局部注射神经生长因子及碱性成纤维生长因子对大鼠胫骨骨折愈合的影响

Effect of local injection of nerve growth factor and basic fibroblast growth factor on the healing of tibial fracture in rats

目的探讨局部注射神经生长因子(NGF)及碱性成纤维生长因子(BFGF)对大鼠胫骨骨折愈合的影响及其机制。方法选取12周龄清洁级成熟雄性SD大鼠48只,用数字表法随机分为NGF组、BFGF组、NGF+BFGF组、对照组4组,各12只;每组大鼠按观察时间点不同随机分为术后2周及术后4周2个亚组,每亚组6只。48只大鼠均建立左胫骨骨折模型,并行1 mm克氏针髓内内固定。术后第3天于骨折断端周围局部经皮注射给药:NGF组注射NGF 0.8μg+0.3 mL生理盐水,BFGF组注射BFGF 1.2μg+0.3 mL生理盐水,NGF+BFGF组注射NGF 0.8μg+BFGF 1.2μg+0.3 mL生理盐水,对照组注射0.3 mL生理盐水;每天注射1次,连续注射7 d。术后2周、4周分别行X线摄片,并采集各组大鼠股动脉血液标本后,采用颈椎离断法处死大鼠。取各组大鼠左胫骨骨痂标本,采用酶联免疫吸附试验(ELISA)检测碱性磷酸酶(ALP)含量;将骨痂组织制成病理切片,行HE染色,观察组织学特点;应用多功能真彩色细胞图象分析管理系统计算切片中骨组织的骨小梁表面成骨细胞指数(IOB)、骨小梁宽度(TW)、骨小梁面积百分比(TV);采用蛋白芯片技术检测大鼠血清中骨形成蛋白-2(BMP-2)、血管内皮生长因子(VEGF)、胰岛素样生长因子-1(IGF-1)的含量。结果(1)术后2周、4周X线摄片:NGF、BFGF、NGF+BFGF三组大鼠骨折愈合均较对照组快,以NGF+BFGF组愈合最快。(2)ELISA检测:对照组、NGF组、BFGF组、NGF+BFGF组术后2周骨痂组织中ALP的含量分别为(110.02±1.92)、(140.87±2.22)、(136.12±1.23)、(187.44±0.90)ng/mL,术后4周骨痂组织中ALP的含量分别为(91.23±1.47)、(106.62±1.64)、(104.83±1.05)、(130.59±1.18)ng/mL;各观察时点4组间比较,NGF+BFGF组含量最高,组间两两比较差异均有统计学意义(P值均<0.05)。(3)组织学观察:不同时间点上NGF、BFGF、NGF+BFGF三组大鼠在骨痂的生长、骨小梁形成均较对照组明显,且NGF+BFGF组优于其他组。骨小梁形态学指标测量显示,不同时间点4组间比较,对照组、NGF组、BFGF组、NGF+BFGF组大鼠骨痂内骨小梁IOB、TW、TV均呈上升趋势,NGF+BFGF组最高;组间两两比较,除NGF组与BFGF组各指标差异均无统计学意义(P值均>0.05),其他指标组间差异均有统计学意义(P值均<0.05)。(4)蛋白芯片检测:不同时间点4组间比较,除VEGF含量在术后4周时差异无统计学意义(P>0.05),VEGF术后2周及BMP-2、IGF-1术后2周、4周时,NGF组、BFGF组、NGF+BFGF组均高于对照组,NGF+BFGF组含量最高,组间两两比较,差异均有统计学意义(F=198.285、368.060、2006.017、33.332、292.643,P值均<0.05)。结论NGF及BFGF均能促进大鼠胫骨骨折愈合,且两者联合应用有协同作用。其机制可能与能够促进骨折愈合过程中VEGF、BMP-2、IGF-1的表达有关。

Objective To investigate the effects and mechanism of local injection of nerve growth factor(NGF)and basic fibroblast growth factor(BFGF)on tibial fracture healing in rats.Methods Forty-eight clean mature male SD rats were randomly divided into four groups:NGF group,BFGF group,NGF+BFGF group and control group.Each group was randomly divided into two subgroups(2 weeks after operation and 4 weeks after operation)according to different observation time points,six rats in each subgroup.The left tibial fracture model was established in 48 rats,and intramedullary fixation with 1 mm Kirschner wire was performed.On the third day after surgery,local percutaneous injection was given around the fracture end.The NGF group was injected with 0.8μg+0.3 mL normal saline,the BFGF group received local injection of BFGF 1.2μg+0.3 mL normal saline,the NGF+BFGF group received local injection of NGF 0.8μg+BFGF 1.2μg+0.3 mL normal saline,and the control group received 0.3 mL normal saline.Each group was injected once a day for 7 consecutive days.X-ray films were taken at 2 weeks and 4 weeks after the operation,and blood samples of femoral artery of rats in each group were taken,then the rats were killed by cervical vertebra disconnection.The content of alkaline phosphatase(ALP)was detected by enzyme-linked immunosorbent assay(ELISA).The callus tissue was made into pathological sections,and HE staining was performed to observe the histological characteristics.Osteoblastic index(IOB),trabecular width(TW)and trabecular area percentage(TV)of bone tissue were calculated by multifunctional true-color cell image analysis and management system.The contents of bone morphogenetic protein-2(BMP-2),vascular endothelial growth factor(VEGF)and insulin-like growth factor-1(IGF-1)in serum of rats were detected by protein chip technique.Results(1)X-ray film:the fracture healing of NGF,BFGF and NGF+BFGF group was faster than that of the control group,and that of NGF+BFGF group was the fastest.(2)ELISA:the content of ALP at 2 weeks after surgery in the callus of the control group,NGF group,BFGF group and NGF+BFGF group were(110.02±1.92),(140.87±2.22),(136.12±1.23)and(187.44±0.90)ng/mL,respectively.The ALP contents in callus were(91.23±1.47),(106.62±1.64),(104.83±1.05)and(130.59±1.18)ng/mL,respectively,at 4 weeks after surgery.The content of ALP in the callus of the NGF+BFGF group was the highest,and the difference between the groups was statistically significant(all P values<0.05).(3)HE staining:histological observation showed that the growth of callus and the formation of trabecula in NGF,BFGF and NGF+BFGF groups were significantly higher than those in the control group at different time points,and NGF+BFGF group was superior to other groups.The morphological indexes of bone trabeculae showed that compared with the four groups at different time points,IOB,TW and TV of bone trabeculae in the control group,NGF group,BFGF group and NGF+BFGF group all showed an increasing trend,and the NGF+BFGF group had the highest values.Pairwise comparison between the two groups showed no statistical significance in all indicators except NGF group and BFGF group(all P values>0.05),and all other indicators had statistical significance between the two groups(all P values<0.05).(4)Protein chip detection:compared between the four groups at different time points,except for VEGF content,there was no statistically significant difference at 4 weeks after surgery(P>0.05).The VEGF content at 2 weeks after surgery,and the BMP-2 and IGF-1 content at 2 weeks and 4 weeks after surgery of NGF group,BFGF group and NGF+BFGF group were all higher than those of the control group,and the content of NGF+BFGF group was the highest.Pairwise comparison between groups showed statistically significant differences(F=198.285,368.060,2006.017,33.332,292.643,all P values<0.05).Conclusions Both NGF and BFGF can promote the healing of tibial fracture in rats,and the combination of them has synergistic effects.The mechanism may be related to the better promotion of VEGF,BMP-2 and IGF-1 expression in the process of fracture healing.