醣質體之質譜分析應用於人類骨髓間質幹細胞及其分化之細胞

Mass spectrometry-based glycomics of human bone marrow mesenchymal stem cells and their differentiated progenies

郭玉岱(Yu-Dai Kuo)

指導教授 ： 邱繼輝

國立臺灣大學/生命科學院/生化科學研究所/博士(2017年)

https://doi.org/10.6342/NTU201700317

摘要

人類間質幹細胞屬於一種具有多功能性分化能力的成體幹細胞，在人體中主要存在於骨髓、脂肪、臍帶血或胎盤等部位，同時也具有能夠在體外被培養及分化成數種細胞的能力，包含硬骨、軟骨細胞、脂肪、肌肉及神經細胞等，因此被廣泛地應用在再生醫學與組織工程等方面的研究上。目前已有不少臨床治療的試驗研究嘗試使用間質幹細胞重建與治療疾病但由於間質幹細胞移轉到受傷部位的效果不彰，使得治療效果十分有限。有些研究認為，細胞膜表面的醣化修飾，或許是影響間質幹細胞移轉效率的因素之一，然而我們對於間質幹細胞以及由其分化的數種細胞表面醣化修飾的了解，仍然有限。
另外位於周邊淋巴組織，其特化的高內皮靜脈系統外圍，分布一種稱為纖維網狀的細胞，具有支撐結構的物理性功能，同時也分泌數種生物因子，包含介白素-7 (IL-7)與紅血球生成素等，因此纖維網狀細胞在周邊淋巴系統中也扮演了舉足輕重的角色，但截至目前為止，由於其不易被取得以及在體外難以被分化的性質，使得我們對於其細胞表面的醣質化結構仍然一無所知。
目前我們已知纖維網狀細胞可以於體外培養的情況下，由人類間質幹細胞所分化而得，因此此論文的主要目的是希望應用我們先前所建立的硫酸化醣質體分析系統，搭配高敏感度、高精確性的質譜儀 (Mass spectrometry, MS)，來分析人類骨髓間質幹細胞、以及其所分化成的纖維網狀細胞表面的醣質化修飾結構，包含N與O型醣鏈、以及其硫酸化醣質修飾結構；另一方面，在此論文當中我們會一併研究人類間質幹細胞分化成的硬骨細胞及脂肪細胞的醣質化結構，作為一種比較及依據，尤其將重點放在尚未被研究過的硫酸化醣質體結構上。
總體而言，人類間質幹細胞在經由不同的特定生物激素刺激下，在分化14天之後可分別形成硬骨細胞以及脂肪細胞，另外分化成gp38+ 的似纖維網狀幹細胞，則需要7天的時間。接著我們取下細胞表面蛋白質上的醣類，利用質譜儀分析其結構、進行比較。我們發現基本上，這4種類型細胞的醣質化修飾結構很相似，其N型醣鏈主要由高甘露醣鏈組成，而complex type N型醣鏈則大多在其還原端的乙醯基葡萄糖胺(GlcNAc)上帶有一個岩藻醣 (fucose)，且具有1-5個乙醯基乳糖胺分支；在其非還原末端上，可分別帶有1-4個的唾液酸，少量的幾種N型醣鏈可同時擁有2個岩藻醣，形成主要帶有H type 2與LeX的末端醣質修飾結構。另外，大多數這些N型醣鏈的非還原端被發現可同時攜帶1-2個硫酸基，且主要的末端醣質結構是帶有1個硫酸基的乙醯基乳糖胺 (sulfated LacNAc)。在4組細胞的比較當中，gp38+ 的似纖維網狀幹細胞表現較多的雙岩藻醣化及唾液酸化，然而在其他方面，這4組細胞並未存在著太顯著的差異。O型醣鏈的部分則主要是由Core 1和Core 2及其延伸的結構所組成，主要帶有1-2個唾液酸，並且少量帶有1個唾液酸的Core 2結構亦可被單岩藻醣化，形成帶有H type 2、LeX，以及sLeX的末端醣質修飾結構。這些主要的O型醣鏈亦可攜帶1個硫酸基，在其非還原端末端形成帶有單個硫酸基的乙醯基乳糖胺、岩藻醣化乙醯基乳糖胺的末端結構，且硫酸基的位置主要在乙醯基葡萄糖胺的6號碳位置上，同時我們也發現有末端硫酸化唾液酸的存在。
綜言之，此論文透過分析人類間質幹細胞以及其所分化的似纖維間質幹細胞、硬骨細胞以及脂肪細胞的醣質化和硫酸醣質化修飾結構，希望藉此在結構工程學的研究方面，能夠提供一個整體的醣質體圖譜概念，使其能夠改善人類間質幹細胞在再生醫學臨床應用上的效能。

