CAJ | 학술논문

目的通过淋巴细胞输注诱导自身免疫性再生障碍性贫血动物模型,探讨CD28/B7信号在淋巴细胞异常活化中的作用及可能机制.方法将来自父本的淋巴细胞悬液(40×106个几左右)通过尾静脉注入CBYB6 F1代受鼠体内,采用CFDA-SE标记法跟踪淋巴细胞在体内的分布,尾静脉注射CD80和CD86阻断性单克隆抗体(单抗),不同时段检测受体小鼠外周血象的变化,病理切片观察骨髓及主要脏器的变化.将骨髓造血细胞与淋巴结淋巴细胞进行共培养,通过计数造血细胞的集落形成数来观察不同数量淋巴结淋巴细胞对骨髓造血细胞的影响.体外测试不同浓度环孢菌素A(cyclosporine A,CsA)对骨髓造血细胞的影响.结果输注淋巴细胞后可诱导受体小鼠在第5天出现骨髓衰竭的表现,主要是白细胞、血红蛋白、血小板下降,21 d左右受体鼠出现死亡.不同时间段受体小鼠主要脏器冰冻切片显示荧光标记的淋巴细胞主要分布在骨髓组织中;病理切片显示有骨髓组织的破坏.同时注射CD80及CD86阻断性单抗的受体鼠同样出现上述表现;体外集落形成试验证实B6淋巴结淋巴细胞数量和F1造血细胞为5:1时,红系集落形成单位(CFU-E)和粒细胞集落形成单位(CFU-G)集落形成数目与空白组比较差异无统计学意义(P>0.05);将比例提高至10:1,CFU-E集落形成数目明显减少(P<0.05);至50:1时,则可完全抑制CFU-E集落的形成,CFU-E和CFU-G集落形成数日的减少呈现淋巴细胞剂量依赖性,加入CsA可显著提高CFU-E和CFU-G形成率.结论通过模型证实T细胞在再生障碍性贫血的发生过程中起重要作用,仅通过阻断CD28/B7信号并不能阻断T淋巴细胞的异常活化.
목적 통과림파세포수주유도자신면역성재생장애성빈혈동물모형,탐토CD28/B7신호재림파세포이상활화중적작용급가능궤제.방법 장래자부본적림파세포현액(40×106개궤좌우)통과미정맥주입CBYB6 F1대수서체내,채용CFDA-SE표기법근종림파세포재체내적분포,미정맥주사CD80화CD86조단성단극륭항체(단항),불동시단검측수체소서외주혈상적변화,병리절편관찰골수급주요장기적변화.장골수조혈세포여림파결림파세포진행공배양,통과계수조혈세포적집락형성수래관찰불동수량림파결림파세포대골수조혈세포적영향.체외측시불동농도배포균소A(cyclosporine A,CsA)대골수조혈세포적영향.결과 수주림파세포후가유도수체소서재제5천출현골수쇠갈적표현,주요시백세포、혈홍단백、혈소판하강,21 d좌우수체서출현사망.불동시간단수체소서주요장기빙동절편현시형광표기적림파세포주요분포재골수조직중;병리절편현시유골수조직적파배.동시주사CD80급CD86조단성단항적수체서동양출현상술표현;체외집락형성시험증실B6림파결림파세포수량화F1조혈세포위5:1시,홍계집락형성단위(CFU-E)화립세포집락형성단위(CFU-G)집락형성수목여공백조비교차이무통계학의의(P>0.05);장비례제고지10:1,CFU-E집락형성수목명현감소(P<0.05);지50:1시,칙가완전억제CFU-E집락적형성,CFU-E화CFU-G집락형성수일적감소정현림파세포제량의뢰성,가입CsA가현저제고CFU-E화CFU-G형성솔.결론 통과모형증실T세포재재생장애성빈혈적발생과정중기중요작용,부통과조단CD28/B7신호병불능조단T림파세포적이상활화.
Objective To explore the role and possible mechanism of CD28/B7 molecules in T cell ab-normal activation by establishing a mouse model of the autoimmune aplastic anemia. Methods Unmanipulated B6D2F1 or CByB6F1 hybrid mice were infused with about 40 × 106 lymph node (LN) cells from their C57BL/6 (B6) parent. Distribution of the injected T cells was assayed by CFDA-SE fluorescent staining. Anti-D80 and anti-CD86 monoclonal antibodies were used to block CD28/B7 signal transduction pathways and to test the change of peripheral blood of F1 mice at different times. Damage was assessed by histological staining. Bone marrow (BM) cells and LN iymphocytes were cultured to observe the effect of different number of lymphocytes in the LN on BM cells' hematopoiesis by the count of hematopoietic colony-forming cells, and to test the effect of cyclosporine A of different concentration on BM cells' hematopoiesis. Results Infusion of about 40 × 106/mouse B6 LN cells led to the development of BM failure in the fifth day: anemia, neutropenia and thrombocytopenia. At 21st day recipients began to appear death. Frozen section revealed the injected lymph node major in myeloid tissue. Pathological sec-tion revealed obvious immune-induced marrow destruction and tissue destruction. There was similar performance of the above in the recipients infused with anti-D80 and anti-CD86 monoclonal antibodies. B6 LN five times the num-ber of lymphocytes in the blood cells F1, CFU-E and CFU-G colony formation of a blank group difference was not significant (P >0.05); When B6 LN 10 times the number of lymphocytes in the blood cells F1, CFU-E colony forming significantly reduce the number of (P < 0.05) ; When B6 LN lymphocytes 50 times in F1 hematopoietic cells, not observed CFU-E colony formation. CFU-E and CFU-G colony formation reducing the number of lympho-cytes showed a dose-dependent. Cyclosporine A can significantly increase the CFU-E and CFU-G colony forming rate. Conclusion This mouse model indicates that activated lymphocytes play important roles in marrow destruc-tion in lymphocyte infusion-induced BM failure. Only blocking the CD28/B7 signal transduction can not block the abnormal T-cells activated.