世界生命科学前沿动态周报（七十九）

（6.10-6.16/2012）
美宝国际集团:陶国新 

　　主要内容：再生细胞能够恢复因角膜异常造成的视觉障碍；AMPK促进代谢压力下肿瘤细胞的存活；内源性逆转录病毒作用开启胚胎干细胞的全能性；炎症是如何导致癌症的；多靶点的系统干预抗癌更有效；发育时期的内脏细菌数量调节大脑的血清素水平。

　　焦点动态：内源性逆转录病毒作用开启胚胎干细胞的全能性。

1. 再生细胞能够恢复因角膜异常造成的视觉障碍

【动态】角膜内皮是角膜内表面的一单层细胞，维持角膜的透明性。角膜内皮功能障碍是严重视觉障碍的一个主要原因。日本科学家最近发现在移植培养的角膜内皮细胞时伴以低分子量的Rho关联激酶（ROCK）抑制剂，能够成功恢复角膜的透明性。以前的注射到角膜组织上的角膜内皮细胞会被眼房水冲掉，附着很差，而有研究表明ROCK信号途径干预此附着过程。因此他们体外培养了兔子的角膜内皮细胞注射到角膜内皮损坏的兔子眼前房，结果与ROCK抑制剂一同注射的兔子的角膜在注射48小时后完全恢复了透明性，而没有同时注射ROCK抑制剂的兔子的角膜模糊不清肿胀严重。这种细胞治疗没有观察到并发症，在有ROCK抑制剂存在下重建的角膜内皮呈现出正常的六角形细胞形态。之后用与人更相近的猴子所做的同样试验也取得了同样的结果，恢复了单层六角形细胞及角膜长期的透明性。

【点评】该研究表明ROCK抑制剂调节下的角膜内皮细胞移植可能对人角膜内皮功能障碍有效，鉴于手术治疗替换受伤的角膜内皮受制于捐献角膜的短缺，该种结合ROCK抑制剂的细胞疗法有可能最终提供医生一种新的再生医学的治疗方式。但是在真正应用于人体取得成功之前，这还只是一种推测。

【参考论文】   
The American Journal of Pathology, 2012 DOI: 10.1016/j.ajpath.2012.03.033
A ROCK Inhibitor Converts Corneal Endothelial Cells into a Phenotype Capable of Regenerating In Vivo Endothelial Tissue
N. Okumura, N. Koizumi, M. Ueno, et al.
Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal transplantation. We previously reported that the adhesion of corneal endothelial cells (CECs) to a substrate was enhanced by the selective ROCK inhibitor Y-27632. It is hypothesized that the inhibition of ROCK signaling may manipulate cell adhesion properties, thus enabling the transplantation of cultivated CECs as a form of regenerative medicine. In the present study, using a rabbit corneal endothelial dysfunction model, the transplantation of CECs in combination with Y-27632 successfully achieved the recovery of corneal transparency. Complications related to cell injection therapy, such as the abnormal deposition of the injected cells as well as the elevation of intraocular pressure, were not observed. Reconstructed corneal endothelium with Y-27632 exhibited a monolayer hexagonal cell shape with a normal expression of function-related markers, such as ZO-1, and Na+/K+-ATPase, whereas reconstruction without Y-27632 exhibited a stratified fibroblastic phenotype without the expression of markers. Moreover, transplantation of CECs in primates in the presence of the ROCK inhibitor also achieved the recovery of long-term corneal transparency with a monolayer hexagonal cell phenotype at a high cell density. Taken together, these results suggest that the selective ROCK inhibitor Y-27632 enables cultivated CEC-based therapy and that the modulation of Rho-ROCK signaling activity serves to enhance cell engraftment for cell-based regenerative medicine.

