increasedwith the prolonged incubation time, two weeks later the frequency reached peak. Whencultured for third week, the Matrigel’s shape reduced than before and spontaneouspulsation began to weak than previous. The immunofluorescence and hematoxylin-eosinstaining results showed that the cell density in the1:1mixed group was relatively uniformand the connections between cells was widely exist.
Conclusion: Co-culture of the bone marrow-derived endothelial progenitor cells andembryo-derived pacemaker cells can successfully build vascularized tissue engineeringsinus node in vitro, and the1:1ratio was the ideal rate of the cells.
Part four: In vivo research of vascularized tissueengineering sinus node
Objective: To observe the morphological structure, angiogenesis and the change of regionalflow in transplant by transplant the vascularized tissue engineering sinus node into the rats’heart.
Materials and Methods:60healthy adult SD rats(any male or female, weighing200-250g)were randomly divided into3groups (n=20). The rats in normal control group withoutany treatment, transplant constructed in vitro of the tissue engineering sinus node into rat’sleft ventricular wall of epicardium of heart in non-vascularized tissue engineering sinusnode group, transplant constructed in vitro of the vascularized tissue engineering sinusnode into rat’s left ventricular wall of epicardium of heart in vascular tissue engineeringgroup.The electrocardiogram of the rats were regularly monitored.4and8weeks later, thelaser Doppler flowmetry, hematoxylin-eosin staining and Western Blot method were used todetect the regional volume of blood flow, vasifaction and the expression of VEGFR-2, PI3K,AKT and other factors in transplantation area of different groups.
Results: The surface ECG monitoring results indicate that compared with the normalcontrol group, both the non-vascularized tissue engineering sinus node group and vasculartissue engineering sinus node group have ventricular premature beat of ectopic pacemaking.The regional blood flow in vascular tissue engineering sinus node group was higher thanother group, the HE staining results showed that the ascular tissue engineering sinus node group had more neovascularization than other group, and the Western Blot results showedthat the expression of VEGFR-2, PI3K and AKT in vascularized tissue engineering sinusnode group were higher than other group.
Conclusion: Our experimental method in constructing vascularization tissue engineeringsinus node in vitro can make the sinus node has a long-term survival after implanted intobody, and can produce ectopic pacemaker and neovascularization. The mechanism of thistissue engineering tissue’s vascularization may be related to the PI3K/Akt signal pathway.
目前，构建生物心脏起搏器的方法主要有三种：利用基因转染的方法调节自体心肌细胞离子电流触发起搏；向受体心脏种植起搏细胞或工程化的起搏细胞；利用组织工程技术构建组织工程化起搏组织。组织工程化窦房结是指选取适当的起搏细胞和支架材料，在体外应用组织工程的方法构建功能稳定的组织工程化起搏组织。组织工程化窦房结的形态结构更接近生理状态窦房结，细胞在三维支架上更易生长、不易分散，能够形成统一节律搏动。本实验室前期已经在体外成功构建了组织工程化窦房结，并证实该窦房结具有较好的起搏活性、体内移植出现异位起搏点。
