多發(fā)性骨髓瘤患者外周血和骨髓液微泡的流式檢測(cè)及其臨床意義
發(fā)布時(shí)間：2019-01-14 13:07
【摘要】：目的:通過(guò)檢測(cè)骨髓瘤患者不同階段特異性微泡(Microvesicles,MVs)的數(shù)量及比例變化情況,掌握不同疾病狀態(tài)MVs的變化規(guī)律,為骨髓瘤診斷及預(yù)后判斷尋找新的監(jiān)測(cè)指標(biāo)。方法:1收集初治、緩解、復(fù)發(fā)多發(fā)性骨髓瘤患者及性別年齡匹配的健康志愿者外周血、骨髓液標(biāo)本。2差異離心法提純分離外周血及骨髓液中的MVs標(biāo)本。3透射電鏡觀察MVs的形態(tài)和大小。4流式細(xì)胞術(shù)檢測(cè)MVs的數(shù)量及特異性MVs的比例,分析臨床病人不同階段漿細(xì)胞來(lái)源MVs(CD38+CD138+)、內(nèi)皮細(xì)胞來(lái)源MVs(CD105+CD45-)及血小板來(lái)源MVs(CD41+CD105-)的構(gòu)成比。BECKMAN流式細(xì)胞儀可以清晰分辨1.0um、3.0um標(biāo)準(zhǔn)微球,最低檢測(cè)下限為200nm,應(yīng)用1.0um標(biāo)準(zhǔn)微球設(shè)定流式細(xì)胞儀FSC和SSC參數(shù),利用3.0um標(biāo)準(zhǔn)微球作為計(jì)數(shù)內(nèi)參。我們用Calcein-AM染液標(biāo)記具有完整膜結(jié)構(gòu)的MVs,并設(shè)定直徑小于1.0um為MVs。5統(tǒng)計(jì)分析MVs數(shù)量及特異性MVs的比例在健康志愿者、緩解多發(fā)性骨髓瘤患者、初治多發(fā)性骨髓瘤患者及復(fù)發(fā)多發(fā)性骨髓瘤患者之間的差異,分析評(píng)估特異性MVs比例與多發(fā)性骨髓瘤臨床特征的相關(guān)性。結(jié)果:1經(jīng)透射電鏡觀察多發(fā)性骨髓瘤患者外周血血清來(lái)源MVs形態(tài)結(jié)構(gòu),多發(fā)性骨髓瘤細(xì)胞來(lái)源的MVs在透射電鏡下觀察均為直徑0.1-1um大小不一、均質(zhì)的雙層磷脂膜包裹的囊泡狀結(jié)構(gòu),這與目前為止國(guó)內(nèi)外所報(bào)道的其他細(xì)胞分泌的MVs在形態(tài)、大小上相一致。2應(yīng)用BECKMAN流式細(xì)胞儀可以比較準(zhǔn)確計(jì)數(shù)標(biāo)本中MVs的數(shù)量。FACS結(jié)果顯示,多發(fā)性骨髓瘤患者外周血及骨髓液中MVs數(shù)量明顯高于健康對(duì)照組(P0.05),復(fù)發(fā)患者與初治患者相比MVs數(shù)量更多(P0.05),且初治患者M(jìn)Vs數(shù)量比高于緩解患者(P0.05)。骨髓上清液MVs濃度明顯高于外周血上清液MVs濃度,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3用CD38和CD138兩個(gè)表面標(biāo)記多發(fā)性骨髓瘤細(xì)胞中漿細(xì)胞來(lái)源的MVs,多發(fā)性骨髓瘤患者外周血和骨髓液MVs中CD38+CD138+-MVs比例明顯高于健康對(duì)照組(P0.05),復(fù)發(fā)患者CD38+CD138+-MVs比例明顯高于初治患者(P0.05),且初治患者CD38+CD138+-MVs比例明顯高于緩解患者(P0.05)。CD38+CD138+-MVs比例與臨床上常用提示腫瘤負(fù)荷及預(yù)后的指標(biāo)β2微球蛋白、LDH呈正相關(guān)(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),提示可用于檢測(cè)腫瘤預(yù)后。CD38+CD138+-MVs比例與MM分型、分期之間暫無(wú)相關(guān)性。4用CD105+CD45-來(lái)標(biāo)記內(nèi)皮細(xì)胞分泌的MVs,用CD41+CD105-來(lái)標(biāo)記血小板分泌的MVs,多發(fā)性骨髓瘤患者外周血和骨髓液MVs中內(nèi)皮細(xì)胞來(lái)源的MVs比例(CD105+CD45-)及血小板來(lái)源MVs(CD41+CD105-)比例均明顯高于健康對(duì)照(P0.05),復(fù)發(fā)患者內(nèi)皮細(xì)胞來(lái)源MVs比例及血小板來(lái)源MVs比例均明顯高于初治患者(P0.05),且初治患者內(nèi)皮細(xì)胞來(lái)源MVs比例及血小板來(lái)源MVs比例均明顯高于緩解患者(P0.05),暫未統(tǒng)計(jì)到內(nèi)皮細(xì)胞來(lái)源的MVs(CD105+CD45-)比例及血小板來(lái)源的MVs(CD41+CD105-)比例與腫瘤負(fù)荷指標(biāo)β2微球蛋白、LDH、MM分型及分期之間的相關(guān)性。結(jié)論:1多發(fā)性骨髓瘤患者外周血及骨髓液MVs的數(shù)量及特異性MVs的比例與多發(fā)性骨髓瘤患者疾病狀態(tài)密切相關(guān)。2多發(fā)性骨髓瘤患者外周血及骨髓液內(nèi)皮細(xì)胞來(lái)源MVs及血小板來(lái)源MVs數(shù)量及比例升高,提示內(nèi)皮細(xì)胞和血小板的活化對(duì)疾病的發(fā)生與進(jìn)展可能有影響,是骨髓瘤患者血栓高發(fā)的潛在可能原因。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.3
本文編號(hào)：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 江紅;黃玲莎;;多發(fā)性骨髓瘤實(shí)驗(yàn)室診斷與進(jìn)展[J];中華實(shí)用診斷與治療雜志;2010年11期
，本文編號(hào)：2408723
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