 2. AMPK促进代谢压力下肿瘤细胞的存活

【动态】美国科学家最近发现AMP激活的蛋白激酶（AMPK）在肿瘤形成时不止会限制细胞增殖也会帮助癌细胞存活，对癌细胞而言AMPK既是“朋友”又是“敌人”。而以前的研究发现AMPK抑制培养的癌细胞的生长，使其被看作是开发新化疗药物的有希望的潜在靶点。但是该新研究发现当细胞处于代谢压力下时（葡萄糖摄取不足），会激活AMPK促进细胞存活。其机制是AMPK通过调节控制脂肪酸合成和氧化的酶而间接调节NADPH的抗氧化功能。这也有助于解释以前的一项意外的发现，即有AMPK缺陷或激活AMPK的酶LKB1缺陷的细胞对癌变有抵抗性。

【点评】 AMPK由于其抑制细胞增殖的作用可能依然会成为开发新化疗药物的有希望的潜在靶点，但是该研究说明此种策略若不同时阻断AMPK对能量短缺时细胞的保护作用，那么很可能会失败。

【参考论文】   
Nature, 2012; DOI:10.1038/nature11066
AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress
Sang-Min Jeon, Navdeep S. Chandel, Nissim Hay. 
Overcoming metabolic stress is a critical step for solid tumour growth. However, the underlying mechanisms of cell death and survival under metabolic stress are not well understood. A key signalling pathway involved in metabolic adaptation is the liver kinase B1 (LKB1)–AMP-activated protein kinase (AMPK) pathway. Energy stress conditions that decrease intracellular ATP levels below a certain level promote AMPK activation by LKB1. Previous studies showed that LKB1-deficient or AMPK-deficient cells are resistant to oncogenic transformation and tumorigenesis, possibly because of the function of AMPK in metabolic adaptation. However, the mechanisms by which AMPK promotes metabolic adaptation in tumour cells are not fully understood. Here we show that AMPK activation, during energy stress, prolongs cell survival by redox regulation. Under these conditions, NADPH generation by the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. The inhibition of the acetyl-CoA carboxylases ACC1 and ACC2 by AMPK maintains NADPH levels by decreasing NADPH consumption in fatty-acid synthesis and increasing NADPH generation by means of fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas the activation of ACC1 or ACC2 attenuates these processes. Thus AMPK, in addition to its function in ATP homeostasis, has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo.

 3. 内源性逆转录病毒作用开启胚胎干细胞的全能性

【动态】胚胎干细胞的治疗作用潜力巨大，但是太多的科学和非科学方面的阻碍使得科学家们无法将其用于疾病治疗。美国科学家最近发现在胚胎发育早期，胚胎干细胞周期性的进出一种“神奇状态”，在这种状态下，一连串的细胞潜能必需的基因被激活，这种称作全能性的状态使得胚胎干细胞具有变成体内任何类型细胞的独特能力，使其成为极具吸引力的治疗靶点。在胚胎发育初期，还只有5-8个细胞时，这些干细胞是全能的能够变成任何类型细胞。3-5天后胚胎发育成囊胚，此时的干细胞是多能的，能够变成几乎所有类型细胞。这些科学家通过RNA序列检测，监测了老鼠未成熟的卵母细胞和两细胞阶段的胚胎，发现了一系列与全能性紧密相关的基因以及这些基因是被与这些干细胞相邻的逆转录病毒所激活。人类基因组的近8%是由先祖时期发生的古代病毒感染的遗迹构成，代代相传但不会产生病毒感染。该研究发现细胞利用了某些这类病毒调节自身基因的开关。在胚胎发育早期的某个很短的特定时间，利用严密控制的古病毒的残余打开数百基因使得细胞有能力发育成身体的任何组织。

【点评】该研究深化了对于细胞发育潜能重要的基因网络的了解，发现了将胚胎干细胞调回更年轻状态（可塑性更强）的机制。基因中古病毒遗迹的被利用也说明以前被忽视的或被认为是垃圾的很多东西可能具有意想不到的关键作用。

【参考论文】            
Nature, 2012; DOI:10.1038/nature11244
Embryonic stem cell potency fluctuates with endogenous retrovirus activity
Todd S. Macfarlan, Wesley D. Gifford, Shawn Driscoll, et al.
Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.

