自身γδT细胞治疗乙型肝炎的临床观察

Clinical observation on adoptive immunotherapy with Autologous γδT cells for the patients with hepatitis B

目的观察γδT细胞和HepG212115细胞共培养后对其HBV复制和HBeAg表达的影响。评估用γδT细胞过继回输治疗乙型肝炎的疗效。方法在体外用异戊希焦磷酸法和IL-2激活人外周血PBMCs,细胞经10 d培养后用流式细胞术检测培养前后γδT细胞含量和Perforin、GranB、NKG2D表达量;将γδT细胞与HepG212115细胞按一定比例共孵育培养后,ELISA法测定上清中HBeAg的表达。将培养10 d的γδT细胞回输给患者;患者一个疗程接受γδT细胞总数在0.8×1010～7.0×1010。结果培养10 d时γδT细胞数为91.27%,其细胞表达的Perforin、GranB、NKG2D分别为和62.8%、72.7%和60.8%。所扩增的γδT细胞能够部分抑制HepG212115细胞中HBeAg的表达。治疗前138例HBeAg阳性者,治疗后有76例HBeAg(55.1%)转阴。总有效率为77.51%。结论γδT细胞能够降低HepG212115细胞中HBeAg表达;用自身γδT细胞过继回输治疗乙型肝炎后患者的HBeAg+HBV-DNA转阴率达74%。该治疗无毒副作用,对乙型肝炎是一种安全有效的治疗方法。

Objective To investigate the inhibitory effect of peripheral blood mononuclear cells （PBMCs） stimulated by IPP on hepatitis B virus （HBV） replication and the expression of HBeAg in vitro and evaluate the effect of adop- tive tansfer of γδT cells in patients with hepatitis B. Methods The peripheral blood mononuclear cells were co-cul- tured with isopentenyl pyrophosphate （IPP） and recombinate rhIL-2 as stimulating reagents for 10 days in vitro. The expansion of γδT cells was analysed by flowcytometry. PBMCs stimulated by IPP were added to HepG212115 cells in certain ratio. HBeAg was measured by ELISA, and HBV-DNA was detected by fluorogenic quantitative PCR method in the supernatant. The numbers of transferred γδT cells at 0.8×10^10-7.0×10^10 were given to per patient in one course of treatment. Results TST cells were cultivated for 10 days which proliferation ratio increased by 86.15% after the ex- pression of Pefforin, GranB, NKG2D levels in the supematant （62.8%, 72.7%, 60.8%） increased significantly. Activat- ed γδT cells significantly reduced HBeAg secretion of HepG212115 cells. Out of 138 patients before receiving γδT cells immunotherapy, the clearance rate of HBeAg, HBV-DNA was 5S.1% at the end of treatment and the total effec- tive rate （the clearance rate HBeAg and/or HBV-DNA ） was 77.51%. Conclusion γδT cells can reduce the expression of HBeAg in HepG212115 cells. Adoptive immunotherapy of γδT cells can significantly enhance the clearance rate of HBeAg＋HBV-DNA of 74% after treatment without side effects, which is a safe and effective treatment for patients with hepatitis B.