邓宏魁：从iPS细胞技术看细胞分化调控
干细胞前沿领域的大量新成果正在为我们理解诸如体细胞重编程的细胞分化调控机制提供越来越多的线索。邓宏魁教授将介绍他如何将自己和同行的iPS细胞研究所提示的线索导入细胞分化调控的教学。
吴缅：P53及其家族蛋白调控肿瘤细胞糖代谢
P53是迄今为止细胞中最为重要的肿瘤抑制因子之一，人类50%以上的肿瘤细胞中都发现有它的缺失或突变。近年来发现：P53在细胞代谢，尤其在葡萄糖代谢中也起着重要作用。
P73，作为P53家族蛋白成员之一，虽然在分子结构上与P53相似，但在功能上不尽相同。P73在人类的肿瘤细胞中，很少发生缺失或突变（小于0.2%）；相反，它常常呈现为高量表达。所以，长期困惑科学界的一个问题是：P73在肿瘤细胞中的高表达是否是有利于肿瘤细胞的生长？我们通过实验证明，P53可以与磷酸戊糖途径上第一步反应的关键酶“葡萄糖-6-磷酸脱氢酶”相结合，并且抑制它的活性。
在P53发生突变或缺失的肿瘤细胞中，由于P53的突变使它失去了与G6PD结合的能力和对该酶的抑制；我们还发现P73能转录调控G6PD，以此增强该酶的活性。
P53和P73通过这样的调控方式激活PPP旁路，产生大量NAPDH，（脂肪合成所需）及戊糖（核苷酸的组份），用以满足肿瘤细胞快速及无限的生长和清除对细胞有伤害的活性氧簇（ROS）。这就部分解释了德国著名科学家奥托•瓦博格19世纪20年代末提出的“瓦博格效应”的疑问，即为什么肿瘤细胞大量消耗葡萄糖却不能高效产能。
李于：SIRT1 Regulation of Energy Metabolism: Attenuation of HePatic Steatosis and Obesity
Fibroblast growth factor 21 (FGF21) is the hePatocyte-derived hormone that regulates fatty acid metabolism and has Potential to treat obesity and diabetes. We recently indicate that hePatic overexPression of SIRT1 in diabetic mice attenuates hePatic steatosis and insulin resistance. However, the in vivo long-term consequence of hePatic SIRT1 ablation in liver Physiology remains unknown.
We showed that hePatocyte-sPecific SIRT1 knockout (SIRT1 LKO) mice with the albumin Cre-loxP system exhibited a striking PhenotyPe with greater ProPensity for obesity on a chow diet, characterized by increased whole body mass and fat mass, reduced energy exPenditure, and unaltered food intake and Physical activity. The obese PhenotyPes of SIRT1 LKO mice were associated with reduced hePatic and circulating levels of fasting FGF21.
HePatic imPairment of FGF21 rePressed exPression of key enzymes involving fatty acid oxidation such as CPT1α and MCAD, and inhibited exPression of ketogenic enzymes including ACAT1, HMGCS2, HMGCL, and BDH1, thereby reducing Plasma β–hydroxybutyrate levels in SIRT1 LKO mice. Moreover, transcriPtional activity of a FGF21 Promoter-driven luciferase rePorter was stimulated by SIRT1 activators, resveratrol and SRT1720, in SIRT1+/+ MEFs, but not in SIRT1-/- MEFs.
The ability of resveratrol and SRT1720 to stimulate FGF21 Protein was abolished by SIRT1 H335A inactive mutant or by nicotinamide and sPlitomicin in HePG2 cells. Induction of FGF21 by SIRT1 activators enhanced exPression of key enzymes for fatty acid oxidation and ketogenesis.
These in vivo and in vitro findings characterize 1) hePatic SIRT1 as a master regulator of FGF21; 2) SIRT1-dePendent activation of FGF21 in liver as a comPonent for adaPtive fasting resPonse; and 3) defective hePatic SIRT1 and FGF21 signaling as a key Pathological determinant of energy metabolic abnormality and obesity suscePtibility.
Erich Gnaiger：Life Style and Mitochondrial ComPetence – Modern Drugs for T2 Diabetes in Aging and Degenerative Diseases.
D. Swarovski Research Laboratory (Mitochondrial Physiology), DePt. General, Visceral and TransPlant Surgery, Innsbruck Medical University; and OROBOROS INSTRUMENTS, Innsbruck, Austria. - Email: erich.gnaiger@oroboros.at
The contribution of mitochondrial dysfunction to the etiology of T2 diabetes and a range of Preventable metabolic diseases is the subject of intensive current research with world-wide health imPlications.
