编者按：美国心脏协会（AHA）2015科学年会上，血管疾病相关专题中展现了大量最新研究进展与治疗方法。作为一种有前景的疗法，干细胞成为近年来的研究热点。本届AHA年会现场，关于干细胞的研究及应用问题，《国际循环》记者采访了John P. Cooke教授。Cooke教授为美国休斯顿卫理公会研究所心血管科学部门主任，休斯顿卫理公会狄贝凯心脏和血管中心主任，他的研究方向为采用小分子或干细胞疗法恢复或刺激内皮功能如血管舒张和血管生成。他的临床研究团队正在探讨使用血管生成制剂和成人干细胞治疗外周动脉疾病（PAD）。Cooke教授近期的研究关注人诱导多潜能干细胞（iPSCs）源性内皮细胞的生成和特征，并探讨其在血管生成及再生中的作用。

　　International Circulation: Dr. Cooke， one of your research focuses is generating and characterizing endothelial cells derived from human iPSCs and exploring their role in angiogenesis and vascular regeneration. What discoveries have you made?

　　《国际循环》：Cooke教授您好，您的研究领域之一是关于人诱导多潜能干细胞（iPSCs）源性内皮细胞的生成和特征，并探讨其在血管生成及再生中的作用。目前，您在这些方面都有哪些发现？

　　Dr. John Cooke: Ok， well， I have been studying endothelial cells for about 25 years now. Endothelial cells form the lining of all of our blood vessels， it is like the Teflon lining on a non-stick non-adherent lining of the blood vessel. When the endothelium is healthy， that lining of the blood vessel， when that is healthy， blood flows smoothly and blood does not stick， plaque does not build up. When the endothelium is not healthy， that causes disease， causes inflammation and it causes vascular lesions to form. And ultimately， it causes atherosclerosis and hardening of the arteries. That is one of the major vascular diseases. Generating healthy endothelial cells could be useful for conditions associated with poor blood vessels， with poor perfusion， or with vascular disease. Individuals that have poor blood flow to their heart， or to their legs， or to their brain could potentially be benefitted by an approach that would generate healthy endothelial cells. One approach that we have taken is to generate induced pluripotent stem cells. These are cells that are derived from somatic cells like skin cells or fat cells or blood cells. These induced pluripotent stem cells can be transdifferentiated into anything. They can be differentiated into endoderm， ectoderm， and mesoderm， any cell in the body. We are making endothelial cells from the iPSCs to study endothelial behavior and to determine if these cells have any therapeutic use.

　　John Cooke 教授：我研究血管内皮细胞已经有25年的时间。内皮细胞负责形成人体所有血管的管壁。内皮健康时，血流可顺畅流动而不会粘滞，因此不会形成斑块。但是，当内皮不健康时，则会导致疾病。具体来说，内皮不健康可导致炎症、形成血管病变，最终形成动脉粥样硬化，这是其所导致的主要血管疾病之一。生成健康的内皮细胞对改善血管情况较差、灌注较差相关疾病或血管疾病非常有用。心脏、下肢或大脑供血较差者可能从生成健康内皮细胞的干预方法中获益。我们目前所采用的一种方法是生成诱导多潜能干细胞。这些诱导多潜能干细胞来源于皮肤细胞、脂肪细胞或血细胞等体细胞，可分化为人体的内胚层、外胚层、中胚层细胞。我们采用iPSCs分化形成内皮细胞来研究其行为并确定这些细胞能否用于治疗。

　　International Circulation: Where are we now? What have you discovered?

　　《国际循环》：现在研究到了哪种阶段？有何发现？

　　Dr. John Cooke: Well， I think the most important thing that we have discovered， we published in 2012， and that was the finding that activation of innate immunity causes epigenetic flexibility. When inflammatory signaling is activated in any cell， it causes the chromatin in that cell， the DNA to open up. It is almost as though when a cell is confronted by a challenge or a pathogen， or by damage， it causes this inflammatory signaling that opens up the chromatin and opens up the DNA， so that the cell can reach back and full out whatever it needs to survive or adapt to the challenge. We discovered this pathway and knowing that that pathway exists we can now manipulate it so we can open up the DNA in the cell， increase chromatin openings so that we can manipulate the cell phenotype. We did that in the 2012 paper we published in Cell to make iPSCs and more recently in a Circulation paper we showed that we could open up the chromatin to increase the flexibility of fibroblasts and transform them to endothelial cells and that could potentially have some therapeutic clinical benefit.

