卡介苗热休克蛋白70基因修饰的树突状细胞疫苗体内抗瘤作用的研究

Antitumor Effects of Dendritic Cell Vaccine Modified by BCG HSP70 Gene in Vivo

目的:探讨卡介苗热休克蛋白70 (BCG HSP70)基因修饰的树突状细胞(DCs)疫苗体内抗瘤效应。方法:从急性白血病患儿骨髓中培养未成熟树突状细胞(imDCs),形态学观察及流式细胞术鉴定后,利用脂质体2000将BCG HSP70基因转入imDCs细胞表面,免疫荧光单克隆抗体进行细胞表面修饰鉴定。取健康BALB/C裸鼠,第0天皮下接种HL-60细胞,第7~10天可形成皮下结节的白血病裸鼠模型,将其随机分为6组,移植瘤内分别接种PBS液、不做任何处理的imDC (imDC组);空载体(pDisplay)转染的imDC (imDC-neo组);重组载体(pDisplay-HSP70)转染的imDC (HSP70组);rhTNF-α (20 ng/ml)诱导的imDC (TNF-α组);以及pDisplay-HSP70转染 + rhTNF-α (20 ng/ml)诱导的imDC (HSP70 + TNF-α组)。每周1次,共2次,于给药第14天处死裸鼠。观察各组肿瘤体积变化,绘制肿瘤生长曲线;计算裸鼠体重的变化情况;以及HE染色观察肿瘤组织切片。结果:1) 骨髓培养的细胞形态特点及细胞表面标志均符合imDCs特征。共聚焦显微镜观察检测证实BCG HSP70表达在转染后的imDCs细胞表面。2) HSP70 + TNF-α组HLA-DR、CD80、CD86阳性率分别为(78.42 ± 8.01)%,(83.00 ± 6.75)%和(88.51 ± 4.44)%,明显高于HSP70组((55.13 ± 4.84)%,(59.93 ± 3.88)%和(62.33 ± 5.42)%)、TNF-α组((57.28 ± 5.63)%,(60.31 ± 6.20)%和(63.70 ± 3.78)%);HSP70组与TNF-α组比较无明显差异(p > 0.05)。3) 体内杀瘤效应:HSP70-DC组肿瘤体积变化(40.94)明显小于PBS对照组、imDC组及imDC-neo组(185.72,181.16和180.18),但大于HSP70 + TNF-α组(17.60) (p < 0.05)。HSP70-DC组、TNF-α组及HSP70 + TNF-α组肿瘤切片染色可见不同程度的核固缩、碎裂及溶解、空泡现象,以HSP70 + TNF-α组最明显。结论:BCG HSP70基因修饰的DC疫苗在小鼠体内可诱导出较强的杀瘤效应,且与细胞因子联合作用更强。

Objective: To investigate anti-tumor effects of dendritic cells (DCs) vaccines modified by heat shock protein 70 of BCG (BCG HSP70) gene. Methods: The immature DCs (imDCs) were cultured from bone marrow in children with acute leukemia and identified by the morphological observation and flow cytometry. Then BCG HSP70 gene was transfected onto the surface of imDCs by lipofectamine 2000 and detected by fluorescence microscope. All of BALB/C nude mice were inoculated with HL-60 cells subcutaneously on day 0. After 7 to 10 days, leukemia xenografts in nude mice with formation of subcutaneous nodules were randomly divided into six groups: PBS, imDC (imDC without special processing), imDC-neo (imDCs transfected with pDisplay vector), HSP70 (imDCs transfected with pDisplay-HSP70 vector), TNF-α (imDCs induced with rhTNF-α), and HSP70 + TNF-α respectively. The DCs activated by different agents were intratumorally administered into nude mice. The changes in tumor volume were observed, the changes of mice weight rate were calculated and tumor sections were assessed by hematoxylin-eosin staining. Results: 1) The cultured cells exhibited typical char-acteristics of surface markers and cell morphology consistent with imDCs. After BCG HSP70 gene transfection, the yellow-green fluorescence on the cells surface was observed under the confocal microscope. 2) The positive rates of HLA-DR, CD80 and CD86 of HSP70 + TNF-α group, which were (78.42 ± 8.01)%, (83.00 ± 6.75)% and (88.51 ± 4.44)%, were significantly higher than HSP70 group ((55.13 ± 4.84)%, (59.93 ± 3.88)% and (62.33 ± 5.42)%) and TNF-α group ((57.28 ± 5.63)%, (60.31 ± 6.20)% and (63.70 ± 3.78)%). There was no difference between HSP70 group and TNF-α group (p > 0.05). 3) Antitumor effects: The changes of tumor volume in HSP70-DC group (40.94) were signifi-cantly smaller than PBS group, imDC group and imDC-neo group (185.72, 181.16 and 180.18), but greater than HSP70 + TNF-α group (17.60). The tumor cells in HSP70-DC group, TNF-α group and HSP70 + TNF-α group showed a large number of nuclear condensation, fragmentation and dissolu-tion, among which HSP70 + TNF-α group was the most obvious. Conclusion: DC vaccine modified by BCG HSP70 gene could induce a stronger anti-tumor effect in vivo, and the combined application ef-fect with cytokines is more obvious.