基于胶原/Matrigel支架材料的工程化心肌组织构建及体内移植的实验研究
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摘要
缺血性心脏疾病是威胁人类健康的头号杀手之一。心肌梗死伴随着功能性心肌细胞大量死亡,最终导致心力衰竭。针对心衰晚期患者,心脏移植手术是目前临床治疗的唯一选择。但因存在供体器官短缺、免疫排斥反应及一系列移植后并发症等问题,而无法在临床广泛应用,急需寻找新的有效治疗方法和手段。组织工程这门交叉学科的兴起,为根治心脏疾病带来了希望。
近年来,心肌组织工程研究进展迅速。国内外相继以新生大鼠心肌细胞、间充质细胞、胚胎干细胞(ESC)源心肌细胞为种子细胞,在体外成功再造了工程化心肌组织,再造心肌组织具有节律性收缩特性,并在组织结构方面与正常新生在体心肌组织相类似。尽管如此,目前心肌组织工程仍面临种子细胞来源选择、如何优化心肌细胞-支架材料体外培育环境,以及如何提高再造心肌组织的质量等一系列关键问题。
从国内外研究现状分析,Zimmermann等利用胶原/Matrigel为支架体外构建心肌这一实验体系最为成功。但是该体系作为心肌体外再造模型采用的是动态力学拉伸,作为体外心肌再造研究模型的稳定性和可靠性尚有待提高。此外,随着相关研究的不断深入,研究人员对如何实现工程化组织体外血管化、以及针对再造心肌组织中非心肌细胞成分对再造心肌组织质量和心肌细胞体外三维重构的影响给予了越来越多的关注。与此同时,本室以往利用该实验体系,以ESC源心肌细胞为种子细胞,虽然成功在体外制备了心肌组织,但因获得种子细胞实验流程复杂、费时长而影响了再造心肌效率与质量。
针对以上分析,本研究利用胶原/Matrigel实验体系,开展了基于静态力学拉伸模型下的心肌组织体外再造研究,并通过添加血管内皮细胞的方式,研究了血管内皮细胞提高再造心肌组织的质量的能力;与此同时,利用胶原/Matrigel实验体系,进行了ESC分化发育的研究,重点观察了ESC自发分化发育及向心肌细胞定向诱导分化情况,在此基础上,利用已探索的定向诱导分化条件,研究ESC在本实验体系下进一步分化形成心肌组织的能力,并对其动物心梗修复能力进行研究。
本论文的主要研究内容与结果简要概括如下,共分为以下四个部分:
第一部分:基于静态力学拉伸模型下的心肌组织体外再造研究再造心肌组织的质量与微环境和非心肌细胞的作用关系密切。其中力学微环境对于再造心肌组织的质量至关重要。目前国内外常见的体外力学拉伸条件以动态力学拉伸为主,该力学拉伸条件存在一定的局限性。相比之下,再造心肌作为心肌体外发育研究模型,静态力学拉伸具有明显的优势,其稳定性和可靠性均较高。此外,近年来发育生物学研究表明,心肌中非心肌细胞成分对心肌细胞三维重构与心脏功能的发挥均起着重要作用,尤其是针对Telocytes细胞在心脏发育中作用的研究,是目前研究的热点之一。
在本部分研究中,首先建立了基于胶原/Matrigel三维立体静态力学拉伸体系;其次,利用该体系,以新生大鼠心肌细胞为种子细胞,成功在体外构建了工程化心肌组织片层。在此基础上,通过组织学染色及免疫组织化学染色,对体外再造心肌组织中是否存在Telocytes细胞进行检测,并对其在心肌细胞三维重构与心脏功能发挥中的作用进行探讨。研究结果分别简要介绍如下。
在基于胶原/Matrigel三维立体静态力学拉伸体系建立方面:研究结果表明,静态力学拉伸抵抗了胶原凝胶在细胞存在时的强烈收缩,避免了由于过度收缩而引起的组织变厚,防止了材料中央部位细胞的大量死亡。在实施静态力学拉伸后,再造心肌组织在体外始终维持一定的形状和厚度,形成较均一的组织片层。
在工程化心肌组织片层制备方面:研究结果表明,心肌细胞在胶原/Matrigel三维立体静态力学拉伸体系中,排列形成致密的肌纤维束,沿着静态拉伸力的方向充分伸展。激光共聚焦观察发现,可见心肌细胞相互连接部位部分区域存在Connexin43阳性染色,说明心肌细胞之间形成功能合胞体。此外,透射电镜结果表明工程化心肌组织中,心肌细胞肌小节排列具有方向性,并且可以观察到与细胞机械收缩和电信号传导密切相关的超微结构,如缝隙连接、桥粒等。
在工程化心肌组织中是否存在Telocytes细胞方面:组织学染色可以观察到再造心肌组织中存在一种长梭形细胞,具有两个或三个长细胞突起,形态上与Telocytes细胞相似。此外,免疫组织化学染色结果表明,这类长梭形细胞表达SMA阳性、Vimentin阳性和CD117阳性。根据以上实验结果,我们初步判定在工程化心肌组织中可能存在Telocytes细胞。
第二部分:组织工程化心肌体外血管化研究
再造组织血管化研究一直是组织工程包括心肌组织工程研究关注的焦点。近年来国内外研究报道表明,血管内皮细胞在三维支架材料中可形成血管样结构,并且血管内皮细胞还对心肌细胞体外空间重构、存活和提高再造心肌组织的质量发挥重要作用。本部分研究,在前期建立基于胶原/Matrigel支架材料构建工程化心肌组织的基础上,进一步在凝胶体系中将血管内皮细胞与心肌细胞复合,深入研究了血管内皮细胞对心肌组织血管化能力和提高心肌组织质量的促进作用。
研究结果表明,复合了血管内皮细胞的组织工程化心肌中,血管样结构形成明显增加,可见较多vWAg阳性的血管内皮细胞排列形成条索样结构。此外,血管内皮细胞还对心肌细胞空间排布上产生影响,cTnT和vWAg荧光双标记激光共聚焦检测发现,心肌细胞与血管内皮细胞在空间上相互靠近,心肌细胞更偏向于在血管内皮细胞网中存在。此外,加入血管内皮细胞还可以稳定再造心肌组织结构,提高再造心肌组织的收缩能力。
第三部分:ESC在胶原/Matrigel支架材料中分化发育的实验研究
在ESC分化发育研究中,拟胚体(EBs)的形成对于ESC分化发育发挥着重要作用。现有ESC分化发育研究体系多是将ESC形成EBs与EBs进一步分化发育这一连续过程分割为两个阶段:首先是在二维或三维环境中形成EBs,随后将所形成的EBs二维贴壁诱导分化或接种在三维支架材料中进一步诱导分化。
在本研究中,基于胶原/Matrigel这一实验体系在心肌再造方面取得的良好结果和建立的平台条件,将ESC以单细胞形式直接接种在胶原/Matrigel支架材料中,进而观察EBs形成以及后续分化发育情况,从而探讨胶原/Matrigel对ESC在三维立体环境中直接形成EBs的可行性。重点针对ESC接种密度对EBs形成的影响、不同支架材料组成对EBs形成与分化的影响以及ESC在本体系下自发分化情况进行了研究。
