骨髓间充质干细胞过表达碱性成纤维细胞生长因子对骨质疏松性骨折大鼠的治疗作用

Therapeutic effect of bone marrow mesenchymal stem cells overexpressing fibroblast growth factor on osteoporotic fracture in rats

目的观察骨髓间充质干细胞（BMSC）过表达碱性成纤维细胞生长因子（bFGF）对骨质疏松性骨折大鼠的治疗效果。方法选取80只3个月龄雌性sD大鼠，分为对照组、模型组、BMSC组和BMSC＋bFGF组，模型组、BMSC组和BMSC＋bFGF组大鼠采用切除卵巢去势法建立骨质疏松骨折模型，模型组、BMSC组和BMSC＋bFGF组大鼠全部制作胫骨骨折模型。BMSC组和BMSC＋bFGF组分别于术后第1、14、21天移植BMSC和过表达bFGF的BMSC，对照组和模型组大鼠给予等体积的生理盐水注射。采用Western blot分析BMSC中bFGF蛋白表达水平；采用骨密度仪测定各组大鼠骨密度；采用X线评分评价骨折愈合程度；采用三点弯曲实验分析胫骨生物力学参数变化；采用反转录．聚合酶链反应（RT—PCR）分析碱性磷酸酶（ALP）、Runt相关基因2（Runx2）和骨形态发生蛋白-2（BMP-2）mRNA水平。结果Western blot结果显示BMSC＋bFGF组细胞bFGF表达水平明显上调。与模型组大鼠X线评分和骨密度（2．02±0．37、0．051±0．002）比较，BMSC组（3．12±0.46、0．800±0．004）和BMSC＋bFGF组（4．43±0．51、0．128±0．006）大鼠x线评分和骨密度明显增加（P=0．032、0．013；P=0．000、0．000），生物学参数（最大载荷、弹性载荷、刚度和最大挠度）显著改善（P=0．000）。与BMSC组比较，BMSC＋bFGF组大鼠X线评分和骨密度显著增加（P=0．010、0．010），生物学参数（最大载荷、弹性载荷、刚度和最大挠度）显著改善（P=0．002、0．000；P=0．000、0．000）。与模型组ALP、Runx2和BMP-2mRNA水平（1.41±0.19、1．31±0．20、1．43±0．24）比较，BMSC组（2．16±0．33、2．48±0．31、3．17±0．33）和BMSC＋bFGF组（4．48±0．48、5．13±0．41、4．89±0．49）大鼠骨痂组织ALP、Runx2和BMP-2 mRNA水平明显增加（P=0．000）。与BMSC组比较，BMSC＋bFGF组大鼠骨痂组织ALP、Runx2和BMP-2 mRNA水平增加更为显著（P=0．000）。结论BMSC中过表达bFGF可提高与骨形成相关蛋白的表达，增加骨密度，促进骨质疏松性骨折的愈合，增强骨折生物学应力。

Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cells （BMSC） overexpressing basic fibroblast growth factor （bFGF） on osteoporotic fracture in rats. Meth- ods 80 female SD rats were randomly divided into control group, model group, BMSC group and BMSC ＋ bFGF group. The osteoporotic fracture model was established by resection of ovariectomized in model group, BMSC group and BMSC ＋ bFGF group. In group BMSC and group BMSC ＋ bFGF, BMSC and bFGF overexpressing BMSC were transplanted at 1, 14 and 21 d after operation respectively, while rats in the control and model groups were given the same volume of saline injection. The protein level were analyzed by Western blotting in BMSC group and BMSC ＋ bFGF group. Determination of bone density of rats in four group were detected by bone densitometry and fracture healing was evaluated by X - ray. The changes of bone biomechanical parameters were analyzed using three point bending test. alkaline phosphatase （ALP） , runt related transcription factor - 2 （ Runx2 ） and bone morphogenetic protein - 2 （ BMP - 2 ） mRNA level in four groups were analyzed by reverse transcriptase - polymerase chain reaction （ RT - PCR ）. Results Western blot showed that the expression of bFGF factor was up - regulated in BMSC ＋ bFGF group. Compared with the model group [2. 02 ±0. 37, 0. 051±0. 002] , the X - ray scores and bone mineral density significantly increased in the BMSC [ 3. 12 ±0. 46, 0. 800 ± 0. 004 ] and BMSC ＋ bFGF groups [ 4. 43 ± 0. 51, 0. 128 ±0. 006 ], and the biological parameters （maximum load, elastic load, stiffness and maximum deflection） significantly improved （ P = 0. 032, 0. 013 ; P = 0. 000, 0. 000 ）. Compared with the BMSC group, the X - ray score and bone mineral density in the BMSC ＋ bFGF group significantly increased （P =0. 010, 0. 010） and the biological parameters （maximum load, elastic load, stiffness and maximum deflection） significantly improved （ P = 0. 002, 0. 000; P = 0. 000, 0. 000）. Compared with the model group （1.41 ±0. 19, 1.31 ± 0. 20, 1.43 ± 0. 24）, the levels of ALP, Runx2 and BMP- 2 mRNA in callus tissue of BMSC group （2. 16± 0. 33, 2. 48±0. 31, 3.17 ±0. 33 ） and BMSC ＋ bFGF group （4. 48 ±0.48, 5.13 ±0. 41,4. 89±0. 49） significantly increased （P =0. 000）. Compared with group BMSC, the levels of ALP, Runx2 and BMP - 2 mRNA in callus tissue of BMSC ＋ bFGF group more significantly increased （P = 0. 000）. Conclusion Overexpression of bFGF in BMSC can increase the expression of bone morphogenetic protein, increase bone mineral density, promote the healing of osteoporotic fracture, and enhance the biological stress of fracture.