腺病毒介导的人骨形成蛋白2基因治疗的免疫学研究

AN IMMUNOLOGICAL STUDY ON ADENOVIRUS MEDIATED HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE THERAPY

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 host immune reaction against adenovirus mediated human bone morphogenetic protein 2 （Adv-hBMP 2） gene therapy in repair of tibial defects. Methods Twelve goats were made 2-1 cm segmental defects in the tibial diaphysis and divided into 2 groups. Adv-hBMP-2 transfected marrow mesenchymal stem cells（MSCs） and untransfected MSCs were implanted into the defect sites of transfected group（n=7） and untransfected group （n=5）, respectively. The defect repair was observed by X-ray films after 4, 8, 16 and 24 weeks of transplantation and cellular and humoral immune reactions to adenovirus were assayed before implantation and after implantation. Results More bony callus was found in the bone defects of transfected group. The healing rates were 6/7 in transfected group and 2/5 in untransfected group, respectively at 24 weeks after implantation. The mixed culture of lymphocytes and MSCs showed that the lymphocytes stimulation indexes （SI） increased 14 days after implantation, and there was significant difference between the transfected group （4. 213±1. 278） and the untransfectedgroup（-0. 310±0. 147,P〈0.05）; SI decreased after 28 days, but there was no significant difference between the transfected group （2. 544±0. 957） and the untransfected group （3. 104±0.644,P〉0.05）. After 14, 28, 49, and 120 days of treatment, the titer values of neutralizing antibody against Adv-hBMP-2 （log0.1） were 2. 359±0. 226, 2. 297±0.200, 2. 214±0. 215 and 2. 297±0. 210 in transfected group, and -0.175±0.335, -0.419±0.171, 0±0.171 and 0. 874 ± 0. 524 in untransfected group, being significant differences between two groups （P 〈 0.05）. Conclusion Adenovirus mediated BMP 2 gene therapy can cause cellular and humoral immune reactions against adenovirus, which can eliminate the influence of adenoviral genes and proteins within a certain period.