　　John Cooke 教授：我认为，我们最重要的发现是2012年发表的关于激活先天免疫可导致表观遗传学变化。任何细胞中的炎症信号被激活后，都会导致细胞内的染色质及DNA开放。当细胞遭受病原体侵袭或损伤时，细胞中的炎症信号会使染色质及DNA开放，从而使细胞全面出击借以生存或适应挑战。我们发现了该通路，现在可以对其进行调控，打开细胞内的DNA及染色质，进而调控细胞表型。我们2012年发表于Cell杂志的一篇研究是关于该内容，并提取出了iPSCs。近期我们发表在Circulation上的一篇文章显示，打开染色质使成纤维细胞的可塑性增加，能够分化为内皮细胞，具有一定的治疗价值。

　　International Circulation: Could you briefly introduce how angiogenic and cell therapies modulate the vascular response to limb ischemia?

　　《国际循环》：请您简要介绍血管生成和细胞疗法如何调节血管对肢体缺血的反应？

　　Dr. John Cooke: Yes， I have been taking care of people with peripheral arterial disease and poor blood flow to their legs. I have been doing this for about 25 years. These individuals have difficulty walking because they have poor blood flow to the limbs and they may also get ulcers or gangrene. They may lose their limbs because of poor blood flow to their limbs. We try to improve the perfusion and the blood flow to the limbs. One of the ways to do this is surgery. You can do a bypass with the patient’s own vein or with plastic piping material， flexible plastic piping material， so you can improve blood flow to the limbs so that that person’s pain can be released， so they can walk farther， and so the ulcers can heal. Another approach would be to allow the body to heal itself by enhancing biological bypasses and enhancing the ability of the person to make their own blood vessels. That process is called therapeutic angiogenesis. There are growth factors that can induce blood vessel growth. There are cells that can enhance blood vessel growth， so there has been much work done in animal models showing that certain factors like vascular endothelial growth factors can enhance blood vessel growth in animal models. There has been work being done with cells and cells from the bone marrow， cells from fat， adipose derived mesenchymal cells for example can enhance angiogenesis， which is blood vessel growth. These approaches are being tested in humans. To date， though the trials have been somewhat disappointing with angiogenic factors， I think it is because we don’t understand all of the details. We do not understand the best way to improve blood vessel growth in the human body using cells or growth factors， but I do believe we will get there.

　　John Cooke 教授：从25年前，我就开始治疗下肢血流较差的外周动脉疾病患者。这些患者会因肢体血流不畅、溃疡或坏疽而出现行走困难甚至截肢。我们需要努力改善其肢体的血液灌注。方法之一是外科手术。我们可采用患者自身静脉或具有弹性的塑料管实施搭桥术来改善肢体血流进而缓解患者疼痛，治愈溃疡，使其能够行走更长距离。方法之二是通过促进生物搭桥及增加患者自身血管生成使其自愈，即所谓的治疗性血管生成。有些生长因子可诱导血管生长，有些细胞可促进血管生长。大量动物模型研究显示，血管内皮生长因子等某些细胞因子可促进血管生长。另外，有研究发现，来源于骨髓、脂肪的细胞如脂肪间充质干细胞可促进血管生成即血管生长。目前，人们正在开展相关人体研究。但到目前为止，有关血管生成因子的人体试验结果有些令人失望。我想这可能与我们还没有完全了解血管生长的所有细节、尚不知道采用细胞或生长因子改善人体血管生长的最佳方法有关，但相信我们最终一定会实现。

　　International Circulation: What about the latest research progress of therapeutic transdifferentiation in regenerative approaches to limb salvage and wound care for PVD patients?

　　《国际循环》：治疗性转分化在外周血管疾病患者的保肢和伤口处理的再生治疗中的最新研究进展如何？

　　Dr. John Cooke: There are now several trials going on to see the benefit of different cells and different angiogenic growth factors. We are learning a little bit more about how to administer these growth factors， how to administer these cells. The trials are still under way， some promising results we need to do a little more work before these things can get into the clinic and treat patients.