组织工程化组织或器官的存活需要血管提供氧气、营养物质，并带走代谢产物，且血管内皮细胞对组织的成熟和分化也有一定的影响。未进行血管化构建的组织工程化组织或器官，移植体内后的存活必须依赖宿主的血管长入，但是，大多数宿主血管长入的速度都不能满足有一定体积的移植体的需求，特别是在植入的早期。因此，决定三维的组织工程化组织或器官移植成功的关键是能否有效、快速的形成功能血管。目前，组织工程组织或器官血管化的方法主要有三种：体内血管预构建；利用血管生成因子促进血管发生，和血管的体外预构。目前，组织工程化窦房结血管化的研究尚未见报道。
基于以上观点，本实验拟在我们前期组织工程化窦房结构建的研究基础上，选用同种异体胚胎源性起搏样细胞与骨髓源性内皮祖细胞联合种植，进行血管化组织工程化窦房结的体外构建和体内移植，通过形态学、免疫组织化学、Western Blot等方法探讨血管化组织工程化窦房结构建心脏生物起搏器的可行性及其可能机制，以进一步探讨组织工化窦房结移植构建心脏生物起搏器的可行性，并确定一种可行的血管化组织工程化窦房结的构建方法。
第一部分胚胎心脏祖细胞的分离、培养、
诱导及鉴定
研究目的：获取具有生理功能的大鼠胚胎源性起搏样细胞。
材料和方法：分离受孕11.5天的SD大鼠胚胎心管，采用胰酶消化的方法接种培养心脏祖细胞。培养1周，用内皮素-1诱导培养3天。观察诱导后细胞的搏动情况和细胞形态，取生长状态良好的细胞用免疫细胞荧光化学法检测起搏细胞特异性标志物HCN2、HCN4、α-actin、TnT及Cx43的表达情况，进行细胞鉴定。
结果：培养的大鼠胚胎心脏祖细胞贴壁生长，呈梭形、三角形，细胞自发搏动。内皮素-1诱导后，可见诱导后起搏样细胞生长密集、成簇生长、部分区域可出现成团搏动。免疫细胞荧光化学检测显示，起搏细胞特异性标志物HCN2、HCN4、Cx43、α-actin和TnT均为阳性表达，阳性细胞率达90%以上。
结论：内皮素-1诱导，可以获得具有生理功能的胚胎源性起搏样细胞。
第二部分骨髓源性内皮祖细胞的分离、
培养及鉴定
研究目的：获取活性好、纯度高的骨髓源性内皮祖细胞。
材料和方法：分离成年SD大鼠双侧股骨、胫骨骨髓，等量淋巴细胞分层液分离，收集单个核细胞，通过EGM-2MV条件培养基进行骨髓源性内皮祖细胞的培养。观察诱导培养后细胞的形态，取生长状态良好的细胞用免疫细胞化学的方法检测内皮祖细胞表面标志物CD31、CD34和CD133的表达，进行细胞鉴定。
结果：大鼠骨髓源性内皮祖细胞贴壁生长，多数细胞呈梭形、三角形、纺锤形或不规则形。5-7天，EPCs细胞集落出现，中间为圆形细胞群，边缘细胞为梭形、出芽，呈单层生长，类似血岛；9-11天，细胞出现“铺路石样”结构；呈现EPCs典型形态特征。免疫细胞化学法检测显示，表面标志物CD31、CD34和CD133阳性表达，阳性细胞达90%以上。
结论：等量淋巴细胞分层液分离、EGM-2MV条件培养基培养，可以获得活性好、纯度高的大鼠骨髓源性内皮祖细胞。
第三部分血管化组织工程化窦房结的
体外构建
研究目的：体外构建血管化组织工程化窦房结。
材料和方法：选择Matrigel基质胶为支架材料，将标记后的骨髓源性内皮祖细胞与胚胎源性起搏样细胞以不同的比例（2:1/1:1/1:2）与液态Matrigel基质胶混合，在37℃、5%CO2培养箱内进行体外共培养。分别在18h、3d、5d、7d、9d、11d、13d、15d、17d、19d、21d观察不同实验组细胞复合体的形态及搏动频率，免疫荧光显微镜下和苏木精-伊红染色方法观察两种细胞的分布情况。
结果：胚胎源性起搏样细胞与骨髓源性EPS细胞2:1和1:1混合组培养18h出现内皮细胞典型的“成血管现象”；1:2混合组“成血管现象”不明显。1:1和1:2混合组培养第3天，复合体大部分区域开始出现统一的搏动，且随着培养时间的延长，频率逐渐增加，培养到2周左右时频率达到最高峰；但培养至3周左右时Matrigel基质胶体积较前减小，自发搏动较前减弱。2:1混合组统一搏动不明显。免疫荧光显微镜下观察和苏木精-伊红染色结果显示，1:1混合组两种细胞密度分布相对均匀，细胞间连接广泛存在。
结论：以骨髓源性内皮祖细胞与胚胎源性起搏样细胞联合种植，可以构建血管化组织工程化窦房结；两种细胞以1:1的比例效果较好。
第四部分血管化组织工程化窦房结的在体研究
研究目的：血管化组织工程化窦房结体内移植，观察移植体功能发挥、局部血流变化，和VEGFR-2、PI3K、AKT等因子的表达情况。
材料和方法：健康成年SD大鼠60只，雌雄不限，体重200-250g，随机分为3组，每组20只。正常对照组大鼠不做任何处理，非血管化组织工程化窦房结组将体外构建的组织工程化窦房结移植入大鼠心脏的左室壁心外膜下，血管化组织工程化窦房结组将体外构建的血管化组织工程化窦房结移植入大鼠心脏的左室壁心外膜下。定期监测各组大鼠心电图情况；术后4周、8周，激光多普勒血流仪在体测定左室壁血流量；HE染色观测移植组织血管形成； Western Blot方法检测移植区域VEGFR-2、PI3K、AKT等因子的表达情况。
结果：非血管化组织工程化窦房结组和血管化组织工程化窦房结组出现异位起搏的室性早搏。术后4周，血管化组织工程化窦房结组移植局部血流量高于正常对照组，非血管化组织工程化窦房结组低于正常对照组；术后8周，三组心室壁血流量无差异。术后4周，血管化组织工程化窦房结中有大量新生血管形成，非血管化组织工程化窦房结组新生血管较少；术后8周，血管化组织工程化窦房结组和非血管化组织工程化窦房结组中新生血管情况无差别。术后4周血管化组织工程化窦房结组中VEGFR-2、PI3K、AKT等因子在移植区域的表达水平高于非血管化组织工程化窦房结组和正常对照组；术后8周，三种因子的表达在3个实验组中无差异。
结论：用本实验方法构建的血管化组织工程化窦房结植入体内可长期存活，产生异位起搏点；血管化组织工程化窦房结可较早期建立血液供应；发生血管化的机制可能与PI3K/Akt信号通路有关。
Bradyarrhythmia such as Sick sinus, severe atrial-ventricular block etc is a commoncardiovascular diseases that endanger human health.With the rapid development of genetherapy, cell therapy, as well as bioengineering technology, biological pacemaker hasbecome a research hotspot of cardiac pacing.
