術(shù)前放化療和未治療口腔鱗癌患者TIL增殖力的比較

1、    術(shù)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(shù)前做過放化療口腔鱗癌患者TIL在體外與rIL-2共培養(yǎng)是否具有繼續(xù)增殖的能力。方法從15例口腔鱗癌原發(fā)灶中分離TIL，用1000u/ml rIL-2與其培養(yǎng)，比較術(shù)前放化療和未治療患者TIL的增殖能力。結(jié)果術(shù)前未治療組比治療組擴(kuò)增快，提前1周達(dá)增殖高峰，但4周以后術(shù)前治療組也顯示出繼續(xù)擴(kuò)增的趨勢。結(jié)論從術(shù)前放化療患者腫瘤組織中分離的TIL，在體外經(jīng)rIL-2刺激后仍能繼續(xù)擴(kuò)增?！娟P(guān)鍵詞】

2、鱗狀細(xì)胞癌； 口腔； 腫瘤浸潤淋巴細(xì)胞； 增殖【中分類號(hào)】R739.81【文獻(xiàn)標(biāo)識(shí)碼】A【文章編號(hào)】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(yán)重。特別是經(jīng)歷了放化療后機(jī)體的免疫功能可能會(huì)進(jìn)一步降低。如果從術(shù)前放化療患者手術(shù)標(biāo)本中分離腫瘤浸潤淋巴細(xì)胞（Tumor Infiltrating Lymphocyte,TIL)，在體外與rIL-2共培養(yǎng)能恢復(fù)增殖能力及殺傷活性，那么將TIL輸入體內(nèi)，將會(huì)提高機(jī)體的免疫功能，是常規(guī)治療極好的補(bǔ)充。本文擬對(duì)此設(shè)

7、想的可行性進(jìn)行初步探討。材料與方法1.標(biāo)本來源1997.31998.2間住院手術(shù)患者，經(jīng)病理證實(shí)為口腔鱗癌，共15例。術(shù)前放化療6例，未治療9例。2.方法2.1TIL的分離與培養(yǎng)用酶消化和不連續(xù)梯度密度的淋巴細(xì)胞分離液將腫瘤細(xì)胞和腫瘤浸潤淋巴細(xì)胞分離。用10胎牛血清的RPMI-1640培養(yǎng)液調(diào)節(jié)細(xì)胞濃度為2.5×105ml，按1000u/ml加入rIL-2，置37，5CO2培養(yǎng)箱中，每隔34天調(diào)整細(xì)胞濃度，并補(bǔ)充rIL-2以維持培養(yǎng)濃度。2.2TIL增殖取初始分離的TIL細(xì)胞懸液1滴和2臺(tái)盼蘭液1滴混合，置3分鐘，顯微鏡下計(jì)數(shù)200個(gè)細(xì)胞，活細(xì)胞不著色，死細(xì)胞核呈藍(lán)色，計(jì)算活細(xì)胞比例

8、。用血細(xì)胞計(jì)數(shù)板計(jì)數(shù)，按下式計(jì)算：原液每次換液時(shí)計(jì)數(shù)1次，觀察其增殖。結(jié)果TIL在培養(yǎng)的24小時(shí)內(nèi)即開始聚集成團(tuán)塊，多呈懸浮生長。第57天數(shù)目顯著增加。術(shù)前作過治療的TIL（6例）比未作治療的TIL（9例）擴(kuò)增慢，后者在第3周時(shí)即達(dá)高峰，前者到第4周才達(dá)高峰，擴(kuò)增延遲。兩組TIL增殖情況見1。1術(shù)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(zhì)內(nèi)，以T細(xì)胞為主，在多數(shù)病例中，CD+8T細(xì)胞多于CD+4T細(xì)胞，TIL中有部分NK細(xì)胞，所有TIL細(xì)胞在腫瘤原位一般處于免疫抑制狀態(tài)。研究發(fā)現(xiàn)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(tài)。過繼免疫治療中轉(zhuǎn)輸足夠的TIL的細(xì)胞數(shù)是獲得較好療效的重要因素13。TIL經(jīng)rIL-2激活后，一般在含有500u-1000umlrIL-2的完全培養(yǎng)基中可持續(xù)增殖，在不斷補(bǔ)充養(yǎng)分和rIL-2時(shí)TIL可長期培養(yǎng)。未經(jīng)活化的TIL幾乎不增殖，活化后的TIL一般條件下可擴(kuò)增幾十至幾百倍4，5。有研究表明，腫瘤病人經(jīng)放化療后，外周血中CD4T、CD8T細(xì)胞顯著減少，且放化療后，外周血中淋巴細(xì)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(fù)其功能并進(jìn)行過繼免疫治療，我們從術(shù)前經(jīng)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(jié)果與我們預(yù)料的相反，未經(jīng)放化療的TIL其增殖高峰較治療患者的TIL提前1周，說明術(shù)前經(jīng)放化療患者TIL增殖能力恢復(fù)較慢，但在4周后仍可繼續(xù)增殖。提示：對(duì)術(shù)前經(jīng)放化療患者，如果TIL的殺傷活性也能恢復(fù)，那么轉(zhuǎn)輸TIL也是可行的，特別是對(duì)放化療不敏感的患者應(yīng)用，受益會(huì)更大。術(shù)前經(jīng)放化療患者TIL的殺傷活性的恢復(fù)有待于進(jìn)一步的研究。作者單位：楊宏宇(中山醫(yī)科大學(xué)附屬第三醫(yī)院口腔頜面外科 510630廣州)劉國萍(中山醫(yī)科大學(xué)附屬第三醫(yī)院口腔頜面外科 510630廣州)黃偉民(中山醫(yī)科大學(xué)附屬第三醫(yī)院口腔頜面外科 510630廣州)李金榮(湖北醫(yī)科大學(xué)口腔醫(yī)學(xué)院)羅娟(深圳市中心醫(yī)院口腔科)

11、參考文獻(xiàn)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