论文标题:乳腺癌干细胞的分离与鉴定及其自我更新和耐药机制的研究
Separation and Identification with Breast Cancer Stem Cells and Study on Proliferation and Drug-resistance of Breast Cancer Stem Cells
论文作者
论文导师 王国斌；黄韬,论文学位 博士,论文专业 外科学
论文单位 华中科技大学,点击次数 820,论文页数 73页File Size17117K
2006-04-01论文网 http://www.lw23.com/lunwen_115662/
stem cell;breast cancer;self-renewal;drug-resistance;MDR1
目的：了解在不同的情况下，对乳腺癌干细胞自我更新的影响和乳腺癌干细胞比例的变化及其意义；以及乳腺癌干细胞逃逸化疗，导致乳腺癌复发的机制。方法：1．以华中科技大学附属协和医院乳腺甲状腺外科在2004年1月～2005年6月期间收治并接受“乳腺癌根治术”的乳腺癌患者为研究对象，在无菌条件下从手术标本中获取乳腺癌组织，并采用机械分离和混合胶原酶消化的方法将组织标本制备成单细胞悬液，然后用流式细胞仪分选出乳腺癌干细胞，最后用动物移植乳腺癌重建模型对分选出的乳腺癌干细胞进行鉴定。2．用流式细胞技术从MCF-7中分选出表达CD44~+CD24~-的细胞和其它细胞，并对两群细胞的端粒酶活性以及在体外的自我更新情况和克隆形成情况进行对比观察，对两群细胞裸鼠种植后的成瘤率、成瘤时间、成瘤大小进行对比观察。3．采用流式细胞技术检测在不同的培养条件下MCF-7中以及乳腺癌组织原代细胞中的CD44和CD24表达情况，来观察不同情况下乳腺癌干细胞的含量变化情况。4．测定人乳腺癌标本和MCF-7中不同细胞亚群的MDR1表达情况；并测定化疗药物在体外对人乳腺癌原代细胞和MCF-7中不同细胞亚群的杀伤作用，以及在化疗药物干预后，乳腺癌干细胞的含量和MDR1表达的变化情况。结果：我们对乳腺癌组织获得了良好的分离效果，每1cm~3组织大约可收获0.5～1×10~7个细胞，原代乳腺癌细胞主要由Lin~-CD44~+CD24~-和Lin~-CD44~-两群细胞组成，只有表达Lin~-CD44~+CD24~-的细胞能在裸鼠中重建出乳腺癌，而MCF-7细胞则由CD44~+CD24~-和CD44~+CD24~+两群细胞组成，虽然这两群细胞都能在裸鼠中重建出乳腺癌，但是CD44~+CD24~-细胞重建乳腺癌所需的时间更短，几率也更高。随着乳腺组织的性质由正常→良性→恶性，其中表达Lin~-CD44~+CD24~-的细胞所占的比例呈现增加趋势，MCF-7中表达CD44~+CD24~-的细胞所占比例会随培养过程中细胞数量的增加而逐渐下降；而且当化疗药物干预后导致细胞数量下降时，人乳腺癌原代细胞和MCF-7中干细胞的比例都会明显上升。人乳腺癌原代细胞中的乳腺癌干细胞MDR1的表达高于Lin~-CD44—的乳腺癌细胞，同时化疗药物对人乳腺癌原代细胞中的乳腺癌干细胞的杀伤率低于Lin~-CD44—的乳腺癌细胞，但是MDR1在MCF-7两群细胞中的表达没有差异，它们在体外对化疗药物的敏感性也相似；而化疗后乳腺癌干细胞不但比例明显上升，而且乳腺癌干细胞的MDR1表达也明显增强。结论：我们从人乳腺癌组织中分离出表达Lin~-CD44~+CD24~-的细胞是乳腺癌干细胞，MCF-7中表达CD44~+CD24~+的细胞则可能是乳腺癌组细胞；乳腺癌干细胞在乳腺组织中的含量可以反映乳腺组织的性质和乳腺上皮细胞的自我更新速度，同时它也受微环境中生长信号的调控；乳腺癌干细胞在乳腺癌化疗中占有重要的地位，它不但比其它乳腺癌细胞更容易在化疗中发生治疗逃逸，并且能够通过变异获得耐药性，而化疗后乳腺癌干细胞的自我更新加速将促进乳腺癌的复发。
Objective: To know how to change about self-renewal and the proportion of breast cancer stem cells in diverse environment,and how to evade from chemotherapy about breast cancer stem cells. Methods: 1.Study on the patients with breast cancer that gained therapy in Department of Breast and Thyroid Center,Union Hospital during January,2004～ June,2005. To obtain mammary cancer tissue after operation,and prepare single cell suspensions by collagenase,then gain breast cancer stem cells by flow cytometry.Finally we identitified breast cancer cells by model of SOD/SCID mice with reconstruction tumor.2. To separate breast cancer cells with expressing CD44~+ CD24～ and other breast cancer cells from MCF-7 by flow cytometry. Then to mensurate the activity of telomerase of different cells,and observate their ability of self-renewal and clone in vitro,and their ability of reconstruction tumor in vivo.3. To mensurate the expression of CD44 and CD24 of human breast cancer primary cells and MCF-7 breast cancer cell line in diverse incubation