- 學位論文
人類臍帶幹細胞在短期低氧預處理條件下的研究及IL-6/IL-6R 免疫調控探討
Research on Human Umbilical Cord Stem Cells Under Short-term Hypoxic Pretreatment Conditions and Discussion on Immunoregulation of IL-6/IL-6R in the Stem Cells
摘要
人類臍帶幹細胞 (hUCMSCs) 是細胞治療的重要細胞，本研究目標為探討 “短時間”在低氧 (< 5%) 與常氧濃度 (21%) 下培養的臍帶幹細胞的細胞增殖及免疫的抑制效果的比較，並對化學性低氧培養的可行性進行分析， 最後針對臍帶幹細胞中對免疫影響多元性的細胞因子IL-6 進行探討。研究內容分成以下三部分： 第一部分: 人類臍帶幹細胞在短時間缺氧預處理的免疫功能 隨著疾病的嚴重度，對hUCMSCs的需求量倍增。如何快速增殖以及維持有效的細胞功能是本部份研究的目的。文獻顯示，在低氧環境下培養hUCMSCs對幹細胞的增殖是有助益的。但是，低氧的細胞培養時間與低氧氧氣濃度的培養環境，尚未達到最佳化。且在 “短時間” 的低氧環境對hUCMSCs 影響著墨幾乎為零。本部分研究主要在短時間低氧對hUCMSCs的生長與免疫調控進行分析與探討。為了評估預處理的有效性，進行了低短時間低氧預處理與體外常氧培養下hUCMSCs的增殖和免疫功能比較。另外，測量 hUCMSCs 的外泌體對周邊血單核細胞 (PBMC)、 抑制試驗以及 hUCMSCs/PBMCs 共培養下的細胞因子/趨化因子，以探究 hUCMSCs 的免疫抑制作用。為書寫便利，內文研究中將短時間低氧預處理縮寫為 (HPT)，常氧下處理縮寫為 (N)。研究結果得知，HPT-hUCMSCs的有效預處理方式，可以提供快速、高產量的細胞增殖能力和刺激產生細胞因子的分泌達到有效的免疫抑制功能。此外，無論在低氧還是正常氧濃度下hUCMSCs 的產物外泌體，對PBMCs 都有抑制的效果，但是需要的數量及作用時間點遠超過hUCMSCs 與 PBMCs 的直接細胞接觸作用 (cell to cell contact)。 第二部分: 物理性低氧及化學性低氧對幹細胞培養的差異性探討 上一部分研究證實，低氧的環境有助於hUCMSCs的增殖與免疫功能增強，然而物理性低氧的設備繁雜，實驗室需要擴充的儀器設備與經費高昂，並非所有實驗室都可以為此建置。本研究的目的是證明 CoCl2 在誘導 hUCMSCs 缺氧方面的可行性及其局限性。將不同濃度的 CoCl2 添加到培養基中，記錄不同時間的氧含量。並用不同濃度的CoCl2刺激 hUCMSCs，進行細胞生長計數 及HIF-1α的測定。 最後，將 hUCMSCs 與 PBMCs 共培養。 觀察 hUCMSCs 的免疫調節作用。 在化學和物理缺氧的氧濃度相等的情況下，兩種環境下表現出相似的 HIF-1α 蛋白表達和免疫調節作用相似。 由於 CoCl2 價格便宜，操作方便，它證實了在缺氧條件下培養 hUCMSCs 的可行性。 第三部分: 臍帶幹細胞中IL-6 在發炎與抑制發炎機轉分析探討 根據本研究的第一部分，研究結果顯示，HPT-hUCMSCs 相較於N-hUCMSCs可以分泌較高的細胞因子，結果呈現顯著差異。其中IL-6 的增加相較於其他細胞因子尤為顯著，更以ng/ml呈現，而其他的因子則在pg/ml範圍。因此，第三部分研究，深入探討了 IL-6 在人臍帶幹細胞在治療中的作用。在hUCMSCs 與PBMCs 共培養作用下， 對IL-6/IL-6R 的影響進行各項分析。細胞數的計數、 IL-6的分泌濃度計量、IL-6/IL-6R mRNA與蛋白質的分析、IL-6 中和作用、及IL-6R 破壞等作用分析得出以下結論: IL-6主要的分泌是由hUCMSCs 產生， IL-6R 主要位於PBMCs 上，而發炎的抑制作用是經由hUCMSCs與PBMCs 作用的時間點與分泌高濃度的IL-6 (1000 ng)，方能達到發炎的抑制效果; 若是對hUCMSCs 的IL-6 分泌中和，或是對PBMCs 膜上的IL-6R 進行破壞，hUCMSCs 對PBMCs 的抗發炎效果則不會發生。換言之，hUCMSCs 的細胞治療需經由分泌高劑量的IL-6與PBMCs 的IL-6R接合來進行發炎反應的抑制效果，不同於 TCZ 的拮抗發炎效果。 IL-6 與 IL-6R 在抗發炎反應中都有存在的必要性。
並列摘要
Part I. An effective short-term hypoxic pretreatment for human umbilical stem cells Human umbilical cord MSCs (hUCMSCs) have been widely used in research and clinical applications. For further therapeutic application, rapid proliferation and improvement of the suppressive functions of hUCMSCs are required. Therefore, in this study, an optimized culture condition and time of hUCMSCs were investigated, and preconditioned hUCMSCs were applied to colitis mice to study their therapeutic potential. To evaluate the efficient preconditioning, the proliferation and immune function of hUCMSCs in the early stage under < 5% O2 hypoxia pretreatment were assessed and compared with those of hUCMSCs cultured under normoxia in vitro. Peripheral blood mononuclear cell (PBMC) suppression assays of exosomes and hUCMSCs and the cytokine/chemokine levels of hUCMSC/PBMC cocultures were measured to estimate the immunosuppressive effect of hUCMSCs. For simplicity, the pretreatment of short time under <5% O2 hypoxia is abbreviated as (HPT) and that under normoxia as (N) for the rest of this paper. The results demonstrated that under <5% O2 hypoxic treatment, the proliferation, exosome secretion, and immune functions of hUCMSCs were already effectively responded to at higher levels than those cultured under normoxia in the first few hours. Part II. CoCl2 induces hUCMSC chemical hypoxia and its immunomodulation The objective of this study was to demonstrate the feasibility of CoCl2 in inducing