其中,在ESC接种密度对EBs形成影响的研究方面,结果表明ESC在胶原/Matrigel支架材料中可以直接启动EBs形成,并且ESC接种浓度对EBs形成具有明显影响,其最佳接种密度在105个/mL至106个/mL之间。在这一密度范围内,ESC所形成的EBs数量多、体积大。在不同支架材料组成对EBs形成与分化的影响研究方面,将ESC分别在单纯胶原凝胶及胶原/Matrigel不同体系中培养发现,支架材料中的胶原凝胶成分主要影响EBs形成,而Matrigel成分则对EBs分化发育与发挥重要作用。在ESC自发分化研究方面,将ESC在胶原/Matrigel三维立体环境中培养,在不施加任何诱导条件的情况下,通过组织学染色结果表明,体外培养21d后EBs自发分化形成了与在体结构类似的各种组织结构,如有心肌细胞合胞体样结构、管腔样结构形成。针对CK18、CD31、cTnT、Nestin等免疫组织化学染色结果证实,ESC可以分化为cTnT阳性的心肌细胞、CK18阳性的上皮细胞、CD31阳性的内皮细胞以及Nestin阳性细胞等。
第四部分:基于ESC为种子细胞的心肌组织构建及体内移植的实验研究
在本部分研究中,利用我室在心肌组织工程研究方面既有的研究平台和技术体系,在前期利用胶原/Matrigel进行ESC定向体外分化研究的基础上,探索了ESC进一步定向诱导分化并形成心肌组织的能力及其动物心梗修复能力。
ESC在本体系下是否可以通过特定诱导条件而向心肌细胞分化进而形成心肌组织是本论文研究关注的焦点之一。在本部分研究中,通过添加抗坏血酸作为ESC向心肌细胞分化的诱导剂,进行了ESC体外定向诱导分化及心肌组织三维再造研究,并对再造组织进行了组织学及免疫组织化学鉴定。在此基础上,将所构建的工程化心肌组织移植到心梗部位,观察其在心梗部位存活、分化,及对心功能的改善情况。
研究发现ESC在胶原/Matrigel支架材料中培养10-11d时(诱导3-4d)分化形成大量跳动的细胞,并且随着培养时间的延长,跳动细胞的数量和面积逐渐增多,14d(诱导7d)左右可见形成大面积跳动区域,体外培养19d(诱导12d)左右出现一致性跳动区域。对其进行免疫组织化学染色证实,再造心肌组织中含有大量表达cTnT、Nkx2.5和GATA4阳性的心肌细胞。与向心肌细胞自发分化相比,诱导分化后cTnT阳性细胞的大量增多,提示抗坏血酸显著促进了ESC向心肌细胞分化。并且,随着再造心肌组织中心肌细胞逐渐成熟,可见心肌细胞相互连接部位部分区域存在Connexin43阳性染色。免疫组织化学染色同时还揭示了在再造心肌组织中存在ESC分化来源的Nestin阳性细胞和CD31阳性细胞。
将上述体外再造心肌组织进行心梗部位移植,并将单纯心梗组和非收缩片层移植组作为对照组。移植术后4w进行心功能检测,结果表明,与单纯心梗组和非收缩片层移植组相比,工程化心肌组织移植组可以显著改善梗死心肌的收缩及舒张功能。组织切片染色观察显示工程化心肌组织片层紧密贴附于梗死心肌部位,并已在结构上与宿主心肌发生整合,两者之间界限不明显。此外,针对心肌特异性标志cTnT,Nkx2.5,GATA4,connexin43免疫组织化学检测结果显示,再造工程化心肌组织中存在具有分化心肌细胞表型的细胞,并且Connexin43染色结果表明工程化心肌组织及工程化心肌组织与宿主心肌细胞之间形成了较广泛的细胞间连接。CD31染色结果显示在移植的工程化心肌组织中存在较丰富的血管内皮细胞,并且形成了管腔样结构。在此基础上,对工程化心肌组织移植后畸胎瘤形成情况进行了检测。移植4w后,开胸观察畸胎瘤形成。工程化心肌组织移植组移植后会引起畸胎瘤发生,发生率在25%(3+/12),形成的畸胎瘤位于心肌梗死部位,具有一定肉眼可见的体积,而在动物胸腔和腹腔均未检测到畸胎瘤形成。所形成的畸胎瘤包含典型的三胚层结构。
综上所述,本论文以胶原/Matrigel为支架材料,在体外建立了三维立体静态力学拉伸体系,并利用该体系,以新生大鼠心肌细胞为种子细胞,体外构建了工程化心肌组织片层,并对再造心肌组织中是否存在Telocytes细胞进行研究。此外,通过将血管内皮细胞与心肌细胞复合在胶原/Matrigel支架材料中,进一步研究了再造心肌组织血管化能力。在此基础上,基于静态力学拉伸体系,以胶原/Matrigel为支架材料,建立了ESC体外分化发育模型,开展了ESC三维立体分化与体外心肌再造研究,并将所构建的工程化心肌组织移植到心梗部位探索其动物心梗修复能力,结果表明,工程化心肌组织片层能够与宿主发生整合,并改善受损心肌功能。
The ischemic heart disease is a leading killer of human beings. Myocardial infarction occurs when a large number of function cardiac cells die, and it eventually induces the heart failure. Currently, for a patient of heart failure in late stage, heart transplantation is the only feasible therapy. However, due to the shortage of donor organ, the immunologic rejection reaction, and a series of post-transplantation complications, clinically heart transplantation is not able to be applied extensively. Therefore, it is urgent to develop new effective therapies and means. The upsurge of tissue engineering, an interdisciplinary, becomes a hope for heart diseases.
