【作者】 杨超；

【导师】 王士杰； 刘运江；

【作者基本信息】 河北医科大学 ， 外科学， 2010， 博士

【摘要】 目的:肿瘤干细胞是存在于肿瘤组织中具有自我更新和分化潜能的细胞,是肿瘤形成的起始细胞,维持肿瘤生长,是肿瘤复发转移的根源。通过对肿瘤干细胞的分选鉴定可以使我们能更清晰认识其特征,从而寻找新的方法来治疗肿瘤,提高治愈率。肿瘤干细胞理论认为肿瘤是由肿瘤干细胞异常增殖、分化而形成的,常规治疗仅能消灭增殖期的非致瘤性肿瘤细胞,从而使肿瘤缩小甚至消失,但是当治疗停止后,具有耐药性的肿瘤干细胞再次增殖形成肿瘤,并像“蒲公英”一样到处播散。如果我们能去除乳腺癌干细胞,肿瘤将会永远消退,从而达到根治肿瘤的目的。近年来,免疫治疗正逐渐成为肿瘤综合治疗的研究热点,有望成为肿瘤治疗的又一主要手段。免疫治疗中常用的活性细胞有:淋巴因子激活的杀伤细胞(LAK)、肿瘤浸润淋巴细胞(TIL)、细胞因子诱导的杀伤细胞(CIK)和特异性细胞毒T淋巴细胞(CTL)。有报道提出,利用树突状细胞(dendritic cell, DC)的高抗原递呈活性,将乳腺癌细胞抗原肽负载DC,从而激活机体免疫反应,可促进抗瘤效应和特异性。树突状细胞作为唯一的专职抗原递呈细胞(antigen processing cell, APC),处于免疫反应的中心地位。非成熟的DC捕获抗原后被激活成为成熟的DC,其细胞表面表达MHCⅠ类和Ⅱ类分子、共刺激分子B7-1(CD80)和B7-2(CD86)、黏附分子CD54(ICAM-1)和CD50(ICAM-3)等,并将抗原肽递呈给CD4+和CD8+T细胞,诱导其成为特异性细胞毒性T细胞(CTL),分泌细胞因子(如IL-12等),产生Th1型免疫应答而发挥抗肿瘤作用。本研究拟采用流式细胞仪测定乳腺癌MCF-7细胞系中CD55高表达(CD55hig)细胞平均荧光密度值,并分选CD55hig细胞,分析其是否具有肿瘤干细胞特性,并以此来检测原发性乳腺癌组织中乳腺癌干细胞的比例,从而寻找一种从乳腺癌组织中分离肿瘤干细胞的方法;术中摘取乳腺癌患者腋窝淋巴结,从腋窝区域引流淋巴结单个核细胞中,诱导培养DCs和肿瘤抗原特异性CTLs,通过体外杀伤实验,为乳腺癌干细胞的治疗提供新的方法和理论依据。方法:第一部分乳腺癌MCF-7细胞系中CD55hig亚群生物学特性分析培养乳腺癌MCF-7细胞系,加入核酸染料Hoechst33342和Verapami,流式细胞仪检测SP亚群和MP亚群细胞比例,并分离SP亚群和MP亚群细胞,以CD55单克隆抗体标记SP干细胞和MP亚群细胞,测定SP干细胞和MP亚群细胞的CD55平均荧光密度值,再以CD55单克隆抗体标记未分选MCF-7细胞,测定CD55hig细胞所占比例,并分选收集CD55hig细胞,检测其细胞贴壁率、克隆形成率和细胞周期等生物学特性,测定CD55hig细胞是否具备肿瘤干细胞特性。第二部分化学药物及免疫治疗对乳腺癌MCF-7细胞系中CD55hig亚群的杀伤研究用不同血药浓度(10%、25%、50%、75%和100%)的化疗药物多西他赛、表阿霉素和氟尿嘧啶杀伤乳腺癌MCF-7细胞,以杀伤效果最高的血药浓度的药物来杀伤CD55hig细胞和CD55low细胞,酸性磷酸酶法测定活细胞数检测其杀伤效应;抽取人外周血,淋巴细胞分离液分离收集单个核细胞,以IFN-γ(1500 U/ml)、抗CD3单抗(100 ng/ml)、IL-1α(100 U/ml)和IL-2(1000 U/ml)诱导培养,流式细胞检测CIK细胞的表型分子CD3、CD4、CD8和CD56表达,培养14天后收集CIK细胞,以效应细胞比靶细胞40:1杀伤CD55hig细胞和CD55low细胞,酶标检测仪检测杀伤率;术中严格无菌摘取乳腺癌引流淋巴结1~2枚,用机械法获取淋巴细胞悬液,并用淋巴细胞分离液分离单个核细胞,以RPMI1640完全培养基重悬、培养2h,贴壁细胞以rhGM-CSF(1000U/ml), rhIL-4(200U/ml)和TNF-α(200U/ml)联合培养诱导DCs;非贴壁细胞,加入rhIL-2(200U/ml)培养为TDLNCs。分选收集MCF-7细胞中CD55hig细胞,通过冻融法制备肿瘤抗原,负载DCs,将后者与TDLNCs共培养,以诱导肿瘤特异性CTLs。分别培养至第1天和第7天收获DCs,流式细胞分析仪检测其CD1a、CD83和CD86细胞表型,用非放射性细胞毒分析试剂盒,检测CTLs细胞对CD55hig细胞和CD55low细胞体外杀伤活性,分析经诱导的DCs功能和CTLs杀伤特异性。第三部分原发性乳腺癌组织中CD55hig细胞检测及其临床意义选取2008年2月至2009年1月在我科收治的乳腺癌患者45例。术中切取部分乳腺癌组织,机械法获得细胞悬液,加入抗CD55抗体和抗CK19抗体,对原发性乳腺癌单细胞悬液进行免疫荧光标记,以流式细胞学方法进行检测CD55高表达(CD55hig)群细胞的比例。结合患者的临床资料,分析乳腺癌患者CD55高表达与临床病理学指标的关系。结果:1 MCF-7细胞染色Hoechst3342染料后,流式细胞分析SP比例为4.34±0.59%,而加入维拉帕米后显著减低为0.1±0.07%。2收集SP和MP细胞,分别标记CD55单克隆抗体后,流式细胞术测定SP细胞CD55的平均荧光密度值为100.85±4.57,MP细胞CD55的平均荧光值为50.51±4.75;CD55单克隆抗体标记未分选的MCF-7细胞,流式细胞仪分析CD55hig细胞比例为2.12±0.39%。3 CD55hig和CD55low细胞接种后6h、12h和18h,贴壁率分别为29.53±1.20%、46.40±2.99%、77.2±1.07%和40.87±1.55%、55.40±2.46%、85.10±3.36%,CD55hig细胞贴壁率低于CD55low亚群细胞,P<0.05,差异有统计学意义;接种24h后,CD55hig细胞贴壁率为95.5±1.1%,CD55low细胞贴壁率为96.3±0.96%,P>0.05,无统计学差异。接种12h后,显微镜下观察CD55hig细胞多为球形呈悬浮状态,而CD55low细胞已贴壁为梭形。4 CD55hig细胞和CD55low细胞均具有一定克隆形成的能力,CD55hig细胞在培养一周后克隆形成率为20.04±1.07%,低于CD55low细胞27.14±1.07%,P=0.000,差异有统计学意义。5流式分析发现,CD55hig细胞中G0/G1静止期细胞占85.4±3.37%,明显高于CD55low细胞(58.6±2.55%)及MCF-7细胞(70.73±4.21%),P值均<0.05 ,有统计学差异。CD55hig细胞处于增殖期的S期细胞仅占13.93±3.45% ,明显低于CD55low细胞( 40.36±2.56% )及MCF-7细胞(24.93±2.86%),P值均<0.05,差异有统计学意义。6不同血药浓度化疗药物对MCF-7细胞杀伤效果不一,多西他赛组5个浓度中以10%血药浓度对MCF-7细胞杀伤效果最高57.3±4.75%,表阿霉素组和氟尿嘧啶组以100%血药浓度最高,分别为40.2±2.46%和26.6±2.44%,高于其他浓度杀伤效果。