【作者】 刘秋亮；

【导师】 王家祥； 范应中；

【作者基本信息】 郑州大学 ， 小儿外科， 2004， 硕士

【摘要】 背景与目的： 肿瘤生物治疗是通过肿瘤宿主防御机制或生物制剂的作用以调节机体自身生物学反应，从而抑制或消除肿瘤生长的治疗方法。肿瘤生物治疗是当前肿瘤治疗的前沿和热点。以肿瘤免疫治疗为代表肿瘤生物治疗已成为继手术、化疗和放疗后肿瘤治疗的第四种模式。过继性细胞免疫治疗(adoptire cellularimmunotherapy，ACI)或过继性免疫治疗(adoptive immunotherapy，AIT)是肿瘤生物治疗的一种，是指向肿瘤患者转输具有抗肿瘤活性的免疫细胞，直接杀伤肿瘤细胞或激发机体免疫效应，从而达到治疗肿瘤的目的。目前，ACI／AIT已被认为是有诸多优势的恶性肿瘤的治疗方法。 LAK(lymphokine activated killer)细胞和TIL(tumor infiltratinglymphocyte)细胞都是ACI／AIT的一种，但是LAK和TIL都因其有比较明显多系统的毒副作用，或其前体细胞分离过程复杂，或培养时间长等原因而限制了它们应用。CD3AK(anti-CD3 antibody induced activated killer cells)是抗CD3单克隆抗体(anti-CD3 antibody，CD3McAb)和重组人白细胞介素-2(rIL-2)诱导产生的免疫效应细胞，目前CD3AK细胞正因其快速扩增，长期存活，高抗肿瘤活性及低毒副作用的优点而倍受重视。但是哪一种来源的细胞作为CD3AK细胞的前体细胞，扩增更快，杀伤肿瘤细胞的活性更强却少有研究，同一种来源的CD3AK细胞对不同肿瘤的杀伤活性有何区别，杀伤机制有何不同也都少有研究。本实验比较脐血(cord blood，CB)和外周血(peripheral blood，PB)来源的CD3AK细胞在扩增速度、杀伤活性及杀伤机制方面的区别，并比较CB来源的CD3AK细胞对不同肿矛粼洲大学2004年硕毖研疙全毕业葱之不间块穿C刀3月K细对功徽瘤细胞的杀伤活性的差异。材料和方法: 脐带血来自正常分娩的健康胎儿胎盘血，共16例;PB取自健康成年人，共16例。CB及PB经淋巴结细胞分离液密度梯度离心获得脐血单个核细胞(cordblood mononuClear eell，CMNC)和外周血单个核细胞(peripheral bloodmononuclear cell，PMNC)，在CD3MeAb和rIL一2联合诱导下获得CD3AK细胞，计算并比较不同培养时期的CD3AK细胞的扩增倍数;用2种 CD3AK细胞作用于不同的肿瘤细胞株，MTT法比较CD3AK细胞对不同肿瘤细胞株的杀伤活性，电镜下观察被杀伤后肿瘤细胞的超微结构变化。统计学分析:应用SPSSIO.0统计软件包的t检验和重复测量的方差分析对统计数据进行分析，a=0 .05为显著性水准。结果: (1) CD3AK细胞增殖动力学观察和比较:CB与PB来源的CD3AK细胞在培养的第4天扩增速度前者稍快，二者相比较差异无统计学意义(乃0.05);以后随培养时间的延长，CB CD3AK细胞的扩增倍数均大于PB来源的CD3AK细胞，二者比较差异具有统计学意义(尸<0.05)，CD3AK细胞的增长趋势至少可以维持3周。 (2)不同来源的CD3AK细胞杀瘤活性的比较:CD3AK细胞与食管癌细胞株Ecal09共育结果发现，培养至第4天的CD3AK细胞既有了一定的杀伤活性，培养至第12天杀伤活性达到高峰，其杀伤活性可维持3周以上。CB来源的CD3AK细胞对Ecal09肿瘤细胞株细胞的杀伤活性在不同培养时间均高于PB来源的CD3AK细胞，二者比较差异有统计学意义(尸<0.05)，二者的杀伤活性均可维持3周以上。 (3)CB CD3AK细胞对不同的肿瘤细胞株(食管癌细胞株Eca109，胃癌细胞株BGC823，肺癌细胞株A549，白血病细胞株HL一60)的杀伤活性是不同的，CB CD3AK细胞对3种实体肿瘤细胞株的杀伤率无明显差异(乃0.05)，而对HL一60的杀伤率显著高于对3种实体瘤细胞株的杀伤率有明显的差异，其差异有统计学意义(P<0.05)。 粼种丈.学2004字烫亡解芜全月毕业老戈不词璐零c刀及刁K潮撇时粼办犷著冲决份籽璧必斑穷 (4)不同来源的CD3AK细胞对肿瘤细胞株细胞杀伤机制的比较:CB来源的CD3AK细胞可能主要以直接杀伤肿瘤细胞株细胞从而使其发生坏死的方式为主;而PB来源的CD3AK细胞主要以诱导肿瘤细胞株细胞发生调亡的方式为主，二者比较差异具有统计学意义(尸<0.01)。结论: (1) CB来源的CD3AK细胞在同样条件下的扩增速度和杀伤活性均明显高于PB来源的CD3AK细胞。 (2)2种CD3AK细胞杀伤肿瘤细胞的机制是有区别的，CB CD3AK细胞以使肿瘤细胞发生坏死为主，而PB CD3AK细胞则以诱导肿瘤细胞株细胞发生凋亡为主。 (3)同一来源的CD3AK细胞对不同的肿瘤细胞株细胞的杀伤活性是不同的，不同组织起源的肿瘤细胞株细胞对同一CD3AK细胞的敏感性是不同的。更多还原

