【作者】 冯海凉；

【导师】 李汇华； 刘玉琴； 李利民；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 本论文分为两个部分：第一部分：CD133在人结肠癌细胞分化中的作用在本研究中,首先我们用Western blotting检测了44种人肿瘤细胞系中CD133的表达,其中8种细胞呈CD133阳性表达,5个来源于结肠癌。接着我们分选了结肠癌细胞系HCT116中CD133high/+和CD133的亚群,发现CD133high/+的HCT116细胞在体外生长快于CD133的HCT116细胞,并有更高的克隆形成率。CD133high/+亚群和CD133-的HCT1 16亚群细胞相比,S期和G2／M期细胞比例更高。但是在BALB/c-nu/nu鼠成瘤实验中,CD133high/+和CD133-的HCT1 16细胞并没有明显的区别。将分选后的CD133high/+和CD133-的HCT1 16细胞体外培养,CD133的分布趋向于和未分选前的HCT1 16细胞相同。我们的研究结果提示不能单用CD133一个分子标记来富集结肠癌细胞系中的肿瘤干细胞。我们进一步研究了CD133的表达和结肠癌分化的关系。我们按照CD133的表达量,将结肠癌细胞HT29分为了四个亚群,以ALP活性作为分化程度的指标进行了检测,结果显示不同亚群的分化状态和CD133的表达呈负相关。我们还发现用丁酸钠诱导HT29和HCT1 16分化后,流式检测这两种细胞表面的CD133／1和CD133／2的免疫活性都呈现时间和剂量依赖的下降。但是Western Blotting却未检测到细胞中CD133总蛋白水平的改变。我们还设计了三对针对人CD133分子的siRNA,其中两对可以在体外有效干扰CD133表达。但是干扰CD133的表达对HT29和HCT116细胞的增殖、克隆形成、周期分布和分化状态都没有明显的影响。CD133可能不是细胞生长和分化的调控基因。总的来说,CD133不是结肠癌细胞系HCT1 16特异的肿瘤干细胞的标记,而只是结肠癌分化相关的一个指标。第二部分：荧光标记肿瘤转移体内模型的建立本研究通过转染pEGFP-Nl质粒,药物筛选后无限稀释得到了四株强表达绿色荧光蛋白的单克隆细胞株-人宫颈癌细胞HeLa-GFP、人结肠癌细胞HCT116-GFP、人乳腺癌细胞MDA-MB-231-GFP和小鼠宫颈癌细胞U14-GFP。我们把HeLa-GFP以8×106个／只的剂量皮下接种于BALB/c-nu/nu裸鼠,成瘤潜伏期是3-5天,成瘤率为100%。利用Photometrics活体荧光成像系统连续观察,可以清楚直观的观察表达GFP的HeLa-GFP移植瘤在体内的生长。接种后第60天处死全部荷瘤鼠,解剖后大体成像仅有一只可见同侧腋窝下淋巴结的转移。取U14-GFP以8×106个／只接种C57BL/6J小鼠,移植成瘤的潜伏期是2-4天,成瘤率1 00%。利用Photometrics活体荧光成像系统连续观察,可分别在第22天、28天、37天和52天观察荷瘤小鼠肿瘤转移的发展过程。在U 14-GFP原发瘤体积≥5 cm3时,肺部和淋巴结转移率分别为67%和1 00%。我们还将HCT116-GFP进行了皮下移植和尾静脉注射,用Berthold活体成像仪观察了肿瘤的生长和转移。利用Berthold活体成像系统可见HCT116-GFP移植瘤在体内仍可强表达GFP。HCT116-GFP尾静脉注射后第43天,可用Berthold活体成像系统检测到GFP阳性的皮下转移肿瘤。最后,我们还检测了两种常见的转移相关分子CD44和E-cadherin在HeLa-GFP和U14-GFP细胞移植瘤中的表达。免疫组化检测显示在两种移植瘤中都有CD44的表达,而无E-cadherin的表达。总之,我们成功建立了四株表达绿色荧光蛋白的单克隆细胞株,体内移植建立了荧光标记的肿瘤转移模型。该模型可用于可视化肿瘤体内的研究。更多还原

【Abstract】 The thesis includes two parts:Part 1. Expression of CD133 correlates with differentiation of human colon cancer cellsIn this study, we tested the CD 133 expression in 44 widely used human cancer cell lines. Interestingly, five of 8 CD133 positive cell lines were colon cancer cells. We sorted the CD133high/+ and CD133- subpopulation of HCT116 cells and found that the CD133high/+ HCT116 cells grew quicker (in vivo) and generated more colonies after 14 days incubation. CD133high/+ HCT116 cells had higher percentages of cells in S and G2/M phases than their CD133- counterparts. In ongoing xenograft tumor formation experiments in BALB/c-nu/nu mice, we did not find a difference in tumorigenic potential of CD133high/+ versus CD133-subpopulations. Cultivation of both CD133high/+ and CD133-HCT116 cells resulted in a redistribution of antigen expression to its original proportion. Our results suggest other markers rather than CD 133 alone should be used to enrich for colon CSCs, especially in cultured colon cancer cell lines.Next we explored the relationship between CD133 expression and differentiation. HT29 cells were