Recently, great progress has been made in the myocardial tissue engineering. Cardiomyocytes, mesenchymal cells and embryonic stem cell (ESC)-derived cardiomyocytes have been used as seeding cells to regenerat engineered heart tissues in vitro successfully both home and abroad. They are of rhythmic contracting characteristics, similar to the normal newborn myocardial tissue in vivo in histology. However, there are still a series of key issues suspended to solve in myocardial tissue engineering, such as the choosing cell source, issues on how to optimize the in vitro cultivation environment of cardiomyocytes-scaffold materials and how to improve the regenerated myocardium quality.
Analyzing from the engineering status quo in the word, the experiment system of myocardial tissue construction invitro set up by Zimmermann etc is more successful, which is based on the collagen/Matrigel scaffold. However as a myocardial tissue in vitro regenerating model, the dynamic stretching and its stability and reliability are required to improve. In addition, following the deep going of relevant studies, more and more attentions are paid on the in vitro vascularization of engineered tissue and the impact of non-myocardium cells in a regenerated myocardial tissue on the quality of regenerated myocardium as well as on the in vitro three-dimensional reconstruction of myocardium cells by researchers. Furthermore, the efficiency and quality of regenerated myocardium are affected due to the complicated and time-consuming experimental process on acquiring the seeding cells, although we have once successfully constructed the myocardial tissue by using the experiment system and taking the embryonic stem cells as seeding cells.
For the above analysis, in this study, we have carried out an experiment of in vitro myocardial tissue regenerating based on a static stretching model and under the collagen/Matrigel experiment system. Through adding vascular endothelial cells, we have studied the ability of the vascular endothelial cell improving the regenerated myocardium quality. Meanwhile, the study of embryonic stem cells differentiation and development has been made under the collagen/Martrigel experiment system, to emphatically observe the embryonic stem cell spontaneous differentiation and development as well as its induced differentiation into cardiomyocytes. Base on the above, a research has been carried out on the embryonic stem cells further differentiating into a myocardium under the experiment system and the induced differentiation condition explored. An investigation into the repairing animal myocardial infarction by the myocardium has been made too.
The main content and results of the paper, including four sections, are briefed as follows:
Section one: Research on the myocardial tissue regeneration in vitro based on the static stretching model The quality of regenerated myocardial tissue is closely depended upon the microenvironment and the non-myocardial cells, and especially the mechanical microenvironment is crucial to the quality. The dynamic stretching is common in the in vitro stretching conditions home and abroad, which has certain limits. Comparatively, the static stretching with high stability and reliability has obvious strengths for the regenerated myocardium as a myocardium in vitro development model. Moreover, it is shown by recent studies in development biology that the non-myocardium cells play important roles in the cardiomyocyte three-dimensional reconstruction and the heart function. Especially the role of Telocytes in a heart development is one of hot topics currently.
In this section, firstly a three-dimensional static stretching system based on the collagen/Matrigel has been set up. Then, an engineered heart tissue has been successfully constructed in vitro under the system, taking cardiomyocytes of a newborn rat as seeding cells. Based on the above, by the histological staining, and immunohistochemistrical staining, the Telocytes existing in the in vitro regenerated myocardial tissue has been detected, and the roles of Telocytes in the cardiomyocytes three-dimensional reconstruction and in the heart functions have been explored too. The results of study are briefly introduced as follows.
While establishing the three-dimensional static stretching system based on the collagen/Matrigel, it is shown that the static stretching resists the strong contracting of collagen gel when cells exist. It consequently avoids the tissue growing thinker induced by over contracting and the mass central cells necroses. After implementing the static stretching, the regenerated myocardial tissue always keeps in a certain thickness and volume in vitro, and forms a uniform tissue patch structure.
While preparing the engineered myocardial tissue patch, it is shown that the cardiomyocytes are arranged tightly to form a dense muscle bundle which extends fully in the direction of the static stretching under the collagen/Matrigel three-dimensional static stretching system. It is discovered through the laser con-focal that Connexin43 positive myocardium cells exist between cardiomyocytes, which demonstrates the connection of cardiomyocytes. Additionally, it is showed by results observed through a transmission electron microscope that the cardiomyocytes sarcomeres are arranged in some obvious directions in the engineered myocardium. The ultra-structures, such as gap junction and bridge corpuscle, close interrelated with the cells mechanical contracting and the electronic conductivity, are also observed clearly.
On the issue that whether Telocytes exist in the engineered myocardium or not, a kind of spindle cell with two or three cell processes, similar to Telocytes in shape, is observed through the histological staining. Meanwhile, immunohistochemistrical staining results show that the SMA, Vimentin and CD117 of the spindle cell are expressed positively. We can conclude preliminarily that there exist Telocytes cells in the engineered myocardium.
Section two: Research on the vascularized engineered heart tissue in vitro
Study on the regenerated vascularized tissue is always the focus of the tissue engineering including the myocardial tissue engineering. Recent years, studies at home and abroad show that the vascular endothelial cells are able to form vascular-like structure in the three-dimensional scaffold materials, and the endothelial cells play important roles in the in vitro space reconstruction, the survival and the quality of cardiomyocytes. In this section, on the basis of the constructed engineered myocardium established previously and based on the collagen/Matrigel scaffolds, we further add vascular endothelial cells with cardiomyocytes in the gel system, and deeply study the promoting roles of vascular endothelial cells on the myocardium vascularization and the myocardium quality.
It is demonstrated that the vascular-like structure are formed increasingly in the engineered myocardium blended with vascular endothelial cells, and a large number of vWAg-positive endothelial cells are arranged to form a vessel-like structure. Additionally, the endothelial cells give an impact on the cardiomyocytes space arrangement. It is discovered through the cTnT and vWAg double-labeled con-focal fluorescence detection that cardiomyocytes and endothelial cells approach each other in space and the cardiomyocytes s tend to exist in the endothelial cells network. Moreover, the addition of endothelial cells can stabilize the regenerated myocardial tissue texture and promote the consistent contracting of regenerated myocardial tissue.