At present, there are three main methods of constructing biological pacemaker:by usingthe method of gene regulation of autologous cardiomyocytes ion current trigger pacing;planting pacemaker cells or engineering to receptor cardiac pacemaker cells;structuringtissue engineering pacemaker tissue by tissue engineering.Tissue engineering sinus node isto select appropriate pacemaker cells and support material, for tissue engineering in vitrotissue engineering method to build functional stability pacing organizations. Themorphology of the tissue engineering sinus node are closer to the physiological state of thesinus node, cells in the three dimensional support are more easily to grow, not easy todisperse, able to form a unified rhythm pulsation. In addition, the tissue transplants to theheart can establish a new pacemaker, adapt to the neurohumoral regulation and with thedevelopment of body to growth. Currently,our experiment have successfully constructed invitro tissue engineering sinus node, the organization can maintain good sinus nodepacemaker activity,via in vivo transplantation experiments found that the tissue intransplantation area can appear ectopic pacemaker.
Tissue engineered tissue or organ survival need blood vessels to provide oxygen andnutrients and take away the metabolites, and vascular endothelial cells also have a certainimpact on maturation and differentiation of the organizations. Before vascularizationconstruction of tissue-engineered tissues or organs transplantation in vivo survival mustrely on the host's vascular ingrowth. However, most of the speed of the host vascularingrowth can not meet the demand of a certain thickness transplant body, especially in theearly days of implantable. Therefore, it was decided that the key to the success of thethree-dimensional tissue engineered tissue or organ is effective, rapid formation offunctional vascular. Currently, tissue engineering of tissue or organ vascularization methodmainly have three: pre-built of the blood vessels; angiogenic factors to promoteangiogenesis and vascular in vitro prefabricated. At present, the sinus node vascularizationof tissue engineering has not been reported.
Based on the above point of view, the present study proposed in previous tissueengineered sinus node built as base, choose allogeneic embryonic origin of pacemaker cells and bone marrow-derived endothelial progenitor cells combined planting, vasculartissue engineering sinus node constructed in vitro and in vivo transplantation,byimmunohistochemistry, Western Blot and other methods to explore the feasibility ofvascularized tissue engineering sinus build cardiac biological pacemaker and its possiblemechanism, in order to develop a viable vascularized tissue engineering sinus nodeconstruction method.
Part one: The isolation, culture, induction and appraisal ofEmbryonic cardiac progenitor cells
Objective: To obtain embryonic endogenous pacemaker cells, which had physiologicfunction.
Materials and Methods: SD rat embryo heart tube conception11.5days was separated bytrypsin digestion method of inoculation culture of cardiac progenitor cells. After one weekof culture, endothelin-1-induced cultured for3days. Observed the beating and cellmorphology of cells after induction in good condition to take the growth of cells withimmunofluorescence chemical method to detect specific markers of HCN2pacemaker cells,HCN4, α-actin, TnT and Cx43expression, cells were identified.
Results: The embryonic endogenous pacemaker cells were adherent cells, which werein fusiform and triangular shape, the cells also had spontaneous cells beating. Application ofendothelin-1-induced embryonic cardiac progenitor cells, it can be seen the growth ofpacemaker cells in dense clusters growth after the induction, some part of the regions canappear lumps pulsation. Immune cell fluorescent chemical testing results showed that thepacemaker cells’ specific markers: HCN2, HCN4, Cx43, α-actin and TnT were all positiveexpression, and the masculine cell rate was above90%.Conclusion: Through the induce of endothelin-1, embryonic origin of pacemaker cells canbe successfully obtained.
