BMP4过表达转基因C57BL/6品系小鼠子代造血干细胞归巢能力观察

Homing and reconstitution of hematopoietic stem cells in transgenic newborn mice of C57BL/6 strain with overexpressed BMP4

目的 目的观察骨形态发生蛋白4(BMP4)过表达转基因C57BL/6品系小鼠造血干细胞(HSC)的归巢能力.方法 ①构建BMP4过表达的C57BL/6品系小鼠转基因模型,设计特异引物对转基因新生小鼠进行筛选,筛选子代BMP4阳性鼠.取筛选后的子代阳性鼠5只,另取野生型C57BL/6品系小鼠5只,流式细胞仪分选出两种小鼠骨髓HSC.另取野生型C57BL/6品系小鼠20只,以10 Gy/20 g的钴60进行致死性辐照,后随机分为4组各5只,转基因1组小鼠经尾静注子代阳性鼠骨髓HSC和CFSE,野生型1组静注野生型小鼠骨髓HSC和CFSE,阳性对照组静注HSC体外CFSE追踪染色,阴性对照组只注射等量PBS.体内追踪16 h后提取骨髓有核细胞数(BMNC),流式细胞仪上机,检测CFSE所在阳性区域为HSC的归巢效率.②随机取转基因BMP4子代阳性鼠和野生型C57BL/6品系小鼠各3只(分别计为转基因2组和野生型2组),采用RT-PCR法检测造血归巢趋化因子(CX-CL12),Western blotting法检测整合素α4(ITGA4)和血管内皮细胞黏附分子1(VCAM-1)蛋白.结果 转基因1组、野生型1组、阳性对照组、阴性对照组归巢效率分别为(0.3520±0.02230)%、(0.7210±0.0320)%、(37.2000±0.5560)%、(0.1013±0.0180)%,转基因组与野生组、阴性组比较,野生组与阴性组比较,P均<0.05.转基因2组、野生型2组CXCL12 mRNA分别为(2.547.±0.126)%、(100.110±3.811)%,ITGA4蛋白分别为(231.0±2.49)%、(163.9±0.38)%,VCAM-1蛋白分别为(88.47±8.33)%、(92.33±1.98)%,两组CXCL12 mRNA、IT-GA4蛋白比较,P均<0.05.结论 BMP4过表达转基因C57BL/6品系转基因子代小鼠HSC归巢效率与野生型相比是降低的,机制可能是通过CXCL12和ITGA4进行调控的,升高的ITGA4无法补偿CXCL12的下调,从而导致了归巢效率的降低.

Objective To observe the effect of bone morphogenetic protein 4 (BMP4 ) overexpression on homing ability of C57BL/6 mouse hematopoietic stem cells (HSCs) and to explore its possible mechanism. Methods We constructed the transgenic model mice of C57BL/6 strain with overexpression of BMP4 and designed the specific primers to screen transgenic newborn mice. Those screened mice were used for the subsequent experiments. Five offspring BMP4 positive mice and five wild-type C57BL/6 mice were selected, and the bone marrow HSCs of the two kinds of mice were separated by flow cytometry. Next, another 20 wild-type C57BL/6 mice were irradiated with 10 Gy/20 g cobalt 60, then they were randomly divided into 4 groups with 5 mice in each. The transgenic mice in the group 1 were injected intravenously with HSC of progeny BMP4 positive mice and 5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester (CFSE), the mice in the wild-type group 1 were injected with wild-type mice bone marrow HSCs and CFSE, the mice in the negative control group were injected with the same amount of PBS, and the mice in the positive control group were injected with HSC in vitro CFSE tracing staining. After 16 hours of in vivo tracking, bone marrow nucleated cells (BMNCs) were extracted after in vivo tracking for 16 h, and the positive area of CFSE was detected by flow cytometry to determine the homing efficiency of HSC. Three transgenic BMP4 offspring positive mice and three wild-type C57BL/6 strain mice (transgenic group 2 and wild-type group 2) were randomly selected and hematopoietic homing chemokine (CXCL12) was detected by RT-PCR. We detected the integrin a 4 (ITGA4 / and vascular endothelial cell adhesion molecule 1 (VCAM-1) protein by Western blotting. Results Homing efficiency of the transgenic group 1 , wild-type group 1 , positive control group and negative control group was 0. 352 0%±0.0223 0%, 0.721 0%±0.032 0%, 37.200 0%±0.556 0%, and 0.101 3%±0.018 0%, respectively;there was significant difference between the transgenic group and wild group and negative group ( all P < 0. 05). The relatively mRNA of CXCL12 in the transgenic group 2 and wild-type group 2 was 2. 547%± 0. 126% and 100. 110%± 3. 811%,and the relative protein of ITGA4 of the transgenic group 2 and wild-type group 2 was 231. 0%± 2. 49% and 163.9%± 0. 38%, and the VCAM-1 protein was 88. 47%± 8. 33% and 92. 33%± 1.98%;and there were significant differences in the CXCL12 mRNA and ITGA4 protein between the transgenic group 2 and the wild-type group 2 ( both P <0. 05). Conclusions The homing efficiency of BMP4 overexpressed C57BL/6 strain of transgenic mice HSMs is lower than that of the wild type ones. The mechanism may be regulated by CXCL12 and ITGA4,and the increased ITGA4 can not compensate for the down-regulation of CXCL12,resulting in the decreased homing efficiency.