术前放化疗和未治疗口腔鳞癌患者TIL增殖力的比较

1、    术前放化疗和未治疗口腔鳞癌患者TIL增殖力的比较        【摘要】目的探讨术前做过放化疗口腔鳞癌患者TIL在体外与rIL-2共培养是否具有继续增殖的能力。方法从15例口腔鳞癌原发灶中分离TIL，用1000u/ml rIL-2与其培养，比较术前放化疗和未治疗患者TIL的增殖能力。结果术前未治疗组比治疗组扩增快，提前1周达增殖高峰，但4周以后术前治疗组也显示出继续扩增的趋势。结论从术前放化疗患者肿瘤组织中分离的TIL，在体外经rIL-2刺激后仍能继续扩增。【关键词】

2、鳞状细胞癌； 口腔； 肿瘤浸润淋巴细胞； 增殖【中分类号】R739.81【文献标识码】A【文章编号】10031634（2000）04022002 Comparable study of proliferation of Tumor Infiltrating Lymphocytes from OSCC previously receiving and without treatmentYang Hongyu, Li Jinrong,Luo Zhuan(Department of Oral and Maxillofacial Surgery, Third Affiliated Hospital,

3、Sun Yat-sen University of Medical Science,Guangzhou,510630)【Abstract】ObjectiveTo understand whether TIL obtained from patients receiving radiotherapy and/or chemotherapy before operation could continue to proliferate in the presence of rIL-2 in vitro. MethodsTIL were isolated from fresh tumor tissue

4、s in 15 patients with OSCC. Then TIL were cultured in the presence of 1000u/ml rIL-2. The proliferation of TIL from primary OSCC previously treated with radiotherapy or/and chemotherapy and without receiving any treatment was compared. ResultsTIL from patients previously treated expanded slower and

5、reached proliferation peak in delaying one week than from patients without receiving any treatment,but the former also exhibited continuing expansive trend after four weeks. ConclusionTIL obtained from patients receiving radiotherapy and/or chemotherapy before operation could continue to proliferate

6、 in the presence of rIL-2 in vitro.【Key words】Squamous cell carcinoma; Mouth; Tumor infiltrating lymphocyte; Proliferation口腔鳞癌患者的免疫功能缺陷常较其它恶性肿瘤更为严重。特别是经历了放化疗后机体的免疫功能可能会进一步降低。如果从术前放化疗患者手术标本中分离肿瘤浸润淋巴细胞（Tumor Infiltrating Lymphocyte,TIL)，在体外与rIL-2共培养能恢复增殖能力及杀伤活性，那么将TIL输入体内，将会提高机体的免疫功能，是常规治疗极好的补充。本文拟对此设

7、想的可行性进行初步探讨。材料与方法1.标本来源1997.31998.2间住院手术患者，经病理证实为口腔鳞癌，共15例。术前放化疗6例，未治疗9例。2.方法2.1TIL的分离与培养用酶消化和不连续梯度密度的淋巴细胞分离液将肿瘤细胞和肿瘤浸润淋巴细胞分离。用10胎牛血清的RPMI-1640培养液调节细胞浓度为2.5×105ml，按1000u/ml加入rIL-2，置37，5CO2培养箱中，每隔34天调整细胞浓度，并补充rIL-2以维持培养浓度。2.2TIL增殖取初始分离的TIL细胞悬液1滴和2台盼兰液1滴混合，置3分钟，显微镜下计数200个细胞，活细胞不着色，死细胞核呈蓝色，计算活细胞比例

8、。用血细胞计数板计数，按下式计算：原液每次换液时计数1次，观察其增殖。结果TIL在培养的24小时内即开始聚集成团块，多呈悬浮生长。第57天数目显著增加。术前作过治疗的TIL（6例）比未作治疗的TIL（9例）扩增慢，后者在第3周时即达高峰，前者到第4周才达高峰，扩增延迟。两组TIL增殖情况见1。1术前治疗与未治疗组TIL增殖比较讨论TIL在肿瘤原位主要存在于肿瘤间质内，以T细胞为主，在多数病例中，CD+8T细胞多于CD+4T细胞，TIL中有部分NK细胞，所有TIL细胞在肿瘤原位一般处于免疫抑制状态。研究发现新鲜分离的TIL和肿瘤原位TIL相仿。新鲜分离的TIL免疫活性比PBL低。在肿瘤原位和新鲜

9、分离的TIL均处于免疫抑制状态。过继免疫治疗中转输足够的TIL的细胞数是获得较好疗效的重要因素13。TIL经rIL-2激活后，一般在含有500u-1000umlrIL-2的完全培养基中可持续增殖，在不断补充养分和rIL-2时TIL可长期培养。未经活化的TIL几乎不增殖，活化后的TIL一般条件下可扩增几十至几百倍4，5。有研究表明，肿瘤病人经放化疗后，外周血中CD4T、CD8T细胞显著减少，且放化疗后，外周血中淋巴细胞的功能也受影响6，7。为了弄清放化疗后的患者TIL是否可以被激活、增殖和恢复其功能并进行过继免疫治疗，我们从术前经过放化疗的6例鳞癌患者中分离出TIL，并与未治疗的TIL相比。有趣

10、的是，其结果与我们预料的相反，未经放化疗的TIL其增殖高峰较治疗患者的TIL提前1周，说明术前经放化疗患者TIL增殖能力恢复较慢，但在4周后仍可继续增殖。提示：对术前经放化疗患者，如果TIL的杀伤活性也能恢复，那么转输TIL也是可行的，特别是对放化疗不敏感的患者应用，受益会更大。术前经放化疗患者TIL的杀伤活性的恢复有待于进一步的研究。作者单位：杨宏宇(中山医科大学附属第三医院口腔颌面外科 510630广州)刘国萍(中山医科大学附属第三医院口腔颌面外科 510630广州)黄伟民(中山医科大学附属第三医院口腔颌面外科 510630广州)李金荣(湖北医科大学口腔医学院)罗娟(深圳市中心医院口腔科)

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