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学科主题: 肿瘤学
题名:
α2δ1阳性胃癌细胞的分离及其肿瘤干细胞样能力的验证研究
作者: 张自然
答辩日期: 2016-05-19
导师: 沈琳
专业: 肿瘤学
授予单位: 北京大学
授予地点: 北京大学临床肿瘤学院
学位: 博士
关键词: 胃癌干细胞 ; α2δ1 ; 分子标记物 ; 致瘤性 ; 自我更新
其他题名: Identification of Gastric Cancer Stem Cells Using Cell Surface Marker α2δ1
分类号: R735.2
摘要:

第一部分 α2δ1在胃癌中的表达差异分析

目的 检测分析α2δ1在不同胃癌细胞系及胃癌组织中的表达情况。

方法 选择9种胃癌细胞系、9例胃癌人源化移植瘤模型组织以及10例晚期胃癌患者腹水原代培养细胞作为实验样本,以标有荧光素的α2δ1特异性单克隆抗体1B50-1标记各样本,通过流式分析及激光共聚焦技术分析α2δ1在不同胃癌细胞及组织中的表达水平差异。

结果 检测分析显示α2δ1在各胃癌细胞系中表达呈现显著异质性,HGC-27细胞系中α2δ1阳性比例为80.0%-95.4%,SGC-7901细胞系中α2δ1阳性比例为0.7-1.0%,其余受检测胃癌细胞系α2δ1阳性比例均低于1.0%;对胃癌人源化移植瘤模型组织及临床晚期胃癌患者腹水培养细胞的检测分析显示α2δ1在其中的表达同样呈现出明显异质性。

结论 α2δ1在胃癌细胞系、胃癌人源化移植瘤模型肿瘤组织以及临床晚期胃癌患者腹水细胞中存在不同程度表达,其表达规律呈现明显异质性。

第二部分 α2δ1阳性胃癌细胞生物学特性的鉴定

目的 鉴定α2δ1阳性胃癌细胞成瘤能力、成球能力、自我更新能力、分化能力、侵袭能力等方面的生物学特性。

方法 选择HGC-27细胞系及SGC-7901细胞系,通过免疫荧光流式细胞分选技术分离获得α2δ1阳性及α2δ1阴性细胞,通过球体形成实验比较阳性及阴性细胞体外成球能力;将分选后细胞接种至NOD/SCID小鼠皮下进行体内成瘤实验观察比较细胞致瘤能力;将体外成球细胞球体及体内成瘤肿瘤组织连续传代观察比较细胞自我更新能力;分选后细胞于完全培养基中培养后,经流式分析回测α2δ1阳性比例观察比较细胞分化能力;通过Transwell实验观察比较细胞侵袭能力。

结果 体外球体形成实验显示HGC-27细胞系和SGC-7901细胞系中á2ä1阳性细胞与相应的α2δ1阴性细胞比较具有更强的成球能力;NOD/SCID小鼠体内成瘤实验显示α2δ1阳性细胞具有高度致瘤性,α2δ1阳性细胞体内成瘤比例明显高于α2δ1阴性细胞;自我更新能力实验显示α2δ1阳性细胞所形成细胞球体在体外传代后仍可连续成球,α2δ1阳性细胞于NOD/SCID小鼠体内成瘤组织经连续传代接种至第二代NOD/SCID小鼠皮下后仍可继续成瘤,而α2δ1阴性细胞不具备相似能力;分化实验显示á2ä1阳性细胞可以分化为α2δ1阴性细胞,而α2δ1阴性细胞不能分化为α2δ1阳性细胞;Transwell实验显示α2δ1阳性细胞比α2δ1阴性细胞具有更强的侵袭性。

结论α2δ1阳性胃癌细胞具有高度体内致瘤性、体外成球能力、自我更新能力以及不对称分化能力及高度侵袭能力,提示α2δ1阳性胃癌细胞具有肿瘤干细胞特性,其侵袭能力提示其可能参与肿瘤的侵袭和转移。

 

英文摘要:

Background: Gastric cancer is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. To date, there is a lack of efficient therapeutic protocols for gastric cancer. Recent studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and resistance to anticancer therapies. Thus, therapies that target gastric CSCs are a promising way to eradicate cancer. However, CSCs in human gastric adenocarcinoma have not been fully described. There is to date no valid surface marker to identify gastric cancer stem cells. α2δ1 is a subunit of voltage dependent calcium channel expressed on cell membrane. Recent research proves α2δ1 as a proper candidate for indentifying cancer stem cell in various tumors as hepatocarcinoma, pancreas cancer and lung cancer. Our purpose is to carry on research whether α2δ1 is expressed in gastric cancer, and whether it could be a candidate for identifying gastric cancer stem cells.
Methods:  Cell lines and  PDX models and gastric cancer acites cells were detected for α2δ1 expression. Later experiments were carried on with HGC-27 and SGC-7901 cell lines. By immunofluorescence flow cytometry sorting technology separation, α2δ1 positive and α2δ1negative cells are acquired. Sphere formation experiment compared sphere formation ability of positive and negative cells in vitro.The sorted cells were inoculated to NOD/SCID mice subcutaneously to test the tumorigenic ability and self-renewal ability. Transwell compared the invasive ability of α2δ1 positive and negative cells.
Results:  α2δ1expression profiles in gastric cell lines and  models and gastric cancer acites cells are hetergeous. Sphere formation in vitro showed α2δ1 positive cells possessed higher sphere formation ablity compared with the corresponding α2δ1 negative cells; α2δ1 positive cells also possessed higher tumorigenic ablity and self-renewal ability in vivo than α2δ1 negative cells. α2δ1 postive cells possessed higher invasive abllity than α2δ1 negtive cells; α2δ1 could deffrentiated into α2δ1 negative cells but not vice versa.
Conclusion: α2δ1 positive gastric cancer cells demonstrated high tumorigenic ability in vivo and sphere formation ability in vitro, as well as self-renewal capacity and asymmetry differentiation ability. Thus, α2δ1 positive gastric cancer cells possess cancer stem cell abilty and α2δ1 could be a candidate cancer stem cell marker for further research.

 

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125698
Appears in Collections:北京大学临床肿瘤学院_学位论文

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作者单位: 北京大学临床肿瘤学院

Recommended Citation:
张自然. α2δ1阳性胃癌细胞的分离及其肿瘤干细胞样能力的验证研究[D]. 北京大学临床肿瘤学院. 北京大学. 2016.
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