Section three: The experimental research on the differentiation and development of embryonic stem cell (ESC) in the collagen / Matrigel scaffold materials
The formation of embryoid bodies (EBs) plays important roles in ESC differentiation and development. In the current ESC differentiation and development system, a continuous process, ESC forming EBs and the EBs further differentiating and developing, is mostly divided into the two stages: First the EBs is formed in a two-dimensional or three-dimensional environment. And then the EBs is induced to differentiate by two-dimensional adherent culture, or it is inoculated in three dimensional scaffold materials to further induce to differentiate.
In the study, based on the sound results and the platform conditionsgained in the myocardium regenerating on the basis of the collagen/Matrigel experiment system, ESC, as a single cell form, is directly inoculated in the collagen/Matrigel scaffold materials to observe the forming of EBs and next EBs’differentiation and developion. Thus, for collagen/Matrigel, the feasibility of ESC direct forming EBs in a three-dimensional environment is further explored. Especially the impact of ESC inoculating density on the forming of EBs, the impact of different scaffold composition on the forming and differentiating of EBs and the spontaneous differentiating of ESC in the system are emphatically studied.
As for impacts of ESC inoculating density on the forming of EBs, it is shown that ESC can directly start the formation of EBs in the collagen/Matrigel scaffold materials, and the ESC inoculating density can obviously affect the formation of EBs. Besides, the best inoculating density range is from 105cells/mL to 106cells/mL. In this range, the EBs formed by ESC is numerous and large.
As for impacts of different scaffold materials composition on the forming and differentiating of EBs, it is discovered through culturing ESC separately in the pure collagen gel system and the collagen/Matrigel system, that the collagen gel in the scaffold materials mainly affects the forming of the EBs while Matrigel plays important roles on the differentiating and developing of the EBs.
As for the ESC spontaneous differentiating, by culturing ESC in the collagen/Matrigel three-dimensional environment and under implanting no inducing condition, it is shown through the histological staining that EBs spontaneous differentiates to form various tissue structures, such as the myocardium cell synplasm-like structure and tube-like structure, which are similar to the in vivo structures, after culturing in vitro for 21 days. It proved, through immunohistochemistrical staining such as CK18, CD31, cTnT and Nestin, that ESC can differentiate into cTnT-positive myocardium cells, CK18-positive epithelial cells, CD31-positive endothelial cells and Nestin-positive cells.
Section four: Experiment researches on the in vivo transplantation and the myocardial tissue construction based on ESC as seeding cells
In this section, ESC further inducing to differentiate into the myocardium and the repairing animal myocardial infarction are explored, using the research platform and the technology system, which is established by our lab in the myocardial tissue engineering and is based the embryonic stem cell induction and differentiation system set up earlier as well as based on the liquid collagen I/Matrigel gel composite scaffold materials.
Whether ESC can differentiate into cardiomyocytes or not under the specified inducing conditions is one of focuses of the study. In this section, ESC in vitro was induced to differentiate into cardiomyocytes in vitro by adding the ascorbic acid as an inductive agent, and reconstruct three-dimensional engineered heart tissue. Moreover, histological and immunohistochemistrical verifications of the regenerated tissue have been performed too. Based on the above, the engineered heart tissue is transplanted into the myocardial infarction, and the survival and differentiating of the tissue in the part as well as the improvement of heart function by the tissue are observed.
It is discovered by studies that ESC differentiate into a large number of beating cells in the collagen/Matrigel scaffold materials. Otherwise the number and area of beating cells gradually increase as the culture time extending. A large area of consistent beating zone has been formed after culturing for 14 days, and the consistent beating has been formed after culturing for 19 days. It proved, by the immunohistochemistrical staining of the patch, that the regenerated myocardium contains a large number of cardiomyocytes expressing cTnT, Nkx2.5 and GATA4. Comparing with the spontaneous differentiating into the cardiomyocytes, the large increase of the cTnT-positive cells as inducing to differentiate indicates that the ascorbic acid promotes markedly the ESC differentiating into the myocardial cells. Commexin43 in the myocardium peripheral connecting tissue can be recognized that the regenerated myocardial cells mature gradually. The staining also indicates that there exist Nestin-positive cells and CD31-positive cells.
The above mentioned in vitro regenerated myocardium is transplanted into the cardiac infarction part, taking the pure cardiac infarction group and the noncontractile material as the control groups. The heart functions are tested after 4w from transplantation. The results show that, compared with the pure cardiac infarction group as well as with the noncontractile material, the contracting and relaxing functions of the infarction myocardium have been tremendously improved after the engineered myocardium transplanting. Through staining tissue sections, it is observed that the myocardium patch densely attaches to the myocardium infraction part. Furthermore it has integrated with the host myocardium in texture and there is not an obvious boundary. In addition, as for the myocardium specific cTnT, NKx2.5, GATA4 and Connexin43, immunohistochemically, it is indicated that there are a large number of cells with differentiated phenotype of myocardium cells in the myocardium patch. Connexin43 staining showed that there were a large number of intercellular junctions in the myocardium patch, as well as between the myocardium patch and the myocardium cells of the host. CD31 staining showed that in the transplanted engineered cardiac tissue, there were many vascular endothelial cells, which has formed into a tube structure. On this basis, detection was conducted on the formation of teratoma after the transplantation of the engineered cardiac tissue. The formation of teratoma was observed to begin from the fourth week after the transplantation. The transplantation will lead to the occurrence of teratoma in the myocardium patch transplantation group with a incidence rate of 25% (3+/12), the teratoma locates at the location of myocardial infarction with certain visible size, and teratoma in the animal’s thoracic cavity and abdominal cavity was not detected. The teratoma contained the typical tridermic structure.
To sum up, in the study, the three-dimensional static stretching system has been established based on the collagen/Matrigen scaffold materials. The engineered myocardium patch has been constructed in vitro under the system, taking the cardiomyocytes of newborn rat as seeding cells. Meanwhile, the study has been carried out to judge the Telocytes existing in the regenerated myocardium. Furthermore, vascularization of the regenerated myocardium has been further studied by blending the vascular endothelial cells and the cardiomyocytes into the collagen/Matrigel scaffold materials. Based on the above, under the static stretching system, ESC in vitro differentiation and development model has been set up using collagen/Matrigel as scaffold materials. The ESC three-dimensional differentiating and the myocardium regenerating are studied. The engineered myocardium constructed is transplanted into a cardiac infarction part to explore the repairing ability of infarction. The results show that the engineered myocardium patch can integrate with the host and improve the function of damaged myocardium.
