细胞外的生命现象（三）

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无细胞生命现象

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　　无细胞发酵（cell free fermentation ）发酵指微生物分解有机物质的过程，它可以在有氧条件下进行，也可以在无氧条件下进行，但没有细胞即没有微生物这个主体发酵被认为是是不能进行的。
. K' G  q$ M9 _　　1897年德国化学家Biichner兄弟证明无细胞酵母提取液仍然能使蔗糖发酵为酒精，告诉我们这种发酵因子虽然由活细胞所产生，但是在离开细胞后仍起作用。
8 s4 L4 P8 D. A" U" Z: w1 b　　The death knell of vitalism：Chemist Eduard Buchner of the University of Berlin produces evidence of ‘cell-free’ enzyme-driven fermentation. The work creates a considerable stir within the life science community. It deals final blows to biological ‘vitalism’ and the ‘life cycle’ theory of fermentation, and reinforces the conceptual foundations of modern biochemistry: Buchner’s work demonstrates how to analyze biochemical phenomena as a series of discrete steps, and to conceptualize metabolic processes as movements through pathways or stages regulated by enzymatic reactions.German chemist. He won a1907 Nobel Prize for the discovery of cell-less fermentation. . i3 L6 i; \  [( o9 l6 p1 Q
　　
& U5 v! g! O; L  `　　无细胞核酸合成体系——PCR(聚合酶链式反应）一种生物体外的特殊DNA复制，是利用DNA在体外摄氏95度时解旋，55度时引物与单链按碱基互补配对的原则结合，再调温度至72度左右，DNA聚合酶沿着磷酸到五碳糖(5'-3')的方向合成互补链。由PCR技术制造的PCR仪实际就是一个温控设备，能在95℃，55℃，72℃之间很好地进行控制。
1 ~+ s0 [4 k: ?9 e5 N7 ^1 a　　Khorana (1971)等最早提出核酸体外扩增的设想：“经DNA变性，与合适的引物杂交，用DNA聚合酶延伸引物，并不断重复该过程便可合成tRNA基因”。但由于当时基因序列分析方法尚未成熟，热稳定DNA聚合酶尚未报道以及引物合成的困难，这种想法似乎没有实际意义。加上70年代初分子克隆技术的出现提供了一种克隆和扩增基因的途径，所以，Khorana的设想被人们遗忘了。1985年，Kary Mullis在Cetus公司工作期间，发明了PCR。Mullis要合成DNA引物来进行测序工作，却常为没有足够多的模板DNA而烦恼。1983年4月的一个星期五晚上，他开车去乡下别墅的路上,猛然闪现出“多聚酶链式反应”的想法。1983年12月，Mullis用同位素标记法看到了10个循环后的49 bp长度的第一个PCR片段；1985年10月25日申请了PCR的专利，1987年7月28日批准（专利号4,683,202 ），Mullis是第一发明人；1985年12月20日在Science杂志上发表了第一篇PCR的学术论文，Mullis是共同作者；1986年5月，Mullis在冷泉港实验室做专题报告，全世界从此开始学习PCR的方法。. H0 e) H$ T8 U) [( x0 G/ ?5 E
　　
4 R2 ]6 B' |" a" T, }　　无细胞转录（cell-free transcription）绝大部分RNA 聚合酶Ⅱ的无细胞转录体系来自哺乳动物或果蝇的组织培养细胞的提取物。 9 U! z  Y7 |: A" R0 u( \& B$ L
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9 b( N+ ~1 n  e0 N4 Y) {　　无细胞翻译系统（cell-free translation system）就是没有完整细胞的体外蛋白质翻译合成系统，提供翻译所需的mRNA、核糖核蛋白、ATP、tRNA及氨基酸，在非细胞环境中合成蛋白质的过程 。通常利用无细胞提取物提供所需要的核糖体、转移核糖核酸、酶类、氨基酸、能量供应系统及无机离子等，在试管中以外加的信使核糖核酸(mRNA)指导蛋白质的合成。常用的无细胞提取物有兔网织红细胞裂解物和麦胚抽提物等。 / ?  Y, @+ K% |/ G5 q2 j8 m
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　　无细胞蛋白合成系统（The cell-free protein synthesis system）是一种以外源mRNA或DNA为模板，在细胞抽提物的酶系中补充底物和能量来合成蛋白质的体外系统。与传统的体内重组表达系统相比,体外无细胞合成系统具有多种优点,如可表达对细胞有毒害作用或含有非天然氨基酸(如D-氨基酸)的特殊蛋白质,能够直接以PCR产物作为模板同时平行合成多种蛋白质,开展高通量药物筛选和蛋白质组学的研究。 9 T- D& E/ t% Y8 i/ j7 R5 f1 A
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" d; e. ?1 m' ~9 O& W+ o& Q: J　　芯片上的基因表达（gene express on chip）2013年3月以色列魏茨曼科学研究所材料与界面系的研究团队在玻璃芯片上创建了一个二维的类细胞系统。这一系统是由细胞中的一些基本生物分子如DNA、RNA和蛋白质构成的，能够执行一个活细胞的中心功能：基因表达，基因中存储的信息正是通过这一过程被“翻译”成蛋白质的。  
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9 Q0 r' D# ?, U1 y; i　　 无细胞病毒复制（cell-free of virus）
2 w0 {8 N& F6 F: ]( Q: P2 R: k9 `, Y       Ali N, Tardif KD, Siddiqui A.  ：J Virol.2002 Dec;76(23):12001-7.  Cell-free replication of the hepatitis C virus subgenomic replicon.
