世界生命科学前沿动态周报（五十五）

（8.29-9.4/2011）
美宝国际集团:陶国新  

   主要内容：端粒延长替代机制不依赖端粒酶就可以维持端粒长度；人体结肠干细胞的分离和体外增殖；微环境的脂肪细胞调节皮肤干细胞活性；内质网小管标记线粒体分裂位点；不能产生和调动Lon蛋白对抗氧化压力促使细胞衰老；体外利用自身干细胞分化和培养红细胞用于输血。

焦点动态：人体结肠干细胞的分离和体外增殖。

1. 端粒延长替代机制不依赖端粒酶就可以维持端粒长度
【动态】
人类癌症亚型中存在一种维护端粒的端粒延长替代机制（ALT）。大约10%到15%的人类癌症检测不到端粒酶活性，其中一些通过被称为端粒延长替代机制的不依赖端粒酶的端粒维护机制维持端粒长度。ALT表型在肉瘤和星形细胞瘤中相对比较普遍，而在上皮癌变中鲜有报道。不过，ALT的普遍性还没有在所有癌症类型中得到彻底鉴定。美国科学家就此在广泛的人类癌症中对ALT表型进行了综合调查。总共两个独立的样本群包括来自94种不同癌症亚型的6110例原发癌症，541例良性肿瘤，和264例正常组织通过端粒特异性的荧光原位杂交和免疫荧光标记PML蛋白相结合的技术进行了鉴定。总体上，在所有癌症样本中3.73%（228/6110）观察到ALT，在良性肿瘤和正常组织中都没有观察到ALT。这是第一例报道ALT出现在膀胱、子宫颈、子宫内膜、食道、胆囊、肾、肝和肺部的癌症中。此外，这也是第一例报道成神经管细胞瘤、少突神经胶质瘤、脑膜瘤、神经鞘瘤和小儿恶性胶质瘤中出现ALT。先前的研究显示ALT状态和某些癌症的预后有关系，因此，需要进一步研究评估ALT阳性的肿瘤的潜在预后意义和独特生物学。

【点评】
能够利用ALT途径的癌症预期会耐受抗端粒酶治疗，因此该研究的这些结果可能会对癌症治疗产生影响，深入研究和理解 ALT途径的分子机理可能对设计针对利用ALT途径的癌症的药物有重要作用。

【参考论文】
The American Journal of Pathology, 2011, 179(4), DOI: 10.1016/j.ajpath.2011.06.018
Prevalence of the Alternative Lengthening of Telomeres Telomere Maintenance Mechanism in Human Cancer Subtypes
Christopher M. Heaphy, Andrea P. Subhawong, Seung-Mo Hong, et al. 
Approximately 10% to 15% of human cancers lack detectable telomerase activity, and a subset of these maintain telomere lengths by the telomerase-independent telomere maintenance mechanism termed alternative lengthening of telomeres (ALT). The ALT phenotype, relatively common in subtypes of sarcomas and astrocytomas, has rarely been reported in epithelial malignancies. However, the prevalence of ALT has not been thoroughly assessed across all cancer types. We therefore comprehensively surveyed the ALT phenotype in a broad range of human cancers. In total, two independent sets comprising 6110 primary tumors from 94 different cancer subtypes, 541 benign neoplasms, and 264 normal tissue samples were assessed by combined telomere-specific fluorescence in situ hybridization and immunofluorescence labeling for PML protein. Overall, ALT was observed in 3.73% (228/6110) of all tumor specimens, but was not observed in benign neoplasms or normal tissues. This is the first report of ALT in carcinomas arising from the bladder, cervix, endometrium, esophagus, gallbladder, kidney, liver, and lung. Additionally, this is the first report of ALT in medulloblastomas, oligodendrogliomas, meningiomas, schwannomas, and pediatric glioblastoma multiformes. Previous studies have shown associations between ALT status and prognosis in some tumor types; thus, further studies are warranted to assess the potential prognostic significance and unique biology of ALT-positive tumors. These findings may have therapeutic consequences, because ALT-positive cancers are predicted to be resistant to anti-telomerase therapies.

