在人类胚胎干细胞的线粒体复杂的活动初探
Summary
在这段视频中，我们将向您可以分析人类胚胎干细胞的线粒体呼吸链复合物如何使用凝胶活性测定。
Abstract
线粒体是细胞质的细胞器，有一个细胞代谢和动态平衡的主要作用，细胞信号转导网络调控，和程序性细胞死亡。线粒体产生ATP，调节细胞质的氧化还原状态和Ca2 +的平衡，分解代谢脂肪酸，合成血红素，核苷酸，类固醇激素，氨基酸，并帮助组装蛋白的铁硫簇。线粒体也有产热的重要作用。线粒体基因组的突变引起人类疾病的几种类型。 mtDNA突变的积累与衰老相关，怀疑是在癌症发展的重要作用。由于他们的作用极为重要，在所有类型的细胞，线粒体的功能，还必须干细胞的关键。干细胞活力，增殖和分化能力的认识已经取得了重要进展。但干细胞的功能活动，尤其是他们的能量状态，尚未被详细研究。几乎没有什么是已知的人类胚胎干细胞（胚胎干细胞），和他们的区别的易制毒化学后代线粒体属性。线粒体在人类胚胎干细胞的功能和发展潜力作用来认识和评价的方法之一是直接测定线粒体呼吸复合物的活性。在这里，我们描述了高分辨率的清晰的原生凝胶电泳凝胶作为活动的可视化分析五年的人类胚胎干细胞呼吸链复合物的方法和后续。
Protocol
高分辨率清除母语凝胶Electophoresis线粒体复合物（hrCNE）活性测定 – 凝胶单层ˠ照射或丝裂霉素C处理过的小鼠胚胎成纤维细胞（MEFs）然后切换MEF空调介质中，使用标准的条件下5天的基质胶上的增长，对维持人类胚胎干细胞（胚胎干细胞）。为了去除污染MEFs，人类胚胎干细胞被重新镀上基质胶和MEF空调额外3天的中期增长。 Oct – 4和SSEA – 4的多能性标志物免疫荧光显微镜是用来确?…
Discussion
在这段视频中，我们已经描述了从人类胚胎干细胞，他们高分辨率的清晰本地凝胶电泳（hrCNE）分离，以及随后的分析凝胶的催化活性检测线粒体蛋白质复合物的提取。 hrCNE解析线粒体膜蛋白复合物，并在一项决议，与蓝色本地凝胶电泳凝胶活性测定优越。可以采用这种技术不仅量化线粒体呼吸复合物IV也分析，无论是在人类胚胎干细胞和其衍生的后代的任何线粒体膜蛋白复合物的亚基组成。
Acknowledgements
目前支持人类胚胎干细胞研究在Teitell实验室由加州再生医学研究所（CIRM）种子批RS1 – 00313。我们感谢卡拉克勒博士（加州大学洛杉矶分校）和克勒实验室成员在这些问题和进一步的研究提供意见和见解。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 0.5M EDTA | Fisher Scientific | BP120-1 | adjust pH to 8.0 with NaOH | |
| 1M NaCl | Sigma | S3014-500MG | ||
| 1M Sodium succinate | Fisher Scientific | S413-500 | ||
| 0.1M Sodium phosphate buffer pH 7.2 | Fisher Scientific | BP332-1 and BP329-1 | Mix 68.4 ml of 1M Na2HPO4 and 31.6 ml of 1M NaH2PO4 to prepare 0.1M sodium phosphate buffer with pH 7.2 at 25 C | |
| 1M Tricine, pH 7.0 (adjusted with NaOH) | EMD | 9010 | store at 4 C | |
| 1M imidazole/HCl, pH 7.0 | Sigma | I-2399 | store at 4 C | |
| 1M Tris-HCl, pH 7.4 | Fisher Scientific | BP153-1 | ||
| 20% (wt/vol) dodecyl-b-D-maltoside (DDM) | Fluka | 44205-500MG | Dissolved in water. Store 1 ml aliquots at –20 C. | |
| 10% (wt/vol) digitonin (>50% purity, used without recrystallization) | Fluka | 37008-1G | Dissolved in water. Store 0.1–1 ml aliquots at –20 C. Should be warmed up to 95 C before use. | |
| 2M 6-aminohexanoic acid | Fluka | 07260-100G | store at 4 C | |
| 10x Ponceau S/glycerol stock solution | Sigma | P-3504 and G6279-1L | 0.1% Ponceau S, 50% glycerol, (wt/vol) in water. | |
| 10% w/v sodium deoxycholate | Fluka | 30970-25G | Dissolved in water. Must be protected from light. Adjust pH to 7.0 with HCl, filter and store at room temperature. | |
| 250 mM phenazine methasulfate (PMS) | TCI America | P0083 | Dissolved in DMSO and aliquoted. Store at –20 C. | |
| Nitro-blue tetrazolium (NBT) | Amresco | 0329-1G | ||
| 3,3’-diaminobenzidine tetrahydrochloride (DAB) | TCI America | D0078 | ||
| Pb(NO3)2 | Fisher Scientific | L62-100 | ||
| MgSO4 | Fisher Scientific | BP213-1 | ||
| Tris base | Fisher Scientific | BP152-10 | ||
| Glycine | Fisher Scientific | G45-212 | ||
| Adenosine 5’-triphosphate disodium salt (ATP) | Sigma | A2383-5G | Grade I, minimum 99% | |
| beta-nicotineamide adenine dinucleotide (NADH) reduced form | Sigma | N-8129 | ||
| Cytochrome c From horse heart | Sigma | C2506-250MG | ||
| Sucrose | Fisher Scientific | BP220-212 | ||
| 100x protease inhibitor cocktail | Sigma | P8340 | Store at – 20 C. | |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | D128-500 | ||
| Trypan blue Stain 0.4% | Gibco | 15250-061 | ||
| 10x Trypsin (0.5%) | Gibco | 15400-054 | ||
| Trypsin Inhibitor | Gibco | 17075-029 |
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Tags
Mitochondrial Complex ActivityHuman Embryonic Stem CellsCellular MetabolismHomeostasisCell Signaling NetworkProgrammed Cell DeathATP ProductionCytoplasmic Redox StateCa2+ BalanceFatty Acid CatabolismHeme SynthesisNucleotide SynthesisSteroid Hormone SynthesisAmino Acid SynthesisIron-sulfur Cluster AssemblyHeat ProductionMitochondrial Genome MutationsAging CorrelationCancer Development CorrelationStem Cell ViabilityStem Cell ProliferationStem Cell Differentiation CapacityEnergy Status Of Stem CellsMitochondrial Properties Of Human Embryonic Stem Cells (hESCs)Precursor Progeny DifferentiationMeasurement Of Mitochondrial Respiratory Complexes
Cite This Article
Khvorostov, I., Zhang, J., Teitell, M. Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells. J. Vis. Exp. (16), e724, doi:10.3791/724 (2008).