7 (10%血药浓度)多西他赛、(100%血药浓度)表阿霉素和(100%血药浓度)氟尿嘧啶对CD55hig亚群细胞杀伤率为28.5±0.04%、18.4±0.02%和12.4±0.01%,明显低于其对MCF-7细胞杀伤率,P值均小于0.05,差异有统计学意义;对CD55low亚群细胞杀伤率为58.8±0.06%、45.4±2.28%和34.3±0.01%,P值均大于0.05,无统计学差异。8 CIK细胞对三组细胞都有杀伤,CIK细胞对MCF-7、CD55hig亚群细胞和CD55low亚群细胞杀伤率为39.15±3.30%、42.72±4.36%和46.41±4.67%, CD55low亚群细胞组杀伤率与MCF-7组(P=0.000)和CD55hig亚群细胞组杀伤率(P=0.046)相比,P值均小于0.05,差异有统计学意义;MCF-7组和CD55hig亚群细胞组杀伤率相比,P=0.053,无统计学差异。9诱导前的TDLNCs中,CD3+和CD8+细胞含量分别为73.93±2.18和32.78±3.21%;诱导后DC-Ag-TDLNC组中CD3+和CD8+细胞含量分别为82.67±2.79%和62.54±2.51%,诱导后CD3+、CD8+T细胞含量均明显升高, P<0.01。诱导前TDLNCs中CD4+细胞含量为27.3±2.58%;CD4+细胞含量无明显变化,P>0.05。10特异性CTL对CD55hig、CD55low和MCF-7细胞杀伤率分别为52.86±4.45%、22.41±2.83%、21.67±4.15%,CD55hig组明显高于CD55low和MCF-7组细胞的杀伤率,P<0.001,CD55low和MCF-7组细胞的杀伤率相比较无统计学意义,P=0.629。11多西他赛(10%血药浓度)、CIK细胞和特异性CTL细胞对CD55hig细胞的杀伤率分别为:28.5±0.04%、42.72±4.36%和52.86±4.45%;特异性CTL细胞组与CIK细胞组和多西他赛(10%血药浓度)组相比较,P均为0.000,CIK细胞组与多西他赛(10%血药浓度)组相比较,P=0.000。12本实验测得原发性乳腺癌肿瘤组织中CD55hig比例为0.21±0.20%,较乳腺癌细胞株MCF-7所测CD55hig的比例2.12±0.39%低。13乳腺癌患者组织中CD55hig比例随着淋巴结转移数目增加而增高,0个腋淋巴结转移组、1～3个腋淋巴结转移组和≥4个腋淋巴结转移3组间比较,P =0.000,差异有统计学意义,三组之间两两比较,均有统计学差异。14乳腺癌患者组织中C-erbB2(++-+++)组CD55hig比例为0.277±0.034%,较C-erbB2(-)组的CD55hig比例(0.028±0.005%)有明显的升高,P=0.000,差异有统计学意义。15 CD55hig比例在乳腺癌不同病理类型之间、不同临床分期之间、ER(-)与ER(+-+++)组之间、PR(-)与PR(+-+++)组之间、Ki67(-),Ki67(+-+++)组之间的差异均无统计学意义,P>0.05。16本研究发现,CD55hig比例在术前化疗组与未化疗组的比较中无统计学差异,P =0.448。结论:1以核酸染料Hoechst33342标记乳腺癌细胞株MCF-7细胞,流式细胞检测发现有淡染的SP细胞,verapamil阻断后此部分细胞明显减少,SP细胞为肿瘤干细胞。2 CD55单克隆抗体标记分选出的SP和MP细胞,检测发现SP细胞CD55荧光值高于MP细胞的荧光值,以此值检测CD55单克隆抗体标记的未分选的MCF-7细胞,发现存在少量的CD55hig细胞。3 CD55hig细胞大多数处于静止期、不贴壁呈悬浮状态和克隆遗传性,CD55hig细胞具有肿瘤干细胞生物学特性。4三种化疗药物对乳腺癌细胞系MCF-7、CD55hig和CD55low细胞均有杀伤效果,但是由于CD55hig细胞绝大多数处于细胞分裂静止期,化疗药物对其杀伤效果不显著。5 CIK细胞对乳腺癌细胞系MCF-7、CD55hig和CD55low细胞杀伤效应有显著差异,CIK细胞杀伤效果高于化疗药物,但是没有特异性杀伤效果。6经CD55hig细胞冻融抗原刺激诱导的DCs可以使TDLNCs增殖、分化为肿瘤抗原特异性CTLs,针对CD55hig细胞具有较强的杀伤效应,而对其他类型的肿瘤细胞无明显杀伤效应。7特异性CTL细胞对CD55hig细胞具有特异性杀伤效果,其杀伤效果高于CIK细胞和化疗药物,为乳腺癌干细胞的治疗提供新的方法和依据。8流式细胞检测乳腺癌组织中CD55hig细胞比例为0.21±0.20%。9乳腺癌组织中CD55hig细胞比例与腋淋巴结转移的数量、C-erbB2的表达呈正相关联性,与病理类型、临床分期、ER、PR、KI67表达及术前化疗无明显相关,推测CD55hig比例与乳腺癌的转移和病情的进展有相关联性。更多还原

【Abstract】 Objective: Tumor stem cells, which reside in tumor tissue with self renewal and differentiated potentiality, are the initiator cells of tumor’s formation and maintain tumor growth,induce recurrence and metastatic. Trough separation and identifacation on breast cancer stem cell, we could recognise those characteristic clearer and find new method to therapy tumor and elevate cure rate. In theory of tumor stem cell, tumor forms of tumor stem cell’s abnormal generation and differentiation. Conventional therapy only eliminate the non-tumorigenic tumour cell in multiplication period to make the tumor diminute even disappear. However, when stop treating, the tumor stem cell with chemical sproof could generate to tumor again and disseminate like dandelion. IF we remove the tumor stem cell, the tumor will extinct forever and radical cure the tumor. Recently, immunotherapy will become another major method of cancer