近年来,心肌组织工程研究进展迅速。国内外相继以新生大鼠心肌细胞、间充质细胞、胚胎干细胞(ESC)源心肌细胞为种子细胞,在体外成功再造了工程化心肌组织,再造心肌组织具有节律性收缩特性,并在组织结构方面与正常新生在体心肌组织相类似。尽管如此,目前心肌组织工程仍面临种子细胞来源选择、如何优化心肌细胞-支架材料体外培育环境,以及如何提高再造心肌组织的质量等一系列关键问题。
从国内外研究现状分析,Zimmermann等利用胶原/Matrigel为支架体外构建心肌这一实验体系最为成功。但是该体系作为心肌体外再造模型采用的是动态力学拉伸,作为体外心肌再造研究模型的稳定性和可靠性尚有待提高。此外,随着相关研究的不断深入,研究人员对如何实现工程化组织体外血管化、以及针对再造心肌组织中非心肌细胞成分对再造心肌组织质量和心肌细胞体外三维重构的影响给予了越来越多的关注。与此同时,本室以往利用该实验体系,以ESC源心肌细胞为种子细胞,虽然成功在体外制备了心肌组织,但因获得种子细胞实验流程复杂、费时长而影响了再造心肌效率与质量。
针对以上分析,本研究利用胶原/Matrigel实验体系,开展了基于静态力学拉伸模型下的心肌组织体外再造研究,并通过添加血管内皮细胞的方式,研究了血管内皮细胞提高再造心肌组织的质量的能力;与此同时,利用胶原/Matrigel实验体系,进行了ESC分化发育的研究,重点观察了ESC自发分化发育及向心肌细胞定向诱导分化情况,在此基础上,利用已探索的定向诱导分化条件,研究ESC在本实验体系下进一步分化形成心肌组织的能力,并对其动物心梗修复能力进行研究。
本论文的主要研究内容与结果简要概括如下,共分为以下四个部分:
第一部分:基于静态力学拉伸模型下的心肌组织体外再造研究再造心肌组织的质量与微环境和非心肌细胞的作用关系密切。其中力学微环境对于再造心肌组织的质量至关重要。目前国内外常见的体外力学拉伸条件以动态力学拉伸为主,该力学拉伸条件存在一定的局限性。相比之下,再造心肌作为心肌体外发育研究模型,静态力学拉伸具有明显的优势,其稳定性和可靠性均较高。此外,近年来发育生物学研究表明,心肌中非心肌细胞成分对心肌细胞三维重构与心脏功能的发挥均起着重要作用,尤其是针对Telocytes细胞在心脏发育中作用的研究,是目前研究的热点之一。
在本部分研究中,首先建立了基于胶原/Matrigel三维立体静态力学拉伸体系;其次,利用该体系,以新生大鼠心肌细胞为种子细胞,成功在体外构建了工程化心肌组织片层。在此基础上,通过组织学染色及免疫组织化学染色,对体外再造心肌组织中是否存在Telocytes细胞进行检测,并对其在心肌细胞三维重构与心脏功能发挥中的作用进行探讨。研究结果分别简要介绍如下。
在基于胶原/Matrigel三维立体静态力学拉伸体系建立方面:研究结果表明,静态力学拉伸抵抗了胶原凝胶在细胞存在时的强烈收缩,避免了由于过度收缩而引起的组织变厚,防止了材料中央部位细胞的大量死亡。在实施静态力学拉伸后,再造心肌组织在体外始终维持一定的形状和厚度,形成较均一的组织片层。
在工程化心肌组织片层制备方面:研究结果表明,心肌细胞在胶原/Matrigel三维立体静态力学拉伸体系中,排列形成致密的肌纤维束,沿着静态拉伸力的方向充分伸展。激光共聚焦观察发现,可见心肌细胞相互连接部位部分区域存在Connexin43阳性染色,说明心肌细胞之间形成功能合胞体。此外,透射电镜结果表明工程化心肌组织中,心肌细胞肌小节排列具有方向性,并且可以观察到与细胞机械收缩和电信号传导密切相关的超微结构,如缝隙连接、桥粒等。
在工程化心肌组织中是否存在Telocytes细胞方面:组织学染色可以观察到再造心肌组织中存在一种长梭形细胞,具有两个或三个长细胞突起,形态上与Telocytes细胞相似。此外,免疫组织化学染色结果表明,这类长梭形细胞表达SMA阳性、Vimentin阳性和CD117阳性。根据以上实验结果,我们初步判定在工程化心肌组织中可能存在Telocytes细胞。
第二部分:组织工程化心肌体外血管化研究
再造组织血管化研究一直是组织工程包括心肌组织工程研究关注的焦点。近年来国内外研究报道表明,血管内皮细胞在三维支架材料中可形成血管样结构,并且血管内皮细胞还对心肌细胞体外空间重构、存活和提高再造心肌组织的质量发挥重要作用。本部分研究,在前期建立基于胶原/Matrigel支架材料构建工程化心肌组织的基础上,进一步在凝胶体系中将血管内皮细胞与心肌细胞复合,深入研究了血管内皮细胞对心肌组织血管化能力和提高心肌组织质量的促进作用。
研究结果表明,复合了血管内皮细胞的组织工程化心肌中,血管样结构形成明显增加,可见较多vWAg阳性的血管内皮细胞排列形成条索样结构。此外,血管内皮细胞还对心肌细胞空间排布上产生影响,cTnT和vWAg荧光双标记激光共聚焦检测发现,心肌细胞与血管内皮细胞在空间上相互靠近,心肌细胞更偏向于在血管内皮细胞网中存在。此外,加入血管内皮细胞还可以稳定再造心肌组织结构,提高再造心肌组织的收缩能力。
第三部分:ESC在胶原/Matrigel支架材料中分化发育的实验研究
在ESC分化发育研究中,拟胚体(EBs)的形成对于ESC分化发育发挥着重要作用。现有ESC分化发育研究体系多是将ESC形成EBs与EBs进一步分化发育这一连续过程分割为两个阶段:首先是在二维或三维环境中形成EBs,随后将所形成的EBs二维贴壁诱导分化或接种在三维支架材料中进一步诱导分化。
在本研究中,基于胶原/Matrigel这一实验体系在心肌再造方面取得的良好结果和建立的平台条件,将ESC以单细胞形式直接接种在胶原/Matrigel支架材料中,进而观察EBs形成以及后续分化发育情况,从而探讨胶原/Matrigel对ESC在三维立体环境中直接形成EBs的可行性。重点针对ESC接种密度对EBs形成的影响、不同支架材料组成对EBs形成与分化的影响以及ESC在本体系下自发分化情况进行了研究。
