人類胃幹細胞於胃癌致癌機制之研究

Human Gastric Stem Cells in Carcinogenesis Studies

楊淵傑(Yuan-Chieh Yang)

指導教授 ： 吳登強

共同指導教授 ： 黃友利(Yeou-Lih Huang)

高雄醫學大學/醫學院/醫學研究所/博士(2009年)

https://doi.org/10.6832/KMU.2009.00052

摘要

許多的證據顯示，癌症的發生可能來自幹細胞的特化，而此種特化可能是致癌因子突變或基因表現之異常所造成。於是，胃上皮幹細胞的取得及培養，對胃癌致癌機制之探討，是極其重要的。
胃上皮組織具有很高的細胞新陳代謝率，一般認為這類組織需相當數量之幹細胞來維持細胞數之恆定。然而至目前為止，有關人類胃幹細胞之分離與體外培養卻未有正式的論文發表。從以往的組織學觀察中可知，不分化（undifferentiated）之人類胃前驅細胞（progenitor）又可稱為多潛能小顆粒細胞（pluripotent mini-granule cell），其位於pit與gland compartments間之isthmus區域，主要功能可持續性的補充及再生胃上皮細胞。為進行胃組織再生或分化機制之探討及建立本土細胞株供胃癌致癌及抑癌機制之研究，分離胃幹細胞及建立體外培養模式是有其必要性的。在我們研究中，主要以胃內視鏡採集胃黏膜組織，進行胃幹細胞之分離與培養，在已進行的56例組織檢體中，共有118個生長能力較好的細胞株被分離，其中Clone KMU-GI2（GI2）的分析，結果顯示其具多項幹細胞之特性，如有較長的生長期（CPDL=37）、於培養過程中能觀察到不同分化型態的細胞（epithelial-like cells, glial-like cells, neuron-like cells）、能表現Oct-4抗原等。另外於GI2之次代培養中，我們分離出了自然轉型（spontaneous transformed）之細胞株GI2CS-（KMU-CSN; CSN），特別的是此株細胞經一段時間之培養後（cpdl=132），得到另一細胞株KMU-CS12（CS12），其於染色體核型之分析中，已發現第十二對染色體出現異常，並且可於裸鼠移植實驗中證實CS12細胞株已具有致癌性（tumorigenic）。本研究論文主要以前述之細胞株（GI2, CSN, CS12）為實驗材料，並分成三部分來探討，第一部份為“自人類胃部組織分離與定性具幹細胞特性之胃上皮細胞株”，此部分除了探討GI2細胞株的分離方法及培養方式，也以特定的分子標誌進行免疫染色、cpdl的計算、細胞型態的觀察等，分析此細胞株之特性。第二部分“人類胃幹/前軀細胞於胃幽門螺旋桿菌致癌機制之應用”，主要將GI2細胞株應用於胃幽門螺旋桿菌（Helicobacter pylori; Hp）致癌機制之探討上，實驗中我們發現在致癌物N-methyl-N’-nitro -N- nitrosoguanidine（MNNG）及特定Hp的刺激及感染下，GI2細胞株有端粒脢表現量上升的現象。第三部分“ 衍自胃上皮幹細胞之轉形細胞株其特性與胃癌致癌機制相關性之分析”，此部分，我們除了對衍自GI2之轉型株CSN及可於裸鼠生成腫瘤之CS12細胞株進行特性分析外，也以Spectral karyotyping（SKY）確定CS12之第12對染色體異常主要為其長臂多了一段DNA複製自第7對染色體短臂，另外我們將細胞染色體核型變化前後（CSN v.s. CS12）以Microarray進行基因表現的比較，結果顯示有1145個基因表現量上升，890基因表現量下降。經分析後，我們發現位於第七對染色體短臂中之HOXA基因組（HOXA4,5,7,9,13）有明顯高度表現的現象，而這些分子可能於CS12致癌機制中扮演重要角色。
我們相信此人類胃細胞株及其轉型株的建立，不僅於胃癌致癌機制分析及抗癌藥物研究上，提供一個極佳的實驗材料外，更有助胃幹細胞特性及其分化機制之探討。特別的是，由於CS12轉形自幹細胞，我們的實驗間接引證了幹細胞致癌理論，而CS12細胞株是否就是Gastric cancer stem cells，則值得進一步實驗探討與證明。