2. 人体结肠干细胞的分离和体外增殖
【动态】
在人体一生中，结肠干细胞每周再生更新一次大肠内层。科学家几十年来证明存在结肠干细胞但一直无法识别和鉴定它们。西班牙科学家最近精确定位了结肠干细胞的存在位置并开发出了一套方法来分离和体外培养它们，第一个报道了人体结肠上皮干细胞的分离和体外培养增殖。细胞表面B型蝶素受体（EPHB2）的不同含量使得能够从人体结肠粘膜活组织切片中分离不同类型的细胞。细胞表面含量最高的EPHB2 对应着具有最长端粒和高表达的肠干细胞（ISC）标志基因的结肠上皮细胞。而且，利用创造ISC微生态环境的培养条件，确实有一部分有高含量EPHB2的细胞能够在体外增殖成为不分化的多能细胞群。

【点评】
成功分离出结肠干细胞这种成体干细胞并体外培养长期维持增殖不分化或定向分化，对于用成体干细胞建立体外模型研究胃肠道细胞的增殖分化开启了大门，也为探索更好的成体干细胞培养条件及其实际临床应用的转化打下了基础。

【参考论文】
Nature Medicine, 2011; DOI:10.1038/nm.2470
Isolation and in vitro expansion of human colonic stem cells
Peter Jung, Toshiro Sato, Anna Merlos-Suárez, et al.
Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.

3. 微环境的脂肪细胞调节皮肤干细胞活性
【动态】
哺乳动物皮肤中需要多种类型成员细胞来形成有功能的组织并支撑组织的稳定和再生。组成上皮干细胞微生态环境的细胞还不完全了解。美国科学家最近在皮肤中鉴定出脂肪前体细胞并证明它们的动态的再生平行于皮肤干细胞的激活。患有脂肪形成障碍的老鼠的以及移植实验中的脂肪细胞的功能分析表明真皮内的脂肪细胞是激活囊泡干细胞的充要条件。而且，未成熟脂肪细胞表达的PDGF可能在调节囊泡干细胞活性上起作用。这些数据凸显出生成脂肪的细胞是正向调节皮肤干细胞活性的皮肤微生态环境的组成细胞并提示临床上脂肪细胞可能会改变上皮干细胞的功能。

【点评】
男性形态性秃发患者在毛囊根部还有干细胞但是它们失去了启动毛发再生的能力，该研究证明形成皮肤干细胞微生态环境的脂肪细胞能够调节皮肤干细胞的活性，如果有办法使这些脂肪细胞能够影响毛囊根部的干细胞，也许会促使毛发重新生长。另一方面，该研究的启示是皮肤干细胞的适合的生命环境是脂肪细胞构成的，皮肤干细胞的增殖分化需要的营养和信号也是脂肪细胞提供的。

【参考论文】
Cell, 2011; 146 (5): 761-771 DOI: 10.1016/j.cell.2011.07.019
Adipocyte Lineage Cells Contribute to the Skin Stem Cell Niche to Drive Hair Cycling
Eric Festa, Jackie Fretz, Ryan Berry, et al.
In mammalian skin, multiple types of resident cells are required to create a functional tissue and support tissue homeostasis and regeneration. The cells that compose the epithelial stem cell niche for skin homeostasis and regeneration are not well defined. Here, we identify adipose precursor cells within the skin and demonstrate that their dynamic regeneration parallels the activation of skin stem cells. Functional analysis of adipocyte lineage cells in mice with defects in adipogenesis and in transplantation experiments revealed that intradermal adipocyte lineage cells are necessary and sufficient to drive follicular stem cell activation. Furthermore, we implicate PDGF expression by immature adipocyte cells in the regulation of follicular stem cell activity. These data highlight adipogenic cells as skin niche cells that positively regulate skin stem cell activity, and suggest that adipocyte lineage cells may alter epithelial stem cell function clinically.

4. 内质网小管标记线粒体分裂位点
【动态】
线粒体通过分裂和融合调节自身结构和分布。线粒体分裂受控于Dnm1/Drp1，一个围绕线粒体形成螺旋调节分裂的与发动蛋白相关的蛋白。在线粒体网络中什么决定了分裂位点还很不清楚。内质网和线粒体表现出紧密耦合的动力学和广泛的接触。加州大学的科学家研究了内质网是否在线粒体分裂中起作用。他们发现线粒体分裂发生在内质网小管接触线粒体并调节招募Drp1前的收缩的位置。因此，内质网小管可能在界定线粒体分裂位点中起积极的作用。

【点评】
线粒体维持正常功能对于细胞代谢是至关重要的，了解线粒体正常分裂的参与因素对于线粒体分裂障碍造成的功能缺陷的治疗有重要意义。

【参考论文】
Science, 2011; DOI: 10.1126/science.1207385
ER Tubules Mark Sites of Mitochondrial Division
Jonathan R. Friedman, Laura L. Lackner, Matthew West, et al. 
Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction prior to Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.
 

