| In vitro Chondrogenic Hypertrophy Induction of Mesenchymal Stem Cells
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Author:
Date:
2016-12-05
[Abstract] To investigate underlying mechanism of chondrogenic hypertrophy, we need proper in vitro hypertrophic model of mesenchymal stem cells (MSCs). This protocol describes our defined method for induction of in vitro chondrogenic hypertrophy of human umbilical cord blood-derived MSCs (hUCB-MSCs). By adding thyroid hormone (T3; triiodothyronine) and minimum osteogenic-inducing factors to culture medium, we could induce hypertrophy of hUCB-MSCs in vitro. Hypertrophic induction was validated using immunohistochemical analysis, Western blotting and reverse transcriptase polymerase chain reaction.
[摘要] 为了研究软骨形成性肥大的基础机制，我们需要适当的体外间充质干细胞（MSC）的增生模型。该协议描述了我们定义的诱导人脐带血衍生的MSC（hUCB-MSC）的体外软骨形成性肥大的方法。通过将甲状腺激素（T3;三碘甲状腺原氨酸）和最小成骨诱导因子添加到培养基中，我们可以在体外诱导hUCB-MSCs的增生。使用免疫组织化学分析，Western印迹和逆转录酶聚合酶链反应验证肥大性诱导。
关键词：间充质干细胞，体外软骨形成性肥大，成软骨分化，三碘甲腺原氨酸;
[背景] ...
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| Laser Microirradiation and Temporal Analysis of XRCC1 Recruitment to Single-strand DNA Breaks
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Author:
Date:
2016-03-05
[Abstract] The DNA molecule is exposed to a multitude of damaging agents that can compromise its integrity: single (SSB) and double strand breaks (DSB), intra- or inter-strand crosslinks, base loss or modification, etc. Many different DNA repair pathways coexist in the cell to ensure the stability of the DNA molecule. The nature of the DNA lesion will determine which set of proteins are needed to reconstitute the intact double stranded DNA molecule. Multiple and sequential enzymatic activities are required and the proteins responsible for those activities not only need to find the lesion to be repaired among the millions and millions of intact base pairs that form the genomic DNA but their activities have to be orchestrated to avoid the accumulation of toxic repair intermediates. For ...
[摘要] DNA分子暴露于多种损害剂，其可损害其完整性：单链（SSB）和双链断裂（DSB），链内或链间交联，碱基丢失或修饰等。 >许多不同的DNA修复途径共存于细胞中以确保DNA分子的稳定性。 DNA损伤的性质将决定重组完整的双链DNA分子需要哪组蛋白质。需要多个和顺序的酶活性，负责那些活性的蛋白质不仅需要在形成基因组DNA的数百万和数百万个完整碱基对中找到待修复的损伤，而且它们的活性必须被协调以避免毒性修复中间体。例如在单链断裂（SSB）的修复中，将需要蛋白质PARP1，XRCC1，聚合酶β和连接酶III，并且它们的活性协调以确保损伤的正确修复。
此外，DNA不是自由的在核中但组织在具有不同压实水平的染色质。因此DNA修复蛋白质处理这种核组织以确保有效的DNA修复。在损伤诱导后研究核中DNA修复蛋白质的分布的一种方式是使用激光微辐射，其中可以在细胞核的局部区域中诱导特定类型的DNA损伤。所使用的激光的波长和强度将决定被诱导的主要类型的损伤。重要的是注意，在微照射部位也可以产生其他损伤。
用荧光蛋白XRCC1-GFP转染的活细胞在共聚焦显微镜下进行微照射，并在1分钟内跟踪荧光蛋白的募集动力学。在我们的协议中，使用405nm激光来诱导SSB。
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| Primer Extension Analysis of HBV DNA with Strand-Specific Primers
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Author:
Date:
2015-08-05
[Abstract] We performed primer extension assay to determine which steps of HBV DNA synthesis (i.e., minus- and plus-strand DNA synthesis and circularization of RC DNA) are affected by phosphoacceptor site mutations in C protein. In these experiments, we used several specific oligonucleotide primers. For quantitation, the level of extended DNA (ED) was normalized to the level of a single internal standard (IS) DNA.
[摘要] 我们进行引物延伸测定以确定HBV DNA合成的哪些步骤（即，负链和正链DNA合成和RC DNA的环化）受C蛋白中的磷酸受体位点突变的影响。 在这些实验中，我们使用几种特异性寡核苷酸引物。 为了定量，将延伸DNA（ED）的水平标准化为单个内标（IS）DNA的水平。
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