As_2O_3对U266细胞DKK-1表达、hFOB1.19细胞分化的影响及MM患者DKK-1水平与临床关系的研究
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摘要
[目的]
探讨三氧化二砷(Arsenic tri oxide,As203)对多发性骨髓瘤(Multiple myeloma,MM)细胞表达DKK-1的影响,及通过Wnt信号通路对成骨细胞分化的影响。探讨131例多发性骨髓瘤患者临床特点与预后的关系,及分析39例患者血清DKK-1水平与临床特点的关系。
[方法]
以0-125umol/L的As203处理U266、hFOB1.19细胞株1-3天,MTT法检测对该两种细胞的增殖抑制作用；RT-PCR检测U266细胞中DKK-1mRNA及hFOB1.19细胞中β-catenin mRNA表达水平,采用ELISA检测U266细胞培养上清液中DKK-1蛋白浓度,Western blot检测hFOB1.19细胞中β-catenin蛋白表达,采用细胞碱性磷酸酶(Alkaline phosphatase, ALP)染色及检测细胞ALP相对活性评估hFOB1.19细胞中ALP的表达。临床研究方面,回顾了我院131例多发性骨髓瘤患者的临床资料,并采用ELISA方法检测39例患者血清DKK-1蛋白水平。
[结果]
1.As203在1-125μmol/L浓度范围对U266细胞均有不同程度的增殖抑制作用,并随着剂量的增加、作用时间的延长,抑制率逐渐增加。不同浓度As203作用hFOB 1.19细胞后,在0.1-1umol/L浓度范围内,细胞增殖抑制不明显,且在作用24、48h后有促进增殖的作用。浓度大于10umol/L以上后,表现为不同程度的细胞增殖抑制。
2. DKK1mRNA在U266细胞中表达。加入不同浓度As203后,DKK1 mRNA的表达水平与As203浓度呈负相关,As203浓度在0.1-2μmol/l时,DKK-1蛋白水平抑制不明显。
3.As203在1-2umol/L时,hFOB1.19成骨细胞ALP表达增加。β-catenin mRNA的表达水平与As203浓度呈正相关。Western blot结果显示As203浓度范围在1.0-2.0μmol/L时,hFOB1.19细胞中β-catenin表达较对照组明显增加。
4.分析131例MM患者的各临床特点,年龄、骨髓中浆细胞比例、Hb、白蛋白、β2-MG、ISS分期、DS分期与生存时间显著相关。并证实MM患者血清DKK-1水平较对照组明显升高,且骨髓浆细胞比例与DKK-1水平高低相关。
[结论]
1.As203在体外对多发性骨髓瘤细胞系U266具有不同程度的增殖抑制作用,且在临床治疗浓度范围内(0.1-1umol/L),对成骨细胞hFOB 1.19促进增殖的作用。
2.骨髓瘤U266细胞中表达DKK-1,As203在基因水平抑制DKK-1表达,但是在蛋白水平抑制不明显。
3.As203能够促进hFOB1.19细胞中β-catenin、ALP的表达。
4.MM患者血清DKK-1蛋白水平较对照组明显升高,且骨髓浆细胞比例与DKK-1水平高低相关。
[Objective] We investigated the effect of the DKK-1 expression in U266 cells by arsenic trioxide and the differenation of hFOB1.19 cells through the Wnt singal pathway. In clinical researches, we retrospectively studied the relationship between various clinical features and the prognosis of 131 patients with multiple myeloma, as well as the DKK-1 levels of 39 patients.
[Methods] MTT method was adopted to observe the inhibition effect that arsenic trioxide had on the proliferation of U266 and hFOB1.19 cells at the dose level of 1-125μmol/L for 1-3 days. Both the DKK-1 mRNA levels in U266 cells and theβ-catenin mRNA levels in hFOB1.19 cells were detected by RT-PCR. ELISA was used to detect the expression of DKK-1 protein, while the expression ofβ-catenin was measured by Western blotting. The alkaline phosphatase expression was assessed by alkaline phosphatase staining and the evaluation of relative activity of alkaline phosphatase in hFOB1.19 cells. In clinical researches, we retrospectively analyzed 131 patients with multiple myeloma,of whom 39 patients'DKK-1 levels were detected.
