間質幹細胞於嚴重再生不良性貧血與敗血症所扮演的角色

兒童嚴重再生不良性貧血的致病機轉仍未完全明瞭，而間質幹細胞有缺陷致使骨髓造血微環境改變與功能失常是可能的原因之一；此次的研究比較五位嚴重再生不良性貧血病童與五位對照病童的骨髓間質幹細胞，並試圖進一步找出嚴重再生不良性貧血病童的間質幹細胞是否有不正常的基因表現。我們發現雖然體外培養時兩者的細胞形態相似，並表現一致的細胞表面標記，但嚴重再生不良性貧血病童之骨髓間質幹細胞體外增生的潛能較差、有提早老化的現象、骨頭與脂肪分化的潛能也較差。微陣列分析顯示嚴重再生不良性貧血病童之骨髓間質幹細胞大致有兩種不同的基因表現模式，而DNA polymerase epsilon 2 (POLE2)、hepatocyte growth factor (HGF)、kinesin family member 20A (KIF20A)、thymidine kinase 1 (TK1)、interleukin 18 receptor 1 (IL18R1)、KIT ligand (KITLG)、fibroblast growth factor 18 (FGF18)、ribonucleotide reductase M2 polypeptide (RRM2)、TTK protein kinase (TTK)、CXC motif ligand 12 (CXCL12)、discs large homolog 7 (DLG7)、DNA topoisomerase II alpha (TOP2A)、NDC80 kinetochore complex component of S. cerevisiae homolog (NUF2)、thymidylate synthetase (TYMS) 這14個與DNA合成、細胞激素或生長因子相關的基因表現較低；當將對照組骨髓間質幹細胞CXCL12、HGF、IL-18R1、FGF18或RRM2的基因表現抑制後，其表現會與嚴重再生不良性貧血病童之骨髓間質幹細胞類似，包括：對抗細胞毒殺的耐受能力、脂肪分化的潛能，尤其當CXCL12表現受抑制後這些改變最為明顯；且進一步將CXCL12基因重新置入受抑制之對照組骨髓間質幹細胞後其表現可明顯地恢復。我們以上的研究證實：兒童嚴重再生不良性貧血之骨髓間質幹細胞確實有缺失，而CXCL12基因的表現減少可能造成骨髓微環境的改變，這可能是嚴重再生不良性貧血發生的原因。敗血症仍是目前重要的死因，而敗血症時厲害的免疫反應雖有助於清除病菌，但也可能造成對自身組織的傷害；間質幹細胞有很強的免疫調節能力，而給予敗血症動物間質幹細胞是有益的。此次研究以盲腸結紮穿刺手術造成大鼠產生敗血症，並給予人類骨髓間質幹細胞或臍帶間質幹細胞，藉由分析週邊血單核細胞的組成與測量血清中感染發炎相關細胞激素的濃度來探討間質幹細胞對敗血症動物的免疫調節機轉。結果顯示：與只給予PBS的對照組大鼠相較，接受盲腸結紮穿刺手術後有給予間質幹細胞的大鼠其週邊血中調節T細胞的比例增加、調節T細胞與T細胞的比值升高、血清白細胞介素-6與腫瘤壞死因子-α的濃度降低，且可使調節T細胞的免疫抑制功能恢復。另外我們發現：臍帶間質幹細胞與骨髓間質幹細胞體外培養都呈現紡錘狀的細胞形態，也都表現一致的細胞表面標記，但臍帶間質幹細胞有較強的骨頭分化潛能但較弱的脂肪分化能力，而對於敗血症動物的功效兩者之間沒有明顯的差異。我們的研究證實，給予敗血症大鼠間質幹細胞可以改變調節T細胞在週邊血液的數量和功能，並提供動物敗血症時調節T細胞與間質幹細胞兩者免疫調節功能相關的有力證據。

The mechanisms of idiopathic severe aplastic anemia (SAA) in children are not completely understood. Insufficiency of the bone marrow microenvironment, in which mesenchymal stem cells (MSCs) are an important element, can be a potential factor associated with hematopoietic impairment. Here we compared the properties of MSCs from 5 SAA children with those of MSCs from 5 controls, and studied whether aberrant gene expression could be found in SAA MSCs. In vitro, MSCs from SAA children and controls were similar in morphology and surface marker expression. However, SAA MSCs had lower proliferative capacity and differentiation potential. Further, two different patterns of global gene expression were detected in the SAA MSCs by microarray analysis. Fourteen genes related to DNA synthesis, cytokines or growth factors were significantly down-regulated, including DNA polymerase epsilon 2 (POLE2), hepatocyte growth factor (HGF), kinesin family member 20A (KIF20A), thymidine kinase 1 (TK1), interleukin 18 receptor 1 (IL18R1), KIT ligand (KITLG), fibroblast growth factor 18 (FGF18), ribonucleotide reductase M2 polypeptide (RRM2), TTK protein kinase (TTK), CXC motif ligand 12 (CXCL12), discs large homolog 7 (DLG7), DNA topoisomerase II alpha (TOP2A), NDC80 kinetochore complex component of S. cerevisiae homolog (NUF2), and thymidylate synthetase (TYMS). Using the shRNA-containing lentivirus mentod, knockdown of CXCL12, HGF, IL-18R1, FGF18 or RRM2 expression compelled MSCs from the controls to behave like those from the SAA children, with decreased survival and differentiation potential. Among them, inhibition of CXCL12 gene expression had the most profound effects on the behavior of MSCs. Further experiments regarding re-introduction of the CXCL12 gene could largely recover the survival and differentiation potential in MSCs with inhibition of CXCL12 expression. These findings provided evidence that defects in bone marrow MSCs of SAA children do exist, and down-regulation of CXCL12 gene may be associated with alterations in the bone marrow microenvironment.Sepsis remains an important cause of death, and vigorous immune responses during sepsis could be beneficial for bacterial clearance but at the price of collateral damage to self tissues. MSCs have been found to modulate the immune system and attenuate sepsis. Here MSCs derived from bone marrow and umbilical cord were used and compared. The mechanisms of MSC-mediated immunoregulation during sepsis were studied by determining circulating inflammation-associated cytokine profiles and peripheral blood mononuclear cells 18 hours after cecal ligation and puncture (CLP). Bone marrow-derived MSCs (BMMSCs) and umbilical cord-derived MSCs (UCMSCs) showed similar in vitro morphology and surface marker expression, but UCMSCs had stronger potential for osteogenesis but lower for adipogenesis. Compared with rats receiving PBS only after CLP, the percentage of circulating regulatory T (Treg) cells and the ratio of Treg cells/T cells were elevated significantly in rats receiving MSCs. Further experiment regarding Treg cell function demonstrated that the immunosuppressive capacity of Treg cells from rats with CLP-induced sepsis was decreased, but could be restored by administration of MSCs. Compared with rats receiving PBS only after CLP, serum levels of interleukin-6 and tumor necrosis factor-α were significantly lower in rats receiving MSCs after CLP. This work provides the first in vivo evidence for the immunomodulatory association of Treg cells and MSCs during sepsis.