Rundle Charles H, Wang Xiaoguang, Sheng Matilda H-C, Wergedal Jon E, Lau K-H William, Mohan Subburaman
Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Administration Medical Center (151), 11201 Benton Street, Loma Linda, CA 92357, USA.
Bone. 2008 Nov;43(5):880-8. doi: 10.1016/j.bone.2008.07.239. Epub 2008 Jul 29.
This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had approximately 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and "Bik-like" pro-apoptotic gene increased approximately 1.5-fold and approximately 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2alpha1 and 9alpha1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair.
本研究旨在通过比较Bax基因敲除（KO）小鼠和野生型C57BL/6J（背景品系）小鼠的股骨骨折愈合情况，确定促凋亡基因Bax在骨折愈合中的作用。与C57BL/6J小鼠的骨折相比，Bax基因敲除小鼠的骨折块更大，骨矿物质含量更多，在骨折愈合的第一周和第二周，每骨痂面积的软骨面积大约大2倍，并且在骨折愈合的第三周和第四周，破骨细胞表面积增加。Bax基因敲除小鼠骨折骨痂中软骨面积的增加是由于前肥大软骨细胞和肥大软骨细胞数量的增加。TUNEL分析显示，在骨折后7天或14天，Bax基因敲除小鼠和C57BL/6J小鼠的骨折中，软骨细胞或非软骨细胞凋亡细胞的数量没有显著差异，这表明Bax基因敲除小鼠骨折中软骨细胞数量的增加不是由于细胞凋亡减少。对凋亡基因表达的分析表明，虽然在骨折后7天或14天，Bax基因敲除小鼠和C57BL/6J小鼠之间Bcl-2和Bcl-xL的表达水平没有差异，但与C57BL/6J小鼠的骨折相比，在骨折后7天和14天，Bax基因敲除小鼠骨折中仅含BH3结构域的Bak和“Bik样”促凋亡基因的表达分别增加了约1.5倍和约2倍，这表明Bax基因敲除小鼠中Bak和Bik样促凋亡基因的上调可能在细胞凋亡方面弥补了Bax功能的缺失。通过将溴脱氧尿苷体内掺入骨折组织中的软骨细胞进行分析表明，与C57BL/6J小鼠的骨折相比，在第7天，Bax基因敲除小鼠骨折中的软骨细胞增殖显著增加。在早期愈合过程中，Bax基因敲除小鼠骨折中胶原蛋白2α1和9α1基因表达的增加与软骨细胞增殖的增加一致。总之，本研究首次证明Bax在骨折愈合的早期阶段具有重要作用，并且Bax基因敲除小鼠骨折中骨痂大小和软骨面积的增加是由于软骨细胞增殖增加，而不是由于细胞凋亡减少或软骨细胞肥大增加。Bax缺乏对软骨细胞增殖的意外影响暗示了Bax在骨折修复过程中对软骨细胞增殖具有新的调节功能。