[Results]
1. MTT assay showed that U266 cells proliferation was inhibited in different degrees by As2O3 with the concentration from 1 to 125umol/L. The inhitory rate increased along with the arising concentration and duration of As2O3. hFOB 1.19 cells were cultured with different concentrations of As2O3. The proliferation of cells was not significantly inhibited within 24 hours when the concentration was from 0.1 to lumol/L, but was promoted after 24 hours or 48h. However, when the concentration of As2O3 raised to lOumol/L, the proliferation of hFOB 1.19 cells was inhibited in different degrees.
2. The DKK-1 mRNA could expressed in U266 cells. DKK-1 mRNA level was negatively correlated with the concentration of As2O3. The expression of DKK-1 protein in the cell culture could not be inhibited by the treatment of 1-2umol/L As2O3.
3. The expression of ALP in hFOB1.19 cells increased after the treatment of 1-2umol/L As2O3. Moreover, the expression ofβ-catenin mRNA was positively correlated with the concentration of As2O3.
4. After analyzing 131 clinical cases, we found that some factors, including age, the proportion of bone marrow plasma cells, Hb, albumin,β2-MG, ISS stage and DS stage, were significantly correlated with the survival time. Meanwhile, it was confirmed that the serum DKK-1 level, which was much higher than the control group, was closely related to the ratio of plasma cells in bone marrow.
[Conclusion]
1. In vitro, As2O3 could inhibit the proliferation of multiple myeloma cell line (U266) and could promote the proliferation of osteoblast hFOB 1.19 within the clinical serum concentration range (0.1-1umol/L).
2. DKK-1 protein could express in U266 cells. As2O3 can inhibited the expression of DKK-1 mRNA, but not the expression of protein.
3. Since As2O3 could promote the expression ofβ-catenin and ALP protein in osteoblast hFOB 1.19 cells, it can be concluded that the Wnt signaling pathway might, at least in part, play a role in the treatment of As2O3.
4. In studies performed on the clinical casese, we showed that the serum DKK-1 level, which was significantly higher than the control group, was closely related to the level of DKK-1.
探讨三氧化二砷(Arsenic tri oxide,As203)对多发性骨髓瘤(Multiple myeloma,MM)细胞表达DKK-1的影响,及通过Wnt信号通路对成骨细胞分化的影响。探讨131例多发性骨髓瘤患者临床特点与预后的关系,及分析39例患者血清DKK-1水平与临床特点的关系。
[方法]
以0-125umol/L的As203处理U266、hFOB1.19细胞株1-3天,MTT法检测对该两种细胞的增殖抑制作用；RT-PCR检测U266细胞中DKK-1mRNA及hFOB1.19细胞中β-catenin mRNA表达水平,采用ELISA检测U266细胞培养上清液中DKK-1蛋白浓度,Western blot检测hFOB1.19细胞中β-catenin蛋白表达,采用细胞碱性磷酸酶(Alkaline phosphatase, ALP)染色及检测细胞ALP相对活性评估hFOB1.19细胞中ALP的表达。临床研究方面,回顾了我院131例多发性骨髓瘤患者的临床资料,并采用ELISA方法检测39例患者血清DKK-1蛋白水平。
[结果]
1.As203在1-125μmol/L浓度范围对U266细胞均有不同程度的增殖抑制作用,并随着剂量的增加、作用时间的延长,抑制率逐渐增加。不同浓度As203作用hFOB 1.19细胞后,在0.1-1umol/L浓度范围内,细胞增殖抑制不明显,且在作用24、48h后有促进增殖的作用。浓度大于10umol/L以上后,表现为不同程度的细胞增殖抑制。
2. DKK1mRNA在U266细胞中表达。加入不同浓度As203后,DKK1 mRNA的表达水平与As203浓度呈负相关,As203浓度在0.1-2μmol/l时,DKK-1蛋白水平抑制不明显。
3.As203在1-2umol/L时,hFOB1.19成骨细胞ALP表达增加。β-catenin mRNA的表达水平与As203浓度呈正相关。Western blot结果显示As203浓度范围在1.0-2.0μmol/L时,hFOB1.19细胞中β-catenin表达较对照组明显增加。
4.分析131例MM患者的各临床特点,年龄、骨髓中浆细胞比例、Hb、白蛋白、β2-MG、ISS分期、DS分期与生存时间显著相关。并证实MM患者血清DKK-1水平较对照组明显升高,且骨髓浆细胞比例与DKK-1水平高低相关。
[结论]
1.As203在体外对多发性骨髓瘤细胞系U266具有不同程度的增殖抑制作用,且在临床治疗浓度范围内(0.1-1umol/L),对成骨细胞hFOB 1.19促进增殖的作用。
2.骨髓瘤U266细胞中表达DKK-1,As203在基因水平抑制DKK-1表达,但是在蛋白水平抑制不明显。
3.As203能够促进hFOB1.19细胞中β-catenin、ALP的表达。