Part two: The isolation, culture and appraisal of Bonemarrow-derived endothelial progenitor cells
Objective: To obtain good activity and pure bone marrow-derived endothelial progenitorcells.
Materials and methods: Separation adult SD rats bilateral femur, tibia bone marrow, theequivalent lymphocytes layered liquid separation, collection of mononuclear cells, bonemarrow-derived endothelial progenitor cells were cultured in the EGM-2MV conditionsmedium. Observe cellular morphology which are after induced cultured, and take the cellswhich growth in good condition to detect the expression of endothelial progenitor cellsurface marker VIII factor, CD31, CD34, and CD133by immunocytochemistry, thenidentified the cells.
Results: Most of the cells were showed in fusiform, triangular, spindle-shaped or irregularshape. About5th-7th days, the colony of EPCs appeared, which intermediate was circularcell populations and border cells send forth as spindle budding and appeared monolayergrowth, this structure was similar to blood islands. At9th-11th days, the cells showed atypical "cobblestone" structure, which was the typical morphological characteristics ofEPCs. The immunocytochemistry assay results showed that the surface markers factorCD31, CD34, and CD133were all in positive expression, and the masculine cell rate wasabove90%.
Conclusion: Through the induction culture of conditioned medium, good activity and purebone marrow-derived endothelial progenitor cells can be successfully obtained.
Part three: In vitro construct of vascularized tissueengineering sinus node
Objective: To construct a vascularized tissue engineering sinus node in vitro.
Materials and Methods: Select Matrigel as support material, bone marrow-derivedendothelial progenitor cells and embryo-derived pacemaker cells in different proportions(2:1/1:1/1:2) with liquid Matrigel matrix glue mixed in a5%CO2incubator at37℃forin vitro co-culture. Respectively in18h,3d,5d,7d,9d,11d,13d,15d,17d,19d,21dinverted phase contrast microscope to observe the morphology and the beating rate of thedifferent experimental group cells complex, immunofluorescence microscopy observationand hematoxylin-eosin staining method to observe the distribution of two kinds of cells.
Results: Inverted phase contrast microscope observation shows that co-culture differentproportions of cells mixed with Matrigel, the2:1and1:1mixed group appeared typical“vascular phenomenon” of endothelial cells18hafter cultured, while the “vascularphenomenon” was not obvious in the1:2mixed group. The1:1and1:2mixed group appeared the phenomenon of unified beating in most of the area of the complex after thecultivate, the unified pulsation of2:1mixed group was not obvious. In the third days, inmost area of the complex began to appear unified beating, frequency gradually increasedwith the prolonged incubation time, two weeks later the frequency reached peak. Whencultured for third week, the Matrigel’s shape reduced than before and spontaneouspulsation began to weak than previous. The immunofluorescence and hematoxylin-eosinstaining results showed that the cell density in the1:1mixed group was relatively uniformand the connections between cells was widely exist.
Conclusion: Co-culture of the bone marrow-derived endothelial progenitor cells andembryo-derived pacemaker cells can successfully build vascularized tissue engineeringsinus node in vitro, and the1:1ratio was the ideal rate of the cells.
Part four: In vivo research of vascularized tissueengineering sinus node
Objective: To observe the morphological structure, angiogenesis and the change of regionalflow in transplant by transplant the vascularized tissue engineering sinus node into the rats’heart.
Materials and Methods:60healthy adult SD rats(any male or female, weighing200-250g)were randomly divided into3groups (n=20). The rats in normal control group withoutany treatment, transplant constructed in vitro of the tissue engineering sinus node into rat’sleft ventricular wall of epicardium of heart in non-vascularized tissue engineering sinusnode group, transplant constructed in vitro of the vascularized tissue engineering sinusnode into rat’s left ventricular wall of epicardium of heart in vascular tissue engineeringgroup.The electrocardiogram of the rats were regularly monitored.4and8weeks later, thelaser Doppler flowmetry, hematoxylin-eosin staining and Western Blot method were used todetect the regional volume of blood flow, vasifaction and the expression of VEGFR-2, PI3K,AKT and other factors in transplantation area of different groups.
Results: The surface ECG monitoring results indicate that compared with the normalcontrol group, both the non-vascularized tissue engineering sinus node group and vasculartissue engineering sinus node group have ventricular premature beat of ectopic pacemaking.The regional blood flow in vascular tissue engineering sinus node group was higher thanother group, the HE staining results showed that the ascular tissue engineering sinus node group had more neovascularization than other group, and the Western Blot results showedthat the expression of VEGFR-2, PI3K and AKT in vascularized tissue engineering sinusnode group were higher than other group.
