| 摘要: |
| 目的:分离并鉴定C57BL/6 小鼠肺组织中内质网线粒体结构偶联(ERMC)。方法:选取8~10 周C57BL/6 雄性小鼠10 只，处死小鼠后，取1 g 肺组织匀浆后，采用梯度离心法分别分离出肺组织细胞内的内质网、粗提线粒体、ERMC和纯化线粒体，然后采用Western blotting 方法对上述结构进行鉴定。结果:通过梯度离心在离心管中间一层白色条带中含有ERMC，底层白色沉淀物为纯化的线粒体，随后将分离纯化的ERMC 和其他组分进行鉴别，结果发现蛋白质二硫键异构酶(PDI)主要表达在内质网和ERMC，Cyto C 主要分布在线粒体，而钙连蛋白主要表达在内质网，三磷酸肌醇受体- Ⅰ(IP3R-1)作为内质网上的钙离子通道，在内质网和ERMC 均有表达，此外，Sigma-1 受体分子伴侣(Sig-1R)多表达在ERMC 和线粒体中。结论:上述特征性蛋白分子表达分布特点说明本次研究分离提纯肺组织内ERMC 的方法有效，可将其应用于后续的实验。 |
| 关键词: 小鼠 肺组织 内质网线粒体结构偶联 |
| DOI：10.3969/j.issn.1007-6948.2020.02.003 |
| 投稿时间：2019-09-18 |
| 基金项目:天津市自然科学基金重点项目(18JCZDJC35400)；国家自然科学基金(81772106，81601707) |
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| Isolation and Certification of Endoplasmic Reticulum–mitochondria Contacts (ERMC) from Mouse Lung |
| Tissues DONG Shu-an,YU Jian-bo,WU Jian-hua |
| Department of Anesthesiology, Tianjin Nankai Hospital,Tianjin (300100), China. |
| Abstract: |
| Objective To isolate and identify ERMC from lung tissue of C57BL/6 mice. Methods Ten8-10 weeks old C57BL/6 male mice were selected. After killing the mice, 1 g of lung tissue homogenate was taken. Endoplasmic reticulum, crude mitochondria, ERMC and purified mitochondria were separated by gradient centrifugation. The above structures were identified by Western blotting. Results Gradient centrifugation contained ERMC in a white band in the middle of the centrifugal tube. The bottom white sediment was purified mitochondria. Subsequently, we identified the purified ERMC and other components. The results showed that PDI was mainly expressed in endoplasmic reticulum and ERMC, Cyto C was mainly distributed in mitochondria,while calcineurin was mainly expressed in endoplasmic reticulum, IP3R-1 was the calcium dissociation in endoplasmic reticulum. Subchannels were expressed in endoplasmic reticulum and ERMC, and Sig-1R was mostly expressed in ERMC and mitochondria. Conclusion The results of the molecular expression distribution of the characteristic proteins mentioned above are consistentthe literature, indicating that our method of isolating and purifying ERMC in lung tissue is effective and can be used in future experiments. |
| Key words: Mice lung ERMC |