其中,在ESC接种密度对EBs形成影响的研究方面,结果表明ESC在胶原/Matrigel支架材料中可以直接启动EBs形成,并且ESC接种浓度对EBs形成具有明显影响,其最佳接种密度在105个/mL至106个/mL之间。在这一密度范围内,ESC所形成的EBs数量多、体积大。在不同支架材料组成对EBs形成与分化的影响研究方面,将ESC分别在单纯胶原凝胶及胶原/Matrigel不同体系中培养发现,支架材料中的胶原凝胶成分主要影响EBs形成,而Matrigel成分则对EBs分化发育与发挥重要作用。在ESC自发分化研究方面,将ESC在胶原/Matrigel三维立体环境中培养,在不施加任何诱导条件的情况下,通过组织学染色结果表明,体外培养21d后EBs自发分化形成了与在体结构类似的各种组织结构,如有心肌细胞合胞体样结构、管腔样结构形成。针对CK18、CD31、cTnT、Nestin等免疫组织化学染色结果证实,ESC可以分化为cTnT阳性的心肌细胞、CK18阳性的上皮细胞、CD31阳性的内皮细胞以及Nestin阳性细胞等。
第四部分:基于ESC为种子细胞的心肌组织构建及体内移植的实验研究
在本部分研究中,利用我室在心肌组织工程研究方面既有的研究平台和技术体系,在前期利用胶原/Matrigel进行ESC定向体外分化研究的基础上,探索了ESC进一步定向诱导分化并形成心肌组织的能力及其动物心梗修复能力。
ESC在本体系下是否可以通过特定诱导条件而向心肌细胞分化进而形成心肌组织是本论文研究关注的焦点之一。在本部分研究中,通过添加抗坏血酸作为ESC向心肌细胞分化的诱导剂,进行了ESC体外定向诱导分化及心肌组织三维再造研究,并对再造组织进行了组织学及免疫组织化学鉴定。在此基础上,将所构建的工程化心肌组织移植到心梗部位,观察其在心梗部位存活、分化,及对心功能的改善情况。
研究发现ESC在胶原/Matrigel支架材料中培养10-11d时(诱导3-4d)分化形成大量跳动的细胞,并且随着培养时间的延长,跳动细胞的数量和面积逐渐增多,14d(诱导7d)左右可见形成大面积跳动区域,体外培养19d(诱导12d)左右出现一致性跳动区域。对其进行免疫组织化学染色证实,再造心肌组织中含有大量表达cTnT、Nkx2.5和GATA4阳性的心肌细胞。与向心肌细胞自发分化相比,诱导分化后cTnT阳性细胞的大量增多,提示抗坏血酸显著促进了ESC向心肌细胞分化。并且,随着再造心肌组织中心肌细胞逐渐成熟,可见心肌细胞相互连接部位部分区域存在Connexin43阳性染色。免疫组织化学染色同时还揭示了在再造心肌组织中存在ESC分化来源的Nestin阳性细胞和CD31阳性细胞。
将上述体外再造心肌组织进行心梗部位移植,并将单纯心梗组和非收缩片层移植组作为对照组。移植术后4w进行心功能检测,结果表明,与单纯心梗组和非收缩片层移植组相比,工程化心肌组织移植组可以显著改善梗死心肌的收缩及舒张功能。组织切片染色观察显示工程化心肌组织片层紧密贴附于梗死心肌部位,并已在结构上与宿主心肌发生整合,两者之间界限不明显。此外,针对心肌特异性标志cTnT,Nkx2.5,GATA4,connexin43免疫组织化学检测结果显示,再造工程化心肌组织中存在具有分化心肌细胞表型的细胞,并且Connexin43染色结果表明工程化心肌组织及工程化心肌组织与宿主心肌细胞之间形成了较广泛的细胞间连接。CD31染色结果显示在移植的工程化心肌组织中存在较丰富的血管内皮细胞,并且形成了管腔样结构。在此基础上,对工程化心肌组织移植后畸胎瘤形成情况进行了检测。移植4w后,开胸观察畸胎瘤形成。工程化心肌组织移植组移植后会引起畸胎瘤发生,发生率在25%(3+/12),形成的畸胎瘤位于心肌梗死部位,具有一定肉眼可见的体积,而在动物胸腔和腹腔均未检测到畸胎瘤形成。所形成的畸胎瘤包含典型的三胚层结构。
综上所述,本论文以胶原/Matrigel为支架材料,在体外建立了三维立体静态力学拉伸体系,并利用该体系,以新生大鼠心肌细胞为种子细胞,体外构建了工程化心肌组织片层,并对再造心肌组织中是否存在Telocytes细胞进行研究。此外,通过将血管内皮细胞与心肌细胞复合在胶原/Matrigel支架材料中,进一步研究了再造心肌组织血管化能力。在此基础上,基于静态力学拉伸体系,以胶原/Matrigel为支架材料,建立了ESC体外分化发育模型,开展了ESC三维立体分化与体外心肌再造研究,并将所构建的工程化心肌组织移植到心梗部位探索其动物心梗修复能力,结果表明,工程化心肌组织片层能够与宿主发生整合,并改善受损心肌功能。
The ischemic heart disease is a leading killer of human beings. Myocardial infarction occurs when a large number of function cardiac cells die, and it eventually induces the heart failure. Currently, for a patient of heart failure in late stage, heart transplantation is the only feasible therapy. However, due to the shortage of donor organ, the immunologic rejection reaction, and a series of post-transplantation complications, clinically heart transplantation is not able to be applied extensively. Therefore, it is urgent to develop new effective therapies and means. The upsurge of tissue engineering, an interdisciplinary, becomes a hope for heart diseases.
