利用C-CHD患者来源的hMSCs和VEGF/bFGF修饰的胶原支架构建工程化心肌补片的实验研究

Engineered heart tissues (EHTs) have been regarded as the most promising alternative to present inert, synthetic materials and autologeous mesenchymal stem cells (MSCs) are widely used as seeding cells in tissue engineering due to their theoretic advantages. But, until now, it remains vacancy if their autologeous MSCs can serve as seeding cells for patients with cyanotic congenital heart disease (C-CHD). In clinic, it is a common sense that aortopulmonary collateral circulation is generally more abundant in patients of C-CHD than their acyanotic controls, accompanying by the elevated vascular endothelial growth factor (VEGF) in serum; and also, in bioscientific field, researchers are in complete agreement that hypoxic preconditioning can improve the cell survival after implantation by modulating some proangiogenetic (eg: VEGF) and anti-apoptotic (eg: Bcl2, Caspase 3) growth factor expression. Besides, in previous study, we developed a novel platform by covalently conjugating 2 pro-angiogenetic cytokines (VEGF+basic fibroblast growth factor, bFGF) into collagen scaffold with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride chemistry（EDC）. The cytokines in patch released gradually and stimulated the cell proliferation in vitro and improve cell survival and patch revascularization in vivo. Thus, we hypothesize that hMSCs from donors of C-CHD might be more anti-apoptic and pro-angiogenetic after seeding on the patch because they have been "preconditioned" in naturally hypoxic surroundings and the immobilized cytokines can interact with seeding cells and accelerate patch vascularization. Fortunately, our preliminary data have partially identified the hypothesis. In hypoxic cultivating condition (1%O2, 5%CO2, 94%N2), C-CHD patients-derived-hMSCs grew faster than their A-CHD controls; and also proliferated more obviously to exogenous stimulation after seeding on the cytokine-conjugated scaffold. Additionally, the cell source exhibit stronger capability to resist apoptosis and death induced by ischemia and hypoxic state in vitro. In this issue, we will further compare their biological activities (eg: cell proliferation, colony-formation ability, anti-apoptosis and differentiation) and explore the possible mechanism. Furthermore, we will seed the 2 kinds of hMSCs into the collagen scaffolds with or without growth factors to generate 4 kinds of EHTs. We will repair the right ventricular outlet tract (RVOT) of immunosuppressive rats (mimicking clinical surgery of patients with C-CHD) with the 4 groups of EHTs. At the endpoint of the study, we will compare their cardiac function of right ventricle with pressure-volume catheter, and the morphological and histological changes among the 4 groups of rats. We hope the novel EHT (C-CHD-derived hMSCs + cytokine-conjugated scaffolds) would provide a practical and individual choice for patients with complex, cyanotic congenital cardiac disease.

紫绀组先心病（C-CHD）患儿的骨髓干细胞（hMSCs）可否作为工程化心肌补片(EHTs)的种子细胞现仍是一项研究空白，尽管乏氧能诱导干细胞内血管内皮生长因子（VEGF）的表达上调和紫绀患者体内侧枝循环丰富早已是学界共识。在预实验中，我们发现：与非紫绀患者相比，C-CHD患者来源的hMSCs具有更好的增殖和抗凋亡能力。我们推测：这是由于C-CHD患者体内的天然乏氧状态诱导自体hMSCs产生了更多的VEGF和抗凋亡因子。在本课题中，我们将在体外构建的乏氧环境中对比两种细胞的生物活性及其相关因子变化，并将两组细胞与已经或未经生长因子修饰的胶原补片复合，用以修补大鼠的右室流出道，并多角度评判其在体生物学行为。我们期望， C-CHD来源的hMSCs和生长因子修饰的胶原补片能从种子细胞和支架平台两个角度加速这种新型EHTs的再血管化进程，为复杂先心病患儿的手术治疗提供一种更理想的个体化修补材料。

当前，随着心外科及相关辅助技术的不断成熟,许多罹患重症的紫绀型先天性心脏病（C-CHD）患儿已可在婴幼儿甚至新生儿时期即获得有效纠治。但由于临床修补材料均缺乏生长能力，许多此类患儿日后仍然面临二次手术的风险，故构建理论上具有生物活性的工程化心肌补片因便成为解决上述问题的希望所在。自体来源的MSCs因其固有的天然优势（易获得、易扩增、没有免疫原性、多分化潜能等）是组织工程学界最为青睐的种子细胞来源，但C-CHD患儿自身来源的hMSCs能否承担种子细胞的职责目前尚无人证实。本研究就是利用构建的常氧及低氧环境，分别比较C-CHD及A-CHD患者来源的hMSCs在细胞增殖、克隆形成及细胞凋亡等方面的差异，进而探究其可能的发生机制。体内实验则是将低氧培养下的两种细胞分别种植于已经过或未经过生长因子修饰的胶原补片，利用构建的4种EHTs修补大鼠的右室流出道，并从形态学、组织学等角度比较4组补片的在体生物学行为。我们的研究结果显示：1.低氧可诱导hMSCs增殖加快，克隆形成增加，这与其体内VEGFA、VEGFR2表达上调有关，C-CHD来源的细胞对这种低氧刺激更为敏感；2.乏氧应激条件下，C-CHD患者来源的hMSCs可分泌更多的促血管生成因子VEGF, bFGF, PDGF；3. C-CHD患者来源的hMSCs可更好的抵抗低氧诱导的细胞凋亡，这与其细胞内Bcl2表达增加，Bax及Caspase3表达降低密切相关；4. 将低氧培养下的紫绀细胞（HC）与VEGF/ bFGF共价修饰的胶原补片复合，构建的EHTs行右室流出道修补后，可更好的维持补片形态，改善细胞生存，促进EHTs的再血管化进程。总之，在本研究中，我们利用一种最新构建的生长因子的缓释平台，以来源于C-CHD患者的hMSCs作种子细胞，构建了一种修复紫绀型先心病患儿心脏缺损的个体化的EHTs。我们期望通过细胞平台和种子细胞两方面的因素加速这种EHTs的再血管化进程，为当今复杂紫绀型先心病的纠治提供一种更加理想的EHTs的选择。