转染IL-21的CD34^＋脐血造血干细胞抗裸鼠卵巢癌作用研究

Study on anti ovarian cancer efficacy elicited by CD34 ^＋ umbilical cord blood hematopoietic stem cells transfected with IL-21 in nude mouse model

目的探讨IL-21转染的脐血造血干细胞（CD34^＋ UBSC—IL-21）对荷卵巢癌裸鼠的治疗作用。方法从脐血分离CD34^＋造血干细胞，体外培养扩增后用于重组体pIRES2-IL-21-EGFP转染。以肿瘤大小、荷瘤鼠生存期判断CD34^＋UBSC·IL-21对荷瘤裸鼠的治疗效应。以RT—PCR、免疫荧光、ELISA、Westernblot、脾细胞增殖试验及免疫组化法分别鉴定CD34^＋UBSC和肿瘤组织中IL-21的表达及活性。裸鼠脾细胞中NK细胞含量及脾细胞的杀伤效应、血清中IFN—γ和TNF—α水平分别用FCM与ELISA检测。结果pIRES2-IL-21-EGFP成功转染CD34^＋UBSC。CD34^＋UBSC—IL-21能抑制肿瘤生长，延长荷瘤裸鼠生存期，治疗鼠肿瘤局部能表达IL-21、血清IFN-γ1和TNF-α水平升高，NK细胞含量及NK细胞杀伤活性明显增强，与其他组相比，差异有统计学意义（P〈0．01）。结论转染IL-21的CD34^＋UBSC有良好的抗裸鼠卵巢癌作用，该结果为临床使用UBSC为载体的基因治疗卵巢癌研究奠定了基础。

Objective To investigate the CD34 ^＋ umbilical cord blood hematopoietic stem cells （CD34 ^＋ UBSC） transfected with interleukin 21 （IL-21） against the ovarian cancer effect in tumor-bearing nude mice. Methods CD34 ^＋ UBSC were obtained from the UBSC by a magnetically activated cell sorting technique and then transfected with recombinant plasmid pIRES2-IL-21-EGFP after the CD34 ^＋ UBSC were proliferated in vitro. The therapeutic effect was evaluated by the size of the tumor and the life span in nude mice treated with the CD34 ＋ UBSC-IL-21. The expression of IL-21 and its bioactivity in CD34 ^＋ UBSC-IL-21 and in local neoplasitc tissues were respectively detected by reverse transcription-polymerase chain reaction （RT-PCR）, immune fluorescence technique, ELISA, Western blot, immunohistochemistry and splenocyte proliferative activity. The NK cell cytotoxicity and the numbers of NK cells, serum level of IFN-γ and TNF-α were simultaneouly detected by FCM and ELISA, respectively. Results CD34 ^＋ UBSC were cultured and transfected with plRES2-IL-21-EGFP successfully. CD34 ^＋ UBSC-IL-21 could inhibit the tumor growth and extended nude mice life span compared with other groups（P 〈 0.01 ）. The expression of IL-21 in the neo- plastic tissue, serum level of IFN-γ and TNF-α , NK cell activity and the numbers of NK cells of mice origin and of human origin in splenocytes were increased significantly in the nude mice treated with CD34 ^＋ UBSC- IL-21 compared with other groups（P 〈0.01）. Conclusion The CD34 ^＋ UBSC transfected with IL-21 have competent against ovarian cancer in tumor-bearing nude mice. The findings may establish a foundation for gene therapy of the ovarian cancer by CD34 ^＋ UBSC-IL-21 in clinic application.