多发性骨髓瘤患者外周血和骨髓液微泡的流式检测及其临床意义
发布时间：2019-01-14 13:07
【摘要】：目的:通过检测骨髓瘤患者不同阶段特异性微泡(Microvesicles,MVs)的数量及比例变化情况,掌握不同疾病状态MVs的变化规律,为骨髓瘤诊断及预后判断寻找新的监测指标。方法:1收集初治、缓解、复发多发性骨髓瘤患者及性别年龄匹配的健康志愿者外周血、骨髓液标本。2差异离心法提纯分离外周血及骨髓液中的MVs标本。3透射电镜观察MVs的形态和大小。4流式细胞术检测MVs的数量及特异性MVs的比例,分析临床病人不同阶段浆细胞来源MVs(CD38+CD138+)、内皮细胞来源MVs(CD105+CD45-)及血小板来源MVs(CD41+CD105-)的构成比。BECKMAN流式细胞仪可以清晰分辨1.0um、3.0um标准微球,最低检测下限为200nm,应用1.0um标准微球设定流式细胞仪FSC和SSC参数,利用3.0um标准微球作为计数内参。我们用Calcein-AM染液标记具有完整膜结构的MVs,并设定直径小于1.0um为MVs。5统计分析MVs数量及特异性MVs的比例在健康志愿者、缓解多发性骨髓瘤患者、初治多发性骨髓瘤患者及复发多发性骨髓瘤患者之间的差异,分析评估特异性MVs比例与多发性骨髓瘤临床特征的相关性。结果:1经透射电镜观察多发性骨髓瘤患者外周血血清来源MVs形态结构,多发性骨髓瘤细胞来源的MVs在透射电镜下观察均为直径0.1-1um大小不一、均质的双层磷脂膜包裹的囊泡状结构,这与目前为止国内外所报道的其他细胞分泌的MVs在形态、大小上相一致。2应用BECKMAN流式细胞仪可以比较准确计数标本中MVs的数量。FACS结果显示,多发性骨髓瘤患者外周血及骨髓液中MVs数量明显高于健康对照组(P0.05),复发患者与初治患者相比MVs数量更多(P0.05),且初治患者MVs数量比高于缓解患者(P0.05)。骨髓上清液MVs浓度明显高于外周血上清液MVs浓度,差异有统计学意义(P0.05)。3用CD38和CD138两个表面标记多发性骨髓瘤细胞中浆细胞来源的MVs,多发性骨髓瘤患者外周血和骨髓液MVs中CD38+CD138+-MVs比例明显高于健康对照组(P0.05),复发患者CD38+CD138+-MVs比例明显高于初治患者(P0.05),且初治患者CD38+CD138+-MVs比例明显高于缓解患者(P0.05)。CD38+CD138+-MVs比例与临床上常用提示肿瘤负荷及预后的指标β2微球蛋白、LDH呈正相关(骨髓液β2微球蛋白:r=0.601,P=0.005)(骨髓液LDH:r=0.720,P=0.000)(外周血β2微球蛋白:r=0.729,P=0.029)(外周血LDH:r=0.621,P=0.018),提示可用于检测肿瘤预后。CD38+CD138+-MVs比例与MM分型、分期之间暂无相关性。4用CD105+CD45-来标记内皮细胞分泌的MVs,用CD41+CD105-来标记血小板分泌的MVs,多发性骨髓瘤患者外周血和骨髓液MVs中内皮细胞来源的MVs比例(CD105+CD45-)及血小板来源MVs(CD41+CD105-)比例均明显高于健康对照(P0.05),复发患者内皮细胞来源MVs比例及血小板来源MVs比例均明显高于初治患者(P0.05),且初治患者内皮细胞来源MVs比例及血小板来源MVs比例均明显高于缓解患者(P0.05),暂未统计到内皮细胞来源的MVs(CD105+CD45-)比例及血小板来源的MVs(CD41+CD105-)比例与肿瘤负荷指标β2微球蛋白、LDH、MM分型及分期之间的相关性。结论:1多发性骨髓瘤患者外周血及骨髓液MVs的数量及特异性MVs的比例与多发性骨髓瘤患者疾病状态密切相关。2多发性骨髓瘤患者外周血及骨髓液内皮细胞来源MVs及血小板来源MVs数量及比例升高,提示内皮细胞和血小板的活化对疾病的发生与进展可能有影响,是骨髓瘤患者血栓高发的潜在可能原因。
[Abstract]:Objective: To study the variation of the number and proportion of microvesicles (MVs) in different stages of the patients with myeloma and to master the change of MVs in different disease states, and to find new monitoring index for the diagnosis and prognosis of the myeloma. Methods: 1 The peripheral blood of healthy volunteers with primary treatment, remission, recurrence of multiple myeloma and gender age were collected. The morphology and size of MVs were measured by flow cytometry. The number of MVs and the proportion of specific MVs were measured by flow cytometry, and the plasma cell sources MVs (CD38 + CD138 +) in different stages of clinical patients were analyzed. The composition ratio of the endothelial cell-derived MVs (CD105 + CD45-) and the platelet-derived MVs (CD41 + CD105-). The BECKMAN flow cytometer can clearly distinguish the 1. 0um, 3.0um standard microball, the lowest detection limit is 200nm, and the FSC and SSC parameters of the flow cytometer are set by using the 1. 0um standard microball, and the 3. 0um standard microball is used as the counting internal reference. We used the Calcein-AM dye solution to mark the MVs with intact membrane structure and set the MVs with a diameter of less than 1. 0um. The number of MVs and the specific MVs in the number of MVs and the specific MVs were measured in healthy volunteers, and the difference between the patients with multiple myeloma and the patients with recurrent multiple myeloma was relieved. The correlation between the specific MVs ratio and the clinical characteristics of multiple myeloma was analyzed. Results: 1. The morphological structure of the blood serum from the peripheral blood of the patients with multiple myeloma was observed by transmission electron microscope. The MVs of the multiple myeloma cell source were observed under the transmission electron microscope. This is consistent with the number of MVs secreted by other cells reported at home and abroad, and the number of MVs in the sample can be accurately counted by using the BECKMAN flow cytometer. The results showed that the number of MVs in peripheral blood and