摘要
背景与目的：尿毒症高转化骨病与尿毒症状态下成骨细胞数量及功能的异常增加有密切关系,其确切机制目前尚未明确。骨髓间充质干细胞(BM-MSC)增殖并向成骨细胞分化是骨形成的细胞生物学基础,但目前尚无尿毒症高转化骨病大鼠BM-MSC体外培养及增殖、分化的报道。我们最近首次发现尿毒症高转化骨病大鼠骨髓基质细胞向成骨细胞分化的能力增加。本研究通过体外培养、鉴定尿毒症高转化骨病大鼠BM-MSC,观察其增殖、分化行为特征及相关的信号转导机制改变。
方法：取180-200g Sprague-Dawley雄性大鼠,制做5/6肾切除并高磷饮食(磷1.2%,钙1.0%)喂养的尿毒症高转化骨病组模型大鼠。以正常磷饮食(0.9%,钙1.0%)喂养的相同体重雄性大鼠作为对照组。12周后处死大鼠,取股骨和胫骨骨干,利用骨片贴壁分离筛选法原代培养BM-MSC.取第3代细胞,进行流式细胞仪鉴定BM-MSC分子标志(CD90+/CD34-/CD44+/CD45-)。采用MTT法观察尿毒症高转化骨病组大鼠与对照组大鼠BM-MSC细胞增殖行为改变,并应用流式细胞仪法检测BM-MSC中DNA含量,分析两组细胞之间细胞周期的差异。免疫组化及免疫印迹法(western-blot)检测两组细胞之间增殖相关信号通路分子表达的差异。在含有磷酸甘油及地塞米松的成骨培养基中,诱导BM-MSC分化为成骨细胞,采用免疫组化法,分析比较尿毒症高转化骨病组与对照组BM-MSC分化的成骨细胞表达碱性磷酸酶(ALP)、骨桥蛋白(OPN)的差异,采用von Kossa染色观察两组细胞内外磷酸钙沉积及骨结节形成数目(HE染色)等成骨细胞特征性标志物的差异。
结果：(1)利用骨片贴壁分离筛选法可成功分离、培养尿毒症高转化骨病组大鼠及对照组大鼠的原代BM-MSC。取第3代培养细胞,经流式细胞术鉴定为CD90+/CD34-/CD44+/CD45-细胞,符合BM-MSC的分子标志特征。(2)流式细胞术对DNA的分析发现,尿毒症高转化骨病组大鼠BM-MSC中G1期、S期、G2/M期、细胞比例分别为79.55±3.66%、20.12±1.96%、0.33±0.10%,对照组分别为84.62±3.10%、15.13±0.87%、0.25±0.11%,尿毒症高转化骨病组S期细胞比例明显增加(P<0.05),此结果与MTT法的细胞增殖分析结果一致。(3)免疫组化结果显示,培养的BM-MSC中,ERK1/2信号通路明显激活,表现为p-ERK1/2与总ERK1/2的比值显著增加(P<0.05)。Western-blot分析显示,对照组BM-MSC细胞ERK1/2信号通路的激活从72h开始,并在其后的24h内维持于较高水平；而尿毒症高转化骨病组细胞ERK1/2信号通路激活从48h即已开始,并在其后的48h内一直维持于较高水平；在48h-96h内的各时间点,尿毒症高转化骨病组ERK1/2信号通路的激活水平均高于对照组(P<0.05)。流式细胞术分析发现,使用ERK1/2信号通路阻断剂后,两组细胞增殖能力均显著下降,尿毒症高转化骨病组S期细胞比例的下降程度较对照组更为显著(P<0.05),此结果与MTT法分析结果一致。(4)免疫组化法显示,大鼠BM-MSC于体外向成骨细胞诱导分化后1周,细胞碱性磷酸酶(ALP)表达明显增强；分化后2周,骨桥蛋白(OPN)表达明显增强(P<0.05),von Kossa染色显示,细胞内外基质磷酸钙沉积较对照组显著增多(P<0.05)；分化后3周,骨结节形成数目有明显增加(P<0.05)。
结论：尿毒症高转化骨病大鼠BM-MSC可在体外培养条件下存活,其增殖、分化能力较正常大鼠BM-MSC明显增强,ERK1/2信号通路的激活在其中发挥了重要的促进作用,这对临床防治尿毒症高转化骨病有重要意义。
Background and objectives:The mechanism of high-turnover renal osteodystrophy (ROD) has not been fully clear to date. The differentiation from bone marrow mesenchymal stem cells (BM-MSC) to osteoblast is the biological foundation of bone transformation, which was enhanced in high-turnover ROD uremic rat. In the present study, we cultured primarily BM-MSC from high-turnover ROD uremic rat in vitro and and identified the characteristics and the signaling pathways involved in its proliferation and differentiation.
