同种带瓣主动脉移植免疫抑制治疗及钙化防治的研究

Effects of immunosuppressive treatment in prevention of calcification in aortic valved homograft.experiment with rats

目的研究免疫抑制治疗在防治同种带瓣主动脉移植(AVH)供体组织钙化的作用。方法采用改良大鼠 AVH腹主动脉异位移植模型,研究分为异基因组和同基因组。异基因组分别为接受低温保存的 AVH 移植组,接受低温保存的 AVH 移植后给予环孢素 A(CsA)治疗组,接受树突状细胞(DC)单抗预处理低温保存,移植后给予 DC 单抗治疗组;同基因组为对照组。异基因组 SD 大鼠为供体,Wistar 大鼠为受体;同基因组受体和供体均为 Wistar 大鼠。治疗时间为4周。手术后2、4、8、12、16周用流式细胞仪检测大鼠血液中 T 细胞抗原受体α、β(TCR-α、β),CD28的表达。观察移植物组织结构形态学变化,用免疫组织化学染色方法检测动脉壁 CD54的表达,用原子火焰吸收法测定AVH 组织钙的含量。结果 (1)接受深低温保存 AVH 移植组于术后2、4周其 TCR-α、β表达水平为(52.4±3.3)%、(43.8±6.4)%,CD28的表达水平为(51.7±7.5)%、(66.3±4.4)%;CsA 治疗组TCR-α、β表达为(34.5±3.5)%、(31.6±2.6)%,CD28表达水平为(41.2±1.6)%、(55.1±5.1)%;DC 单抗治疗组 TCR-α、β表达水平为(31.6±2.3)%、(29.5±3.0)%;CD28为(36.6±3.6)%、(51.8±5.6)%;对照组 TCR-α、β表达为(23.2±1.3)%、(21.6±2.3)%;CD28为(30.7±1.4)%、(33.3±0.9)%。(2)异基因组不同时间的供体组织钙含量自移植后4周开始升高,同基因组各时点的钙含量差异无统计学意义(P>0.05)。结论免疫抑制治疗可减轻免疫反应,延缓钙化进程,疗效明显。

Objective To investigate the effects of immunosuppressive treatment in prevention of calcification in aortic valved homograft （AVH）. Methods 120 Wistar rats were randomly divided into 4 equal groups： Group A （ allogene group） undergoing incision of the abdominal aorta and implantation of the AVH with myocardial cuff from SD rats, Group B, injected with cyclosporine A intraperitoneally one day after the implantation, Group C, injected intraperitoneally with anti-dendritic cell monoclonal antibody （DCmAb） one day after the implantation, and Group D （isogenenic or control group）, receiving the AVH of another Wistar rats. All groups were further subdivided into 5 equal subgroups to be sacrificed at different time points： 2, 4, 8, 12, and 16 weeks postoperatively. Blood samples were obtained from the vena cava to detect the expression of T-cell antigen receptor （TCR）-α and β and CD28 by flow cytometry. AVH specimens were obtained to observe the changes of endotheliocytes and smooth muscle cells with light and electron microscopy. The expression of CD54 was detected by immunohistochemistry. The calcium content of the AVH tissue after transplantation was examined by flame atomic absorption spectrophotometry. Results （1） Compared with the isogenic group, the expression levels of TCR-α and β and CD28 in the allogene groups were all significantly higher at all time points （ all P 〈 0.01 ）, peaked 2 ～4 weeks after operation, then gradually decreased, and approached the level of the controls 12 weeks after the implantation. Specifically, the expression levels of TCR-α and TCR-β 2 and 4 weeks postoperatively of Group A were 52.4% ±3.3% and 43.8% ± 6.4% respectively, significantly higher than those of Group B [ （ 34.5 ± 3.5 ） % and （ 31.6 ± 2.6） % respectively ] , Group C [ （ 31.6 ± 2.3） % and （29.5 ± 3.0） % respectively）, and Group D（23.2 ± 1.3） % and （21.6 ± 2.3） % （ all P 〈 0.01 ） ] ; and the CD28 expression level 2 ～ 4 weeks after operation of Group A were （51.7 ±7.5）% and （66.3 ±4.4）% respectively, both significantly higher than those of Group B [ （41.2± 1.6）% and （55.1± 5.1 ）% respectively], Group C [ （36.6 ±3. 6）% and （51. 8 ±5. 6）% respectively], and Group D [30. 7 ±1. 4）% and （33. 3 ± 0. 9）% respectively） ] [ all P 〈0.01 except those levels 12 and 16 weeks after the operation in each subgroup （P 〉 0.05 ） ] And the levels of TCR-α and TCR-β and CD28 of the 2 treatment groups were all significantly lower than those of the untreated group （Group A） （all P 〈0.01 ）. （2） The calcium contents of the AVH tissues of Group A, B, and C significantly increased 4 weeks after the operation and peaked 12 and 16 weeks after operation. No significant difference in calcium level was found in Group D at different time points （ all P 〉 0.05）. The calcium contents in AVH tissues 4 and 8 weeks postoperatively of Groups A, B, and C were （2856±79 ） μg/g and （3587 ± 168） μg/g respectively, （2518 ±73 ） μg/g, （3237 ± 187） μg/g; and （2176 ±210） μg/g and （3089 ± 176） μg/g; all significantly higher than those of Group D （860±60） μg/ g and （870 ± 50） μg/g respectively, all P 〈 0.01. Conclusion Immunosuppressive treatment obviously reduces the immune rejection and delays the course of AVH calcification.