针刺对人羊膜间充质细胞移植治疗去卵巢大鼠骨质疏松的协同效应

Synergistic effect of acupuncture and human amniotic mesenchymal stem cell transplantation on the treatment of osteoporosis in ovariectomized rats

目的探讨针刺联合人羊膜间充质细胞(human amniotic mesenchymal cells,HAMCs)移植对去卵巢骨质疏松协同效应研究。方法选择雌性未孕的Wistar大鼠45只,完全随机分组分为假手术组、模型组及实验组(针刺+人羊膜间充质细胞移植),每组各15只。各组手术伤口恢复后,第7天开行针刺+尾静脉注射HAMCs液(假手术组除外)连续干预12周,于末次干预后24 h,处死大鼠并采集标本。从开始干预至处死,每周测定1次各组大鼠的体质量,双能X线吸收骨密度仪分别测定左侧股骨中点、远端和近端骨密度,用原子吸收法测定其骨钙含量;干预12周后采用ELISA法检测血清25-羟基维生素D(25-HVD)、I型胶原C端肽(CTX-I)、骨特异性碱性磷酸酶(BAP)、抗酒石酸酸性磷酸酶5b(TRAP5b)的表达变化;干预12周后检测各组大鼠生物力学性能的变化。干预12周后RT-PCR技术检测椎骨中转化生长因子β1(TGF-β1)的基因表达情况。结果假手术组大鼠体重逐渐增加,符合动物自然生长规律,模型组于造模后两个月开始,体重却明显高于假手术组,实验组体重渐增,与假手术组相近。模型组比较假手术组的骨密度和骨钙含量均降低,差异具有统计学意义(P<0.05),与模型组较,实验组的骨密度和骨钙含量含量均升高,差异具有统计学意义(P<0.05)。ELISA法显示,血清25-HVD、CTX-I、BAP、TRAP5b的表达,与假手术组比较,模型组显著降低,与模型组比较,实验组显著升高,(P<0.05)。生物法力学检测显示,假手术组的极限载荷、极限应力和弹性模量比模型组有显著增高(P<0.05),实验组的极限载荷和弹性模量比假手术组有显著增高(P<0.05)。RT-PCR检测显示TGF-β1的m-RNA的表达,与假手术组、模型组比较,实验组明显上调(P<0.05)。结论针刺联合人羊膜间充质细胞(HAMCs)移植具有较好的对去卵巢骨质疏松协同效应,可改善去卵巢大鼠骨质疏松症的作用。

Objective To investigate the effect of acupuncture combined with human amniotic mesenchymal stemcells（ HAMCs） transplantation on the osteoporosis in ovariectomized rats. Methods Forty-five healthy female nonpregnant Wistar rats were randomly divided into sham operated group,model group and experimental group（ acupuncture＋ HAMC transplantation）,15 rats in each group. At 7 days after the surgical wound healing, the rats received acupuncture every day and intravenous injection of HAMCs suspension（ except the sham operated group） once a week for consecutive 12 weeks. The body weight was measured every week. 24 hours after the last intervention,the rats were killed and specimens were collected. Specimens of the mid-,proximal- and distal femur were taken to measure the bone mineral density using a dual energy X-ray absorptiometer. At 12 weeks after the intervention,changes of the expression of serum25-hydroxy-vitamin D（ 25-HVD）,C-terminal peptide of type I collagen（ CTX-I）,bone specific alkaline phosphatase（ BAP） and human tartrate-resistant acid phosphatase 5b（ TRACP 5b） were determined by ELISA,the biomechanical properties of the removed femur was measured,and the expression of transforming growth factor-β（ TGF-β） in the vertebrae was assessed by RT-PCR assay. Results Body weight of rats in the sham operation group was increased gradually in accordance with the natural growth of animals,that of the model group was significantly higher than that of the sham operated group since two months after modeling,and the body weight of the experimental group was similar to that of the sham operated group. The bone mineral density and calcium content of the model group were significantly decreased compared with that of the sham operated group（ P〈0. 05）,and the bone mineral density and bone calcium content of the experimental group were significantly higher than that of the model group（ P〈0. 05）. The ELISA showed that the expressions of serum 25-HVD,CTX I,BAP,and TRAP5 b in the model group were significantly lower than that of the sham operated group,and those of the experimental group were significantly higher than that of the model group（ P〈0. 05）.Measurement of biomechanical properties showed that the limit load,limit stress and elastic modulus of the femur of the sham operated group were significantly higher than that of the model group（ P〈0. 05）,and the limit load and elastic modulus of the experimental group were significantly higher than that of the sham operated group（ P〈0. 05）. T-PCR showed that the expression of TGF-β1 m-RNA in the experimental group was significantly up-regulated than that of the sham operated and model groups（ P〈0. 05）. Conclusions Acupuncture combined with human amniotic mesenchymal stem cell（ HAMCs） transplantation has a synergistic effect on the treatment of osteoporosis,and can improve the conditions in ovariectomized rats.