磁性丹参微球对大鼠骨折愈合过程中BMP-2、BMP-7表达的影响

发布时间：2018-02-10 15:45

本文关键词： 丹参股骨骨折磁性微球 BMP2 BMP7　出处：《兰州大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景：骨损伤后愈合障碍是骨科常见的临床疾病,迄今在骨修复中仍存在重大挑战,近年来对骨折愈合的骨生长因子关于细胞分子和基因水平作用的研究越来越多。其中BMP-2、BMP-7在骨折愈合中的作用已得到认可。丹参作为活血化瘀类代表性中药,在促骨愈合的作用机理已有研究,但传统中药给药方式仍存在局限性,开展新的给药途径并用于促骨愈合中的研究较少,本实验通过磁靶向给药系统(Magnetic tageting drug delivery system, MTDDS),观察磁性丹参微球对骨折愈合过程中BMP-2、BMP-7表达的影响及其作用。目的：观察磁性丹参微球(Feromagnetic tanshinol microspheres)在大鼠骨损伤后的体内分布特点及对成骨细胞的增殖及功能分化的影响,探讨磁性微球促骨愈合缓释诱导的作用机制。方法：建立大鼠股骨骨折模型,在磁靶向作用下注射磁性丹参微球制剂,筛选最佳浓度,考察其磁响应性。选用造模成功健康成年SD大鼠,雌雄不计,体重230±30g,使用随机数字表进行随机分为四组：空白组；药物组；磁场组；药物+磁场组。空白组未做任何干预,药物组只给予磁性丹参微球未外加磁场。磁场组只给外加磁场未给予磁性丹参微球；药物+磁场组是指给磁性微球的同时给予外加磁场。无菌条件下,取下术后股骨,用注射器冲取骨髓,离心获取乳白色单个核细胞,成骨诱导培养液进行诱导培养,得到成骨细胞。丹参磁性微球分别给药1天、4天、8天和12天,最后一天给药后2小时处死大鼠并提取骨折组织。MTT法检测骨折组织中第1天、第4天、第8天及第12天成骨细胞的增殖情况,Western blotting检测BMP-2和BMP-7蛋白各时间点的表达情况,RT-PCR检测BMP-2和BMP-7 mRNA的表达。结果：(1)在35000e外加磁场下给予60mg/L的微球制剂后骨折组织的丹参素含量检测达到最大值(P0.05)。(2)MTT试验显示在成骨细胞对数生长期第4天和第8天药物组+磁场组增殖效果明显高于其他三组(P0.01),药物组高于空白组和磁场组(P0.05)。(3)Western blotting检测药物+磁场组BMP-2和BMP-7所有时间点表达量均高于对照组(P0.05),BMP-2、BMP-7药物组表达量高于对照组和磁场组(P0.05)。(4)实时荧光定量PCR检测第12天时BMP-2、BMP-7mRNA表达升高最为明显(P0.05),：药物组和药物+磁场组表达量高于空白组和磁场组(P0.05)。结论：磁性丹参微球大鼠体内有较好的磁响应性,并对成骨细胞的增殖以及骨形态蛋白BMP2和BMP7的表达有促进作用。
[Abstract]:Background: the disorder of healing after bone injury is a common clinical disease in orthopedic department. So far, there are still major challenges in bone repair. In recent years, more and more researches have been made on the effects of bone growth factor on cell molecular and gene levels in fracture healing. The role of BMP-2BMP-7 in fracture healing has been recognized. Salvia miltiorrhiza is a representative Chinese medicine for promoting blood circulation and removing blood stasis. The mechanism of promoting bone healing has been studied, but there are still some limitations in traditional Chinese medicine administration. The effect of magnetic Danshen microspheres on the expression of BMP-2 and BMP-7 during fracture healing was observed by magnetic tageting drug delivery system (MTDDSX). Objective: to observe the effect of magnetic Danshen microspheres Feromagnetic tanshinol microspheres after bone injury in rats. Characteristics of osteoblasts and their effects on proliferation and functional differentiation of osteoblasts, Methods: a model of femur fracture in rats was established. Magnetic Danshen microspheres were injected with magnetic target to screen the best concentration. To investigate its magnetic response, male and female male and female SD rats, weighing 230 卤30 g, were randomly divided into four groups: blank group, drug group, magnetic field group, drug magnetic field group, and blank group without any intervention. The drug group was only given magnetic Salvia miltiorrhiza microspheres without external magnetic field. The magnetic field group only gave magnetic field not magnetic Salvia miltiorrhiza microspheres. In the drug magnetic field group, the magnetic field was given to the magnetic microspheres at the same time as the external magnetic field. Bone marrow was washed with syringe, milky white mononuclear cells were obtained by centrifugation, osteoblasts were cultured in osteoblast induction medium, and Salvia miltiorrhiza magnetic microspheres were given 1 day, 4 days, 8 days and 12 days, respectively. The rats were killed 2 hours after the last day of administration and the fracture tissue was extracted by MTT method to detect the first and fourth day of fracture tissue. Proliferation of osteoblasts on the 8th and 12th day the expression of BMP-2 and BMP-7 protein was detected by Western blotting and the expression of BMP-2 and BMP-7 mRNA was detected by RT-PCR. Results: the bone marrow cells were treated with 60mg / L of microspheres at 35000e magnetic field to detect the expression of BMP-2 and BMP-7 mRNA. The results of MTT assay showed that the proliferative effect of the magnetic field group on the 4th and 8th day of logarithmic growth of osteoblasts was significantly higher than that of the other three groups, and that of the drug group was higher than that of the blank group and the magnetic field group. The expression of BMP-2 and BMP-7 in the magnetic field group was higher than that in the control group at all time points. The expression of BMP-2BMP-7 in the drug group was higher than that in the control group and the magnetic field group.) the expression of BMP-7 mRNA in the drug group and the drug magnetic field group was significantly higher than that in the control group and the magnetic field group on the 12th day. The expression of P0.05 in the field group was higher than that in the blank group and the magnetic field group. Conclusion: the magnetic salvia miltiorrhiza microspheres have better magnetic response in vivo. It also promotes the proliferation of osteoblasts and the expression of bone morphogenetic protein BMP2 and BMP7.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R683.4

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