hypoxia in human hUCMSCs and its limitations. Different concentrations of CoCl2 were added to the culture medium. The oxygen content at different intervals was recorded. hUCMSCs were stimulated with different cobalt chloride concentrations. The MTT assay, western blotting (e.g. HIF-1α) were performed at different intervals. Finally, hUCMSCs were co-cultured with PBMCs. The immune-regulatory effects of hUCMSCs were observed. In equal oxygen concentrations between chemical and physical hypoxia, the cellular hypoxia factor and immuno-regulatory effects were similar under the two environments. hUCMSCs induced by CoCl2 chemical hypoxia demonstrated similar HIF-1α protein expression and coculture results compared with those obtained under physical hypoxia. As CoCl2 is cheap and convenient, it demonstrated feasibility for the culture of hUCMSCs under hypoxic conditions. Part III. Immunoregulation of IL-6/IL-6R in human umbilical Stem Cells IL-6-related therapies were used to treat autoimmune diseases, the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy was explored in this research. Peripheral blood mononuclear cells (PBMCs) were cocultured with hUCMSCs or exogenous IL-6 and subjected to PBMC suppression, IL-6 expression, and IL-6/IL-6R localization assays. The correlation between the anti-inflammatory effects of hUCMSCs and IL-6, IL-6/IL-6R mRNA, and protein expression was analyzed. The main secretion of IL-6 is produced by hUCMSC, IL-6R is mainly located on PBMC, and the inhibitory effect of inflammation is through the time point of the interaction of hUCMSC and PBMC and the secretion of high concentration of IL-6 (1000 ng/mL), to achieve the inhibitory effect of inflammation. The neutralizes the IL-6 secretion of hUCMSC or IL-6R of PBMC block, the anti-inflammatory effect of hUCMSC on PBMC will not occur. The proposed anti-inflammatory cell therapy using hUCMSCs is based on the high production of IL-6. hUCMSCs exert excellent anti-inflammatory effects that are different from those of IL-6 antagonists such as TCZ. We believe that the combination of IL-6 secreted by hUCMSCs and the subsequent macrophage IL-6R antagonist can stimulate the anti-inflammatory process faster and more efficiently.
參考文獻
延伸閱讀
- lin, C. H. (2010). 利用人類臍靜脈內皮細胞探討LPLs調控VEGF-C及淋巴標記表現機制之研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2010.00607
- 朱喬伶（2004）。探討Pyrrolidine Dithiocarbamate影響人類臍帶靜脈內皮細胞株與初級細胞對基質金屬蛋白酵素活化的作用與機轉差異〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0007-1704200714562985
- 李立旋、劉學玫、林烱熙、陳瀅如、邱宗傑（2016）。個體IL-6基因多形性／表現量與輸血後產生紅血球異體抗體之相關性探討。Journal of Biomedical & Laboratory Sciences，28(4)，127-133。https://www.airitilibrary.com/Article/Detail?DocID=10137653-201612-201701100007-201701100007-127-133
- Li, L., Wu, S., Liu, Z., Zhuo, Z., Tan, K., Xia, H., Zhuo, L., Deng, X., Gao, Y., & Xu, Y. (2015). Ultrasound-Targeted Microbubble Destruction Improves the Migration and Homing of Mesenchymal Stem Cells after Myocardial Infarction by Upregulating SDF-1/CXCR4: A Pilot Study. Stem Cells International, 2015(), 206-219-119. https://doi.org/10.1155/2015/691310
- Chen, L. Y. (2011). Ex Vivo expansion of Hematopoietic Stem Cells on Surface-modified Materials having Nano-segments and Extracellular Matrix Proteins from Umbilical Cord Blood [master's thesis, National Central University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0031-1903201314413253