treatment. The effective cells using in immunotherapy include lymphakine active killer cells (LAK), tumor-infiltrating lympjocytes(TIL), cytokine induced killer cells(CIK), and specific cytotoxicity T lymphocytes(CTL). The researches have showed that dendritic cell(DC) can greatly process tumor antigen, DC loaded by tumor antigen can activate host immune, promote the activity and specificity of CTL to kill tumor cells. DC, as the professional antigen processing cells, play a center role in immune reaction. The im-matured DCs will be activated to matured DCs after captured antigen, which express MHC classⅠand classⅡmolecule、co-stimulatory molecule B7-1(CD80) and B7-2 (CD86)、adhesion molecule CD54 (ICAM-1) and CD50 (ICAM-3) in the cell surface. After presenting the antigen peptide to CD4+ and CD8+ T cells, DCs induce the T cells become to specific cytotoxicity T cells,which can excrete cytokines and produce Th1 type immune response to educe the anti-tumor effect. We adopt flow cytometry determing the mean fluorecence density of cell highly exprssing CD55 in breast cancer MCF-7 cell line and sorting CD55hig cells, to analyze whether the CD55hig cells possess characteristics of cancer stem cells, detect the existence and the ratio in the primarily breast cancer and find a new method for isolate breast cancer stem cells in breast cancer tumor tissue. DCs and specific CTLs were induced from single-nucleus cells of axillary lymph nodes and analyzed by killing test in vitro. The aim is hoping to find a new method to treat breast cancer stem cells.Method: Part one: Biological characteristics of CD55hig side population in human breast cancer line MCF-7.In this rsearch, we cultivated the human breast cancer MCF-7 cells and addined nucleic dye Hoechst33342 and verapami, detected the ratio of SP and MP subpopulation cell by flow cytometry and separated them. Then SP and MP cells were labeled with anti-CD55 monoclonal antibody and detected the mean fluorescent intensity. We used anti-CD55 monoclonal antibody labling unsorted MCF-7 cells, detected the proportion of CD55hig side population cells and sorted them to detect their biological characteristics such as cell adherence efficiency, cloning efficiency and cell cycle, to analyze whether the CD55hig cells possess characteristics of cancer stem cells.Part two: Killing activity of chemotherapeutics and immunity on CD55hig side population in human breast cancer line MCF-7.Human breast cancer line MCF-7 cells were killed by docetaxel, epirubicin and fluorouracil in different blood drug level (10%, 25%, 50%, 75% and 100%). Then choosed the maximal killing blood drug level chemotherapeutics to kill CD55hig cells and CD55low cells, detected the lethal effect by Acid Phosphatase Assay; We drew-off human peripheral blood , abrupted and gathered the mononuclearcell with lympholeukocyte separating medium,cultured them with IFN-γ(1500 U/ml)、anti-CD3(100 ng/ml)、IL-1α(100 U/ml) and IL-2(1000 U/ml) and detected the phaenotype molecule of CIK cells