sorted 4 scales according to CD133 staining, our results suggested that differentiation status (ALP activity) was inversely correlated with CD 133/2 expression in HT29 cells. Then, we found that HT29 and HCT116 lose their CD133/1 and CD133/2 reactivity (flow cytometry) in a time-and dose-dependent manner after differentiation induced by SB, but the total protein level (Western Blotting) in the cell did not change. We also synthesized three pairs of siRNAs, two of which could efficiently reduce CD133 expression in vitro. There was no appreciable effect on the proliferation, cell cycle distribution, colony formation and differentiation of HCT116 and HT-29 cells by knocking down the expression of CD133. CD133 seems not to be a prerequisite gene for cell growth and differentiation.In all, CD 133 may be more a differentiation indicator rather than a specific stem cell lineage marker in colon cancer cell lines.Part 2. Establishment of tumor metastasis models expressing green-fluorescent proteinFour cell lines were transfected with the plasmid pEGFP-N1, and limited dilution was employed to screen four monoclonal cell strains expressing GFP:human cervical adenocarcinoma cell HeLa-GFP, human colon carcinoma cell HCT116-GFP, human mammary adenocarcinoma cell MDA-MB-231-GFP and murine cervical carcinoma cell U14-GFP.8×106 HeLa-GFP cells were transplanted into BALB/c-nu/nu mice. The latent period of tumor mass formation was 3-5 days and its tumorigenicity is 100%. The tumor growth of HeLa-GFP was well defined by the Photometries. Only one mouse was shown to harbor lymphatic metastasis by Photometrics 60 days after transplantation.8×106 U14-GFP cells were transplanted into C57BL/6J mice, the latent period of tumor formation was 2-4 days and its tumorigenicity is also 100%. The metastasis process of U14-GFP was depicted through the observation by Photometrics on 22,27,37 and 52 days post-transplantation. The incidence of pulmonary metastasis and lymphatic metastasis of U14-GFP was 67% and 100% respectively when the tumor volume was> 5 cm3. We also transplanted HCT116-GFP subcutaneously and injected them into tail vein of BALB/c-nu/nu mice. The HCT116-GFP tumor stably expressed green flourcence in vivo by Berthold Viviperception Fluorescence Imagining System. Fourty three days after HCT116-GFP tail vein injection, GFP positive metastasis tumors were detected by Berthold.The expression of CD44 and E-cadherin were checked in HeLa-GFP and U14-GFP tumor tissues, CD44 was positive and E-cadherin was negative in both tumors by immunohistochemistry.In conclusion, we successfully established four monoclonal tumor cell strains stably expressing GFP. Transplantation of these cells into mice can establish tumor metastasis models which could be used for future visualized tumor research in vivo.更多还原