急性早幼粒细胞白血病(APL)属于急性髓系白血病（AML）, 是FAB分型中的M3亚型。 部分APL患者形成早幼粒细胞白血病/维甲酸受体融合基因, 即PML-RARα融合基因。 在内外界多种因素的共同作用下, 早幼粒细胞白血病发病。 胚胎干细胞(ESCs)具有多向分化的能力, 在一定诱导条件下, ESCs可以向造血系统分化。 早幼粒细胞位于ESCs分化下游, 为粒系分化阶段的一种细胞。 探索一种非标记的技术方法鉴别不同分化阶段造血细胞具有重要的科研和实践意义。 拉曼光谱技术可用于多种类型疾病的鉴别诊断研究, 近年来应用前景愈加广阔。 实验研究人胚胎干细胞系（ES）、 急性早幼粒细胞白血病细胞系（NB4）和急性早幼粒细胞白血病患者（M3）白血病细胞的拉曼光谱特征, 建立拉曼光谱非标记鉴别不同分化阶段白血病的方法, 为临床实验研究提供基础。 分别收集胚胎干细胞系（ES）、 急性早幼粒细胞白血病细胞系(NB4)和4例M3患者白血病细胞, 使用Horiba Xplora拉曼光谱仪获取拉曼光谱, 每组或每例患者采集25～30个白血病细胞光谱。 结合应用主成分分析法(PCA)、 判别函数分析(DFA)、 系统聚类分析和偏最小二乘判别分析（PLS-DA）, 对三类细胞的光谱进行分析并建立模型, 进而对三类细胞进行鉴别, 应用交互验证法对模型进行验证。 同时结合细胞超微结构分析三种细胞的拉曼光谱特征。 M3, NB4和ES细胞的拉曼光谱差别显著, 主要表现为M3和NB4细胞光谱中对应核酸、 蛋白质及脂类物质的谱峰明显高于ES细胞, 其生物学机制包含了APL与PI3K/Akt/mTOR通路的密切关系。 PI3K/Akt/mTOR通路在急性早幼粒细胞白血病细胞中存在异常激活, 影响白血病细胞的生物大分子代谢； 鉴别建模的总体分类准确率达100%(181/181), 交互验证的分类准确率达98.9%(179/181), 表明鉴别模型预测能力良好。 拉曼光谱分析显示M3细胞和NB4细胞增殖代谢明显高于ES细胞, 根据PCA-DFA、 聚类分析及PLS-DA建立的拉曼光谱鉴别模型能够准确区分3种不同分化阶段白血病相关细胞, 其结果与电镜结果相符。

Acute promyelocytic leukemia (APL) belongs to acute myeloid leukemia (AML) and is an M3 subtype in FAB typing. Some APL patients develop a promyelocytic leukemia/retinoic acid receptor fusion gene, the PML-RARα fusion gene. Under the combined action of various factors inside and outside, promyelocytic leukemia is onset. Embryonic stem cells (ESCs) have the ability to multi-differentiate. Under certain induction conditions, ESCs can differentiate into hematopoietic system. Promyelocytes are located downstream of ESCs differentiation and are a cell in the granulocyte differentiation stage. Exploring a non-labeled technical method to identify hematopoietic cells at different stages of differentiation has important scientific and practical significance. Raman spectroscopy can be used for differential diagnosis of many types of diseases, and its application prospects have become more extensive in recent years. This experiment investigated the Raman spectral characteristics of human embryonic stem cells leukemia cell line (ES), acute promyelocytic leukemia cell line (NB4) and leukemia cells from 4 patients with acute promyelocytic leukemia (M3), established a novel Raman label-free method to distinguish leukemia-related cells of distinct differentiation stages and provided basis for clinical research. Leukemia cells were collected from human embryonic stem cell line, acute promyelocytic leukemia cell line and bone marrow of patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 25~30 cells from each group or each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA), cluster analysis and partial least squares discrimination analysis (PLS-DA), and the spectra of three kinds of cells were analyzed and classified. Characteristics of Raman spectra were discussed combined with ultrastructure of leukemia cells. There were significant differences among Raman spectra of three kinds of leukemia-related cells. Compared with ES cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks which contributed to nucleic acids, proteins and lipids. Its biological mechanism involved the close relationship between APL and the PI3K/Akt/mTOR pathway. The PI3K/Akt/mTOR pathway was abnormally activated in acute promyelocytic leukemia cells, affecting the biomacromolecular metabolism of leukemia cells. The diagnostic models established by PCA-DFA, cluster analysis and PLS-DA could successfully classify these Raman spectra of different cells with a high accuracy of 100% (181/181). The model was evaluated by “Leave-one-out” cross-validation and reached a high accuracy of 98.9% (179/181). The proliferation and metabolism of M3 cells and NB4 cells were higher than those of ES cells. The diagnostic models established by PCA-DFA, cluster analysis and PLS-DA can classify these Raman spectra of different cells with a high accuracy. Raman spectra show consistent result with ultrastructure by TEM.