Recently, great progress has been made in the myocardial tissue engineering. Cardiomyocytes, mesenchymal cells and embryonic stem cell (ESC)-derived cardiomyocytes have been used as seeding cells to regenerat engineered heart tissues in vitro successfully both home and abroad. They are of rhythmic contracting characteristics, similar to the normal newborn myocardial tissue in vivo in histology. However, there are still a series of key issues suspended to solve in myocardial tissue engineering, such as the choosing cell source, issues on how to optimize the in vitro cultivation environment of cardiomyocytes-scaffold materials and how to improve the regenerated myocardium quality.
Analyzing from the engineering status quo in the word, the experiment system of myocardial tissue construction invitro set up by Zimmermann etc is more successful, which is based on the collagen/Matrigel scaffold. However as a myocardial tissue in vitro regenerating model, the dynamic stretching and its stability and reliability are required to improve. In addition, following the deep going of relevant studies, more and more attentions are paid on the in vitro vascularization of engineered tissue and the impact of non-myocardium cells in a regenerated myocardial tissue on the quality of regenerated myocardium as well as on the in vitro three-dimensional reconstruction of myocardium cells by researchers. Furthermore, the efficiency and quality of regenerated myocardium are affected due to the complicated and time-consuming experimental process on acquiring the seeding cells, although we have once successfully constructed the myocardial tissue by using the experiment system and taking the embryonic stem cells as seeding cells.
For the above analysis, in this study, we have carried out an experiment of in vitro myocardial tissue regenerating based on a static stretching model and under the collagen/Matrigel experiment system. Through adding vascular endothelial cells, we have studied the ability of the vascular endothelial cell improving the regenerated myocardium quality. Meanwhile, the study of embryonic stem cells differentiation and development has been made under the collagen/Martrigel experiment system, to emphatically observe the embryonic stem cell spontaneous differentiation and development as well as its induced differentiation into cardiomyocytes. Base on the above, a research has been carried out on the embryonic stem cells further differentiating into a myocardium under the experiment system and the induced differentiation condition explored. An investigation into the repairing animal myocardial infarction by the myocardium has been made too.
The main content and results of the paper, including four sections, are briefed as follows:
Section one: Research on the myocardial tissue regeneration in vitro based on the static stretching model The quality of regenerated myocardial tissue is closely depended upon the microenvironment and the non-myocardial cells, and especially the mechanical microenvironment is crucial to the quality. The dynamic stretching is common in the in vitro stretching conditions home and abroad, which has certain limits. Comparatively, the static stretching with high stability and reliability has obvious strengths for the regenerated myocardium as a myocardium in vitro development model. Moreover, it is shown by recent studies in development biology that the non-myocardium cells play important roles in the cardiomyocyte three-dimensional reconstruction and the heart function. Especially the role of Telocytes in a heart development is one of hot topics currently.
In this section, firstly a three-dimensional static stretching system based on the collagen/Matrigel has been set up. Then, an engineered heart tissue has been successfully constructed in vitro under the system, taking cardiomyocytes of a newborn rat as seeding cells. Based on the above, by the histological staining, and immunohistochemistrical staining, the Telocytes existing in the in vitro regenerated myocardial tissue has been detected, and the roles of Telocytes in the cardiomyocytes three-dimensional reconstruction and in the heart functions have been explored too. The results of study are briefly introduced as follows.
While establishing the three-dimensional static stretching system based on the collagen/Matrigel, it is shown that the static stretching resists the strong contracting of collagen gel when cells exist. It consequently avoids the tissue growing thinker induced by over contracting and the mass central cells necroses. After implementing the static stretching, the regenerated myocardial tissue always keeps in a certain thickness and volume in vitro, and forms a uniform tissue patch structure.
While preparing the engineered myocardial tissue patch, it is shown that the cardiomyocytes are arranged tightly to form a dense muscle bundle which extends fully in the direction of the static stretching under the collagen/Matrigel three-dimensional static stretching system. It is discovered through the laser con-focal that Connexin43 positive myocardium cells exist between cardiomyocytes, which demonstrates the connection of cardiomyocytes. Additionally, it is showed by results observed through a transmission electron microscope that the cardiomyocytes sarcomeres are arranged in some obvious directions in the engineered myocardium. The ultra-structures, such as gap junction and bridge corpuscle, close interrelated with the cells mechanical contracting and the electronic conductivity, are also observed clearly.
On the issue that whether Telocytes exist in the engineered myocardium or not, a kind of spindle cell with two or three cell processes, similar to Telocytes in shape, is observed through the histological staining. Meanwhile, immunohistochemistrical staining results show that the SMA, Vimentin and CD117 of the spindle cell are expressed positively. We can conclude preliminarily that there exist Telocytes cells in the engineered myocardium.
Section two: Research on the vascularized engineered heart tissue in vitro
Study on the regenerated vascularized tissue is always the focus of the tissue engineering including the myocardial tissue engineering. Recent years, studies at home and abroad show that the vascular endothelial cells are able to form vascular-like structure in the three-dimensional scaffold materials, and the endothelial cells play important roles in the in vitro space reconstruction, the survival and the quality of cardiomyocytes. In this section, on the basis of the constructed engineered myocardium established previously and based on the collagen/Matrigel scaffolds, we further add vascular endothelial cells with cardiomyocytes in the gel system, and deeply study the promoting roles of vascular endothelial cells on the myocardium vascularization and the myocardium quality.
It is demonstrated that the vascular-like structure are formed increasingly in the engineered myocardium blended with vascular endothelial cells, and a large number of vWAg-positive endothelial cells are arranged to form a vessel-like structure. Additionally, the endothelial cells give an impact on the cardiomyocytes space arrangement. It is discovered through the cTnT and vWAg double-labeled con-focal fluorescence detection that cardiomyocytes and endothelial cells approach each other in space and the cardiomyocytes s tend to exist in the endothelial cells network. Moreover, the addition of endothelial cells can stabilize the regenerated myocardial tissue texture and promote the consistent contracting of regenerated myocardial tissue.
