【作者】 潘猛；

【导师】 窦骏； 王立新；

【作者基本信息】 东南大学 ， 免疫学， 2006， 硕士

【摘要】 目的:最近研究显示癌症来源于癌干细胞(CSC) ,即肿瘤源发细胞,其有许多特性与干细胞相似。已分离鉴定出人的癌症细胞中存在CSC,而动物源性CSC的分离鉴定仍未见报道。根据人源性肿瘤细胞与动物源性肿瘤细胞有诸多相似的生物学特性,本研究试图从鼠黑色素瘤B16F10细胞中分离与鉴定CSC,为今后对CSC抗药性、靶向治疗及信号传导通路等研究奠定基础。方法:从小鼠黑色素瘤分离瘤细胞,用不同免疫磁珠标记的单克隆抗体筛选出具有特征性标记的瘤细胞,采用流式细胞仪,对分离细胞进行鉴定和纯度评价;体外观察不同CD表型瘤细胞的增殖活性;再经软琼脂培养基或普通培养基形成克隆能力筛选及通过皮下注射特征性CD标记的瘤细胞到C57BL/6小鼠体内,比较其致瘤性,最后综合分析与鉴定B16F10细胞中CSC。结果:1.用不同免疫磁珠标记的单克隆抗体从小鼠B16F10细胞中成功分离出CD133~+、CD133~―、CD44~+、CD44~―、CD44~+CD133~+、CD44~+CD133~―等特征性CD标记的瘤细胞,经流式细胞仪分析显示,用不同免疫磁珠标记的单克隆抗体分离的瘤细胞纯度较高、两者检测结果基本一致。2.经软琼脂培养基或普通培养基形成克隆能力筛选结果显示:CD133~+ B16F10细胞的克隆形成率高于CD133~―B16F10细胞,CD44~+ B16F10细胞克隆形成率高于CD44~―B16F10细胞。CD44~+CD133~+ B16F10细胞克隆形成率高于CD44~+CD133~―B16F10细胞。3. CD133~+ B16F10细胞体外增殖活性要高于CD133―。4. CD133~+、CD44~+、CD44~+CD133~+和CD44~+CD133~+CD24+ B16F10细胞在C57BL/6小鼠体内致瘤性分别强于CD133~―、CD44~―、CD44~+CD133―和CD44~+CD133~+CD24―B16F10细胞。结论:CD133~+ B16F10细胞体外增殖活性较高, CD133~+、CD44~+、CD44~+CD133~+ B16F10细胞在软琼脂培养基或普通培养基上形成克隆能力较强、CD44~+CD133~+ CD24+ B16F10更多还原

【Abstract】 Objective: Recent studies suggest that cancer can arise from a cancer stem cell (CSC), a tumor-initiating cell that has properties similar to those of stem cells. CSC has been identified in several human malignancies, however, it is not reported yet that there are CSC isolated and identified from animal cancer cell. According to the many similar biological features between the cancer cell of human origin and the cancer cell of animal origin, we try our best to isolate and identify murine CSC from the murine melanoma B16F10 cells, and to lay a foundation for research of resistance to chemotherapeutic agents in the CSC, target therapy for CSC, and signaling pathway in carcinogenesis etc in future.Methods: Isolating B16F10 cells from the murine melanomas model and selecting the specific marked B16F10 cell from the cells binding different monoantibody labeled with immune magnetic beads by magnetic activated cell sorting system (MACS), the purity of isolated specific marked B16F10 cells was valuated by flow cytometry (FCM) further, and the proliferation activites of different CD phenotypes B16F10 cells were observed in the cultured B16F10 cells by keeping count of cells in vitro. After the capability of clonegenesis were selected in soft agar culturing or common culture, the potential clonegenesis B16F10 cells with the specific CD phenotypes were inoculated s.c into C57BL/6 mice and the tumorigenic process were compared with the different CD phenotypes B16F10 cells in C57BL/6 mice. The compostive analysis and identification CSC was finally made from a series of experiments results.Results: 1.CD133~+, CD133~―, CD44~+, CD44~―, CD44~+CD133~+,CD44~+CD133~―B16F10 cells were successfully isolated from the murine melanomas cells binding different monoantibody labeled with immune magnetic beads by MACS and these specific marked B16F10 cells were basically consistent with the identified B16F10 cells by FCM. The FCM analysis results suggest that the specific CD phenotypes B16F10 cells are a high purity also. 2. The proliferation activity of CD133~+ phenotype B16F10 cells is stronger than those of CD133~―phenotype B16F10 cells and wild B16F10 cells in vitro. 3. The results of soft agar culture or common culture show that the rate of clonegenesis of CD133~+ phenotype B16F10 cells are prior to CD133~― phenotype B16F10 cells and the rates of clonegenesis of CD44~+, CD44~+CD133~+ phenotypes B16F10 cells are prior to CD44~―, CD44~+CD133~―phenotypes B16F10 cells respectively. 4. The tumorigenesis of CD133~+ phenotype B16F10 cells in更多还原