世界生命科學(xué)前沿動態(tài)周報（五十五）

（8.29-9.4/2011）
美寶國際集團:陶國新  

   主要內(nèi)容：端粒延長替代機制不依賴端粒酶就可以維持端粒長度；人體結(jié)腸干細胞的分離和體外增殖；微環(huán)境的脂肪細胞調(diào)節(jié)皮膚干細胞活性；內(nèi)質(zhì)網(wǎng)小管標記線粒體分裂位點；不能產(chǎn)生和調(diào)動Lon蛋白對抗氧化壓力促使細胞衰老；體外利用自身干細胞分化和培養(yǎng)紅細胞用于輸血。

焦點動態(tài)：人體結(jié)腸干細胞的分離和體外增殖。

1. 端粒延長替代機制不依賴端粒酶就可以維持端粒長度
【動態(tài)】
人類癌癥亞型中存在一種維護端粒的端粒延長替代機制（ALT）。大約10%到15%的人類癌癥檢測不到端粒酶活性，其中一些通過被稱為端粒延長替代機制的不依賴端粒酶的端粒維護機制維持端粒長度。ALT表型在肉瘤和星形細胞瘤中相對比較普遍，而在上皮癌變中鮮有報道。不過，ALT的普遍性還沒有在所有癌癥類型中得到徹底鑒定。美國科學(xué)家就此在廣泛的人類癌癥中對ALT表型進行了綜合調(diào)查?？偣矁蓚€獨立的樣本群包括來自94種不同癌癥亞型的6110例原發(fā)癌癥，541例良性腫瘤，和264例正常組織通過端粒特異性的熒光原位雜交和免疫熒光標記PML蛋白相結(jié)合的技術(shù)進行了鑒定?？傮w上，在所有癌癥樣本中3.73%（228/6110）觀察到ALT，在良性腫瘤和正常組織中都沒有觀察到ALT。這是第一例報道ALT出現(xiàn)在膀胱、子宮頸、子宮內(nèi)膜、食道、膽囊、腎、肝和肺部的癌癥中。此外，這也是第一例報道成神經(jīng)管細胞瘤、少突神經(jīng)膠質(zhì)瘤、腦膜瘤、神經(jīng)鞘瘤和小兒惡性膠質(zhì)瘤中出現(xiàn)ALT。先前的研究顯示ALT狀態(tài)和某些癌癥的預(yù)后有關(guān)系，因此，需要進一步研究評估ALT陽性的腫瘤的潛在預(yù)后意義和獨特生物學(xué)。

【點評】
能夠利用ALT途徑的癌癥預(yù)期會耐受抗端粒酶治療，因此該研究的這些結(jié)果可能會對癌癥治療產(chǎn)生影響，深入研究和理解 ALT途徑的分子機理可能對設(shè)計針對利用ALT途徑的癌癥的藥物有重要作用。

【參考論文】
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. 人體結(jié)腸干細胞的分離和體外增殖
【動態(tài)】
在人體一生中，結(jié)腸干細胞每周再生更新一次大腸內(nèi)層。科學(xué)家?guī)资陙碜C明存在結(jié)腸干細胞但一直無法識別和鑒定它們。西班牙科學(xué)家最近精確定位了結(jié)腸干細胞的存在位置并開發(fā)出了一套方法來分離和體外培養(yǎng)它們，第一個報道了人體結(jié)腸上皮干細胞的分離和體外培養(yǎng)增殖。細胞表面B型蝶素受體（EPHB2）的不同含量使得能夠從人體結(jié)腸粘膜活組織切片中分離不同類型的細胞。細胞表面含量最高的EPHB2 對應(yīng)著具有最長端粒和高表達的腸干細胞（ISC）標志基因的結(jié)腸上皮細胞。而且，利用創(chuàng)造ISC微生態(tài)環(huán)境的培養(yǎng)條件，確實有一部分有高含量EPHB2的細胞能夠在體外增殖成為不分化的多能細胞群。

【點評】
成功分離出結(jié)腸干細胞這種成體干細胞并體外培養(yǎng)長期維持增殖不分化或定向分化，對于用成體干細胞建立體外模型研究胃腸道細胞的增殖分化開啟了大門，也為探索更好的成體干細胞培養(yǎng)條件及其實際臨床應(yīng)用的轉(zhuǎn)化打下了基礎(chǔ)。

【參考論文】
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. 微環(huán)境的脂肪細胞調(diào)節(jié)皮膚干細胞活性
【動態(tài)】
哺乳動物皮膚中需要多種類型成員細胞來形成有功能的組織并支撐組織的穩(wěn)定和再生。組成上皮干細胞微生態(tài)環(huán)境的細胞還不完全了解。美國科學(xué)家最近在皮膚中鑒定出脂肪前體細胞并證明它們的動態(tài)的再生平行于皮膚干細胞的激活?；加兄拘纬烧系K的老鼠的以及移植實驗中的脂肪細胞的功能分析表明真皮內(nèi)的脂肪細胞是激活囊泡干細胞的充要條件。而且，未成熟脂肪細胞表達的PDGF可能在調(diào)節(jié)囊泡干細胞活性上起作用。這些數(shù)據(jù)凸顯出生成脂肪的細胞是正向調(diào)節(jié)皮膚干細胞活性的皮膚微生態(tài)環(huán)境的組成細胞并提示臨床上脂肪細胞可能會改變上皮干細胞的功能。

