阻断可诱导共刺激分子刺激通路抑制大鼠异体肢体移植急性排斥反应的实验观察

Study on inhibition of acute rejection in rat limb allografts by inducible costimulator pathway blockade

目的通过RNA干扰并阻断可诱导共刺激分子（ICOS）刺激通路，观察对大鼠异体肢体移植急性排斥反应的抑制作用。方法成年Wistar、SD大鼠各27只，按体质量将大鼠随机分为排斥组、对照组、干扰组．每组9例。大鼠肢体移植采用改良法，将Wistar大鼠（供体）右下肢体移植到SD大鼠（受体）左下肢体上。移植术后排斥组不给予特殊处置；对照组阴茎背静脉注射梭华-Sofast—pSilencer 4．1空载体复合物：干扰组注射梭华-Sofast—pSilencer 4．1-ICOSshRNA干扰质粒复合物。术后8d每组处死SD大鼠3只．取移植肢体皮肤、肌肉及骨骼进行病理学检查；流式细胞仪及RT—PCR检测淋巴细胞表面ICOS和基因mRNA表达：分别用Wistar、Lewis大鼠脾细胞刺激，进行单向混合淋巴细胞培养，计算细胞刺激指数：ELISA双抗体夹心法检测大鼠血清细胞因子γ干扰素（IFN-γ）和白细胞介素4（IL-4）；另6只大鼠观察生存情况及肢体存活时间。结果排斥组大鼠肢体平均存活时间（11．34±1．21）d，对照组平均存活时间（11．14±1．32）d，干扰组平均存活时间（16．85±1．73）d，组间比较差异有统计学意义（F=8．72，P〈0．05）。排斥组和对照组病理学结果提示，肢体出现了中、重度急性排斥反应，干扰组急性排斥反应轻微。光镜下排斥组和对照组皮肤出现表皮坏死、大泡形成伴有血管周围炎及大量淋巴细胞浸润，肌肉组织有弥散性淋巴细胞浸润，间质水肿同时伴有肌细胞坏死。脾淋巴细胞ICOS基因mRNA表达显示，干扰组（18．75％）明显低于排斥组（100％）和对照组（98．51％），组间比较差异有统计学意义（Х^2=13．57，P〈0．01）。淋巴细胞表面ICOS表达荧光强度，干扰组为45．59±12．87，排斥组为103．72±21．76．对照组为93．47±29．55，组间比较差异有统计学意义（F=6．89，P〈0．05）。单向混合淋巴细胞反应刺激指数（SI）．排斥组（5．26±0．42、5．18±0．29）和对照组（5．37±0．27、4．93±0．44）明显高于干扰组（2．37±0．35、4．87±0．36）．组间比较差异有（无）统计学意义（F=7．29，P〈0．05；F=6．19，P〉0．05）。IFN-γ和IL-4表达，干扰组（230．17±38．47、160．32±59．13）较排斥组（490．73±51．48、230．67±45．21）和对照组（480．15±43．96、240．53±47．36）均降低，组间比较差异有统计学意义（F值分别是7．23、6．75，P〈0．05）。结论体内转染pSileneer 4．1-ICOSshRNA干扰质粒能有效阻断T细胞共刺激通路，抑制急性排斥反应，延长移植肢体存活时间。

Objective To observe the effect of inducible costimulator（ICOS） costimulation pathway blockade in rat limb allografts acute rejection by RNA interference. Methods Twenty-seven cases of modified hind limb allotransplantation were performed from Wistar to SD rats. The rats were divided into 3 groups（each n = 9）： the rejection group not given a special disposal; the control group, consisting of SD rats that received injection of pSilencer 4.1 and Sofast complex by vein post transplantation; and the interference group that received injection of pSilencer 4.1-ICOSshRNA and Sofast complex. On the eighth day posttransplantation, 3 rats were killed to study the pathological changes in each group. The expressions of ICOS gene in vivo were detected by flow cytometry and RT- PCR. The mixed lymphocyte reaction （MLR） was performed and cytokines in blood were measured by ELISA. The rest rats were used to record limb survival time. Results The mean survival time in rats of the rejection and the control groups were（11.34 ±1.21 ） and （11.14 ±1.32） days respectively. In the interference group, the mean survival time of limb allografts was （16.85 ±1.73） days（P〈 0.05）. The rats in the rejection and the control groups experienced moderate to serious acute rejections with skin epidermal necrosis, a large quantity of lymphocyte infiltration, muscle cell necrosis and interstitial edema, while the pathological changes in rats of the interference group were mild. The splenocyte ICOS mRNA expression level in the interference group（18.75%） was significantly lower than that of the rejection group（100%） and the control group（98.51%）. ICOS cell surface expression level as judged by the fluorescence intensity was 45.59 ±12.87 in the interference group, 103.72 ±21.76 in the rejection group, and 93.47 ±29.55 in the control group（F = 6.89, P 〈 0.05）. In stimulation assays, a one-way mixed lymphocyte reaction stimulation index（SI）, with spleen cells from Wistar and Lewisrats, respectively, the rejection group （5.26 ± 0.42,5.18 ± 0.29） and the control group （5.37 ± 0.27,4.93 ± 0.44） had significantly greater reactions than the interference group （2.37 ± 0.35, 4.87 ± 0.36）, respectivily （F = 7.29, P 〈 0.05 ; F = 6.19, P 〉 0.05）. In the IFN-γ and IL-4 expression assays, reactions of the interference group （230.17 ± 38.47,160.32 ± 59.13） were lower than those of the rejection group（490.73 ± 51.48,230.67 ± 45.21 ） and the control group（480.15 ± 43.96, 240.53 ± 47.36）, （F = 7.23,6.75, all P 〈 0.01 ）. Conclusions In vivo transfection of pSilencer 4.1-ICOS shRNA interference plasmid can effectively block T-cell co-stimulation pathway, suppress acute rejection, and prolong limb allografts survival.