CpGODN结合CD28/CD80单抗活化T细胞对结肠癌细胞的杀伤作用

目的研究联合应用CpGODN和CD28/CD80单抗共刺激健康人外周血T淋巴细胞(PBLs)在体外对结肠癌细胞株HT-29的杀伤作用,为结直肠癌的过继免疫治疗可能性提供参考。方法PBLs的分离与体外培养;CD28/CD80共刺激活化PBLs;用含共刺激活化PBMC或/和CpGODN的100ml/L胎牛血清的RPMI1640培养基培养细胞,测生长曲线。MTT法检测活化细胞的体外淋巴细胞毒作用,并用电镜观察效应细胞杀伤的结肠癌细胞超微结构及用流式细胞仪检测结肠癌细胞的相关凋亡情况。结果CpGODN本身对HT-29细胞有一定的杀伤作用(P<0.05),CD28/CD80共刺激活化PBLs在体外对结肠癌细胞株HT-29杀伤作用明显(P<0.01),CpGODN与CD28/CD80共刺激活化PBLs合用,对HT-29的杀伤作用显著大于CpGODN单独对HT-29细胞杀伤作用(抑制率92.31%vs68.00%,P<0.01),亦大于单独应用CD28/CD80共刺激活化PBMC对HT-29细胞的杀伤作用(P<0.05),CpGODN增加了HT-29细胞对共刺激活化PBMC的敏感性。电镜结果显示,效应细胞作用24h肿瘤细胞就部分发生坏死,部分肿瘤细胞可见凋亡。流式细胞仪检测效应细胞HT-29细胞72h明显凋亡。结论联合应用CpGODN可以提高单抗协同诱导的效应细胞对结肠癌细胞株HT-29杀伤作用,而坏死细胞进一步增多说明效应细胞是通过诱导肿瘤细胞坏死及细胞凋亡两条途径来实现抑制杀伤作用从而说明活化的淋巴细胞杀伤结肠癌细胞是通过坏死及凋亡实现的。

Killing effects of CD28/CD80 MoAB combined with CpGODN for costimulation activation of PBLs on the colonic carcinoma cell

Objective To investigate the killing effects of CpGODN combined with CD28 and CD80 MoAB for costimulate activation of PBLs on the human colorectal carcinoma cell lines in vitro. Method The cancer cells were cultured with PBLS and CD28/CD80 MoAb and CpGODN, then the growth curve of cells was obtained. The killing efficiency was measured by MTT method. The ultramicroscopic structure of cells was observed by electron microscope. Apoptosis was verified by a flow cytometer. Results After treated with costimulate activation of PBLs with CD28 and CD80 or CpGODN, the inhibition rate of HT-29 cells was 68.00%, respectively, while two methods were used in combination, the inhibition rate was 92.31%. By MTT method, combination of costimulated activation of PBMC with CD28/CD80 andCpGODN showed enhanced killing effect compared with single therapy (P<0.01). Electron microscope and flow cytometer verified that cells apoptosis after 72h exposion to costimulate activation of PBMC. Conclusion CpGODN can elevate the killing effect of anti-CD28 stimulated cells on colorectal carcinoma. Apoptosis is possibly one of the reasons of the synergistic action. They kill target-cells in two ways:necrosis and apoptosis.