Xiao Kai, Yang Lin, Xie Wei, Gao Xinfeng, Huang Ruokun, Xie Ming
Foot and Ankle Surgery, Wuhan Fourth Hospital, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430033, P.R. China.
Department of Allergy, Tongji Hospital of Tongji Medical College of HUST, Wuhan, Hubei 430033, P.R. China.
Exp Ther Med. 2021 Jul;22(1):736. doi: 10.3892/etm.2021.10168. Epub 2021 May 9.
Bcl-xL is a transmembrane molecule in the mitochondria, with apoptosis-related and pro-metabolic functions, that also plays a role in chondrogenesis and differentiation. A Bcl-xL mutant, in which the GRI sequence is replaced by ELN, has no anti-apoptotic effect, while other biological functions of this mutant remain unchanged. The present study investigated the impact of this Bcl-xL mutant on cartilage differentiation and the expression levels of TGF-β and bone morphogenetic protein (BMP). Human bone marrow mesenchymal stem cells (BMSCs) were transfected with Bcl-xL and Bcl-xL mutant (∆Bcl-xL) overexpression vectors. The cells were divided into four groups: Control (not subjected to any transfection), EV (empty pcDNA3.1-Bcl-xL vector), OV (Bcl-xL overexpression) and ∆OV (∆Bcl-xL overexpression). Saffron and toluidine blue staining was performed to observe cartilage tissue formation. Flow cytometry was conducted to measure BMSC apoptosis. The expression levels of TGF-β and BMP were evaluated using reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Compared with that in the control group, the expression levels of Bcl-xL in the OV group increased significantly (P<0.05). Western blotting and RT-qPCR results revealed that OV and ∆OV treatment increased the expression levels of TGF-β and BMP in transfected cells, compared to their expression in the control and EV groups (P<0.05). Saffron and toluidine blue staining results showed that cartilage formation was increased in the ∆OV and ∆OV + Bax-/Bak-groups to similar degrees. Cell apoptosis in the ∆OV group did not change compared with that in the control group. The Bcl-xL mutant promoted cartilage differentiation of BMSCs and upregulated TGF-β/BMP expression. This enhancement of chondrogenic differentiation was not related to the expression of Bax and Bak. Taken together, these findings provided for improved application of bone tissue engineering technology in the treatment of articular cartilage defects.
Bcl-xL是一种存在于线粒体中的跨膜分子，具有凋亡相关和促代谢功能，在软骨形成和分化中也发挥作用。一种Bcl-xL突变体，其GRI序列被ELN取代，没有抗凋亡作用，而该突变体的其他生物学功能保持不变。本研究调查了这种Bcl-xL突变体对软骨分化以及转化生长因子-β（TGF-β）和骨形态发生蛋白（BMP）表达水平的影响。用Bcl-xL和Bcl-xL突变体（∆Bcl-xL）过表达载体转染人骨髓间充质干细胞（BMSC）。细胞分为四组：对照组（未进行任何转染）、空载体组（空的pcDNA3.1-Bcl-xL载体）、过表达组（Bcl-xL过表达）和∆过表达组（∆Bcl-xL过表达）。进行番红和甲苯胺蓝染色以观察软骨组织形成。采用流式细胞术检测BMSC凋亡。使用逆转录定量PCR（RT-qPCR）和蛋白质印迹法评估TGF-β和BMP的表达水平。与对照组相比，过表达组中Bcl-xL的表达水平显著升高（P<0.05）。蛋白质印迹和RT-qPCR结果显示，与对照组和空载体组相比，过表达组和∆过表达组处理使转染细胞中TGF-β和BMP的表达水平升高（P<0.05）。番红和甲苯胺蓝染色结果表明，∆过表达组和∆过表达组+Bax-/Bak-组的软骨形成增加程度相似。∆过表达组的细胞凋亡与对照组相比没有变化。Bcl-xL突变体促进了BMSC的软骨分化并上调了TGF-β/BMP表达。这种软骨生成分化的增强与Bax和Bak的表达无关。综上所述，这些发现为骨组织工程技术在治疗关节软骨缺损中的更好应用提供了依据。
