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目的评价机体对腺病毒介导的人骨形成蛋白2(adenovirusmediatedhumanbonemorphogeneticprotein2,Ad-hBMP-2)基因治疗的免疫学反应。方法崇明山羊12只,手术截去右侧胫骨中段2.1cm,制备胫骨干缺损模型,随机分为2组。将Ad-hBMP-2转染的山羊骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs,转染组,n=7)及未转染的MSCs(未转染组,n=5)分别植入骨缺损内。于术后4、8、16及24周摄X线片检查骨缺损愈合情况,并对治疗前后机体对腺病毒的细胞和体液免疫反应进行检测。结果X线片示,转染组4～8周,骨缺损内有连续性骨痂形成;24周,有6侧骨缺损完全愈合,部分骨髓腔再通。未转染组4～8周,骨缺损内骨痂形成较少,与骨端界限清楚;24周,仅2侧骨缺损愈合。淋巴细胞与MSCs混合培养示淋巴细胞刺激指数(stimulationindex,SI)于植入后14d升高,转染组4.213±1.278,未转染组-0.310±0.147,且差异有统计学意义(P<0.05);28d后下降,转染组2.544±0.957,未转染组3.104±0.644,差异无统计学意义(P>0.05)。治疗后14、28、49和120d,转染组的血浆腺病毒中和抗体滴度分别为2.359±0.226、2.297±0.200、2.214±0.215和2.297±0.210,未转染组分别为-0.175±0.335、-0.419±0.171、0±0.171和0.874±0.524,两组各时间点差异均有统计学意义(P<0.05)。结论腺病毒介导的基因治疗可引起机体对腺病毒的细胞及体液免疫反应,从而逐渐消除腺病毒基因和相关蛋白的影响。
Objective To evaluate the immunological response to adenovirus-mediated human bone morphogenetic protein 2 (Ad-hBMP-2) gene therapy. Methods Twelve Chongming goats were divided into two groups randomly by clipping the middle of the right tibia by 2.1cm. Ad-hBMP-2 transfected goat bone marrow mesenchymal stem cells (MSCs, transfection group, n = 7) and untransfected MSCs (untransfected group, n = 5) were implanted into the bone defect . At 4, 8, 16 and 24 weeks after surgery, radiographs were taken to examine the healing of bone defects and the cellular and humoral immune response to adenovirus was detected before and after treatment. Results X-ray showed that in the transfection group for 4 to 8 weeks, there was a continuous callus formation in the bone defect. At the 24th week, 6 sides of the bone defect healed completely and part of the marrow cavity recanalized. After 4 to 8 weeks of untransfected group, the callus formation in the bone defect was less, and the border of the bone was clearly defined. At the 24th week, only 2 sides of the bone defect healed. The mixed lymphocyte and MSCs culture showed that the stimulation index (SI) increased on the 14th day after transplantation and was 4.213 ± 1.278 in the transfected group and -0.310 ± 0.147 in the untransfected group (P <0.05) ). After 28 days, there was no significant difference between the two groups (2.544 ± 0.957 in transfection group and 3.104 ± 0.644 in non-transfected group) (P> 0.05). At 14, 28, 49 and 120 days after treatment, the plasma adenovirus neutralizing antibody titers of transfection group were 2.359 ± 0.226,2.297 ± 0.200,2.214 ± 0.215 and 2.297 ± 0.210 respectively, and those of non-transfected group were -0.175 ± 0.335 , -0.419 ± 0.171,0 ± 0.171 and 0.874 ± 0.524 respectively. There was significant difference between the two groups at each time point (P <0.05). Conclusions Adenovirus-mediated gene therapy can cause cellular and humoral immune responses to adenovirus and thus gradually eliminate the effects of adenovirus genes and related proteins.