5 I' J- p- |5 d2 Q( x5 q+ B2 T       Source：Department of Microbiology and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
% M! b/ |5 K4 {- ?      Abstract：The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5' noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3' NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [alpha(32)P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.
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4 q6 c+ [* T( A( z" k% V; G& o       无细胞治疗（Cell-free therapy）
0 V( i' [& }. `1 A      Serena Rubina Baglio1,2*, D. Michiel Pegtel2 and Nicola Baldini1 ： Front. Physiol., 06 September 2012 | doi: 10.3389/fphys.2012.00359           Mesenchymal stem cell secreted vesicles provide novel opportunities in (stem) cell-free therapy7 b2 f0 D2 {' z8 V" p
       1Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna, Italy.  2Department of Pathology, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands1 U% a1 t* Q7 e! m, C" I! m2 Y- K
　　Mesenchymal stem cells (MSCs) are adult multipotent cells that give rise to various cell types of the mesodermal germ layer. MSCs are of great interest in the field of regenerative medicine and cancer therapy because of their unique ability to home to damaged and cancerous tissue. These cells also regulate the immune response and contribute to reparative processes in different pathological conditions, including musculoskeletal and cardiovascular diseases. The use of MSCs for tissue repair was initially based on the hypothesis that these cells home to and differentiate within the injured tissue into specialized cells. However, it now appears that only a small proportion of transplanted MSCs actually integrate and survive in host tissues. Thus, the predominant mechanism by which MSCs participate in tissue repair seems to be related to their paracrine activity. Indeed, MSCs provide the microenvironment with a multitude of trophic and survival signals including growth factors and cytokines. Recent discoveries suggest that lipid microvesicles released by MSCs may also be important in the physiological function of these cells. Over the past few years the biological relevance of micro- and nano-vesicles released by cells in intercellular communication has been established. Alongside the conventional mediators of cell secretome, these sophisticated nanovesicles transfer proteins, lipids and, most importantly, various forms of RNAs to neighboring cells, thereby mediating a variety of biological responses. The physiological role of MSC-derived vesicles (MSC-MVs) is currently not well understood. Nevertheless, encouraging results indicate that MSC-MVs have similar protective and reparative properties as their cellular counterparts in tissue repair and possibly anti-cancer therapy. Thus, MSC-MVs represent a promising opportunity to develop novel cell-free therapy approaches that might overcome the obstacles and risks associated with the use of native or engineered stem cells.
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0 ?# J7 B/ e3 _: y5 k  @       Tatiana Lopatina, Maria Chiara Deregibus, Vincenzo Cantaluppi and Giovanni CamussiPages 11-22 (12)   Current Biotechnology ISSN (Print): 2211-5501 ISSN (Online): 2211-551X VOLUME: 1 ISSUE: 1  DOI: 10.2174/2211550111201010011
; a  C3 I+ ~4 G8 _2 }' k        Stem Cell-Derived Microvesicles: A Cell Free Therapy Approach to the Regenerative Medicine. R+ p) L: A4 d3 M9 d7 z
       Abstract:Microvesicles (MVs) include a heterogeneous population of vesicles released as exosomes from the endosomal compartment or as shedding vesicles from the cell surface of different cell types. The broad spectrum of biological activities displayed by MVs candidate them to a pivotal role in cell-to-cell communication. It is now recognized that they constitute an integral part of the intercellular microenvironment acting as vehicle for information transfer. After receptorligand interaction with target cells, MVs may directly stimulate the cells or may transfer from the cell of origin various bioactive molecules including membrane receptors, bioactive lipids and proteins. In addition, MVs may induce epigenetic changes in target cells by delivering specific subsets of mRNA and microRNA associated with different cell functions such as differentiation of blood cells, metabolic pathways and modulation of immune response. In vivo, MVs released from mesenchymal stem cells may account for the described paracrine action of these cells in tissue regeneration. A bidirectional exchange of genetic information between stem and injured cells could be envisaged: i) transcripts delivered by MVs from injured cells may reprogram the phenotype of stem cells to acquire specific features of the tissue; ii) transcripts delivered by MVs from stem cells may limit tissue injury and induce cell cycle re-entry of resident cells leading to tissue self-repair. This review presents an overview of the many biological actions of MVs produced by stem cells that may be exploited in regenerative medicine to repair damaged tissues as an alternative to stem cell-based therapy.
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7 V, o/ F9 p0 \　　无细胞体系（cell-free system）不具有完整细胞结构，但包含进行正常生物学反应所需的物质组成的系统。
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/ S) a5 s. ^+ ^2 M- \- K  k' g* {细胞外的生命现象（一）http://www.stemcell8.cn/thread-69216-1-1.html
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细胞外的生命现象（二）http://www.stemcell8.cn/thread-69222-1-1.html
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