| 摘要: | 研究目的
特發性嚴重再生不良性貧血（idiopathic severe aplastic anemia; SAA）雖然罕見，但對病童卻有著生命的威脅；其發病的原因主要是喪失骨髓造血的功能，但直至今日真正的致病機轉卻還未能瞭解透徹。骨髓間質幹細胞（mesenchymal stem cells; MSCs）在骨髓造血微環境中扮演舉足輕重的角色，可提供造血幹細胞（hematopoietic stem cells）生長分化成熟之所需。本研究的目的為探討嚴重再生不良性貧血病童之骨髓間質幹細胞基本特質的缺陷。
研究方法
分別自五位嚴重再生不良性貧血的病童及五位對照組兒童取得骨髓抽出液，再將間質幹細胞分離並作體外培養。藉由觀察骨髓間質幹細胞之細胞型態（morphology）、測定其免疫表面標誌的表現（immunophenotyping）、分析其增生能力（proliferative capacity）和分化潛能（differentiation potential），比較兩組之間是否有差異。
研究結果
雖然嚴重再生不良性貧血病童及對照組兒童兩組之骨髓間質幹細胞體外培養的細胞型態均呈現相似之紡錘狀，且免疫表面標誌的表現均為CD45、CD14、CD34陰性和CD105、CD73、CD44陽性；但增生能力的實驗發現，嚴重再生不良性貧血病童之骨髓間質幹細胞的增生速度較慢且累積群體倍增殖（cumulative population doubling）較對照組小，分別為1.83 ± 1.21 與 3.36 ± 0.87（p = 0.046），這意味著嚴重再生不良性貧血病童的骨髓間質幹細胞有著較低的增生能力。經過骨分化誘導（osteogenic induction）之後，嚴重再生不良性貧血病童之骨髓間質幹細胞表現較弱的鹼性磷酸塩酶活性（alkaline phosphatase activity；1.46 ± 0.04 與 2.27 ± 0.32；p = 0.013）、呈現較淡的von Kossa染色和較低的core binding factor-alpha 1基因表現（0.0015 ± 0.0005 與 0.0056 ± 0.0017；p = 0.013）。經過脂肪分化誘導（adipogenic induction）之後，嚴重再生不良性貧血病童之骨髓間質幹細胞呈現較淡染的Oil red O染色（0.86 ± 0.22 與 1.73 ± 0.42；p = 0.013）和較低的lipoproteinlipase基因表現（0.0105 ± 0.0074 與0.0527 ± 0.0254；p = 0.013）。 以real time-PCR分析骨分化與脂肪分化之特定基因表現的結果和特殊染色的發現一致，兩者都代表著嚴重再生不良性貧血病童之骨髓間質幹細胞骨分化與脂肪分化的潛能較差。
研究結論
本研究中，我們首次成功地顯示：嚴重再生不良性貧血病童之骨髓間質幹細胞的增生與分化潛能較差。而骨髓間質幹細胞的改變或許和骨髓造血能力的喪失有著密切的相關，這需要後續的研究進一步來闡明骨髓間質幹細胞與嚴重再生不良性貧血兩者之間的相關性。
Background
Idiopathic severe aplastic anemia (SAA), characterized by failure of hematopoiesis, is rare and potentially life-threatening to children. However, the pathogenesis has not been completely understood, and insufficiency in the hematopoietic microenvironment can be an important factor. Mesenchymal stem cells (MSCs) play an important role in maintaining bone marrow microenvironment. Therefore, we aimed at the intrinsic defects of bone marrow MSCs derived from SAA children.
Materials and Methods
Bone marrow MSCs were obtained from 5 SAA children and 5 controls. The morphology, immunophenotyping, proliferative capacity and differentiation potential of MSCs from SAA children were determined and compared with those of MSCs from controls.
Results
MSCs of SAA and control group shared a similar spindle-shaped morphology in vitro. Both revealed a consistent immunophenotypic profile which was negative for CD45, CD14 and CD34, and positive for CD105, CD73, and CD44. However, SAA MSCs had slower expansion rate and smaller cumulative population doubling from passage 4 to 6 (1.83 ± 1.21 vs 3.36 ± 0.87; p = 0.046), indicating lower proliferative capacity. Besides, only 3 of 5 cultures of SAA group retained the ability to continue expansion till 80%-90% confluent cell layer beyond passage 6, suggesting earlier senescence of SAA MSCs. After osteogenic induction, SAA MSCs showed lower alkaline phosphatase activity (1.46 ± 0.04 vs 2.27 ± 0.32; p = 0.013), less intense von Kossa staining and lower gene expression of core binding factor-alpha 1 (0.0015 ± 0.0005 vs 0.0056 ± 0.0017; p = 0.013). Following adipogenic induction, SAA MSCs showed less intense Oil red O staining (0.86 ± 0.22 vs 1.73 ± 0.42; p = 0.013) and lower lipoproteinlipase expression (0.0105 ± 0.0074 vs 0.0527 ± 0.0254; p = 0.013). The results of real time-PCR analysis for the assessment of lineage-specific genes were consistent with the findings of histochemical stains, and both indicated that SAA MSCs had poor osteogenic and adipogenic potential.
Conclusions
In this study, we demonstrated that bone marrow MSCs from children with SAA had poor potential of proliferation and differentiation. These alterations in MSCs may contribute to the failure of hematopoiesis, and lead to the development of the disease. Further studies are needed to elucidate the relationship between MSCs and SAA. |
