EB1089对年轻初诊系统性红斑狼疮患者骨髓间充质干细胞增殖的影响及其机制

Effects of active Vitamin D analogue EB1089 on proliferation of MSCs in young patients with preliminarily systemic lupus erythematosus

目的 观察活性维生素D类似物EB1089对年轻的初诊系统性红斑狼疮(SLE)患者骨髓间充质干细胞(MSC)增殖的影响,并探讨其机制。方法 收集10例年轻的初诊SLE患者及5例年轻体检健康者的骨髓标本,分离MSC并分别记为A、B组。观察两组MSC的生长速度及细胞形态,记录原代细胞(P0代)、第二代细胞(P1代)培养至融合传代所需时间。取对数生长期的同代A、B组MSC接种于96孔板,分别记为A1、A2、A3、A4、B1、B2、B3、B4组,采用MTT法检测加入不同浓度(0、1、10、100pmol/L)EB1089后各组MSC光密度值,以光密度值表示MSC增殖能力;采用流式细胞术检测加入不同浓度EB1089后各组MSC内活性氧(ROS)荧光强度,以荧光强度表示MSC内ROS水平。结果 两组MSC在形态上相似。A组P0代细胞培养至融合传代需(24.9±0.73)d,B组需(14.2±0.66)d,两组相比,P<0.05;A组P1代细胞培养至融合传代需(14.2±1.48)d,B组需(7.0±0.70)d,两组相比,P<0.05。A1、A2、A3、A4组细胞光密度值分别为0.258±0.004、0.322±0.009、0.372±0.004、0.438±0.007,B1、B2、B3、B4组细胞光密度值分别为0.448±0.007、0.462±0.022、0.473±0.024、0.484±0.012;A1、A2、A3组与B1、B2、B3组相比,P均<0.05;A1、A2、A3、A4各浓度组内两两相比,P均<0.05。A1、A2、A3、A4组ROS荧光强度分别为192.15±10.78、149.69±9.18、139.46±10.05、103.98±11.77,B1、B2、B3、B4组ROS荧光强度分别为75.76±8.34、75.30±7.95、74.61±9.81、74.26±8.46;A1、A2、A3、A4组与B1、B2、B3、B4组相比,P均<0.05;A1、A2、A3、A4各浓度组内两两相比,P均<0.05。结论 年轻的初诊SLE患者骨髓MSC增殖能力降低、传代周期延长;EB1089能够促进SLE患者骨髓MSC增殖,且促进增殖的能力存在浓度依赖性;EB1089促进SLE患者骨髓MSC增殖的机制与ROS水平降低有关。

Objective To investigate the effects of active Vitamin D analogue EB1089 on the proliferation of mesenchymal stem cells (MSCs) in young patients with preliminarily systemic lupus erythematosus (SLE) and to explore the mechanism. Methods Ten cases of bone marrow specimens from SLE patients and 5 healthy bone marrow specimens were collected. The MSCs were isolated and recorded as the groups A and B. The morphology, phenotype of bone marrow MSCs were observed, and the time required for the primary cells (P0 generation) and the second generation cells (P1 generation) to be cultured for fusion passage was recorded. The MSCs of the same generation A and B in the logarithmic growth phase were inoculated into 96-well plates, which were recorded as groups A1, A2, A3, A4, B1, B2, B3, and B4. MTT assay was used to detect the optical density values of MSC with different concentrations (0, 1, 10, and 100 pmol/L) of EB1089 in each group;flow cytometry was used to detect the fluorescence intensity of ROS in each group after adding different concentrations of EB1089, and we expressed the ROS level in MSC by fluorescence intensity. Results MSCs of the two groups were similar in morphology. In the group A, we needed (24.9±0.73) d to culture P0 cells to fusion passage, group B (14.2±0.66) d, with statistically significant difference ( P <0.05);in the group A, we needed (14.2±1.48) d to culture P1 cells to fusion passage, group B required (7.0 ± 0.70) d, with statistically significant difference ( P < 0.05 ). The optical density values of cells in the groups A1, A2, A3 and A4 were 0.258±0.004, 0.322±0.009, 0.372±0.004, and 0.438±0.007, respectively. The optical density values of groups B1, B2, B3, and B4 were 0.448± 0.007 , 0.462±0.022, 0.473±0.024, and 0.484±0.012, respectively;significant differences were found between the groups A1, A2, A3 and groups B1, B2, B3, and between every two groups of groups A1, A2, A3, and A4 (all P < 0.05 ). The ROS fluorescence intensities of groups A1, A2, A3, and A4 were 192.15±10.78, 149.69±9.18, 139.46±10.05, and 103.98±11.77, respectively;the ROS fluorescence intensities in the groups B1, B2, B3, and B4 were 75.76±8.34, 75.30±7.95, 74.61±9.81, and 74.26±8.46, respectively;significant differences were found between the groups A1, A2, A3 and groups B1, B2, B3, and between every two groups of groups A1, A2, A3, and A4 (all P <0.05). Conclusions In young patients with newly diagnosed SLE, the proliferation of bone marrow MSCs decreases and the passage time is prolonged. EB1089 can promote the proliferation of bone marrow MSCs in SLE patients with a concentration-dependent manner. The mechanism of EB1089 promoting bone marrow MSC proliferation in SLE patients is related to the decrease of ROS levels.