经不同途径应用 NK-NKG5-SV 细胞防治肝细胞肝癌的研究

Use of NK cell transducted with NKG5-SV gene to inhibit heaptocellular carcinoma

目的研究 NKG5-SV 导入肝癌病人 NK 细胞对其抗肝细胞肝癌作用的影响。方法Percoll 不连续密度梯度离心法分离肝癌病人外周血 NK 细胞,以逆转录病毒为载体将 NKG5-SV 基因和报告基因 LacZ 导入 NK 细胞。将不同 NK 细胞与 LCI-D20裸鼠人肝癌同时皮下接种或待 LCI-D20肿瘤成瘤后瘤内注射,观察对肿瘤成瘤和生长的影响。术中经切缘或脾内注射不同的 NK 细胞,观察其对 LCI-D20肝癌切除术后转移复发的影响。结果不同 NK 细胞与 LCI-D20肝癌同时皮下接种 NK-NKG5-SV 细胞组成瘤率、肿瘤直径(cm)均低于 NK-LacZ 细胞组(P 均小于0.01)。肿瘤内注射不同 NK 细胞,NK-NKG5-SV 细胞组的肿瘤生长明显低于 NK-LacZ 细胞组(P 均小于0.05)。肝癌切除术中应用 NK 细胞对照组、NK-LacZ 细胞组、NK-NKG5-SV 细胞组切缘复发瘤直径、肝内转移灶数目、转移灶累及肝叶数、转移灶累及腹内部位数分别为切缘注射:1.51±0.47、0.86±0.49、0.50±0.19(P<0.01),2.60±0.70、1.40±0.54、1.00±1.76,2.40±1.80、O.80±0.54、0.40±1.18,4.20±2.16、1.80±0.13、0.60±0.54(P<0.05)。脾内注射:1.18±0.34、0.86±0.40、0.70±0.35,2.40±1.67、0.60±0.89(P<O.05)、0.40±0.89(P<0.05),2.2±1.09、O.4±O.54(P<0.05)、0.2±0.01(P<0.01),4.60±1.14、1.20±1.70(P<0.01)、0.80±1.09(P<0.01)。结论 NK 细胞具有抑制 LCI-D20肝癌生长及防治肝癌切除术后转移复发的作用。NKG5-SV 基因转染 NK 细胞后可提高 NK 细胞的抗肝癌作用。

Objective To investigate the use of NK cell transducted with NKG5-SV gene to in- hibit hepatocellular carcinoma(HCC).Methods NK cell was isolated from peripheral vein blood with Percoll by discontinuous density gradient centrifugation.NKGS-SV gene was transducted into NK cell by retroviral vector.LCI-D20 tumor together with saline,NK-LacZ cell or NK-NKG5-SV cell were inoculated subcutaneously.Tumor formation rate was determined 35 days after inoculation.LCI-D20 tumor was inoculated subcutaneously.When tumor appeared 10 days later,NK-LacZ cell or NK- NKGS-SV cell was injected into the tumor and the tumor growth was observed 35 days after inocula- tion.Liver cancer was resected 22 days after intrahepatic inoculation.In 35 days after the inoculation, mice were killed to observe tumor recurrence at incisal margin,intraheaptic metastasis and extrahepat- ic metastasis.Results Tumor formation rate and tumor growth were lower in NK-NKG5-SV group than in the control group and NK-LacZ group.Size of recurring tumor at the incisal margin,intrahe- patic metastatic nidi,metastasis-involved hepatic lobes,sites with extrahepatic metastatic nidi in the control group,NK-LacZ group and NK-NKGS-SV group were 1.51±0.47,0.86±0.49,0.50±0.19 (P<0.01);2.60±0.70,1.40±0.54,1.00±1.76;2.40±1.80,0.80±0.54,1.18±0.40;4.20± 2.16,1.80±0.13,0.60±0.54(P<0.05),respectively,when NK cells were injected at the incisal margin and 1.18±0.34,0.86±0.40,0.70±0.354;2.40±1.67,0.60±0.89(P<0.05),0.40± 0.89(P<0.05);2.2±1.09,0.4±0.54(P<0.05),0.2±0.01(P<0.01);4.60±1.14,1.20± 1.70(P<0.01),0.80±1.09(P<0.01),respectively,when NK cells were injected into the spleen. Conclusions NK cell can inhibit HCC growth and its postoperative metastasis and recurrence.Mean- while,transduction of NK-NKG5-SV gene into NK cell can enhance its anti-HCC effects.