 4. 炎症是如何导致癌症的

【动态】肝脏、结肠或胃等器官发生癌症的一个最大的风险因素是这些器官因病毒或细菌感染造成的长期发炎。美国MIT的一项最新研究追踪了感染肝螺杆菌的老鼠肝脏和结肠里一系列基因和化学变化，给予我们现今关于这类感染是如何驱使组织癌变的最完整的了解。发炎是身体应对感染或损伤的正常反应，当免疫系统监测到病原体或细胞损伤时，它激活巨噬细胞和中性粒细胞的汇集，这些细胞的任务是吞噬细菌、死细胞和碎片（死亡或损伤细胞释放的蛋白、核酸等分子），在此期间会产生高活性的化学物质帮助分解细菌，但同时这些化学物质也会扩散到组织中，长此以往，炎症很可能引起组织癌变。MIT的实验对形成慢性肝脏和结肠感染的老鼠进行了20周的观察，发现肝脏和结肠对感染的反应不同。只有在结肠里，中性粒细胞会分泌次氯酸，通过氯化严重损害蛋白、DNA和RNA。本来次氯酸是用于杀死细菌的，但泄漏到周围组织里后就损害了结肠的上皮细胞。另一个不同是在经历DAN损伤后，肝脏的DNA修复系统比结肠的更活跃。 这两种不同都对结肠更不利，导致长期炎症更易于引起结肠癌。

【点评】 创新的全面的研究会帮助我们更好的认识癌症，该研究是我们对于长期炎症和癌症的关系有了进一步的了解，有助于采取更好的措施来预防癌症。

【参考论文】  
Proceedings of the National Academy of Sciences, 2012; DOI:10.1073/pnas.1207829109
PNAS Plus: Infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and DNA damage leading to colon cancer
A. Mangerich, C. G. Knutson, N. M. Parry, et al. 
Helicobacter hepaticus-infected Rag2-/- mice emulate many aspects of human inflammatory bowel disease, including the development of colitis and colon cancer. To elucidate mechanisms of inflammation-induced carcinogenesis, we undertook a comprehensive analysis of histopathology, molecular damage, and gene expression changes during disease progression in these mice. Infected mice developed severe colitis and hepatitis by 10 wk post-infection, progressing into colon carcinoma by 20 wk post-infection, with pronounced pathology in the cecum and proximal colon marked by infiltration of neutrophils and macrophages. Transcriptional profiling revealed decreased expression of DNA repair and oxidative stress response genes in colon, but not in liver. Mass spectrometric analysis revealed higher levels of DNA and RNA damage products in liver compared to colon and infection-induced increases in 5-chlorocytosine in DNA and RNA and hypoxanthine in DNA. Paradoxically, infection was associated with decreased levels of DNA etheno adducts. Levels of nucleic acid damage from the same chemical class were strongly correlated in both liver and colon. The results support a model of inflammation-mediated carcinogenesis involving infiltration of phagocytes and generation of reactive species that cause local molecular damage leading to cell dysfunction, mutation, and cell death. There are strong correlations among histopathology, phagocyte infiltration, and damage chemistry that suggest a major role for neutrophils in inflammation-associated cancer progression. Further, paradoxical changes in nucleic acid damage were observed in tissue- and chemistry-specific patterns. The results also reveal features of cell stress response that point to microbial pathophysiology and mechanisms of cell senescence as important mechanistic links to cancer.