【Abstract】 Background and ObjectiveTumor’s biological therapy is one of the methods of suppressing or removing tumor’s growth through tumor’s defense mechanism or the biological pharmaceutics and it has become the focus and front line of tumor’s therapy, now. Tumor’s biological therapy presented by adoptive cellular immunotherapy (ACI/AIT) has become the fourth model of tumor’s treatment following operation, chemotherapy and radiotherapy. ACI/AIT is one of the methods of tumor’s biological therapy, which is to inject activated immunized cells, which can kill tumor cells directly or activate immunity effect, to deal with tumor. Now, tumor’s biological therapy has been thought one of the effective treatments of malignant tumor with such advantages.LAK (lymphokine activated killer) cells and TIL (tumor infiltrating lymphocyte) cells is one of ACI / AIT. But LAK and TIL cells all were limited to be used because of their obvious multi-system-side-effect, or complex process of separating original cells, or lone time of culturine. CD3Ak cells, which is induced bv anti-CD3 monoclonalantibody and rIL-2, is attaching importance based on it’s rapid expansion, long time of living, strong anti-tumor activity and low side -effect. But there is few research on which origin cells is the best choice to obtain rapider speed of expansion, and stronger activity of killing tumor cells, nor does what is the difference of killing activity among different tumor cells line killed by one kind of CDSAk cells, and nor does the killing mechanism. This experiment plans to compare the expansion speed, cytotoxicity between two-origin CD3AK cells, from umbilical cord blood and peripheral blood, and the difference of umbilical cord blood CDSAk cells’cytotoxicity.Material and Methods16 pieces of umbilical cord blood and peripheral blood come from normal healthy fetus’ peripheral and healthy adult respectively. Obtain cord blood mononuclear cells (CMNC) and peripheral blood mononuclear cells (PMNC) by density gradient centrifugation, culture and harvest CDSAK cells induced by CDSMcAb and rIL-2, calculate and compare the expansion times of CDSAK cells from two origins. Put CDSAK cells and different tumors cells line together cultured, and compare the cytotoxicity of them with MTT. Observe super-microstructure and changes of tumor cells killed. Process the data using f-test and analysis of variance of repetive metrical data with SPSS 10.0 statistical software, a (equals 0.05) was considered significant test level.Results(l).CDSAK cells’ dynamics effect comparison: CDSAK cells from two origins, umbilical cord blood CD3AK cells expansion speed is higher, and there is no significant difference (P>0.05)on the fourth day of culturing. With culture time passing by, umbilical cord blood CDSAK cells’ expansion speed is obviously higher than peripheral blood CD3AK cells’, and there is significant difference (8d, 12d, 16d, 21d,), (P<0.05). The expansion effect can maintain at least three weeks.(2).Comparison of CDSAK cells’ cytotoxicity; put two kinds of CDSAK cells andtumor cells line Ecal09 together cultured, the fourth day’s CD3Ak cells shows some cytotoxicity and reachs the highest level at the twelfth day of culturing, umbilical cord blood CD3AK cells’ cytotoxicity are higher than peripheral blood CD3AK cells at 8d, 12d, 16d, 2Id, and there are significant difference (P<0.05). Two kinds of CD3AK cells can maintain their cytotoxicity at least three weeks(3).Comparison of cytotoxicity mechanism: perhaps umbilical cord blood CD3AK cells kill tumor cells to necrosis mainly, and peripheral blood CD3AK cells induced tumor cells to apoptosis mainly. And there is significant difference (P<0.01).(4).Umbilical cord blood CD3AK cells’ cytotoxicity to different tumor cells(esophageal cancer cells line Ecal09,stomach cancer cells line BGC823, lung cancer cells line A549, and leukemia cells line HL-60): To these tumor cells, the cytotoxicity of umbilical cord blood CD3AK cells were different, there is significant difference between leukemia cells line HL-60’s更多还原