肿瘤生物治疗是肿瘤治疗研究最活跃的领域。新的治疗方法不断涌现。CIK细胞治疗正在临床转化过程中。如何提高细胞治疗的特异性及其杀伤活性,仍有很大的研究空间。CIK细胞由一系列细胞因子激活,肿瘤杀伤特异性不强。肿瘤细胞疫苗是肿瘤疫苗的一种,主要由肿瘤细胞和佐剂构成。肿瘤细胞表面抗原激活免疫系统,佐剂辅助增强免疫系统的肿瘤杀伤作用,对免疫细胞的扩增刺激作用有待提高。目前比较成功的肿瘤细胞疫苗是GVAX,是表达GM-CSF的前列腺癌细胞系,已用于转移性黑色素瘤、转移性肾癌和非小细胞肺癌等肿瘤的临床试验。本研究的目的是建立及鉴定稳定表达IL-21的和IL-15的白血病细胞系,并初步检测其刺激活化淋巴细胞的能力及活化淋巴细胞对肿瘤细胞的杀伤功能,探讨肿瘤细胞成为肿瘤细胞疫苗的可能性。本研究首先构建携带白介素和RFP基因的质粒,包装慢病毒,感染4种白血病细胞系,连续传代后,通过荧光显微镜、流式细胞仪、PCR和ELISA法鉴定建立的8种细胞系。然后以稳定分泌白介素的白血病细胞为抗原刺激PBMC增殖,细胞分析计数仪检测增殖倍数,流式细胞仪分析淋巴细胞的表型,CCK8检测增殖后的淋巴细胞对肿瘤细胞的杀伤作用。本研究建立稳定表达IL-21的4种细胞系：K562-IL-21、THP-1-IL-21、Jurkat E6-1-IL-21和RPMI8226-IL-21和稳定表达IL-15的4种细胞系：K562-IL-15、THP-1-IL-15.Jurkat E6-1-IL-15和RPMI8226-IL-15.3份人PBMC在4种分泌IL-21的白血病细胞的作用下都有增殖,增殖倍数在1.147±0.057～2.725±0.345倍之间,淋巴细胞的增殖倍数在3份人PBMC之间无显著统计学差异（P>0.01）。不同数量的工L-21分泌白血病细胞都可激活淋巴细胞增殖,增殖的倍数在1.127±0.152“2.213±0.200之间,增殖的淋巴细胞数量之间亦无显著统计学差异（P>0.01）。流式结果显示增殖后CD3+T淋巴细胞和CD56+NK细胞的比例降低（P<0.05）,CD19+B淋巴细胞比例增加（P<0.05）；由分泌IL-21白血病细胞激活后的淋巴细胞对母系白血病细胞的杀伤作用明显高于对照组。由分泌IL-21白血病细胞激活后的淋巴细胞对母系白血病细胞和其他7种肿瘤细胞均有杀伤作用,杀伤率在（28.68±9.31）%～（78.45±0.61）%之间。3份PBMC在2种分泌IL-15白血病细胞的作用下都有增殖,增殖倍数在1.23±0.227～1.59±0.178倍之间,淋巴细胞的增殖倍数在3份之间无显著性差异（P>0.01）。不同数量的抗原细胞都可激活淋巴细胞增殖,增殖的倍数在1.30±0.277～1.48±0.222之间,增殖的淋巴细胞数量之间亦无显著性差异（P>0.01）。分泌IL-15的白血病细胞刺激增殖后CD3+T淋巴细胞和CD19+B淋巴细胞比例下降,CD56+NK细胞比例增加（P<0.05）。由分泌IL-15白血病细胞激活后的淋巴细胞对母系白血病细胞的杀伤作用明显高于对照组。用分泌IL-15的白血病细胞刺激增殖的淋巴细胞对母系白血病细胞和其他7种肿瘤细胞,杀伤率在（34.84±3.98）%～（78.45±0.61）%之间。本研究证明分泌IL-21的白血病细胞和分泌IL-15的白血病细胞均可刺激PBMC增殖并激活其对肿瘤细胞的杀伤作用,具有成为肿瘤疫苗的可能。

Cancer biotherapy is the most inspiring area of cancer therapy. More and more innovative methods appeared and is clinically tried. Cytokine Induced Killer (CIK) cells are among them. Tumor cell vaccines are composed of tumor cells and adjuvant. Antigens on the surface of tumor cells, with the assistance of adjuvants, can stimulate the immune system and elicit the production of memory cell. The specificity of CIK cells and the the proliferation stimulation of tumor cell vaccines have much to be desired. GVAX, which consist of 2 prostatic tumor cell lines expressing GM-CSF, has proven a potential treatment for prostatic cancer, non-small lung cancer and metastatic melanoma.
In this research, we tried the combination of newly defined cytokines and tumor cells for their tumor associated antigen. Leukemia cell lines and IL-21 and IL-15 were chosen and stable constitutively expression were achieved. The ability of their activation and proliferation on lymphocytes and their potential possibility as tumor vaccines were explored .
At first, leukemia cell lines were infected with lentivirus carrying IL-21 or IL-15 and RFP genes. Then, Fluorescent microscope was used for observing the expression of RFP and flow cytometry was used for analyzing the percentage of RFP-positive cells. The concentration of Interleukins in the medium was detected by ELISA. After stimulation with the Interleukin stable-expressing leukemia cell lines, the amplification folds of PBMC were calculated by cell counter and the surface markers were analyzed by flow cytometry, while their cytotoxicity towards different tumor cells were assessed with CCK8 test.
As a result, four leukemia cell lines stable-expressing IL-21 were established, namely, K562-IL-21, THP-1-IL-21, Jurkat E6-1-IL-21 and RPMI8226-IL-21. Four leukemia cell lines stable-expressing IL-15 were established, namely, K562-IL-15, THP-1-IL-15, Jurkat E6-1-IL-15 and RPMI8226-IL-15.
Stimulated by IL-21 secreting leukemia cells, the lymphocytes amplification of 3 different samples varied from 1.147±0.057 to 2.725±0.345 times and showed no difference (P>0.01) between and there was no difference among the amplification folds of lymphocytes stimulated by different numbers of leukemia cells (p>0.01), which varied from 1.127±0.152 to 2.213±0.200. The percentage of CD3~+T cell and CD56~+NK cell decreased (P<0.05) while CD19~+B cells increased (P<0.05) after stimulation. The killing rates of activated lymphocytes are much higher than that of the control. The activated lymphocytes had powerful killing capacity on their stimulating leukemia cells and 7 other tumor cells and the killing rates ranged from
(28.68±9.31) % to (78.45±0.61) %.
Stimulated by IL-15 secreting leukemia cells, the lymphocytes amplification of 3 different samples varied from 1.23±0.227~1.59±0.178 times and showed no difference (P>0.01) and there was no difference among the amplification folds of lymphocytes stimulated by different numbers of leukemia cells (P>0.01), which varied from 1.30±0.277~ 1.48±0.222. The percentage of CD3~+T cell and CD19~+ B cells decreased (P<0.05) while CD56~+NK cell increased (P<0.05) after stimulation. The killing rates of activated lymphocytes are much higher than the control. The activated lymphocytes had powerful killing capacity on their stimulating leukemia cells and 7 other  tumor  cells   and   the   killing  rates   ranged   from     (34.84±3.98)   %  to
(78.45±0.6l) %.
In conclusion, all six leukemia cell lines stable-expressing Interleukin could activate PBMC to kill different tumor cells and show a potential ability as tumor cell vaccines.