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目的研究外源性神经生长因子(nerve growth factor,NGF)对转基因降钙素基因相关肽(calcitonin gene related peptide,CGRP)骨髓干细胞(mesenchymal stem cells,MSCs)移植治疗骨质疏松大鼠的影响。方法构建CGRP基因表达载体,转染制备转基因CGRP-MSCs靶细胞;60只雌性SD大鼠骨质疏松模型随机均分为空白对照组(A组)、单纯MSCs移植治疗组(B组)、CGRP-MSCs移植治疗组(C组)、CGRP-MSCs+NGF移植组(D组)。在治疗1w、2w、3w后,实时定量荧光PCR和Western blot检测3个时间段每组大鼠股骨骨组织中CGRP基因和蛋白的相对表达水平,评价各组疗效。结果与A组比较,3个时间点,C组和D组中CGRP表达水平均显著上调(P<0.01);治疗1w后,C组和D组上调水平差异明显(P<0.01),D组低于C组;治疗2w后,C组和D组之间仍保持显著差异(P<0.05),D组仍低于C组;治疗3w后,C组和D组上调水平差异明显缩小,二者差异无统计学意义(P>0.05)。CGRP基因和蛋白相对表达水平在各组检测结果差异一致,同上。结论在转基因CGRP-MSCs移植治疗骨质疏松大鼠中联合应用外源性NGF,无累计效应,并可能抑制大鼠股骨骨组织中CGRP的表达,表明外源NGF于转基因CGRP-MSCs移植治疗骨质疏松症中可能并无促进作用,至少在CGRP表达层面无促进作用,而单纯的转基因CGRP-MSCs移植治疗骨质疏松大鼠疗效优于联合应用外源性NGF治疗方案。
Objective To investigate the effect of nerve growth factor (NGF) on the osteoporosis rats treated with transplanted calcitonin gene related peptide (CGRP) and bone marrow mesenchymal stem cells (MSCs). Methods CGRP gene expression vector was constructed and transfected into target cells of transgenic CGRP-MSCs. Sixty female SD rats were randomly divided into blank control group (group A), simple MSCs transplantation group (group B), CGRP -MSCs transplantation group (C group), CGRP-MSCs + NGF transplantation group (D group). After 1w, 2w and 3w treatment, the relative expression levels of CGRP gene and protein in each group were detected by real-time fluorescence quantitative PCR and Western blot. The curative effect of each group was evaluated. Results Compared with group A, the expression of CGRP in group C and group D were significantly increased at 3 time points (P <0.01), and the level of CGRP in group C was significantly higher than that in group D (P <0.05), but still lower than that in group C after 2 weeks of treatment. After 3 weeks of treatment, the differences in up-regulation between groups C and D were significantly reduced. The difference was not statistically significant (P> 0.05). The relative expression levels of CGRP gene and protein were the same in all groups, as above. Conclusion Transplantation of CGRP-MSCs transplantation in the treatment of osteoporosis rats combined with exogenous NGF, no cumulative effect, and may inhibit the expression of CGRP in rat femur bone tissue, indicating that exogenous NGF in transgenic CGRP-MSCs transplantation treatment of bone Osteoporosis may not promote the role, at least in the CGRP expression level did not promote the role of simple transgenic CGRP-MSCs transplantation in the treatment of osteoporosis in rats is better than the combined application of exogenous NGF treatment.