4.MM患者血清DKK-1蛋白水平较对照组明显升高,且骨髓浆细胞比例与DKK-1水平高低相关。
[Objective] We investigated the effect of the DKK-1 expression in U266 cells by arsenic trioxide and the differenation of hFOB1.19 cells through the Wnt singal pathway. In clinical researches, we retrospectively studied the relationship between various clinical features and the prognosis of 131 patients with multiple myeloma, as well as the DKK-1 levels of 39 patients.
[Methods] MTT method was adopted to observe the inhibition effect that arsenic trioxide had on the proliferation of U266 and hFOB1.19 cells at the dose level of 1-125μmol/L for 1-3 days. Both the DKK-1 mRNA levels in U266 cells and theβ-catenin mRNA levels in hFOB1.19 cells were detected by RT-PCR. ELISA was used to detect the expression of DKK-1 protein, while the expression ofβ-catenin was measured by Western blotting. The alkaline phosphatase expression was assessed by alkaline phosphatase staining and the evaluation of relative activity of alkaline phosphatase in hFOB1.19 cells. In clinical researches, we retrospectively analyzed 131 patients with multiple myeloma,of whom 39 patients'DKK-1 levels were detected.
[Results]
1. MTT assay showed that U266 cells proliferation was inhibited in different degrees by As2O3 with the concentration from 1 to 125umol/L. The inhitory rate increased along with the arising concentration and duration of As2O3. hFOB 1.19 cells were cultured with different concentrations of As2O3. The proliferation of cells was not significantly inhibited within 24 hours when the concentration was from 0.1 to lumol/L, but was promoted after 24 hours or 48h. However, when the concentration of As2O3 raised to lOumol/L, the proliferation of hFOB 1.19 cells was inhibited in different degrees.
2. The DKK-1 mRNA could expressed in U266 cells. DKK-1 mRNA level was negatively correlated with the concentration of As2O3. The expression of DKK-1 protein in the cell culture could not be inhibited by the treatment of 1-2umol/L As2O3.
3. The expression of ALP in hFOB1.19 cells increased after the treatment of 1-2umol/L As2O3. Moreover, the expression ofβ-catenin mRNA was positively correlated with the concentration of As2O3.
4. After analyzing 131 clinical cases, we found that some factors, including age, the proportion of bone marrow plasma cells, Hb, albumin,β2-MG, ISS stage and DS stage, were significantly correlated with the survival time. Meanwhile, it was confirmed that the serum DKK-1 level, which was much higher than the control group, was closely related to the ratio of plasma cells in bone marrow.
[Conclusion]
1. In vitro, As2O3 could inhibit the proliferation of multiple myeloma cell line (U266) and could promote the proliferation of osteoblast hFOB 1.19 within the clinical serum concentration range (0.1-1umol/L).
2. DKK-1 protein could express in U266 cells. As2O3 can inhibited the expression of DKK-1 mRNA, but not the expression of protein.
3. Since As2O3 could promote the expression ofβ-catenin and ALP protein in osteoblast hFOB 1.19 cells, it can be concluded that the Wnt signaling pathway might, at least in part, play a role in the treatment of As2O3.
4. In studies performed on the clinical casese, we showed that the serum DKK-1 level, which was significantly higher than the control group, was closely related to the level of DKK-1.
引文
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