中国地方病学杂志
中國地方病學雜誌
중국지방병학잡지
CHINESE JOURNAL OF ENDEMIOLOGY
2008年
4期
405-411
,共7页
陆晓峰%毕郑钢%杨卫良%张风民%刘伟%邵明%朱启亮
陸曉峰%畢鄭鋼%楊衛良%張風民%劉偉%邵明%硃啟亮
륙효봉%필정강%양위량%장풍민%류위%소명%주계량
RNA干扰%移植,同种%下肢%移植物排斥%共刺激通路
RNA榦擾%移植,同種%下肢%移植物排斥%共刺激通路
RNA간우%이식,동충%하지%이식물배척%공자격통로
RNA interference%Transplantation,homologous%Limb,lower%Transplantation rejections%Costimulatary pathway
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-pSileneer 4.1空载体复合物;干扰组注射梭华-Sofagt-pSileneer 4.1-ICOSshRNA干扰质粒复合物.术后8 d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(X2=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,P0.05).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pSilencer4.1-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.
目的 通過RNA榦擾併阻斷可誘導共刺激分子(ICOS)刺激通路,觀察對大鼠異體肢體移植急性排斥反應的抑製作用.方法 成年Wistar、SD大鼠各27隻,按體質量將大鼠隨機分為排斥組、對照組、榦擾組.每組9例.大鼠肢體移植採用改良法.將Wistar大鼠(供體)右下肢體移植到SD大鼠(受體)左下肢體上.移植術後排斥組不給予特殊處置;對照組陰莖揹靜脈註射梭華-Sofast-pSileneer 4.1空載體複閤物;榦擾組註射梭華-Sofagt-pSileneer 4.1-ICOSshRNA榦擾質粒複閤物.術後8 d每組處死SD大鼠3隻,取移植肢體皮膚、肌肉及骨骼進行病理學檢查;流式細胞儀及RT-PCR檢測淋巴細胞錶麵ICOS和基因mRNA錶達:分彆用Wistar、Lewis大鼠脾細胞刺激,進行單嚮混閤淋巴細胞培養,計算細胞刺激指數;ELISA雙抗體夾心法檢測大鼠血清細胞因子γ榦擾素(IFN-γ)和白細胞介素4(IL-4);另6隻大鼠觀察生存情況及肢體存活時間.結果 排斥組大鼠肢體平均存活時間(11.34±1.21)d,對照組平均存活時間(11.14±1.32)d,榦擾組平均存活時間(16.85±1.73)d,組間比較差異有統計學意義(F=8.72,P<0.05).排斥組和對照組病理學結果提示,肢體齣現瞭中、重度急性排斥反應,榦擾組急性排斥反應輕微.光鏡下排斥組和對照組皮膚齣現錶皮壞死、大泡形成伴有血管週圍炎及大量淋巴細胞浸潤,肌肉組織有瀰散性淋巴細胞浸潤,間質水腫同時伴有肌細胞壞死.脾淋巴細胞ICOS基因mRNA錶達顯示,榦擾組(18.75%)明顯低于排斥組(100%)和對照組(98.51%),組間比較差異有統計學意義(X2=13.57,P<0.01).淋巴細胞錶麵ICOS錶達熒光彊度,榦擾組為45.59±12.87,排斥組為103.72±21.76,對照組為93.47±29.55,組間比較差異有統計學意義(F=6.89,P<0.05).單嚮混閤淋巴細胞反應刺激指數(SI),排斥組(5.26±0.42、5.18±0.29)和對照組(5.37±0.27、4.93±0.44)明顯高于榦擾組(2.37±0.35、4.87±0.36),組間比較差異有(無)統計學意義(F=7.29,P<0.05;F=6.19,P0.05).IFN-γ和IL-4錶達.榦擾組(230.17±38.47、160.32±59.13)較排斥組(490.73±51.48、230.67±45.21)和對照組(480.15±43.96、240.53±47.36)均降低,組間比較差異有統計學意義(F值分彆是7.23、6.75,P<0.05).結論 體內轉染pSilencer4.1-ICOSshRNA榦擾質粒能有效阻斷T細胞共刺激通路,抑製急性排斥反應,延長移植肢體存活時間.
목적 통과RNA간우병조단가유도공자격분자(ICOS)자격통로,관찰대대서이체지체이식급성배척반응적억제작용.방법 성년Wistar、SD대서각27지,안체질량장대서수궤분위배척조、대조조、간우조.매조9례.대서지체이식채용개량법.장Wistar대서(공체)우하지체이식도SD대서(수체)좌하지체상.이식술후배척조불급여특수처치;대조조음경배정맥주사사화-Sofast-pSileneer 4.1공재체복합물;간우조주사사화-Sofagt-pSileneer 4.1-ICOSshRNA간우질립복합물.술후8 d매조처사SD대서3지,취이식지체피부、기육급골격진행병이학검사;류식세포의급RT-PCR검측림파세포표면ICOS화기인mRNA표체:분별용Wistar、Lewis대서비세포자격,진행단향혼합림파세포배양,계산세포자격지수;ELISA쌍항체협심법검측대서혈청세포인자γ간우소(IFN-γ)화백세포개소4(IL-4);령6지대서관찰생존정황급지체존활시간.결과 배척조대서지체평균존활시간(11.34±1.21)d,대조조평균존활시간(11.14±1.32)d,간우조평균존활시간(16.85±1.73)d,조간비교차이유통계학의의(F=8.72,P<0.05).배척조화대조조병이학결과제시,지체출현료중、중도급성배척반응,간우조급성배척반응경미.광경하배척조화대조조피부출현표피배사、대포형성반유혈관주위염급대량림파세포침윤,기육조직유미산성림파세포침윤,간질수종동시반유기세포배사.비림파세포ICOS기인mRNA표체현시,간우조(18.75%)명현저우배척조(100%)화대조조(98.51%),조간비교차이유통계학의의(X2=13.57,P<0.01).림파세포표면ICOS표체형광강도,간우조위45.59±12.87,배척조위103.72±21.76,대조조위93.47±29.55,조간비교차이유통계학의의(F=6.89,P<0.05).단향혼합림파세포반응자격지수(SI),배척조(5.26±0.42、5.18±0.29)화대조조(5.37±0.27、4.93±0.44)명현고우간우조(2.37±0.35、4.87±0.36),조간비교차이유(무)통계학의의(F=7.29,P<0.05;F=6.19,P0.05).IFN-γ화IL-4표체.간우조(230.17±38.47、160.32±59.13)교배척조(490.73±51.48、230.67±45.21)화대조조(480.15±43.96、240.53±47.36)균강저,조간비교차이유통계학의의(F치분별시7.23、6.75,P<0.05).결론 체내전염pSilencer4.1-ICOSshRNA간우질립능유효조단T세포공자격통로,억제급성배척반응,연장이식지체존활시간.
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 llmb allotransplantation were performed from Wistar to SD rats. The rats were divided into 3 gronps(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 eytokines 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 infdtration, 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 Lewis rats, 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, P0.05). 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.