Method:High-turnover ROD uremic models were made with male Sprague-Dawley rats (body weight180-200g) by5/6nephrectomy fed a high phosphate diet (1.2%phosphorus and1.0%calcium). Male body-weight-matched rats were fed with normal phosphorus diet (0.9%phosphorus and1.0%calcium) as control group. After12weeks feeding, all rats were executed and BM-MSC was isolated from femur and tibia bone and cultured primarily by adherence method. The3rd passage cells were identified for BM-MSC molecular markers (CD90+/CD34-/CD44+/CD45-) by flow cytometry. The cell viability was investigated by MTT assay, and the proliferation level was measured by propidium iodide DNA staining by flow cytometry. The signaling pathway involved was detected by immunohistochemistry and western blot. Then BM-MSC was induced to differentiate to osteoblast in culture medium containing glycerin and dexamethasone phosphate. The differences in the level of cell proliferation, the degree of ERK1/2protein phosphorylation, and the expression of specific osteoblast markers alkaline phosphatase (ALP), osteopontin (OPN), the calcium phosphate deposits (von Kossa staining) and the number of bone nodule (HE staining) were investigated between high-turnover ROD uremic group and the control group.
Results:(1) BM-MSC from the high-turnover ROD rats was separated and cultured by adherence method. The3rd passage cells were identified as CD90+/CD34-/CD44+/CD45-cells by flow cytometry. Flow cytometry analysis of DNA showed the proportion of G1phase, S phase and G2/M phase cells in the BM-MSC from uremic group was79.55±3.66%,20.12±1.96%and0.33±0.10%, respectively, while84.62±3.10%,15.13±0.87%and0.25±0.11%in the BM-MSC from control group, respectively. The proportion of S phase cells was significantly higher in uremic group than that in control group (P＜0.05), which were consistent with the results of MTT assays.(2) ERK1/2signaling pathway of the cultured BM-MSC was activated obviously, characterized by p-ERK1/2increased significantly (p＜0.05). Western blot showed that the ERK1/2pathway of control group was activated at the72h point and was maintained behind for24h, whereas the activation in uremic group began at the48h point and was maintained behind for as long as48h. The p-ERK1/2level in uremic group was higher than that in control group at all time points from48h to96h (P＜0.05). Flow cytometry and MTT assay showed that BM-MSC proliferation in both groups decreased significantly after adding of ERK1/2blockers, and the proportion of S phase cells decreased more significantly in uremic group than in control group (P＜0.05).(3) Compared with BM-MSC from control group, BM-MSC from uremic group expressed ALP more significantly1week after differentiation to osteoblast, similarly, for OPN (P＜0.05) and calcium phosphate deposits (P＜0.05) after2weeks, and for the formation of bone nodule after3weeks (P＜0.05).
Conclusions:BM-MSC from uremic rat with high-turnover ROD survives in vitro, with an enhanced proliferation and differentiation ability in which the activation of ERK1/2might involed. This study might be valuable for preventing and treating uremia with high-turnover ROD.
引文
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