外源性NGF对转基因CGRP干细胞移植治疗骨质疏松大鼠的影响

The effect of exogenous NGF on the treatment of osteoporosis with CGRP gene transgenic MSCs transplantation in rats

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 investigatetheeffect of exogenous nervegrowth factor（ NGF） on osteoporotic rats under therapy with mesenchymal stem cell（ MSC） transfected with calcitonin generelated peptide（ CGRP） gene. Methods Vectors express CGRP wereconstructed and transfected to CGRP-MSCs target cells. Sixty femaleosteoporotic SD rats wererandomly divided into thecontrol group（ group A）,simpleMSCs transplantation treatment group（ group B）,CGRP transgenic MSCs（ CGRP-MSCs）transplantation treatment group（ group C）,and CGRP-MSCs transplantation with exogenous NGF（ CGRP-MSCs ＋ NGF） treatment group（ group D）. At 1,2,and 3 weeks after thetreatment,CGRP expression levels in thefemurs was measured using real-timePCR and Western blotting. Theefficacy was compared between each group. Results Compared with control group,CGRP expression in Group C and D wereboth significantly up-regulated（ P 0. 01） at all time. After 1- week treatment,thedifferencebetween group C and D was significant（ P 0. 01）. Theexpression in group D was significantly lower than that in thegroup C.After2- week treatment,thedifferencebetween group C and D was still significant（ P 0. 05）,and theexpression in group D was still significantly lower than that in group C. After 3-week treatment,theup-regulation differencein group D and C becamenarrower,and thedifferencewas not statistically significant（ P 0. 05）. Theresults of CGRP geneand protein expression wereconsistent in all groups. Conclusion This study suggests that additional application of exogenous NGF may inhibit CGRP expression when CGRP genetransgenic MSCs transplantation therapy is applied in osteoporosis rat model,indicating that additional application of exogenous NGF has no benefit to theeffect of CGRP,at least on CGRP geneexpression level. CGRP genetransgenic MSCs transplantation therapy without exogenous NGF is better than additional application of exogenous NGF.