利用幹細胞達成唾液腺再生

Application of Stem Cells for Salivary Glands Regeneration

林佳詠(Chia-Yung Lin)

指導教授 ： 陳敏慧

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.01208

摘要

背景: 目前臨床上仍無法徹底解決因頭頸部癌症之放射治療或自體免疫疾病如Sjögren's syndrome等所導致的唾液腺功能低下問題，使唾液分泌減少而造成嚴重齲齒、牙周病、吞嚥消化困難將終身影響病人生活的舒適度。近年許多研究發現骨髓幹細胞有能力分化成不同胚層的許多細胞，因此本研究之假說乃骨髓幹細胞具潛力轉分化成類唾液腺細胞並提供一個細胞療法之大量細胞來源以幫助受損的唾液腺再生。
本研究目的在於 (1) 探討骨髓幹細胞轉分化為唾液腺細胞之能力 (2) 探討利用骨髓幹細胞達成唾液腺再生的效用 (3) 觀察植入小鼠唾液腺內之骨髓幹細胞長期分布之情形 (4) 探討骨髓幹細胞轉分化為唾液腺細胞之整體基因變化。
方法: 首先從體外細胞培養驗證骨髓幹細胞轉分化為唾液腺細胞之能力。從4周大的大鼠之大腿骨取其骨髓幹細胞並將之與頷下腺唾液腺細胞進行間接與直接共同培養。間接共同培養利用雙層培養皿以薄膜將兩種細胞區隔。骨髓幹細胞在底層培養皿而唾液腺細胞在上層培養皿。唾腺細胞所釋放的化學訊號可經由培養基透過薄膜上0.4μm孔洞傳遞。直接共同培養是利用傳統DiI螢光染劑及新型螢光結合之奈米粒子事先標定出骨髓幹細胞，再與唾液腺細胞進行共同培養。藉此可在其後實驗中直接觀察骨髓幹細胞並利用免疫螢光染色來檢驗其變化。結果顯示經由直接與間接共同培養都使骨髓幹細胞轉分化成類似唾液腺細胞一般具有特殊分泌澱粉酶蛋白的表現。這些轉分化後的骨髓幹細胞以下稱為類唾液腺細胞。其次，利用動物實驗在生物體內檢驗骨髓幹細胞與類唾液腺細胞的治療效果與達成唾液腺再生的效用。利用脂質包膜超小極性氧化鐵奈米粒子先標定骨髓幹細胞並經由直接共同培養以得到標定過的類唾液腺細胞。骨髓幹細胞與類唾腺細胞都利用動物實驗來檢視其治療效果。總共140隻免疫功能受限的小鼠分成四組: (1)正常唾液腺功能 (2) 利用放射線照射其頭頸部破壞唾腺功能 (3)將標定過的骨髓幹細胞植入放射線後功能被破壞後的唾腺 (4)將標定過的類唾腺細胞植入放射線後功能被破壞後的唾腺。在其後固定的時間再分別對四組小鼠做巨觀及微觀的唾液腺功能測量。此外，利用多光子顯微鏡合併特殊設計的活體觀測裝置，觀察取自螢光小鼠之骨髓幹細胞植入唾液內之分布情形，並輔佐驗證骨髓幹細胞在體內轉分化為類唾腺細胞之能力。最後，利用基因微陣列檢測經過間接共同培養後之骨髓幹細胞，確認其整體基因變化朝向唾液腺細胞分化之方向。
結果: 經由間接/直接共同培養所得骨髓幹細胞，利用免疫螢光染色及RT-PCR檢視，可發現其表現出唾液腺細胞特殊的澱粉酶蛋白質及基因。直接測試其澱粉酶功能，也的確具有將澱粉實際水解成單醣之功能。在動物實驗方面，結果顯示，植入骨髓幹細胞及類唾腺細胞都可使因放射線萎縮的唾腺重量回復、並且和整體健康相關的全身體重亦重新增加、使原本受損降低的唾液分泌量重新回升，而且發現類唾腺細胞在促進唾液分泌方面明顯骨髓幹細胞之效果好而且具有統計意義上之差別。此外，組織H&E染色亦證實骨髓幹細胞在唾液腺內可直接轉分化成類唾液腺細胞。另一方面，利用特殊裝置可以達成活體觀測小鼠之唾液腺內狀況，而且其後的唾液腺組織切片不需經過染色，即可見標定過的骨髓幹細胞細胞存活於組織中並結合到唾液腺泡中。更進一步，由基因微陣列晶片所分析之結果，經過間接共同培養後之骨髓幹細胞，原本應該往硬骨/軟骨/脂肪分化的基因表現都降低，而唾液腺細胞之基因例如水離子通道基因、鈉鉀離子通道基因之表現都大幅提高，因此可推定原本骨髓幹細胞朝間葉細胞分化之方向被改變成往類上皮細胞唾液腺細胞分化。
結論: 本研究首度證實原本的假說，不論在體外或體內，利用共同培養，由中胚層而來的骨髓幹細胞都有能力轉分化為類唾腺細胞，並且可促進唾液腺再生。唾腺細胞原本則是從外胚層所起源。由骨髓幹細胞轉分化而來的類唾腺細胞從基因到蛋白質的層級都表現出唾液腺細胞特殊的澱粉酶之功能表現。本研究亦由基因微陣列晶片的大量分析結果探測骨髓幹細胞分化成類唾液腺細胞之基因變化。另一方面，從動物實驗的結果，在接受鈷60照射使唾腺受損後，給予細胞療法可發現類唾腺細胞表現出比骨髓幹細胞更優異的細胞治療效果，可幫助唾液腺的組織再生使受損的腺體回復重量、功能恢復並讓減低的唾液量重新恢復、更進一步使得消化咀嚼功能變好而讓全身體重恢復近正常標準。由統計更顯示了利用類唾液腺細胞比起骨髓幹細胞將有更直接快速的治療效果。關於小鼠唾液腺之型態，本研究亦是第一次利用多光子顯微鏡做到活體觀察，追蹤植入幹細胞之分布。

