作　　者： (廖翠玲）; (章淼锋）; (孙继红）; (董江军）; (朱园园）; (叶招明）; (邹飞雁）;

机构地区： 暨南大学生命科学技术学院发育与再生生物学系,广州510632

出　　处： 《中华骨科杂志》 2017年第16期1045-1053,共9页

摘　　要： 目的构建三种rLC3B融合表达载体，探究其在骨肉瘤细胞中的自噬检测应用。方法采用PCR扩增大鼠LC3B基因序列，克隆至pEGFP-C1和pmCherry—C1，分别构建pEGFP-rLC3B和pmCherry-rLC3B融合表达载体；随后以pEG-FP-rLC3B为模板经PCR得到EGFP—rLC3B序列，克隆至pmCherry-C1并构建pmCherry-EGFP—rLC3B融合表达载体。将上述三种质粒转染至U-2OS细胞后，采用饥饿和Rapamycin激活自噬，或Chloroquine和Baf-A1抑制自噬，通过激光共聚焦显微镜观察自噬体和自噬溶酶体的形成。采用Westernblot检测Rapamycin、Chloroquine和Baf-A1作用下的内源性LC3B和外源性EGFP-rLC3B、mCherry，rLC3B及mCherry—EGFP-rLC3B，验证外源性rLC3B的正确表达和自噬检测功能；采用West-erablot检测游离EGFP表达，从而精确检测细胞内的自噬性降解水平。结果经酶切鉴定和测序验证：成功构建三种融合表达载体。采用饥饿和Rapamycin激活自噬，或Chloroquine和Baf-A1抑制自噬，并在激光共聚焦显微镜下观察到明显的自噬体或自噬溶酶体。Westernblot同时检测到内源LC3B和外源rLC3B的表达，并且游离EGFP蛋白的表达随着饥饿时间增加而明显上调，12h组比对照组增加1．05倍，24h组比对照组增加1．56倍，显示自噬性降解水平升高。结论EGFP-rLC3B载体可检测自噬体的形成和反应细胞内的自噬性降解水平；mCherry-rLC3B载体可同时显示自噬体和自噬溶酶体，但不能区分这两者；mCherry-EGFP-rLC3B载体可同时显示和区分自噬体和自噬溶酶体，是检测自噬流的最佳方法。 Objective To monitor the autophagy in osteosarcoma ceils by constructing three rLC3B fusion expression vectors, respectively. Methods Rat LC3B gene sequence was amplified by PCR and cloned into pEGFP-C1 and pmCherry-C1 to construct the fusion expression vector of pEGFP-rLC3B and pmCherry-rLC3B. Subsequently, the EGFP-rLC3B sequence was ob- tained by PCR with the pEGFP-rLC3B as a template, and cloned into pmCherry-C1, so the pmCherry-EGFP-rLC3B fusion expres- sion vector was constructed. Three plasmids were transfected into U-20S cells, and the starvation or Rapamycin was adopted to in- duce autophagy or the chloroquine or Bar-A1 was used to inhibit autophagy, to verify the above plasmids＇ function in autophagy detection by laser scanning eonfocal microscopy. Western blot was used to detect the endogenous LC3B and exogenous EGFP- rLC3B, pmCherry-rLC3B and mCherry-EGFP-rLC3B, and to verify the correct expression of exogenous rLC3B and their function of autophagy detection. Finally, cleaved free EGFP was detected by Western blot to evaluate the level of autophagic degradation. Results Three fusion expression vectors were constructed successfully through sequencing and restriction enzyme digestion vali- dation. The starvation or Rapamycin was adopted to induce autophagy or the chloroquine or Baf-A I was used to inhibit antophagy in transfected U-20S cells. Clear autophagosomes and autolysosomes were observed by laser scanning confocal microscopy. Endogenous LC3B and exogenous EGFP-rLC3B, pmCherry-rLC3B and mCherry-EGFP-rLC3B were detected through western blot. Finally, western blot verified that the expression of cleaved free EGFP was significantly up-regulated with the increase of starva- tion time. 12 h group increased 1.05 times than the control group and 24 h group increased 1.56 times, showing that the levels of autophagic degradation increased. Conclusion EGFP-rLC3B can be used to detect autophagosome and evaluate the level of au- tophagic degradation, mCherry-rLC3B can be used to detect autophagosome and aut

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