such as: CD3, CD4, CD8 and CD56. After 14 days, we collected the CIK cells and kill the CD55hig cells and CD55low cells with effector cell vs target cell 40:1, the kill ratio were detected by with non-radioaction cytotoxic analytical reagent kite; 1 or 2 lymphonodes strictly with asepsis were taked in operation. Then it was separated single-nucleus cells(SNC) in albuginea rete. The SNC from lymphonodes were cultured in 10% FCS RPMI1640. After adherencing 2 hours, the attached cells were cultured with rhGM-CSF(1000U/ml), rhIL-4(200U/ml) and TNF-α(200U/ml) to be induced into DCs. The unattached cells were cultured with rhIL-2(200U/ml) induce into tumor draining lymph node cells(TDLNCs). CD55hig cells seperated and collected from MCF-7 cells were made for the breast cancer freeze-thawing antigen. DCs were stimulated by the cancer freeze-thawing antigen in order to load the tumor antigen, then, were co-cultured with TDLNCs to derivation tumor antigen specific CTLs. DCs suspension were harvested at the 1st day and the 7th day in vitro culturing and co-culturing with PE-CD1a、PE-CD83、FITC-CD86 MoAb, the cytophenotype was detected with flow cytometry(FCM). The cytotoxicity of the CTLs to CD55hig cells and CD55low cells were determined with non-radioaction cytotoxic analytical reagent kite, to analysis the function of differentiated DCs and specificity of the CTLs.Part three: The detection of stem-like cells in primary breast cancer tissue and its clinical significance.Chosen 45 Patients with breast cancer which came from the 1st surgery department in my hospital, from February in 2008 to January in 2009. Small amounts breast cancer tissue were taked in operation, Then it was separated single-nucleus cells (SNC) in albuginea rete. We joined in fluid of both immunofluorescence anti-CD55 and anti-CK19 to label the cells and detected the ratio of the high expression of CD55(CD55hig) by flow cytometry. Combined the clinical data of patients , we analyzed the experimental data. Results:1. After dyed by Hoechst33342,the percentage of SP cells contained in MCF-7 was 4.34±0.59%. The percentage of SP significantly degrated when blocked by verapamil(0.1±0.07%).2. The mean fluorescent intensity of SP and MP cells labeled with anti-CD55 monoclonal antibody was 100.85±4.57 and 50.51±4.75, respectively. Unsorted MCF-7 cells were labeled with anti-CD55 monoclonal antibody. The proportion of CD55hig cells was 2.12±0.39%.3. The adherence efficiency of CD55low cells at 6h, 12h, 18h and 24h were 40.87±1.55%, 55.40±2.46%, 85.10±3.36% and 96.3±0.96%, the adherence efficiency of CD55hig cells at 6h, 12h, 18h and 24h were 29.53±1.20%, 46.40±2.99%, 77.2±1.07% and 95.5±1.1%. The adherence efficiency of CD55low cells were higher than CD55hig cells at 6h, 12h and 18h. There was statistical difference between the two groups, (P<0.05). The adherence efficiency of CD55hig cells and CD55low cells at 24h didn’t appear marked difference( P>0.05); the CD55hig cells were mostly spherica and CD55low cells were spindle