Cx43在造血调控及重建中的作用

目的:采用条件性基因敲除技术构建造血系统间隙连接蛋白43(Cx43)基因敲除(Cx43^-/-)小鼠模型,并探讨Cx43在维持造血细胞自我更新及功能稳定中的作用。方法:将引进的2对转基因小鼠Cx43 loxP/loxP和Lyz-Cre/+杂交,选取F1雌性子代Cx43 loxP/-_Lyz-Cre/+与雄性Cx43 loxP/loxP合笼回配,提取所获得子代小鼠鼠尾组织基因组DNA,采用PCR方法鉴定小鼠基因型,RT-PCR方法筛选Cx43^-/-小鼠,同时分析小鼠不同器官中Cx43基因的表达差异;该类小鼠经5-氟尿嘧啶(5-FU;125 mg/kg)处理,在化疗前及化疗后第5、10和15天经眼球取血分析其血象变化。Cx43^-/-及Cx43^+/+小鼠予7.5 Gy(⁶⁰Co-γ)的致死量照射,剂量率1 Gy/min,照射后6 h分别给予事先准备就序的骨髓细胞,每只3×10~6细胞于尾静脉注入,2周后处死小鼠检测造血是否重建:分离股骨切片后,收集骨髓细胞进行细胞表型分析(选用的单抗为CD45R、Gr-1、CD4、 CD8a、TCRαβ、Mac-1、抗sIgM、TER119、Sca-1及CD117);同时进行体外造血细胞集落实验观察造血细胞的体外增殖能力。结果:本研究通过2种转基因小鼠间杂交和回交,成功获得造血系统选择性Cx43基因敲除小鼠;该类小鼠骨髓及外周血细胞无Cx43表达,参与造血的组织,如肝脏和脾脏中Cx43表达也显著下调(P<0.01),而心脏和肾脏的Cx43表达则无影响,小鼠成年后外周血象分析并无明显异常,但应急代偿能力下降,经5-FU处理后,其造血功能恢复显著减缓,处理15 d后,Cx43^+/+小鼠造血功能已接近正常水平,而Cx43^-/-小鼠仍无明显的恢复迹象,血红蛋白、白细胞及血小板仍处低位,2者差别有统计学显著性(P<0.01);体外集落试验也证实Cx43^-/-小鼠造血干/祖细胞的增殖能力下降,其CFU-GM或CFU-E集落数均明显少于Cx43^+/+小鼠(P<0.01),但流式细胞术结果显示,Cx43^-/-小鼠骨髓中Lin^-/c-Kit^+/Sca-1^+细胞亚群数量与Cx43^+/+小鼠相比差异并无统计学显著性;Cx43^-/-小鼠在化疗或移植后其骨髓造血功能重建均延迟,且化疗15 d后骨髓切片及涂片均证实其骨髓中造血细胞增生程度明显降低,脂肪组织显著增多,而且T、B细胞发育也有异常。此外,其外周血中CD4^+CD8^+细胞比例比野生型小鼠增多(P<0.05),但CD4^+T细胞显著减少(P<0.01),尤其是TCRαβ亚群细胞减少最为明显(P<0.01)。同样,Cx43^-/-小鼠外周血中CD45R^+sIgM^-细胞亚群比例与野生型小鼠相比显著减少(P<0.01)。结论:骨髓中Cx43基因表达在造血干/祖细胞发育(尤其是应急状态时)具重要作用,敲除Cx43基因后造血干/祖细胞增殖减缓,造血及免疫重建功能受损。

Role of connexin 43 in regulation and reconstruction of hematopoiesis

AIM:To establish a conditional connexin 43 (Cx43) gene knock-out mouse model and to explore the role of Cx43 in proliferation and differentiation of developing haematopoietic cells in mice. METHODS:Two pairs of transgenic mice, Cx43 loxP/loxP brought from Jackson Lab, USA and Lyz-Cre/+ brought from Center of Experimental Animal, Jingus, were inbreeded to produce hybrid mice F1. The Cx43 loxP/-_Lyz-Cre/+ female F1 mice were selected to mate with Cx43 loxP/loxP male mice to finally produce the Cx43^-/- mice. The expression of Cx43 in different organ of these mice was detected by PCR. Both Cx43^-/- and Cx43^+/+ mice either received intravenous injection of 5-FU at 125 mg/kg or 7.5 Gy irradiation with ⁶⁰Co-γ ray equipment and bone marrow cells transfusion, the recovery of their hematopoietic tissues was assessed with different assays at different time interval. The change of phenotype of immunologic cells was determined by flow cytometry using different fluorochrome conjugated CD45R, Gr-1, CD4, CD8a, TCRαβ, Mac-1, anti-sIgM, TER119, Sca-1 and CD117. RESULTS:The Cx43 gene knock-out mouse model was successfully established by mating those 2 pairs of transgenic mice. No Cx43 expression on blood cell either from peripheral blood or bone marrow was observed in Cx43^-/- mice. The expression of Cx43 on the liver and spleen cells of these Cx43 gene conditional knock-out mice was decreased significantly as compared with wild type mice (P<0.01). There was no significant diffrernece of Cx43 expression on the heart and renal cells between Cx43^-/- mice and wild type mice. The bone marrow cellularity and peripheral blood counts of Cx43^-/- mice were markedly reduced as analyzed post-5-FU treatment for 15 days, while the wild type mice recovered to normal (P<0.01). Moreover, hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by decreasing proliferation of hematopoietic progenitor content, in which granulomacrophagic colony forming units (CFU-GM) and erythroid forming units (CFU-E) in bone marrow cells of Cx43 deficient mice were greatly decreased (P<0.01). However, the content of Lin^-/c-Kit⁺/Sca-1⁺ in Cx43^-/- bone marrow cells was maintained as compared with Cx43^+/+ bone marrow cells. Cx43 deficiency not only delayed the hematopoiesis reconstruction after chemotherapy or bone marrow transplantation, but also decreased the bone marrow cellularity and increased adipocytes in bone marrow microenvironment as compared with those in Cx43^+/+ mice. The T and B lineage cell developments were abnormal in Cx43^-/- mice. The proportion of CD4⁺CD8⁺ cell increased significantly (P<0.05), while the CD4⁺ T cells, especially th TCRαβ subpopulation markedly reduced in comparison with wild type mice (P<0.01). Moreover, the CD45R⁺sIgM^- subgroup in Cx43 deficiency mice was markedly reduced in comparison with its counterpart. CONCLUSION:The expression of Cx43 on bone marrow microenvironment is critical for developing hematopoietic stem cells, especially in response to hematopoietic stress. The hematopoietic regeneration and lymphoid cell development are blocked in Cx43 deficient mice.