 5. 多靶点的系统干预抗癌更有效

【动态】美国科学家最近的研究表明相比于特异攻击某一疾病病理过程中某一基因或蛋白的高度选择性的特效药物，在已知化合物库中找寻那些为数不多的能够广泛破坏整个病理过程的分子可能是更有效的治疗策略。他们发现这种从针对单一靶点转变为同时针对一系列靶点的策略使得我们在不造成很多副作用的情况下获得好得多的阻止癌症的能力。该策略已产生了两个潜在的药物AD80 和 AD81,比现有抗癌药物凡德他尼（vandetanib）对果蝇的疗效更好毒性更低，已被美国FDA批准用于治疗某一特定类型的甲状腺癌。广义上讲，绝大多数药物只是干预疾病过程中的蛋白和基因的化合物，其破坏关键的疾病过程的能力越强，它就越有效。另一方面，它干扰身体其他部分的作用越大，它的毒性就越大。很多抗癌药物对癌细胞和正常细胞有同样的杀伤力。现在看来，选择性越高越好的观念也许并不正确。对于癌症的治疗，作用于癌症病理过程的多个环节的低选择性的化合物或许会带来更好的效果和更低的毒性。

【点评】 传统的药物设计认为化合物的选择性越高越好，但是该研究的实践表明，这种观念也许过时了，尤其是在癌症治疗领域，选择性低的化合物反而可能更好，意味着特异性干预疾病单一环节的策略向多环节系统干预的策略转移。

【参考论文】   
Nature, 2012; 486 (7401): 80 DOI: 10.1038/nature11127
Chemical genetic discovery of targets and anti-targets for cancer polypharmacology
Arvin C. Dar, Tirtha K. Das, Kevan M. Shokat, Ross L. Cagan. 
The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-drivenDrosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the ‘anti-target’ Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.

 6. 发育时期的内脏细菌数量调节大脑的血清素水平

【动态】爱尔兰科学家最新研究表明正常成年人的大脑功能依赖于发育期间内脏微生物的存在。血清素（5-羟色胺）是涉及情绪调节的主要化学物质，随压力、焦虑、抑郁等改变，大多数临床有效的抗抑郁药也是作用于这一神经化学物质。该研究用无菌老鼠模型表明生命早期缺乏细菌显著影响了成年后大脑的血清素浓度，尤其是对雄性动物影响更大，对大脑功能造成了不可逆的永久印迹。脑-肠-微生物轴的存在是维持正常健康必须的，肠道菌群在内脏和大脑的双向通讯中起了重要作用。

【点评】该研究提供了有趣的机会通过干预肠道菌群来调节大脑的功能障碍，对维护整体健康包括心理健康有多重含意。

【参考论文】   
Molecular Psychiatry, June 12, 2012 DOI:10.1038/mp.2012.77
The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner
G Clarke, S Grenham, P Scully,et al.  
Bacterial colonisation of the intestine has a major role in the post-natal development and maturation of the immune and endocrine systems. These processes are key factors underpinning central nervous system (CNS) signalling. Regulation of the microbiome–gut–brain axis is essential for maintaining homeostasis, including that of the CNS. However, there is a paucity of data pertaining to the influence of microbiome on the serotonergic system. Germ-free (GF) animals represent an effective preclinical tool to investigate such phenomena. Here we show that male GF animals have a significant elevation in the hippocampal concentration of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, its main metabolite, compared with conventionally colonised control animals. Moreover, this alteration is sex specific in contrast with the immunological and neuroendocrine effects which are evident in both sexes. Concentrations of tryptophan, the precursor of serotonin, are increased in the plasma of male GF animals, suggesting a humoral route through which the microbiota can influence CNS serotonergic neurotransmission. Interestingly, colonisation of the GF animals post weaning is insufficient to reverse the CNS neurochemical consequences in adulthood of an absent microbiota in early life despite the peripheral availability of tryptophan being restored to baseline values. In addition, reduced anxiety in GF animals is also normalised following restoration of the intestinal microbiota. These results demonstrate that CNS neurotransmission can be profoundly disturbed by the absence of a normal gut microbiota and that this aberrant neurochemical, but not behavioural, profile is resistant to restoration of a normal gut flora in later life.