circumstance,and observate the proportion of breast cancer stem cell in diverse environment.4. To mensurate the expression of MDR1 of different breast cancer cells from human breast cancer primary cells and MCF-7 breast cancer cell line,and the kill effect of chemotherapy drug with different breast cancer cells in vitro,and the change of the proporation and the expression of MDR1 of breast cancer stem cells after chemotherapy.Results:We gain excellent effect of separation with breast cancer tissues,we could obtain 0.5—1×10~7 cells from 1cm~3 breast cancer tissues, breast cancer primary cells mainly include expressing Lin~-CD44~+CD24～- breast cancer cells and expressing Lin~-CD44~-breast cancer cells, expressing Lin~-CD44~+CD24~- breast cancer cells could reconstruct tumor by transplanting SOD/SCID mice.MCF-7 cells mainly include expressing CD44~+CD24~-breast cancer cells and expressing CD44~+CD24~+ breast cancer cells,although they allhave ability of reconstruction tumor by transplanting SOD/SCID mice, the ability of expressing CD44~+CD24~- breast cancer cells is more stronger. From normal,benign to malignant mammary tissue,the proporation of CD44+CD24-/low mammary cell gradually rise,the proporation of stem cell gradually decrease with the number of MCF-7 gradually increasing in the process of culture,and the proporation of breast cancer stem cells from human breast cancer primary cells and MCF-7 breast cancer cell line markedly increases after chemotherapy. Although the expression of MDR1 of breast cancer stem cells is higher than expressing CD44~ breast cancer cells from human breast cancer primary cells,it is not different in different breast cancer cells from MCF - 7,and the kill effect of chemotherapy drug for different breast cancer cells from MCF - 7 is similar. After chemotherapy, the proporation and the expression of MDR1 of breast cancer stem cells rise all. Conclusion:Expressing Lin~-CD44~+CD24~- breast cancer cells from human breast cancer primary cells are breast cancer stem cells,and Expressing CD44~+CD24~+ breast cancer cells from MCF-7 maybe are breast cancer progenitor cells. The proporation of mammary stem cell that is modulated by diverse factors in microenvironment could reflect property of mammary tissue and self- renewal speed of mammary cell. The role of breast cancer stem cell is very important in chemotherapy.Breast cancer stem cells more easily escape from chemotherapy than other breast cancer cells,and obtain ability of drug resistance by mutation of gene. Speedup of breast cancer stem cells self-renewal after chemotherapy will promote relapse of breast cancer.
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