after planted 12h.4. The Cloning efficiency of CD55hig cells at one week was 20.04±1.07%, which was lower than CD55low cells. There was statistical difference between the two groups(P<0.05).5. The percentage of G0/G1 in CD55hig cells was 85.4±3.37%, which was higher than CD55low cells(58.6±2.55%) and unsorted MCF-7 cells (70.73±4.21%), there was statistical difference between them, P<0.05. The percentage of S in CD55hig cells was 13.93±3.45%,which was lower than CD55low cells(40.36±2.56%) and unsorted MCF-7 cells (24.93±2.86%), there was statistical difference between them, P<0.05;6. The killing effect of chemotherapeutics on MCF-7 cells in different blood drug level were not same. In docetaxel group, the maximal blood drug level(10% blood drug level) killing MCF-7 cells was 57.3±4.75%. In epirubicin and fluorouracil group, the maximal blood drug level(100% blood drug level) killing MCF-7 cells was 40.2±2.46% and 26.6±2.44%. 7. The killing effect on CD55hig cells of docetaxel(10% blood drug level), epirubicin(100% blood drug level) and fluorouracil(100% blood drug level) were 28.5±0.04%, 18.4±0.02% and 12.4±0.01%, which were lower than on MCF-7 cells Obviorsly, there was statistical difference between them, P<0.05. The killing effect on CD55low cells of docetaxel(10% blood drug level), epirubicin(100% blood drug level) and fluorouracil(100% blood drug level) were 58.8±0.06%, 45.4±2.28% and 34.3±0.01%. There were no significant difference, P>0.05.8. The killing ratio on MCF-7 cells, CD55hig cells and CD55low cells of CIK cells were 39.15±3.30%, 42.72±4.36% and 46.41±4.67%. The killing ratio of CD55low cells group compared to MCF-7 cells group(P=0.000) and CD55hig cells group(P=0.046), there was statistical difference between them, P<0.05, there were significant difference. Compared the killing ratio of MCF-7 cells group with CD55hig cells group, P=0.053, there were no significant difference.9. The percentage of CD3+ and CD8+ T cells in TDLNCs were 73.93±2.18 and 32.78±3.21% before stimulation with tumor antigen. and were 82.67±2.79% and 62.54±2.51% after stimulation with rhIL-2 and Ag-DCs. The percentage of CD3+ and CD8+ T cell could be increased by the Ag-DCs. There was statistical difference between them, P<0.01. The percentage of CD4+ T cells in TDLNCs was 27.3±2.58% before stimulation with tumor antigen, however the percentage were 17.49±4.21% after stimulation. The percentage of CD4+ T cells wasn’t heighten after induced by comparison, there were no significant difference, P>0.05.10. The killing ratio on MCF-7 cells, CD55hig cells and CD55low cells of special CTL cells were 52.86±4.45%,22.41±2.83% and 21.67±4.15%. The killing ratio of CD55hig cells group was higher than MCF-7 cells group and CD55low cells group, there was statistical difference between them, P<0.001; Compared MCF-7 cells group with CD55low cells group, P=0.629, there were no significant difference.11. The killing ratio of docetaxel(10% blood drug level), CIK cells and special CTL cells on CD55hig cells