關鍵字

醣質生物學 ； 質譜儀 ； 硫酸化醣質體 ； 甲基化衍生 ； 人類骨髓間質幹細胞 ； 脂肪細胞 ； 骨細胞

並列摘要

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells that have therapeutic potential for regenerative medicine and are easy to induce into several differentiated types of cells, including adipocytes, chondrocytes, osteoblasts and fibroblast reticular cells, providing great potential in regenerative therapy. To date, there is little knowledge on the chemical properties of glycosylated surface markers characterizing MSCs and their various differentiated stages, particularly those of sulfated N- and O-glycans. Besides, fibroblast reticular cells (FRC) play vital roles in the high endothelial venules (HEV) system in peripheral lymph organs, in which they physically support HEV scaffold and secret survival essential cytokines for lymphoid cells, including interleukin-7 (IL-7). However, the glycomics of FRC had never been investigated.

In this study, we aimed to identify several development stage-specific glycans by a mass spectrometry (MS)-based glycomic mapping of the N- and O-glycans of undifferentiated and 14 days adipogenically/osteogenically differentiated human MSCs, as well as gp38+ derived from human MSCs after 7 days of differentiation. We first obtained an overall MS profile of permethylated non-sulfated and sulfated glycans by MALDI-MS analysis in positive and negative ion modes, respectively, followed by advanced nanoLC-MS2/MS3 analysis to identify the glycotopes. We found that the complex type N-glycans were mostly core-fucosylated when carrying a single fucose but Lewis X and H type 2 could be identified. Almost all of the N-glycans including those with mono- to tetra-sialylated and those carrying poly-N-acetyllactosamine structures were found to have mono- and disulfated counterparts carrying sulfates on the GlcNAc or Gal of the LacNAc unit, and primarily the sulfate located at 6 arm of GlcNAc. Moreover, gp38+ cells expressed more di-fucosylated and sialylation level of non-sulfated N-glycans and more fucosylation at mono-sulfated N-glycans compared with other 3 types of cells.

The major O-glycans of MSCs and their differentiated progenies were mostly based on mono- or di-sialylated extended core 1 and 2 structures, some of which additionally fucosylated and most could also be mono-sulfated. The major glycotopes of non-sulfated O-glycans were H type 2 and LeX, as well as sLeX, and the predominant sulfoglycotopes was sulfated LacNAc, while lower level of sulfated fucosyl LacNAc could also be observed. The location of sulfate was primarily on the 6 position of GlcNAc (GlcNAc6S), but minor Gal6S, Gal3S and sulfated sialic acid also existed. Upon induced differentiation to different progenies, gp38+ cells was found to have more mono- and di-sialylation non-sulfated O-glycans, whereas adipocyte showed less of those but more non-sulfated core 1 and core 2 structures. Besides, gp38+ cells seemed to express less mono-sialylated mono-sulfated O-glycans compared with the others.

並列關鍵字

Glycobiology ； Mass spectrometry (MS) ； sulfoglycomics ； permethylation ； human mesenchymal stem cells (MSC) ； osteocyte ； adipocyte

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