Conclusion: Our experimental method in constructing vascularization tissue engineeringsinus node in vitro can make the sinus node has a long-term survival after implanted intobody, and can produce ectopic pacemaker and neovascularization. The mechanism of thistissue engineering tissue’s vascularization may be related to the PI3K/Akt signal pathway.
引文
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[1] Stefano C, Venu C, Mayank M, et al. Endothelial progenitor cells as factors inneovascularization and endothelial repair. World J Cardiol,2010,2(12):411-420.
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[5] MiakeJ, Marban E, Nuss HB. Biological pacemaker created by gene transfer.Nanture,2002,419(6903):132-133．
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[8] Nikolic DK,Jovanovic Z, Turkovic G, Vulovic R, Mladenovic M. Supracondylar misslefractures of the femur. Injury,2002,33:161-166.
[9] Walles T, Herden T, Haverich A, et al. Influence of scaffold thickness and scaffoldcomposition on bioartificial graft survival. Biomaterials.2003;24(7):1233-1239.
[10] Rouwkema J, Rivronand NC, Blitterswijk CA. Vascularization in tissueengineering.Trends in Biotechnology.2008;26(8):8434-8441.
[11] Druecke D, Langer S, Lamme E, et al. Neovascularization of poly(ether ester)blockcopolymer scaffolds in vivo： long-term investigations using intravital fluorescentmicroscopy. J Biomed Mater Res.2004;68(1):A10-18.
[12] Hutmacher DW, Sittinger M, Risbud MV,et al.Scaffold-based tissue engineering：rationale for computer-aided design and solid free-form fabrication systems. TrendsBiotechnol.2004;22(7):354-362.
[13] Cohen DL, Malone E, Lipson H, et al. Direct freeform fabrication of seeded hydrogelsin arbitrary geometries. Tissue Eng.2006;12(5):1325-1335.
[14] Gafni Y, Zilberman Y, Ophir Z, et al. Design of a filamentous polymeric scaffold forin vivo guided angiogenesis. Tissue Eng.2006;12(5):3021-3034.
[15] Post MJ, Laham R, Sellke FW, et al. Therapeutic angiogenesis in cardiology usingprotein formulations. Cardiovasc Res.2001;49(3):522-531.
[16] Segers VF, Tokunou T, Higgins LJ, et al. Local delivery of protease-resistant stormalcell derived factor-1for stem cell recruitment after myocardial infarction. Circulation.2007;116(15):1683-1692.
[17] Ehrbar M, Metters A, Zammaretti P,et al. Endothelial cell proliferation and progenitormaturation by fibrin-bound VEGF variants with differential susceptibilities to local cellularactivity. JControl Release.2005;101(1-3):93-109.
[18] Martino MM, Rothenfluh DA, Rempel SA, et al. Controlling integrin specificity andstem cell differentiation in2D and3D environments through regulation of fibronectindomain stability. Biomaterials.2009;30(6):1089-1097.
[19] Freeman I, Cohen S. The influence of the sequential delivery angiogenic factors fromaffinity-binding alginate scaffolds on vascularization. Biomaterials.2009;30(11):2122-2131.
[20] Kneser U, Polykandriotis E, Ohnolz J, et al. Engineering of vascularizedtransplantable bone tissues： induction of axial vascularization in an osteoconductivematrix using an arteriovenous loop.Tissue Eng.2006;12(7):1721-1731.
[21] Mertsching H, Walles T, Hofmann M, et al. Engineering of a vascularized scaffold forartificial tissue and organ generation.Biomaterials.2005;26(33):6610-6617.
[22] Lesman A, Habib M, Caspi O, et al. Transplantation of a tissue-engineered humanvascularized cardiac muscle. Tissue Eng. Part A.2010;16(1):115-125.
[23] Neumann T, Nicholson BS, Sanders JE. Tissue engineering of perfused microvessels.Microvascular Research.2003;66(1):59-67.
[24] Sasagawa T,Shimizu T,Sekiya S,et al.Design of prevascularized three-dimensionalcell-dense tissues using a cell sheet stacking manipulation technology. Biomaterials.2010;31(7):1646-1654.
[25] Ruhparwar A,Kallenbach K,Klein G,et al.Adenylate-Cyclase VI transforms ventricularcardiomyocytes into biological pacemaker cells. Tissue Eng Part A.2010;16(6):1867-1877.
[26] Fuchs S,Baffour R,ZhouYF,et al.Transendocardial delivery of autologous bonemarrow enhances collateral perfusion and regional function in pig Swith chronicexperimental myocardial ischemia.J Am Coll Cardiol.2001;37:1726-1732.
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