【作者】 张朗；

【导师】 李玉民；

【作者基本信息】 兰州大学 ， 临床医学·外科学， 2012， 博士

【摘要】 研究背景:近年来,国内外学者为了降低胃癌的发病率和死亡率,做了很多有意义的探索,并取得了一定的成果,但胃癌的远期生存率仍不甚理想,究其原因,与胃癌的发生、发展机制不是很清楚相关。有研究表明,恶性肿瘤的很多生物学特点与干细胞基本特征十分相似,并由此提出了肿瘤干细胞学说,目前已经陆续从白血病及众多实体瘤中分离出了成瘤性强的干细胞。在近期的研究中有多篇文献报道乙醛脱氢酶A1(ALDH1A1)和CD44的阳性细胞具有很强的肿瘤干细胞特征,是许多恶性肿瘤的肿瘤干细胞生物标志。在胃癌中,ALDH1A1和CD44的阳性细胞扮演着什么样的角色?与胃癌的生物学特性有什么样的关系?目前研究不是很多,这是我们拟关注、探讨和解决的问题。方法:1.应用免疫组化的方法检测66例不同分化程度人胃癌标本、29例非胃癌标本中ALDH1A1和CD44的表达差异;2.采用免疫荧光的方法检测MKN28、SGC7901、MKN45等高、中、低分化程度胃癌细胞系中ALDH1A1和CD44表达差异;3.运用免疫磁珠分选和流式细胞仪分选相结合的方法分选不同表型的胃癌细胞,将不同表型的胃癌细胞分为5组:A组ALDH1A1+CD44+,B组ALDH1A1+CD44-,C组未分选细胞,D组ALDH1A1-CD44+,E组ALDH1AI-CD44-;4.检测不同表型细胞对化疗药物的耐药性、平板克隆形成能力、软琼脂克隆形成能力和Transwell小室侵袭能力;5.应用Western Blot的方法对CD133、OCT4、ABCG2和MMP2等干细胞通用标志物蛋白表达进行检测;6.检测不同表型细胞在裸鼠体内的成瘤能力。结果:1.不同分化程度的胃癌标本中均有ALDH1A1、CD44的表达,ALDH1A1在高、中、低分化胃癌患者组织中的阳性表达情况分别为18/21、27/30、14/15,胃癌组织中的平均阳性表达率为89.4%(59/66),在非胃癌组织中的阳性表达率为24.1%(7/29);CD44在高、中、低胃癌患者的组织中阳性表达情况分别为19/21、28/30、14/15,胃癌组织中的平均阳性表达率为92.4%(61/66),在非胃癌组织的阳性表达率为41.4%(12/29);2.不同分化程度的胃癌细胞系中均有ALDH1A1、CD44的表达,ALDH1A1在高、中、低胃癌细胞系中阳性细胞所占的比例分别为2.3%、3.4%、6.3%,CD44在高、中、低胃癌细胞中阳性细胞的比例分别为3.1%、5.2%、7.1%;3.分选细胞免疫荧光染色鉴定,各组细胞都能呈现出相应的荧光。流式细胞仪检测各目的细胞的阳性比例,除过SGC7901细胞以外,MKN28,MKN45细胞所获得的目的细胞比例均在95%以上;4.不同浓度化疗药物5-FU对不同组别的细胞具有不同的抑制率,当5-FU浓度达到100ug/ml时对MKN28,M N45分选后所得ALDH1A1+CD44+组细胞抑制率分别是16.7%和21.4%,其它各组均在59.4%以上,差异具有统计学意义;5.MKN28、MKN45分选后所得ALDH1A1+CD44+组细胞在平板克隆形成实验中克隆形成率分别为36%和42%,高于其他各组;软琼脂克隆形成实验中克隆形成率分别为4.3%和4.9%,克隆形成能力均强于其他各组,差异有统计学意义;6.Transwell侵袭实验中,MKN28和MKN45分选的ALDH1A1+CD44+细胞分别有265.8个和293.8个细胞从上室穿透Matrigel膜到达下室,其他各组细胞到达下室的数量在68.8～164.3之间,差异有统计学意义;7.MKN28、MKN45分选后所得ALDH1A1+CD44+组细胞中CD133、OCT4、ABCG2和MMP2等蛋白表达分别是未分选组细胞的1.9、0.9、1.4、1.5倍和2.2、2.6、1.8、2.1倍,差异具有统计学意义;8.MKN28细胞系分选所得的ALDH1A1+CD44+细胞数量在1.5×103、1.5×104、1.5×105时成瘤情况分别为3/5、4/5、5/5,M N45细胞系分选所得的ALDH1A1+CD44+细胞数量在1.5 ×103、1.5X104、1.5X105时成瘤情况分别为3/5、4/5、5/5,成瘤能力较其他各组高,差异有统计学意义。结论:1.不同分化程度的胃癌组织和细胞系中均有不同程度的ALDH1A1和CD44表达;2.胃癌细胞系中具有不同的细胞表型,ALDH1A1+CD44+细胞具有较强的化疗耐受性,增殖能力和侵袭能力;3.ALDH1A1+CD44+细胞高表达CD133、OCT4、ABCG2和MMP2等干细胞通用标志蛋白;4.ALDH1A1+CD44+细胞在裸鼠体内具有较强的成瘤能力;5.ALDH1A1+CD44+细胞具有干细胞特性,可作为胃癌干细胞的候选生物标志。更多还原