5. 不能产生和调动Lon蛋白对抗氧化压力促使细胞衰老
【动态】
美国科学家研究了Lon蛋白诱导的衰老Wi-38成纤维细胞中抵御氧化蛋白积累的保护机制的损坏。线粒体蛋白的氧化损伤被认为促进衰老进程，而Lon蛋白通常降解这些氧化损伤蛋白。在早期的人体Wi-38成纤维细胞，过氧化氢压力会迅速诱导产生Lon蛋白，避免积累氧化损伤蛋白，保护细胞活力。相反的，中期细胞在氧化压力下只表现出迟缓的诱导产生Lon蛋白，氧化损伤蛋白开始积累。后期或衰老的细胞，Lon蛋白基础水平很低，积累的氧化损伤蛋白水平很高，对氧化压力的反应中它们不能诱导产生Lon蛋白，表现出持续增加的氧化损伤蛋白积累。衰老细胞分裂成两群，一群表现出正常的线粒体质量而另一群表现出明显的线粒体丢失。两群细胞的线粒体跨膜电位都减弱了。这些衰老的变化类似于在年轻细胞中关闭Lon基因造成的结果。据此研究者建议Lon蛋白产生的压力诱导性的丧失是使细胞偏向衰老的弱化的压力适应能力模式的一部分。

【点评】
该研究部分解释了造成线粒体蛋白氧化和细胞衰老的原因。Lon蛋白作为重要的线粒体抗氧化蛋白是其中的关键因素。美宝再生物质能够增强线粒体数量和功能，在这个意义上，促进Lon蛋白的产生，避免积累氧化损伤蛋白，保护细胞活力，保持细胞不衰老。

【参考论文】
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2011; DOI: 10.1093/gerona/glr145
Impairment of Lon-Induced Protection Against the Accumulation of Oxidized Proteins in Senescent Wi-38 Fibroblasts 
J. K. Ngo, L. C. D. Pomatto, D. A. Bota, et al.
Oxidative damage to mitochondrial proteins is thought to contribute to the aging process, but the Lon protease normally degrades such proteins. In early-passage WI-38 human lung fibroblasts, Lon expression is rapidly induced during H2O2 stress, which prevents the accumulation of oxidized proteins and protects cell viability. In contrast, middle passage cells exhibit only sluggish induction of Lon expression in oxidative stress, and oxidized proteins initially accumulate. Late-passage, or senescent, cells have low basal levels of Lon and high levels of accumulated oxidized proteins; in response to oxidative stress, they fail to induce Lon expression and exhibit continually increasing accumulation of oxidized proteins. Senescent cells separated into two populations, one exhibiting normal mitochondrial mass and a second displaying significant loss of mitochondria; both populations had diminished mitochondrial transmembrane potential. These senescent changes are similar to the effects of Lon silencing in young cells. We suggest that loss of Lon stress inducibility is part of a pattern of diminishing stress adaptability that predisposes cells to senescence.
 

6. 体外利用自身干细胞分化和培养红细胞用于输血
【动态】
干细胞体外生产红血球可能会是代替传统输血用血的一种选择。这一概念的临床可行性尚未阐明。法国科学家最近的研究就这一问题给出了初步肯定的答案。他们使用一种培养方案允许外周CD34+造血干细胞分化为网织红细胞，生产了同种的培养的红细胞，在变形性、酶含量、其血红蛋白结合和释放氧气的能力以及表达血型抗原方面都有功能。而后在老鼠体内证明培养的红细胞在体内遇到了使其完全成熟所需的条件。临床研究中注射入人体内的培养红细胞存活半衰期类似于天然的红细胞，证明了它们的质量和功能性。

【点评】
体外利用自身干细胞分化和培养红细胞用于输血，可能有助于解决血源问题和免疫排斥问题。

【参考论文】
Blood, 2011; DOI: 10.1182/blood-2011-06-362038
Proof of principle for transfusion of in vitro generated red blood cells
Marie-Catherine Giarratana, Hélène Rouard, Agnès Dumont, et al. 
In vitro red blood cell (RBC) production from stem cells could represent an alternative to classical transfusion products. Up until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBC (cRBC) to survive in human. Using a culture protocol permitting erythroid differentiation from peripheral CD34+ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen and expression of blood group antigens. We then demonstrated in the NOD/SCID mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 1010 cRBC generated under GMP conditions and labeled with 51Cr. The level of these cells in the circulation 26 days after injection lies between 41 and 63 % which compares favorably to the reported half-life of 28 ± 2 days for native RBC. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro generated RBC and path the way towards new developments in transfusion medicine. This study has been registered at clinicaltrials.gov as NCT0929266.