【點評】
男性形態(tài)性禿發(fā)患者在毛囊根部還有干細胞但是它們失去了啟動毛發(fā)再生的能力，該研究證明形成皮膚干細胞微生態(tài)環(huán)境的脂肪細胞能夠調(diào)節(jié)皮膚干細胞的活性，如果有辦法使這些脂肪細胞能夠影響毛囊根部的干細胞，也許會促使毛發(fā)重新生長。另一方面，該研究的啟示是皮膚干細胞的適合的生命環(huán)境是脂肪細胞構(gòu)成的，皮膚干細胞的增殖分化需要的營養(yǎng)和信號也是脂肪細胞提供的。

【參考論文】
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. 內(nèi)質(zhì)網(wǎng)小管標記線粒體分裂位點
【動態(tài)】
線粒體通過分裂和融合調(diào)節(jié)自身結(jié)構(gòu)和分布。線粒體分裂受控于Dnm1/Drp1，一個圍繞線粒體形成螺旋調(diào)節(jié)分裂的與發(fā)動蛋白相關(guān)的蛋白。在線粒體網(wǎng)絡(luò)中什么決定了分裂位點還很不清楚。內(nèi)質(zhì)網(wǎng)和線粒體表現(xiàn)出緊密耦合的動力學(xué)和廣泛的接觸。加州大學(xué)的科學(xué)家研究了內(nèi)質(zhì)網(wǎng)是否在線粒體分裂中起作用。他們發(fā)現(xiàn)線粒體分裂發(fā)生在內(nèi)質(zhì)網(wǎng)小管接觸線粒體并調(diào)節(jié)招募Drp1前的收縮的位置。因此，內(nèi)質(zhì)網(wǎng)小管可能在界定線粒體分裂位點中起積極的作用。

【點評】
線粒體維持正常功能對于細胞代謝是至關(guān)重要的，了解線粒體正常分裂的參與因素對于線粒體分裂障礙造成的功能缺陷的治療有重要意義。

【參考論文】
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. 不能產(chǎn)生和調(diào)動Lon蛋白對抗氧化壓力促使細胞衰老
【動態(tài)】
美國科學(xué)家研究了Lon蛋白誘導(dǎo)的衰老Wi-38成纖維細胞中抵御氧化蛋白積累的保護機制的損壞。線粒體蛋白的氧化損傷被認為促進衰老進程，而Lon蛋白通常降解這些氧化損傷蛋白。在早期的人體Wi-38成纖維細胞，過氧化氫壓力會迅速誘導(dǎo)產(chǎn)生Lon蛋白，避免積累氧化損傷蛋白，保護細胞活力。相反的，中期細胞在氧化壓力下只表現(xiàn)出遲緩的誘導(dǎo)產(chǎn)生Lon蛋白，氧化損傷蛋白開始積累。后期或衰老的細胞，Lon蛋白基礎(chǔ)水平很低，積累的氧化損傷蛋白水平很高，對氧化壓力的反應(yīng)中它們不能誘導(dǎo)產(chǎn)生Lon蛋白，表現(xiàn)出持續(xù)增加的氧化損傷蛋白積累。衰老細胞分裂成兩群，一群表現(xiàn)出正常的線粒體質(zhì)量而另一群表現(xiàn)出明顯的線粒體丟失。兩群細胞的線粒體跨膜電位都減弱了。這些衰老的變化類似于在年輕細胞中關(guān)閉Lon基因造成的結(jié)果。據(jù)此研究者建議Lon蛋白產(chǎn)生的壓力誘導(dǎo)性的喪失是使細胞偏向衰老的弱化的壓力適應(yīng)能力模式的一部分。

【點評】
該研究部分解釋了造成線粒體蛋白氧化和細胞衰老的原因。Lon蛋白作為重要的線粒體抗氧化蛋白是其中的關(guān)鍵因素。美寶再生物質(zhì)能夠增強線粒體數(shù)量和功能，在這個意義上，促進Lon蛋白的產(chǎn)生，避免積累氧化損傷蛋白，保護細胞活力，保持細胞不衰老。

【參考論文】
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. 體外利用自身干細胞分化和培養(yǎng)紅細胞用于輸血
【動態(tài)】
干細胞體外生產(chǎn)紅血球可能會是代替?zhèn)鹘y(tǒng)輸血用血的一種選擇。這一概念的臨床可行性尚未闡明。法國科學(xué)家最近的研究就這一問題給出了初步肯定的答案。他們使用一種培養(yǎng)方案允許外周CD34+造血干細胞分化為網(wǎng)織紅細胞，生產(chǎn)了同種的培養(yǎng)的紅細胞，在變形性、酶含量、其血紅蛋白結(jié)合和釋放氧氣的能力以及表達血型抗原方面都有功能。而后在老鼠體內(nèi)證明培養(yǎng)的紅細胞在體內(nèi)遇到了使其完全成熟所需的條件。臨床研究中注射入人體內(nèi)的培養(yǎng)紅細胞存活半衰期類似于天然的紅細胞，證明了它們的質(zhì)量和功能性。

【點評】
體外利用自身干細胞分化和培養(yǎng)紅細胞用于輸血，可能有助于解決血源問題和免疫排斥問題。

【參考論文】
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.