bone marrow was significantly higher in patients with multiple myeloma (P0.05). The MVs concentration of the supernatant in the bone marrow was significantly higher than that of the supernatant in the peripheral blood (P0.05). The MVs of the plasma cells in the multiple myeloma cells were marked with two surface markers of CD38 and CD138. The proportion of CD38 + CD138 +-MVs in peripheral blood and bone marrow fluid MVs in patients with multiple myeloma was significantly higher than that in healthy control group (P0.05). The proportion of CD38 + CD138 +-MVs in patients with multiple myeloma was significantly higher than that in the patients with primary treatment (P0.05). The proportion of CD38 + CD138 +-MVs in the primary treatment group was significantly higher than that of the patients with remission (P0.05). The proportion of CD38 + CD138 +-MVs was significantly higher than that of the index (2 microglobulin, LDH: r = 0.601, P = 0. 005) (bone marrow fluid LDH: r = 0.720, P = 0.000) (bone marrow fluid LDH: r = 0.729, P = 0.000) (bone marrow fluid LDH: r = 0.720, P = 0.000) (bone marrow fluid LDH: r = 0.729, P = 0.000). P = 0.029 (peripheral blood LDH: r = 0.621, P = 0.018), suggesting that it can be used to detect the prognosis of the tumor. CD38 + CD138 +-MVs ratio and MM type, no correlation between stages. The ratio of MVs (CD105 + CD45-) and platelet-derived MVs (CD41 + CD105-) in the peripheral blood and bone marrow fluid MVs of multiple myeloma patients was significantly higher than that in the healthy control group (P0.05). The proportion of MVs (CD105 + CD45-) and the ratio of MVs (CD41 + CD105-) to the source of the endothelial cells and the proportion of MVs (CD41 + CD105-) of the platelet derived from the endothelial cells were significantly higher than those of the patients with remission (P <0.05), and the proportion of MVs (CD41 + CD105-) and the number of the platelet-derived MVs (CD41 + CD105-) in the endothelial cells were not statistically higher than that of the patients with the remission (P <0.05). The correlation between the types and stages of MM. Conclusion: The number of MVs and MVs in peripheral blood and bone marrow fluid in patients with multiple myeloma are closely related to the condition of multiple myeloma. The number and proportion of MVs and MVs in peripheral blood and bone marrow fluid in patients with multiple myeloma are increased. It is suggested that the activation of endothelial cells and platelets may have an effect on the occurrence and progression of the disease, which is the potential cause of the high risk of the patients with myeloma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3
本文编号：2408723
[Abstract]:Objective: To study the variation of the number and proportion of microvesicles (MVs) in different stages of the patients with myeloma and to master the change of MVs in different disease states, and to find new monitoring index for the diagnosis and prognosis of the myeloma. Methods: 1 The peripheral blood of healthy volunteers with primary treatment, remission, recurrence of multiple myeloma and gender age were collected. The morphology and size of MVs were measured by flow cytometry. The number of MVs and the proportion of specific MVs were measured by flow cytometry, and the plasma cell sources MVs (CD38 + CD138 +) in different stages of clinical patients were analyzed. The composition ratio of the endothelial cell-derived MVs (CD105 + CD45-) and the platelet-derived MVs (CD41 + CD105-). The BECKMAN flow cytometer can clearly distinguish the 1. 0um, 3.0um standard microball, the lowest detection limit is 200nm, and the FSC and SSC parameters of the flow cytometer are set by using the 1. 0um standard microball, and the 3. 