多發性骨髓瘤病患的骨髓間質幹細胞老化之臨床意義與分子機轉

Abstract

骨髓間質幹細胞在多發性骨髓瘤的致病機轉與對治療的反應性都有重要角色，相較於健康人的骨髓間質幹細胞，骨髓瘤病患的骨髓間質幹細胞不論在基因表現、細胞激素表達、蛋白質製造甚至細胞型態、生長速度都有明顯差異。在體外培養下，骨髓瘤骨髓間質幹細胞生長速度比健康人的骨髓間質幹細胞顯著地慢上許多。雖然有代謝活性，但即使處於適當環境細胞也不再分裂，這樣的生理/病理現象稱作老化。骨髓瘤骨髓間質幹細胞較正常族群老化的現象已非新聞，但其中的病理機轉並未經過廣泛研究，對治療、預後的影響也尚未明朗。我們假設，骨髓間質幹細胞的老化可能在骨髓瘤的致病、治療扮演重要的角色並嘗試瞭解其中奧秘。我們蒐集健康骨髓捐贈者及骨髓瘤患者的骨髓間質幹細胞，在體外培養的環境下，比較兩個族群骨髓間質幹細胞老化的程度、訊息傳遞因子表現多寡，並針對老化相關基因進行DNA微陣列分析，同時比較同一檢體、不同分裂代數的間質幹細胞在特定基因表現有無差異，希望藉此找出骨髓瘤細胞造成骨髓間質幹細胞老化的機制；此外，我們也對骨髓間質幹細胞施加特定蛋白(lipocalin-2, prolactin)，觀察這些分子是否能延緩或反轉老化。我們發現，骨髓瘤病患的骨髓間質幹細胞不論老化的表現型或基因型都與健康細胞明顯不同。藉由DNA微陣列分析搭配基因網絡分析軟體(Ingenuity Pathway Analysis)，我們高度懷疑骨髓瘤骨髓間質幹細胞的老化與轉化生長因子-β(Transforming growth factor beta，TGF-β)訊息傳遞有關。此外，SERPINB2基因的表現量隨著細胞分裂次數增加而顯著增加。一般常見於老化分析的基因，如CDKN1A (p21)與CDKN2A (p16)表現則較不顯著。最後，本實驗尚無法重現lipocalin-2, prolactin分子降低老化表現的結果，這或許與細胞暴露藥物的時間不足有關。本研究發現TGF-β訊息傳遞與SERPINB2基因在骨髓瘤骨髓間質幹細胞的老化可能扮演重要角色。未來將針對這些標的進行基因轉殖、剔除等實驗，除了嘗試反轉骨髓瘤骨髓間質幹細胞的老化，也期待改變骨髓間質幹細胞能成為未來多發性骨髓瘤的治療標的之一。

Bone marrow mesenchymal stem cells (BMMSCs) play an important role in pathogenesis and even drug resistance of multiple myeloma (MM). There are several distinctions in BMMSCs between myeloma patients (MM-BMMSCs) and healthy donors (HD-BMMSCs), such as proliferation capacity, ability of osteoblastogenesis, gene expressions, and secretory cytokine profiles (secretome). Cellular senescence is defined as a state of irreversible cell cycle arrest even with adequate supply of growth factors. To date, cellular senescence of MM-BMMSCs has been widely noted while comparing to HD-BMMSCs. However, its molecular mechanism(s) or clinical significance (e.g. impact on treatment outcome) are so far largely unclear. We propose that the senescence and subsequent malfunction of MM-BMMSCs may contribute to the pathogenic abnormalities and lead to different prognosis of MM patients. To answer these questions, we collected 42 MM-BMMSCs and 16 HD-BMMSCs. We compared genotype by using senescence-specific cDNA PCR array between these two groups, as well as phenotype of senescence, expression levels of certain genes in different passages in vitro, levels of lysophosphatidic acid (LPA) and its receptors 1 & 3 (LPAR 1/3). Furthermore, we also treated MM- and HD-BMMSCs with lipocalin-2 and prolactin to see whether reversal of senescence possible. Our preliminary findings indicated that the genotype and phenotype of senescence were quite different between these two groups. By cDNA PCR array and Ingenuity Pathway Analysis (IPA) analysis, we highly suspected that the senescence of MM-BMMSCs might be regulated through TGF-β signaling. We also found expressions of SERPINB2 genes were highly correlated with the senescence of BMMSCs (and there was a trend for higher expression of SERPINB2 in MM-BMMSCs than in HD-BMMSCs). However, these changes were not seen in other senescence related genes, such as CDKN1A and CDKN2A. Finally, the impact of lipocalin-2 and prolactin on senescence were not evident in this study, which might be due to relatively short duration of exposure. Our results point out the role of TGF-β signaling and SERPINB2 gene in the molecular mechanism of senescence in MM-BMMSCs. In the future, we will perform further manipulations (e.g. knock-in or knock-out) to valid these findings and we hope to clarify molecular mechanism of senescence in MM-BMMSC and reverse the senescence if possible will shed light on the novel treatment paradigm in MM.