谈谈重要的研究。 威尼斯人娱乐城 Assistant Professor Maria Spletter’s biology lab is investigating the breakdown of proteins in the body that lead to chronic conditions such as cancer and heart disease. She has lasered in on myotonic dystrophy — or loss of muscle function.
强直性肌营养不良症是最常见的罕见疾病之一，估计每2100个新生儿中就有1个患有此病。 The muscle disease also causes accelerated aging as the regulation of ribonucleic acid, present in all living cells and also called “RNA”, changes and alters muscle control, growth and contraction.
spleter选择了一个在大小上不重要但在效率上很重要的模型：果蝇。
为什么果蝇?
“Drosophila are a very powerful genetic model,” Spletter said. “This means that there are a lot of tools available in the fly that enable us to do experiments that are not possible in mice or rats or humans. Plus, Drosophila grow quickly from an egg to adult fly in 10 days, so you can follow each step during development in a matter of weeks instead of years.”
使用果蝇的另一个好处是它的肌肉是高度保守的，这意味着构建肌肉的蛋白质、肌肉收缩的方式以及肌肉的结构和组织在果蝇中与人类是一样的。 事实上，人类疾病在果蝇身上的模型揭示了相同的机制和相同的肌肉类型。 因此，这些果蝇提供了一个有用的模型，以了解由致病突变引起的肌肉的基本机制和缺陷，从而可以针对人类细胞进行进一步的研究。
这意味着研究小组可以研究导致肌肉纤维丢失或损伤的发育机制，而这在哺乳动物身上是不可能进行详细研究的。 通过跟踪肌肉纤维在发育过程中的初始阶段，他们可以准确地说出组装过程中的哪些步骤有缺陷。 对小鼠或大鼠的研究通常没有这种水平的分辨率，也没有详细关注肌肉结构是如何被破坏的。
使用高性能工具进行测试
“We test muscle function to measure how well flies can fly, jump, climb, flip themselves over after falling on their back and how quickly they are able to clean themselves after being dusted with a fluorescent powder,” Spletter said. “All of these give us insight into live flies on their behavior when it comes to how well their muscles work.”
为了研究这些rna结合蛋白在细胞水平上的功能，实验室用荧光标记标记肌肉的尖端，并用显微镜观察肌肉的运动。 然后，他们通过测量肌肉收缩的频率、收缩时的运动幅度以及收缩的动态来量化运动。 这是在突变果蝇中收缩通常受损和不规则的地方。
然后，研究人员使用高倍显微镜，利用激光对样品成像，观察染色的肌肉，并标记不同的成分。 与传统的显微镜不同，激光能够成像厚度为1微米或更小的单个平面（果蝇约为1毫米厚，而1毫米中有1000微米）。 间接飞行肌细胞的厚度约为100微米，因此可以拍摄至少100张肌肉不同平面的照片，以查看其内部的所有结构。
With the lab’s microscope having four different lasers, four different components of the cell can be viewed at the same time to see where they are located relative to each other. 特别是这些突变体，研究小组可以观察到它们的定位是如何变化的。 这样就可以比较突变果蝇和对照果蝇，看看在细胞结构水平上突变果蝇的结构是如何不同的。
然后，研究小组从对照组和突变果蝇身上提取组织样本，进行分子和生化测试，以找出突变肌肉的基因变化，随后将分子缺陷与细胞结构和肌肉功能的变化联系起来。
这通常是当mRNA-Seq，一种生物化学和生物信息学方法的结合，发生。 从果蝇中分离出mRNA（在细胞中转化为蛋白质的编码蓝图），实验室对肌肉细胞中表达的每个基因和基因变体进行测序。 通常情况下，在任何时候都有大约6000到8000个基因表达，如果你观察整个发育过程，大约有10000个基因改变表达。 数据通常用于单个基因，单个基因内的剪接事件，或在基因表达和剪接的所有变化中全局查看。 所获得的不同水平的数据使实验室能够在系统水平上了解突变肌肉细胞与对照相比发生了什么变化，并在个体基因水平上确定可能解释我们所看到的特定表型的目标。
Spletter’s lab also conducts mass spectrometry, an analytical tool useful for measuring the mass-to-charge ratio of one or more molecules present in a sample, to isolate the proteins from muscle cells and determine the identity of most of the proteins present in the muscle. 通常可以检测到大约4000种蛋白质，但更灵敏的机器可以检测到多达6000种蛋白质。 这种分析方法提供了在我们的突变肌肉中哪些蛋白质发生变化的信息，并允许从mRNA-Seq数据中比较RNA中的蛋白质变化，以准确地找出RNA调节的变化如何导致肌肉纤维和结构的缺陷。
这些发现
Recent research findings from Spletter’s lab, which were published on bioRxiv, revealed how the characteristics in mutant muscle are a domino effect. 在肌肉发育的每一步中出现的小问题都会导致并进一步加剧对肌肉的影响。 与布鲁诺1突变体被添加到肌肉发育的后期相比，这导致了对肌肉的更大破坏。
From the same research findings, Spletter’s lab discovered the potential possibility of testing gene therapy strategies in the flies that are currently in development for possible use in human patients.
Although gene replacement therapy can “normalize” patterns of splicing, patients only have a partial improvement of symptoms. 这意味着基因治疗通常会提高生活质量，并可能延长预期寿命，但不能治愈。
Spletter’s lab was able to gain insight into why exactly this is the case.
“Because the structure of the muscle has defects in the core mechanical structure that allows it to move, just fixing the splicing pattern is not sufficient to fix those defects,” Spletter said. “This suggests that we need better detection methods to find patients before they seek medical help, as the earlier a gene therapy can be administered, the better chance these patients are going to have of maintaining muscle function.”