were 28.5±0.04%, 42.72±4.36% and 52.86±4.45%; Compared special CTL cells group with docetaxel(10% blood drug level) and CIK cells group, P=0.000. Compared docetaxel(10% blood drug level) and CIK cells group, P=0.000.12. The mean of the rate of CD55hig cells in primary breast cancer tissue was 0.21%, which was lower than the rate in breast cancer MCF-7 cells (2.12±0.39%).13. The ratio of CD55hig cells were increased with increased the number of the transferring axillary lymph nodes. There were statistically significance among non-metastatic axillary lymph node group、one to three axillary lymph nodes metastasis group and more than three axillary lymph nodes metastasis group, P =0.000.14. The ratio of CD55hig cells in C-erbB2(++~+++) group was 0.277±0.034%, higher than the ratio of CD55hig cells in C-erbB2(-~+) group the difference was statistical significant, P =0.000.15. There were no relationship between the ratio of CD55hig cells and pathology category, clinical stage, ER(-) and ER(+~+++), PR(-) and PR(+~+++), Ki-67(-) and Ki-67(+~+++), P>0.05.16. In the research, there was no relationship between the rate of CD55hig cells and preoperative chemotherapy,P =0.448. Conclusions:1. Hypochromatic sp cells are detect in human breast cancer line MCF-7 cells dyed by Hoechst33342. The percentage of SP cells obviously decreased when blocked by verapamil. SP cells contain cancer stem cells.2. The mean fluorescent intensity of SP is higher than MP cells when labeled with anti-CD55 monoclonal antibody. Unsorted MCF-7 cells are labeled with anti-CD55 monoclonal antibody by flow cytometer, we find a small amount of CD55hig cells.3. CD55hig cells are more nonadherent, quiensent and clonogenetic. So CD55hig cells have biological characteristics of cancer stem cells. 4. Three chemotherapeutics have certainly killin effect on MCF-7 cells, CD55low cells and CD55hig cells. But most CD55hig cells don’t divide, the killing effect of chemotherapeutics on CD55hig cells is not significant.5. Although the killing effect of CIK cells on MCF-7 cells, CD55low cells and CD55hig cells are significant, which are higher than chemotherapeutics, the special killing effect are not significant.6. DCs induced by CD55hig cells freeze-thawing antigen could make TDLNCs generate to tumor antigen special CTLs, which have significant killing effect on CD55hig cells and non significant killing effect on other category tumor cells.7. Special CTLs have special killing effect on CD55hig cells, heigher than CIK cells and chemotherapeutics.This provids new method to cure breast cancer stem cells.8. The rate of CD55hig cells in breast cancer tissue is 0.21±0.20%.9. The rate of CD55hig cells in breast cancer tissue relates to the axillary lymph nodes metastasis and C-erbB2 protain expressions. There are no relationship to pathology category, clinical stage, expression of ER, PR and Ki-67 and preoperative chemotherapy. Those suppose that the rate of CD55hig cells relates to the metastasis and advancement of breast cancer.更多还原