【Abstract】 Backgrounds:Despite decades of efforts and a diversity of treatments,gastric cancer is still a disease with poor prognosis.The problem mainly lies in the unclear etiological factors and pathogenetic mechanisms in gastric cancer.Research found some malignant tumors cells’biological characteristics are very similar to stem cells.These phenomena might be explained by the so-called tumor-initiating cells or cancer stem cell(CSC)theory.Accumulating evidence has proposed that CSC plays a responsible role for cancer progression and relapse.In recent researches,more and more solid organ tumors’ CSC were discovered and approved.However,either whether gastric cancer possesses cancer stem cells or biomarker of gastric cancer stem cell is still unclear.Aldehyde dehydrogenase 1 A1(ALDH1A1)and CD44 positive cells have recently been suggested as a marker for cancer stem cells or cancer stem-like cells in many human malignancies,but the exact function of ALDH1A1 and CD44 in gastric tumor tissue and cell lines remains largely unknown.The purpose of this study is to investigate the function,biological characteristics and stem cell characteristics of ALDH1A1 and CD44 positive cells in vitro and in vivo.Methods:1.The expression of ALDH1A1 and CD44 in 66 human gastric cancer tissue and 29 normal gastric tissues were evaluated by immunohistochemical method;2.The expressions of ALDH1A1 and CD44 of gastric cancer cell lines MKN28,SGC7901 and MKN45 were investigated by immunofluorescence method;3.Magnetic Activated Cell Sorting(MACS)and fluorescence activated cell sorting(FACS)were used to sorting different phenotype of gastric cancer cell lines.The cells were divide into five groups:Group A,ALDH1A1+CD44+;Group B,ALDH1A1+CD44-;Group C,unsorted cells,Group D,ALDH1A1-CD44+;Group E,ALDH1AI1-CD44-;4.Detected different group cells’ by chemoresistance,Flat colony forming assay,Soft agar clonogenic assay and transwell invasion assay;5.Western Blot assay was used to detected CD133,OCT4,ABCG2 and MMP2 protein expression in different groups;6.Tumorigenicity of different cells was detected in BALB/C nude mice.Results:1.The ALDH1A1 and CD44 expressed in most gastric carcinoma specimens,The outcomes of ALDH1A1+ expression in well-differentiated,moderately-differentiated and poor-differentiated gastric tumor tissues of patients are 18/21,27/30,14/15,respectively.The mean rate of ALDH1A1+ expression in the gastric tumor tissues was 89.4%(59 of 66).The expression rate in none-cancer tissues is 24.1%(7/29);CD44+ expression in well-differentiated,moderately-differentiated and poor-differentiated gastric tumor tissues of patients are19/21,28/30,14/15,respectively.The mean rate of CD44 expression in the gastric tumor tissues was 90.9%(61 of 66),The expression rate in none-cancer tissues is 41.4%(12/29).2.ALDH1A1 and CD44 express differently in different gastric carcinoma cell lines,the positive rate of ALDH1A1 expressions is 2.3%,3.4%,6.3%,respectively.the positive rate of CD44 is 3.1%,5.2%,7.1%,respectively.3.Isolated cells were checked by immunofluorescence cell staining and flow cytometer,except SGC7901 cell line,MKN28 and MKN45 cell lines positive rate was both above 95%.4.Different groups show different resistant to 5-Fu,when concentrations is at 100ug/ml group A with MKN28 or MKN45 inhibition ratio is 21.4%and 16.7%,other groups are all above 59.4%.5.The Flat colony forming efficiency in the ALDH1A1+CD44+ cells from MKN28 and MKN45 is 36%and 42%.Soft agar colony forming efficiency is 4.3%and 4.9%.6.Transwell invasion assay,the number of ALDH1A1+CD44+ cells is 265.8 and 293.8 in lower surface of the membrane,the number of cell between 68.8-164.3 in other groups.7.The expression of CD133,OCT4,ABCG2 and MMP2 in the ALDH1A1+CD44+ cells from MKN28,MKN45 are 1.9,0.9,1.4,1.5 fold and 2.2,2.6,1.8,2.1 fold compared with unsorted isogenic cells.8.The tumorigenicity of ALDH1A1+CD44+ cells from MKN28 is 3/5,4/5,5/5 when cell number were 1.5×103,1.5×104 and 1.5×105.The tumorigenicity of the ALDH1A1+CD44+ cells from MKN45 is 3/5,4/5,5/5 when cell number were 1.5×103,1.5×104 and 1.5×105 in athymic mouse.All the ALDH1A1+CD44+ cells show higher oncogenicity compared with the isogenic cells.Conclusions:1.The ALDH1A1 and CD44 express differently in different stages of human gastric cancer cells.2.The ALDH1A1+CD44+ cells from gastric carcinoma cell lines have higher drug resistance,more colony forming efficiency and invasion ability.3.The expression of CD133,OCT4,ABCG2,MMP2 in the ALDH1A1+CD44+ cells are higher than the isogenic cells.4.The xenograft tumor formation in vivo of the ALDH1A1+CD44+ cells is higher than the isogenic cells.5.The ALDH1A1+CD44+ cells have the characters of stem cells.ALDH1A1 and CD44 may be the candidate marker for stem cells of gastric carcinoma.更多还原