肿瘤患者自体CIK细胞输注增强再次制备时CD3^+CD56^+细胞的体外扩增能力

Autologous CIK cell infusion promotes amplification ability of CD3^+CD56^+ cells when re-preparation from patients with malignant tumors

目的探讨自体细胞因子诱导的杀伤细胞(CIK细胞)输注治疗对再次制备CIK细胞亚群的构成和活性的影响。方法选取经2个疗程CIK细胞治疗的患者201例,分为CIK治疗后90 d内(含90 d)制备检测组和大于90 d制备检测组。台盼蓝拒染法检测细胞增殖;流式细胞术分析CD3、CD4、CD8,CD56和NKG2D受体表达情况的变化;乳酸脱氢酶释放法检测细胞毒活性。结果 CIK细胞输注治疗后90 d内再次制备CIK细胞的CD3+CD56+细胞百分率(16.7±9.1)%,与CIK细胞输注治疗前相比(13.5±8.6)%,显著增加(P<0.01),而且,表面受体NKG2D表达水平((84.1±10.8)%,也比CIK细胞输注治疗前明显增高((81.1±14.8)%)(P<0.05)。与此相反,CIK细胞输注治疗后导致再次制备时,CIK细胞群体中CD3+CD4+百分率(15.2±9.7)%,与CIK输注治疗前(17.6±12.5)%相比,显著下降(P<0.01)。值得注意的是,CIK细胞输注治疗后超过90 d再次制备CIK细胞的CD3+CD56+细胞分布和NKG2D受体表达,与CIK细胞输注治疗前相比均无明显变化,但CIK细胞输注治疗后仍可导致再次制备时,CIK细胞群体中CD3+CD4+百分率(14.5±9.4)%,与CIK细胞输注治疗前相比(18.2±12.9)%,明显下降(P<0.01)。另外,2组再次制备CIK细胞的总数和体外细胞杀伤活性无明显差异。结论 CIK细胞输注治疗有助于提高肿瘤患者CD3+CD56+细胞前体细胞,在相同CIK细胞培养制备体系中增殖、定向分化和活化的能力,该作用持续时间不超过90 d,因此,CIK细胞治疗疗程间隔时间不应超过90 d。

Objective To investigate the effects of the autologous cytokine-induced killer （CIK） cell infusion on the subpopulation distribution and activity of the CIK cells from the patients with malignant tumors when prepared in the same liquid culture system again. Methods A total of 201 patients who gave a written consent and received 2 courses of therapeutic infusion of CIK cells were divided into ≤90-day group in which the secondary preparation of CIK cells was performed in less than 90 days after the primary infusion and 〉90-day group in which the secondary preparation of CIK cells was performed in more than 90 days after the primary infusion. The proliferation and subtypes, including CD3 ＋ cells, CD3＋ CD4 ＋ cells, CD3 ＋ CD8 ＋ cells, and CD3＋ CD56 ＋ cells, of CIK cells were analyzed by hemocytometer with trypan blue exclusion and flow cytometry, respectively. The expression of NKG2D receptor was also detected using flow cytometry. The cytotoxicity against K.562 cells was analyzed using lactate dehydrogenase （LDH） release. Results The percentage of CD3 ＋ CD56 ＋ cell subpopulation in the secondary preparation of CIK cells in ≤90-day group [ （16. 7± 9.1 ）% ] was higher than that in the primary preparation of CIK cells [（13.5 ± 8.6）% ] （P 〈 0.01 ）. Furthermore, the percentage of NKG2D in the secondary CIK cell preparation [ （84.1 ± 10.8）%] was significantly higher than that in the primary CIK cell preparation [ （81. 1 ± 14. 8）% ] in ≤ 90-day group （P〈0.05）. In contrast, the percentage of CD3 ＋ CD4 ＋ cells in the secondary CIK cell preparation [ （15.2 ± 9. 7 ）% ] was significantly lower than that in the primary CIK cell preparation [ （ 17.6 ± 12.5） % ] （ P 〈 O. 01 ）. However, no significant differences in CD3＋ CD56 ＋ cell subpopulation and expression of NKG2D was detected between the primary and secondary CIK cell preparation in 〉 90 d group, although the percentage of CD3＋ CD4＋ cells in the secondary CIK cell preparation [（14.5 ±9.4）%] was significantly lower than that in the primary CIK cell preparation [（18.2±12.9）%] （P〈0.01）. In addition,no significant differences in total cell number and cytotoxic activity against K562 cells between the primary and secondary CIK cell preparation was detected either in 〉90-day group or in ≤90-day group. Conclusion CIK cell infusion can facilitate and enhance the proliferation and differentiation of the precursor cells of the CD3＋ CD56 ＋ subpopulation in the same CIK cell culture system and this effect does not last more than 90 days, suggesting that the secondary CIK cell infusion should be performed within 90 days in order to obtain the better therapeutic efficacy.