0um standard microball is used as the counting internal reference. We used the Calcein-AM dye solution to mark the MVs with intact membrane structure and set the MVs with a diameter of less than 1. 0um. The number of MVs and the specific MVs in the number of MVs and the specific MVs were measured in healthy volunteers, and the difference between the patients with multiple myeloma and the patients with recurrent multiple myeloma was relieved. The correlation between the specific MVs ratio and the clinical characteristics of multiple myeloma was analyzed. Results: 1. The morphological structure of the blood serum from the peripheral blood of the patients with multiple myeloma was observed by transmission electron microscope. The MVs of the multiple myeloma cell source were observed under the transmission electron microscope. This is consistent with the number of MVs secreted by other cells reported at home and abroad, and the number of MVs in the sample can be accurately counted by using the BECKMAN flow cytometer. The results showed that the number of MVs in peripheral blood and bone marrow was significantly higher in patients with multiple myeloma (P0.05). The MVs concentration of the supernatant in the bone marrow was significantly higher than that of the supernatant in the peripheral blood (P0.05). The MVs of the plasma cells in the multiple myeloma cells were marked with two surface markers of CD38 and CD138. The proportion of CD38 + CD138 +-MVs in peripheral blood and bone marrow fluid MVs in patients with multiple myeloma was significantly higher than that in healthy control group (P0.05). The proportion of CD38 + CD138 +-MVs in patients with multiple myeloma was significantly higher than that in the patients with primary treatment (P0.05). The proportion of CD38 + CD138 +-MVs in the primary treatment group was significantly higher than that of the patients with remission (P0.05). The proportion of CD38 + CD138 +-MVs was significantly higher than that of the index (2 microglobulin, LDH: r = 0.601, P = 0. 005) (bone marrow fluid LDH: r = 0.720, P = 0.000) (bone marrow fluid LDH: r = 0.729, P = 0.000) (bone marrow fluid LDH: r = 0.720, P = 0.000) (bone marrow fluid LDH: r = 0.729, P = 0.000). P = 0.029 (peripheral blood LDH: r = 0.621, P = 0.018), suggesting that it can be used to detect the prognosis of the tumor. CD38 + CD138 +-MVs ratio and MM type, no correlation between stages. The ratio of MVs (CD105 + CD45-) and platelet-derived MVs (CD41 + CD105-) in the peripheral blood and bone marrow fluid MVs of multiple myeloma patients was significantly higher than that in the healthy control group (P0.05). The proportion of MVs (CD105 + CD45-) and the ratio of MVs (CD41 + CD105-) to the source of the endothelial cells and the proportion of MVs (CD41 + CD105-) of the platelet derived from the endothelial cells were significantly higher than those of the patients with remission (P <0.05), and the proportion of MVs (CD41 + CD105-) and the number of the platelet-derived MVs (CD41 + CD105-) in the endothelial cells were not statistically higher than that of the patients with the remission (P <0.05). The correlation between the types and stages of MM. Conclusion: The number of MVs and MVs in peripheral blood and bone marrow fluid in patients with multiple myeloma are closely related to the condition of multiple myeloma. The number and proportion of MVs and MVs in peripheral blood and bone marrow fluid in patients with multiple myeloma are increased. It is suggested that the activation of endothelial cells and platelets may have an effect on the occurrence and progression of the disease, which is the potential cause of the high risk of the patients with myeloma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3
【参考文献】
相关期刊论文 前1条
1 江红;黄玲莎;;多发性骨髓瘤实验室诊断与进展[J];中华实用诊断与治疗杂志;2010年11期
，本文编号：2408723
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