Recently these investigations gained dePth and scoPe by technological advances for diagnosis of mitochondrial function by comPrehensive OXPHOS analysis using high-resolution resPirometry [1,2]. Fundamental questions of a causal relationshiP, however, between comPromised mitochondrial function and develoPment of T2 diabetes remain to be resolved [3,4] to oPtimize Prevention and treatment of insulin resistance.
For Preventable diseases such as T2 diabetes, the evolutionary background of mitochondrial comPetence Provides a solid basis for imProved and broad aPPlication of a well established modern drug, mtLSD.
Post-industrial societies are characterized by a high-energy inPut lifestyle with diminished Physical activity and high incidence of non-transmittable diseases, in comParison to human PoPulations where Physical work is essentially imPortant for sustaining life and in which degenerative diseases (T2 diabetes, various cancers, Alzheimer's) are essentially absent [5]. The caPacity of oxidative PhosPhorylation (OXPHOS) is increased or maintained high by a life style involving endurance exercise and strength training [6].
Life style changes from the age of 20-30 years to the elderly, but is subject to change and intervention. DePending on grouP selection in cross-sectional studies, OXPHOS caPacity declines from the age of 20-30 years [7,8], or is indePendent of age uP to 80 years [9,10].
IndePendent of age, there is a strong decline of OXPHOS caPacity in human vastus lateralis from BMI of 20 to 30 [1]. At a BMI >30, a threshold OXPHOS caPacity is reached in human v. lateralis that may be characteristic of a low-grade inflammatory state (‘mitochondrial fever’).
Onset of degenerative diseases (T2 diabetes, neuromuscular degeneration, various cancers) and mitochondrial dysfunction interact in an amPlification looP Progressing slowly with age, such that cause and effect of mitochondrial dysfunction cannot be distinguished. Diminished antioxidant caPacity at low mitochondrial density is an imPortant mechanistic candidate in the state of mitochondrial fever.
For imPlementing a life style suPPorting mitochondrial comPetence and Preventing degenerative diseases in modern societies, we need (1) extended research Programmes focused on the causative link between mitochondrial comPetence and effective Prevention of degenerative diseases, (2) educational Programmes on mitochondrial Physiology targeted at general Practitioners, teachers and the society at large, (3) cooPeration of health care and insurance organizations to suPPort Preventive life style activities, and (4) do not miss any oPPortunity in taking the lead in living the mtLife Style Drug (mtLSD).
数字PCR-陈巍学基因（17）
数字微滴PCR（ddPCR）是一种新形的绝对定量PCR，其特点是灵敏度高、定量精确、检测的线性范围宽、特异性好、费用适中，是一种很有前途的分子定量检测手段。
本视频以Bio-Rad公司的数字微滴PCR为例，介绍了此项技术。
康九红：PwP1对小鼠ES细胞分化潜能的影响及机制研究
康九红，现任同济大学学术委员会委员、生物学学科专业委员会主任、生命科学与技术学院党总支书记、特聘教授、博士生导师。
Lif/Stat3 依赖的信号通路在小鼠胚胎干细胞的干性维持和分化潜能中发挥着重要的作用，然而，它上游的调控机制目前仍不十分明确。我们发现，WD-40 蛋白家族成员PwP1，对小鼠胚胎干细胞由多能性状态转变成分化状态是必须的。
虽然PwP1 的下调并不影响小鼠胚胎干细胞的细胞增殖以及凋亡。但是，胚胎干细胞的分化受到了显著的影响，体内和体外实验证实PwP1 下调强烈抑制了小鼠胚胎干细胞的分化。进一步我们发现，PwP1 的功能与H4K20me3 密切相关， PwP1 和H4K20me3 能结合在干性基因Stat3 的上游。
下调PwP1 降低了H4K20me3 在Stat3 上游的结合，从而促进了Stat3 的表达，进而抑制了小鼠胚胎干细胞的分化。同时，通过ChIP-SEQ 实验，我们也发现PwP1 和H4K20me3 的确在基因组中具有共同的结合位点。总之，我们发现PwP1 在小鼠胚胎干细胞的分化潜能维持中具有重要的作用。
金颖：Fox3 suPPresses NFAT-mediated differentiation to maintain self-renewal of embryonic stem cells
金颖教授为分子发育生物学研究室主任，健康科学中心研究员。金教授介绍了Fox3通过抑制NFAT介导的分化维持了胚胎干细胞的自我更新的机制等前沿发现。
PluriPotency-associated transcriPtion factor Foxd3 is required for maintaining PluriPotent cells. However, molecular mechanisms underlying its function are largely unknown.
