1、    術前放化療和未治療口腔鱗癌患者TIL增殖力的比較        【摘要】目的探討術前做過放化療口腔鱗癌患者TIL在體外與rIL-2共培養是否具有繼續增殖的能力。方法從15例口腔鱗癌原發灶中分離TIL，用1000u/ml rIL-2與其培養，比較術前放化療和未治療患者TIL的增殖能力。結果術前未治療組比治療組擴增快，提前1周達增殖高峰，但4周以后術前治療組也顯示出繼續擴增的趨勢。結論從術前放化療患者腫瘤組織中分離的TIL，在體外經rIL-2刺激后仍能繼續擴增。【關鍵詞】

2、鱗狀細胞癌； 口腔； 腫瘤浸潤淋巴細胞； 增殖【中分類號】R739.81【文獻標識碼】A【文章編號】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口腔鱗癌患者的免疫功能缺陷常較其它惡性腫瘤更為嚴重。特別是經歷了放化療后機體的免疫功能可能會進一步降低。如果從術前放化療患者手術標本中分離腫瘤浸潤淋巴細胞（Tumor Infiltrating Lymphocyte,TIL)，在體外與rIL-2共培養能恢復增殖能力及殺傷活性，那么將TIL輸入體內，將會提高機體的免疫功能，是常規治療極好的補充。本文擬對此設

7、想的可行性進行初步探討。材料與方法1.標本來源1997.31998.2間住院手術患者，經病理證實為口腔鱗癌，共15例。術前放化療6例，未治療9例。2.方法2.1TIL的分離與培養用酶消化和不連續梯度密度的淋巴細胞分離液將腫瘤細胞和腫瘤浸潤淋巴細胞分離。用10胎牛血清的RPMI-1640培養液調節細胞濃度為2.5×105ml，按1000u/ml加入rIL-2，置37，5CO2培養箱中，每隔34天調整細胞濃度，并補充rIL-2以維持培養濃度。2.2TIL增殖取初始分離的TIL細胞懸液1滴和2臺盼蘭液1滴混合，置3分鐘，顯微鏡下計數200個細胞，活細胞不著色，死細胞核呈藍色，計算活細胞比例

8、。用血細胞計數板計數，按下式計算：原液每次換液時計數1次，觀察其增殖。結果TIL在培養的24小時內即開始聚集成團塊，多呈懸浮生長。第57天數目顯著增加。術前作過治療的TIL（6例）比未作治療的TIL（9例）擴增慢，后者在第3周時即達高峰，前者到第4周才達高峰，擴增延遲。兩組TIL增殖情況見1。1術前治療與未治療組TIL增殖比較討論TIL在腫瘤原位主要存在于腫瘤間質內，以T細胞為主，在多數病例中，CD+8T細胞多于CD+4T細胞，TIL中有部分NK細胞，所有TIL細胞在腫瘤原位一般處于免疫抑制狀態。研究發現新鮮分離的TIL和腫瘤原位TIL相仿。新鮮分離的TIL免疫活性比PBL低。在腫瘤原位和新鮮

9、分離的TIL均處于免疫抑制狀態。過繼免疫治療中轉輸足夠的TIL的細胞數是獲得較好療效的重要因素13。TIL經rIL-2激活后，一般在含有500u-1000umlrIL-2的完全培養基中可持續增殖，在不斷補充養分和rIL-2時TIL可長期培養。未經活化的TIL幾乎不增殖，活化后的TIL一般條件下可擴增幾十至幾百倍4，5。有研究表明，腫瘤病人經放化療后，外周血中CD4T、CD8T細胞顯著減少，且放化療后，外周血中淋巴細胞的功能也受影響6，7。為了弄清放化療后的患者TIL是否可以被激活、增殖和恢復其功能并進行過繼免疫治療，我們從術前經過放化療的6例鱗癌患者中分離出TIL，并與未治療的TIL相比。有趣

10、的是，其結果與我們預料的相反，未經放化療的TIL其增殖高峰較治療患者的TIL提前1周，說明術前經放化療患者TIL增殖能力恢復較慢，但在4周后仍可繼續增殖。提示：對術前經放化療患者，如果TIL的殺傷活性也能恢復，那么轉輸TIL也是可行的，特別是對放化療不敏感的患者應用，受益會更大。術前經放化療患者TIL的殺傷活性的恢復有待于進一步的研究。作者單位：楊宏宇(中山醫科大學附屬第三醫院口腔頜面外科 510630廣州)劉國萍(中山醫科大學附屬第三醫院口腔頜面外科 510630廣州)黃偉民(中山醫科大學附屬第三醫院口腔頜面外科 510630廣州)李金榮(湖北醫科大學口腔醫學院)羅娟(深圳市中心醫院口腔科)

11、參考文獻1Tsunoda T,Tanimura H, Yamaue H, et al. Clonal and functional analysis for the augmentation of tumor infiltrating lymphocytes by interleukin-4 J. Br J Cancer, 1996；74:10851089.2Reisser D, Lejeune P,Lagabec P, et al.Interleukin-8 antitumor effect is associated with a local infiltration but not wi

12、th a systematic activation of T Lymphocytes J.Anticancer Res,1994;14:977980.3Wimmenauer S, Keller H, Rahner S,et al. Phenotypical and functional characteristics of tumor-infiltrating lymphocytes from colon carcinomas stimulated with rIL-2 and rIL-4 in vitro:Comparison with lymphocytes of the normal

13、colon mucosa and the peripheral blood J. Anticancer Res,1994;14:963968.4Yagita M, Itoh K, Tusudo M, et al. Involvement of both Tac and non-Tac Interleukin-2-binding peptides in the interleukin-2-dependent proliferation of human tumor-infiltrating lymphocytes J. Cancer Res, 1989;49:11541159.5Trentin L, Zambello K, Bulian P, et al.Functional role of rIL-2 receptors on tumor infiltrating lymphocytes J. Br J Cancer, 1994;69:10461451.6Tisch M, Heimlich F, Daniel V, et al. Cellular immune defect caused by postsurgical radiation therapy in patients with head