關鍵字

幹細胞 ； 胃癌 ； 幽門螺旋桿菌

並列摘要

Cancer has been described as disorganized organogenesis, since a wealth of studies point out the similarity between carcinogenesis and organogenesis. The close link underlying the two seemingly different events is suggested to be tissue stem cells. Therefore, it is important to understand what tissue stem cells are and how they behave under different conditions, which may help to unravel the mystery of carcinogenesis.
Gastric epithelium is a high turn-over tissue, in which stem cells are assumed to be robust. Accordingly, it is an ideal tissue for stem cell study. From histology study, the undifferentiated cells resided in the gastric corpal isthmus were suggested to be the putative gastric stem/progenitor cells, also called pluripotent mini-granule cells. As the study how the putative human gastric stem/progenitor cells may contribute to gastric carcinogenesis, an in vitro model of cultured gastric stem/progenitor cells would be a useful model for further dissection. In my study, I used the gastric specimens obtained by endoscopic biopsy to isolate gastric stem/ progenitor cells. From 56 human specimens, 118 cell clones were isolated. Among them, KMU-GI2 (GI2) showed many characteristics of gastric stem cells, including a high cumulative population doubling level (CPDL=37), a capacity to give rise to cells with different morphology, including epithelial cells, glial cells and neuron cells, and expression of Oct-4. In addition, we isolated a spontaneously transformed subclone, named GI2CS- (KMU-CSN; CSN). After long-term culture (CPDL=132), a subclone, named KMU-CS12 (CS12), in CSN cell was tumorigenic and showed an abnormality in chromosome 12 by karyotyping. With both of normal and transformed gastric epithelial cell clones at hand (GI2, CSN, CS12), it is possible to study how the specific genetic abnormality can contribute to gastric carcinogenesis.
My thesis is comprised of 3 parts. The first part is to show how I isolated and cultured the gastric stem/progenitor cell like-cell clones (KMU-GI2) from human gastric specimens and my approach to characterize their gastric stem/progenitor cells properties, including cumulative population doubling level (cpdl), cell morphology and immunocytochemistry. The second part is to show how this GI2 interacts with carcinogenic environmental cues, like N-methyl-N’-nitro -N- nitrosoguanidine (MNNG) and Helicobacter pylori (Hp), and evaluate their responses. This study was assumed to simulate how stem cells would behave under similar conditions. We found telomerase in the study cells was highly activated under these noxious stimuli, and this increment was correlated with human clinical findings. The third part is to evaluate how transformed gastric stem/progenitor cells can recapitulate carcinogenesis. By Spectral karyotyping (SKY), we confirmed there was an extra fragment, replicated from the short arm of chromosome 7, on chromosome 12 in CS12 clone, which appeared to be tumorigenic in nude mice. There were 1145 genes up-regulated and 890 gene down-regulated by this genetic abnormality, detected by microarray. According to the array data, the HOXA genes, located in chromosome 7, were focused. Further analysis revealed HOXA 4, 5, 7, 9, 13 were significantly over-expressed and this effect might be attributed to the tumorigenecity we observed.
With the creation of in vitro gastric stem/progenitor cell line, a model to approach carcinogenesis and built a preclinical system to test the possible anti-cancer therapy has been established. This study also alludes gastric cancer may derive from transformed gastric stem cells and provides a great chance to further increase our understanding of gastric cancer stem cells.

並列關鍵字

stem cell ； gastric cancer ； Helicobacter pylori

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