Section three: The experimental research on the differentiation and development of embryonic stem cell (ESC) in the collagen / Matrigel scaffold materials
The formation of embryoid bodies (EBs) plays important roles in ESC differentiation and development. In the current ESC differentiation and development system, a continuous process, ESC forming EBs and the EBs further differentiating and developing, is mostly divided into the two stages: First the EBs is formed in a two-dimensional or three-dimensional environment. And then the EBs is induced to differentiate by two-dimensional adherent culture, or it is inoculated in three dimensional scaffold materials to further induce to differentiate.
In the study, based on the sound results and the platform conditionsgained in the myocardium regenerating on the basis of the collagen/Matrigel experiment system, ESC, as a single cell form, is directly inoculated in the collagen/Matrigel scaffold materials to observe the forming of EBs and next EBs’differentiation and developion. Thus, for collagen/Matrigel, the feasibility of ESC direct forming EBs in a three-dimensional environment is further explored. Especially the impact of ESC inoculating density on the forming of EBs, the impact of different scaffold composition on the forming and differentiating of EBs and the spontaneous differentiating of ESC in the system are emphatically studied.
As for impacts of ESC inoculating density on the forming of EBs, it is shown that ESC can directly start the formation of EBs in the collagen/Matrigel scaffold materials, and the ESC inoculating density can obviously affect the formation of EBs. Besides, the best inoculating density range is from 105cells/mL to 106cells/mL. In this range, the EBs formed by ESC is numerous and large.
As for impacts of different scaffold materials composition on the forming and differentiating of EBs, it is discovered through culturing ESC separately in the pure collagen gel system and the collagen/Matrigel system, that the collagen gel in the scaffold materials mainly affects the forming of the EBs while Matrigel plays important roles on the differentiating and developing of the EBs.
As for the ESC spontaneous differentiating, by culturing ESC in the collagen/Matrigel three-dimensional environment and under implanting no inducing condition, it is shown through the histological staining that EBs spontaneous differentiates to form various tissue structures, such as the myocardium cell synplasm-like structure and tube-like structure, which are similar to the in vivo structures, after culturing in vitro for 21 days. It proved, through immunohistochemistrical staining such as CK18, CD31, cTnT and Nestin, that ESC can differentiate into cTnT-positive myocardium cells, CK18-positive epithelial cells, CD31-positive endothelial cells and Nestin-positive cells.
Section four: Experiment researches on the in vivo transplantation and the myocardial tissue construction based on ESC as seeding cells
In this section, ESC further inducing to differentiate into the myocardium and the repairing animal myocardial infarction are explored, using the research platform and the technology system, which is established by our lab in the myocardial tissue engineering and is based the embryonic stem cell induction and differentiation system set up earlier as well as based on the liquid collagen I/Matrigel gel composite scaffold materials.
Whether ESC can differentiate into cardiomyocytes or not under the specified inducing conditions is one of focuses of the study. In this section, ESC in vitro was induced to differentiate into cardiomyocytes in vitro by adding the ascorbic acid as an inductive agent, and reconstruct three-dimensional engineered heart tissue. Moreover, histological and immunohistochemistrical verifications of the regenerated tissue have been performed too. Based on the above, the engineered heart tissue is transplanted into the myocardial infarction, and the survival and differentiating of the tissue in the part as well as the improvement of heart function by the tissue are observed.
It is discovered by studies that ESC differentiate into a large number of beating cells in the collagen/Matrigel scaffold materials. Otherwise the number and area of beating cells gradually increase as the culture time extending. A large area of consistent beating zone has been formed after culturing for 14 days, and the consistent beating has been formed after culturing for 19 days. It proved, by the immunohistochemistrical staining of the patch, that the regenerated myocardium contains a large number of cardiomyocytes expressing cTnT, Nkx2.5 and GATA4. Comparing with the spontaneous differentiating into the cardiomyocytes, the large increase of the cTnT-positive cells as inducing to differentiate indicates that the ascorbic acid promotes markedly the ESC differentiating into the myocardial cells. Commexin43 in the myocardium peripheral connecting tissue can be recognized that the regenerated myocardial cells mature gradually. The staining also indicates that there exist Nestin-positive cells and CD31-positive cells.
The above mentioned in vitro regenerated myocardium is transplanted into the cardiac infarction part, taking the pure cardiac infarction group and the noncontractile material as the control groups. The heart functions are tested after 4w from transplantation. The results show that, compared with the pure cardiac infarction group as well as with the noncontractile material, the contracting and relaxing functions of the infarction myocardium have been tremendously improved after the engineered myocardium transplanting. Through staining tissue sections, it is observed that the myocardium patch densely attaches to the myocardium infraction part. Furthermore it has integrated with the host myocardium in texture and there is not an obvious boundary. In addition, as for the myocardium specific cTnT, NKx2.5, GATA4 and Connexin43, immunohistochemically, it is indicated that there are a large number of cells with differentiated phenotype of myocardium cells in the myocardium patch. Connexin43 staining showed that there were a large number of intercellular junctions in the myocardium patch, as well as between the myocardium patch and the myocardium cells of the host. CD31 staining showed that in the transplanted engineered cardiac tissue, there were many vascular endothelial cells, which has formed into a tube structure. On this basis, detection was conducted on the formation of teratoma after the transplantation of the engineered cardiac tissue. The formation of teratoma was observed to begin from the fourth week after the transplantation. The transplantation will lead to the occurrence of teratoma in the myocardium patch transplantation group with a incidence rate of 25% (3+/12), the teratoma locates at the location of myocardial infarction with certain visible size, and teratoma in the animal’s thoracic cavity and abdominal cavity was not detected. The teratoma contained the typical tridermic structure.
To sum up, in the study, the three-dimensional static stretching system has been established based on the collagen/Matrigen scaffold materials. The engineered myocardium patch has been constructed in vitro under the system, taking the cardiomyocytes of newborn rat as seeding cells. Meanwhile, the study has been carried out to judge the Telocytes existing in the regenerated myocardium. Furthermore, vascularization of the regenerated myocardium has been further studied by blending the vascular endothelial cells and the cardiomyocytes into the collagen/Matrigel scaffold materials. Based on the above, under the static stretching system, ESC in vitro differentiation and development model has been set up using collagen/Matrigel as scaffold materials. The ESC three-dimensional differentiating and the myocardium regenerating are studied. The engineered myocardium constructed is transplanted into a cardiac infarction part to explore the repairing ability of infarction. The results show that the engineered myocardium patch can integrate with the host and improve the function of damaged myocardium.
引文
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