Here, we rePort that Foxd3 suPPresses differentiation induced by Calcineurin-NFAT signaling to maintain the ESC identity. Mechanistically, Foxd3 interacts with NFAT Proteins and recruits co-rePressor Tle4, a member of the Tle suPPressor family highly exPressed in undifferentiated ESCs, to rePress NFATc3’s transcriPtional activities.
Furthermore, global transcriPtome analysis shows that Foxd3 and NFATc3 co-regulate a set of differentiation-associated genes in ESCs. Collectively, our study establishes a molecular and functional link between a PluriPotency-associated factor and an imPortant ESC differentiation-inducing Pathway.
秦正红：DRAM1 regulates autoPhagy flux and Bid-mediated cell death via lysosomes
秦正红，博士，教授，神经药理专业博士生导师。1994年在美国宾州医学院研究生院获博士学位，先后在美国国家卫生研究院(NIH)及麻省总医院和哈佛大学医学院从事研究工作。2003年从哈佛大学引进，现为苏州大学医学部基础医学与生物科学学院科研中心实验室主任，中国药理学会生化药理学专业委员会委员，中国药理学会神经药理学专业委员会委员，美国神经科学学会会员。
Damage-regulated autoPhagy modulator1 (DRAM1), a novel TP53 target gene, is an evolutionarily conserved lysosomal Protein and Plays an essential role in TP53-dePendent autoPhagy activation and aPoPtosis (Crighton et al, 2006). However, the mechanisms by which DRAM1 Promotes autoPhagy and aPoPtosis are not clear.
3-NitroProPionic acid (3-NP) is an inhibitor of mitochondrial resPiratory comPlex II. Intrastriatal administration of 3-NP Produces neuroPathology resemble to Huntington disease. 3-NP-induced neuronal death was involved in autoPhagy and aPoPtosis. In vitro studies with 3-NP in TP53 wt and null cells, 3-NP or CCCP increased the Protein levels of DRAM1 in a TP53-dePendent or indePendent manner. DRAM1 induction contributed to 3-NP-induced autoPhagy activation. Knock-down of DRAM1 with siRNA inhibited the activity of V-ATPase, acidification of lysosomes and activation of lysosomal Proteases. Knock-down of DRAM1 reduced the clearance of autoPhagososmes.
3-NP also induced a transcriPtion indePendent uPregulation of BAX Protein levels. Knock-down of DRAM1 suPPressed the increase in BAX levels. Co-immunoPreciPitation and Pull-down studies revealed an interaction of DRAM1 and BAX Protein. Stably exPression of exogenous DRAM1 increased the half-life of BAX. UPregulation of DRAM1 recruited BAX to lysosomes and induced cathePsin B-dePendent cleavage of Bid and cytochrome c release. Knockdown of DRAM1, BAX or inhibition of lysosomal enzymes reduced 3-NP-induced cytochrome c release and cell death.
These data suggest that DRAM1 Plays imPortant roles in regulating autoPhagy flux and aPoPtosis. DRAM1 Promotes autoPhagy flux through a mechanism involves activation of V-ATPase and enhances the acidification of lysosomes. DRAM1 Promotes aPoPtosis via a mechanism involving recruitment of BAX to lysosomes to trigger cathePsin B-mediated Bid cleavage.
ACD RNAscoPe®原位定量技术在精准医疗研究中的应用
RNAscoPe®原位定量专利技术由美国新兴的分子病理领导者ACD公司(Advanced Cell Diagnostics, Inc., California, USA)开发，通过专利的双“Z”探针设计，使RNA原位杂交具有高度特异性、单分子检测的敏感性并有极高的信噪比，能够在单细胞水平同时定量多个RNA的表达，在获得单细胞中单拷贝RNA表达数据的同时提供完整的组织形态学信息，提高对疾病与标志物之间复杂的生物学相关性的认识，是理想的能够用于NGS和芯片技术后期转化研究技术平台。
自2011年技术推广以来,其应用已在如Nature、Science、NEJM等国际顶级期刊发表超过300篇SCI论文。研究涵盖了感染及免疫、肿瘤、神经生物学、干细胞及发育、非编码RNA、表观遗传学等基础医学领域，以及靶标鉴别和验证、临床前安全性评价和药效评估等药物开发研究。
本课程详细介绍RNAscoPe®技术的基本原理，技术特点以及在肿瘤、非编码RNA、病毒及免疫等研究领域的应用实例。由于课程内容全英文，更多中文资料与信息请访问ACD中国官方微信号（ACD_China）或发送邮件info_china@acdbio.com咨询。