细胞凋亡和继发性坏死的Annexin法定量检测

Annexin V for Flow Cytometric Detection of Phosphatidylserine Expression on Lymphoma Cells Undergoing Apoptosis

目的 探讨分析凋亡与坏死淋巴瘤 (Raji)细胞的定量方法。方法 以 1.0 μm ol/ L 地塞米松(DEX)诱导 Raji细胞凋亡 ,用异硫氰酸荧光素标记的连接素 V(Annexin V)和碘化丙锭 (PI)分别与凋亡细胞膜上的磷脂酰丝氨酸和继发性坏死细胞内降解的 DNA结合 ,并以流式细胞术分析和分选标记细胞 ,用电镜和 DNA凝胶电泳对分选细胞及其 DNA进行鉴定。结果 Annexin+细胞数随 DEX孵育时间的延长而增加 (r=0 .97) ;Annexin+/ PI- 与 Annexin+/ PI+细胞经电镜和 DNA梯形带确证具有凋亡与坏死细胞的特征 ;Annexin法可准确测定 10 5～ 10 6细胞中的标记细胞。结论 Annexin法可快速。

Objective To quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions. Methods The cells of Burkitt lymphoma cell line Raji were incubated with 1.0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detect the exposure of phosphatidylserine (PS) on the out membrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows the analysis of secondary necrotic cells related with cell membrane and DNA damage, then apoptotic cells was quantified by flow cytometry (FMC). Furthermore, Annexin + /PI - and Annexin +/PI + cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electrophoresis. Results The results revealed that the percentage of apoptotic cells was increased and correlated well with incubation time ( r =0.97). The sensitivity of this method was shown by its detection limit 0.02%; the method was reproducible, and the coefficient variance (CV) was 4.2%. Meanwhile, the Annexin +/PI - and Annexin +/PI + cells were identified as apoptotic and necrotic cells under EM, and the DNA extracted from the Annexin +/PI - cells was characterized by “ladder pattern”. Conclusion Annexin V assay for analyzing apoptotic cells is specific, sensitive, accurate, reproducible and quantitative for apoptosis investigation.