蛋白激酶B抑制剂对肝癌组织中肿瘤浸润淋巴细胞生物学活性的影响

Influence of protein kinase B inhibitors on the bioactivity of tumor infiltrating T lymphocytes

目的探讨蛋白激酶B(protein kinase B,Akt)抑制剂对肝癌组织中肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)生物学活性的影响。方法肝癌患者4例,取其手术切除肿瘤组织标本密度梯度离心法分离TIL,并分为白细胞介素(interleukin,IL)-2组(6 000u/mL IL-2)和Akt抑制剂组(1mmol/L Akt抑制剂+6 000u/mL IL-2),培养第10天,2组均加入抗CD3抗体,继续原培养液培养20d。依据细胞增殖倍数绘制生长曲线检测TIL增殖活性;流式细胞法检测2组T淋巴细胞亚型及TIL分泌干扰素-γ(interferon-γ,IFN-γ)的能力;ELISA法检测TIL培养上清液中IFN-γ、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)及IL-10水平。结果 Akt抑制剂组与IL-2组TIL数目在抗CD3抗体刺激前(培养第5、10天)、刺激后(培养第15、20、25、30天)比较差异均无统计学意义(P>0.05);培养第5、10、15、20、25、30天,Akt抑制剂组中央记忆型T细胞占CD8^+T淋巴细胞比率[(9.57±9.14)%、(13.98±3.92)%、(15.74±2.74)%、(16.05±2.36)%、(19.14±3.55)%、(14.33±3.66)%]均明显高于IL-2组[(9.14±3.06)%、(10.07±3.57)%、(11.10±3.32)%、(11.72±2.87)%、(12.80±4.20)%、(9.61±4.23)%](P<0.05);培养第25天,中央记忆型T细胞占CD8^+T细胞比率在Akt抑制剂组、IL-2组均最高;随培养时间延长,2组TIL上清中IL-10、IFN-γ、TNF-α分泌量均逐渐升高,且Akt抑制剂组IFN-γ分泌量自第5天开始明显高于IL-2组(P<0.05),TNF-α分泌量在第25天明显高于IL-2组(P<0.05),2组不同时间IL-10分泌量比较差异均无统计学意义(P>0.05);流式细胞法检测结果显示,培养第25天,Akt抑制剂组分泌IFN-γ的TIL比率[(11.35±0.47)%]明显高于IL-2组[(6.25±0.66)%](P<0.05)。结论 Akt抑制剂可提高肝癌组织TIL中中央记忆型T细胞比率,促进TIL分泌IFN-γ,且不影响TIL增殖活性。

Objective To explore the influence of protein kinase B （Akt） inhibitors on the bioactivity of tumor infiltrating T lymphocyte （TIL）. Methods TILs were separated from the resected tumor samples in 4 patients with hepatic cancer by density gradient centrifugation, and were divided into interleukin （IL）-2 group （6 000 u/mL of IL-2） and Akt inhibitor group （1 mmol/L of Akt inhibitor ± 6 000 u/mL of IL-2）. By day 10 after culture, anti-CD3 antibody was added to both groups and the original culture medium was continued for 20 d. The growth curve of TIL was drawn according to cell proliferation fold to detect the proliferation activity of TIL. Flow cytometry was used to detect T lymphocyte subsets and the ability of TIL to secrete interferon-3＇ （IFN-7）. The levels of IFN-3’, tumor necrosis factor-α （TNF-α and IL-10 in TIL culture supernatants were detected by ELISA method. Results There were no significant differences in TIL number between Akt inhibitor group and IL-2 group before anti-CD3 antibody stimulation （by day 5 and 10 after culture） and after stimulation （by day 15, 20, 25 and 30 after culture） （P〈0.05）. By day 5, 10, 20, 25 and 30 after culture, the percentages of central memory T cell （Tcm） in CD8± T cell were significantly higher in Akt inhibitor group （（9. 574- 9.14）%, （13. 98±3.92）%, （15. 74±2.74）%, （16. 054±2.36）%, （19. 14±3.55）%, （14. 33±3.66）%） than those in IL 2 group （（9.14±3.06）%, （10.07±3.57）%, （11.10±3.32）%, （11. 72±2. 87）%, （12.80±4.20）G, （9.61± 4.23）%） （P〈0.05）. The percentages of Tcm in CD8± T cell were the highest by day 25 after culture both in Akt inhibitor group and IL-2 group. With the prolongation of culture time, the secretion of IL-10, IFN-7 and TNF-a in the supernatant increased gradually in both two groups, and the secretion volume of IFN 7 in Akt inhibitor group was significantly larger than that in IL 2 group by day 5 after culture （P〈 0.05）, the secretion volume of TNF -α was significantly larger than that in IL-2 group by day 25 after culture （P〈0.05）, and there was no significant difference in IL-10 secretion volumes between two groups at different time points （P〈0.05）. The results of flow cytometry showed that the percentage of TIL in IFN-α was significantly higher in Akt inhibitor group （（11.35_--4-0.47）~） than that in IL-2 group （（6. 25±0. 66）%） （P〈0.05）. Conclusion Akt inhibitor could increase the percentage of Tcm in TIL and promote the secretion of IFN-α by TIL, with no influence on the proliferative activity of TIL.