　　John Cooke 教授：现在有几项旨在探讨不同细胞及不同血管生长因子对上述患者的益处。我们已经对如何应用这些生长因子及细胞有了更多了解。这些试验仍在进行之中。若要将上述治疗方法用于临床和患者，尚需开展更多研究以获得有前景的结果。

　　International Circulation: You mentioned that the drawback of the research， could you please elaborate a little bit about the human iPSCs? What about the current limitations and the routes towards therapeutic application of this technique?

　　《国际循环》：人iPSCs源性内皮细胞用于临床有哪些局限性？应如何解决？

　　Dr. John Cooke: All right. The induced pluripotent stem cells have already changed medicine. Yamanaka received the noble prize in 2012 for his discovery of how to change a somatic cell like a skin cell into an induced pluripotent stem cell or iPSCs， because these cells can become any tissue and now that we have these cells， we have been able to do already much with these cells that have taught us a lot about disease. I can take for example， a small piece of skin from you and turn it into an induced pluripotent stem cell， and then that cell basically with that iPSC can be converted into your brain cells or converted into heart cells or pancreatic cells. If you have a brain disease that you do not understand we can now study your brain cells in a dish. We can make these brain cells and study them， so for studying disease， they have already become very useful because obscure diseases and diseases we do not understand， we can generate iPSCs from person’s skin fibroblasts and then make the cells that we need to study whether it is pancreas or gut or brain or heart， we can make those cells in a dish and then we can study them. We can do high throughput screening for drugs that might improve the pathobiology and might improve the health of those cells. We have learned more about the disease， we can do screening for drugs so that is already changing medicine. Every big pharmaceutical company now has an iPSCs program for understanding disease and for screening drugs. Now the question you asked is can we use these cells for therapy. Not yet， I am afraid， because there are still too many unknowns about using these cells as therapy. For example， there are concerns about changes in the DNA when one does nuclear reprogramming and can one induce mutations in the DNA during this process. In addition， if you can make the cells that you need like brain cells， how do you deliver those cells， because our bodies have certain architecture and you cannot just inject cells in willy-nilly and expect them to recreate the organ. Some individuals we heard today， some individuals are making scaffolds. For example， a scaffold for a blood vessel， they make a collagen tube and then you can put the iPSCs derived cells into that tube to make a blood vessel. The use of iPSCs for therapy will require more knowledge about how to differentiate them stably into a healthy cell type and how do they deliver that cell to the body.

　　John Cooke教授：诱导多潜能干细胞已经改变了现有医学。Yamanaka因发现如何将皮肤细胞等体细胞分化为可发育成为任何组织的iPSCs而于2012年荣获诺贝尔奖。现在，我们已经拥有了这些细胞，并通过这些细胞对疾病有了更多认识。举例来说，人体的一小块皮肤可被分化为诱导多潜能干细胞，进而可被分化为脑细胞、心脏细胞或胰腺细胞。对于目前尚不了解的脑部疾病，我们可在培养皿中对脑细胞进行研究，我们可以生成脑细胞并对其进行研究以期对疾病有更多了解。iPSCs非常有用，因为对于一些不明原因或我们不太了解的疾病，可通过采用人体自身皮肤的成纤维细胞生成iPSCs，然后在培养皿中生成我们研究所需要的细胞如胰腺细胞、脑细胞或心脏细胞，进而对其进行研究。我们可以对药物进行高通量筛选，从而改善疾病的病理生理学及细胞健康。我们已经对疾病有了更多了解，并开展药物筛选，极大改变了医学现状。现在几乎每家大型制药公司均设立了iPSCs项目以了解疾病或进行药物筛选。至于你所提问的是否可应用这些细胞进行治疗，我认为目前恐怕还不行，因为采用这些细胞进行治疗尚存很多未知之处。例如，有人担心治疗过程中可能会导致DNA改变，导致核重新编码或诱发DNA突变。此外，在生成所需脑细胞后还存在如何将其输送至体内的问题，因为我们的身体有着特定结构，我们的目的不是单纯将细胞注入体内而希望通过他们重建器官。现在有人正在研究将iPSCs来源的细胞放入胶原试管中来使其生成血管。总之，要想采用iPSCs进行治疗，还需更多了解如何将这些细胞稳定地分化为所需健康细胞类型及如何将其输送至体内。