關鍵字

骨髓幹細胞 ； 類唾液腺細胞 ； 共同培養 ； 細胞轉分化 ； 奈米粒子 ； 多光子雷射螢光顯微鏡 ； 基因微陣列晶片 ； 唾液腺功能低下 ； 唾液腺再生

並列摘要

Background: There are still no effective solutions for atrophic salivary glands caused by autoimmune disease or radiation therapies for cancers. Hyposalivation results in severe dental problems reducing patients’ long-term life quality. Recent reports demonstrated that bone marrow stem cells have a great potential for transdifferentiating into different cells. We hypothesized that bone marrow stem cells can be transdifferentiated into salivary acinar cells for glands regeneration.
The purposes of this study were as follows: (1) to investigate the potentials of bone marrow stem cells transdifferentiating into salivary acinar cells (2) to investigate the effects of cell therapy for glands regeneration (3) to observe long term distributions of bone marrow stem cells transplanted into salivary glands (4) to discover the whole genome change of bone marrow stem cell transdifferentiated into salivary acinar cells.
Methods: bone marrow stem cells were acquired from 4-week-old rats and cocultured with submandibular glands acinar cells. Indirect coculture was preceded by culture inserts to separate these 2 kinds of cells. Bone marrow stem cells were on lower wells and acinar cells were on upper inserts; media could help signal compunctions through 0.4μm insert-membrane pores. Direct coculture was preceded by labeling bone marrow stem cells with DiI or Rhodamine-conjugated nanoparticles for cell-tracking. Immunocytochemical stains were performed to examine the changes after coculture. Next, mouse bone marrow stem cells were labeled with cationic lipid-coated ultra small superparamagnetic iron oxide nanoparticles and direct cocultured with acinar cells to obtain labeled acinar-like cells; both bone marrow stem cells and acinar-like cells were examined their therapeutic effects in the animal study. Total 140 severe combined immune deficiency mice were divided into 4 groups: (1) normal salivary glands mice, (2) mice received irradiation around their head-and-neck areas to cause atrophic glands (3) mice received irradiation and intra-gland transplantation with labeled bone marrow stem cells or (4) labeled acinar-like cells. Evaluations of glands functions were performed in all groups after cell therapy. Besides, distributions of bone marrow stem cells transplanted into glands were explored by specific designed equipments for in-vivo observation with the multiphoton laser fluorescence microscopy. The observation also assisted in examining transdifferentiation of bone marrow stem cells. Finally, microarray analyses were performed to examine the whole genome change of transdifferentiated bone marrow stem cells.
Results: After coculture, bone marrow stem cells were transdifferentiated into acinar-like cells with expressions of α-amylase protein, gene, and starch- digestion function. The animal study showed that damaged salivary glands with reduction in saliva production, body weight loss and gland weight loss due to irradiation can be rescued by cell therapy. For recovery of saliva production, cell therapy with acinar-like cells was significantly better than those with bone marrow stem cells. Transdifferentiation into acinar-like cells in-vivo was also noted by immunocytochemical staining. Besides, we designed specific equipments for in-vivo observation. By multiphoton laser fluorescence microscopy, distributions of transplanted cells could be observed directly. We also found that gene expressions of bone marrow stem cells related with mesenchymal characteristics were down regulated after coculture. In contrast, genes related with acinar cells such as water channels, sodium/potassium ions channels were unregulated.
Conclusion: This study is the first report demonstrating that bone marrow stem cells originated from the mesoderm are capable of transdifferentiating into acinar-like cells in-vitro and in-vivo. Acinar-like cells derived from bone marrow stem cells showed similarities from gene level to true function as salivary acinar cells originated from the ectoderm. Microarray analyses also confirmed this transdifferentiation. From animal study, acinar-like cells have better therapeutic effects in salivary glands regeneration. This study was also the first report for in-vivo observation of mice salivary glands with cell tracking of transplanted bone marrow stem cells.

並列關鍵字

bone marrow stem cells ； acinar-like cells ； coculture ； cell transdifferentiation ； nanoparticles ； multiphoton laser fluorescence microscopy ； microarray analysis ； hyposalivation ； salivary glands regeneration

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