Objective To determine the effect of isopsoralen (ISO) on the healing of tibia fracture in mice and explore its underlying mechanism. Methods Fifty male C57BL/6 mice (2 month old, 20±2 g) were randomly divided into model group and ISO treatment group, with 25 animals in each group. From the 3rd day after modeling, the mice from the ISO group were given an intragastric gavage of 40 mg/kg ISO, once per day for 28 consecutive days, while those of the model group was given same volume of normal saline in same way. On the 7th, 14th, 21st, and 28th day after gavage, the tibia on the surgical side was taken, and the fracture area was quantified by bone volume/total volume (BV/TV) after micro-CT scanning. The healing and shaping of the fracture end were observed through HE staining. ELISA was used to detect the serum contents of bone alkaline phosphatase (BALP) and procollagen type Ⅰ N-terminal peptide (PINP) on the 14th day of gavage. Western blotting was employed to determine the expression levels of Collagen Ⅰ, Runx2, BMP2, OSX, and VEGF in the tibial callus tissue in 7 and 14 d after gavage. Vascular perfusion was applied to observe the callus microvessels in 28 d to quantitatively analyze the vascular volume fraction and vessel diameter. Immunohistochemical staining was conducted to observe the expression of VEGF in the callus in 14 d after gavage. Results HE staining displayed that the ISO group had faster healing process than the model group. Micro-CT quantification results showed that the ISO group had higher BV/TV ratio in 7 d after gavage though no statistical difference, significantly higher ratio in 14 d (P < 0.05), but obviously lower ratio in 21 and 28 d after gavage (both P < 0.05) when compared with the model group. The serum contents of BALP and PINP were also remarkably higher in the ISO group than the model group (P < 0.05). Western blotting results indicated that the expression levels of Collagen Ⅰ, Runx2, BMP2, OSX and VEGF in the ISO group were higher than those in the model group (P < 0.05). The results of angiography revealed that the vascular volume fraction and vessel diameter were notably increased in the ISO group than the model group (both P < 0.05). Immunohistochemical assay showed that the expression of VEGF was higher in the ISO group than the model group (P < 0.05). Conclusion ISO can improve the activity of osteoblasts, increase the expression of osteogenesis-related proteins, and accelerate the angiogenesis to promote fracture healing.

目的 研究木香烃内酯(CT)对生长期大鼠骨质量的影响.方法 1 月龄雌性 SD 大鼠 30 只,体质量(99±3)g,随机分为 3 组:对照组(Control),9 mg/(kg·d)的 CT处理组(CT-9)和 18 mg/(kg·d)的 CT处理组(CT-18),每组 10 只.适应性饲养 3 d后,灌胃给药,Control组每天灌服等体积蒸馏水,每周称一次体质量,每 4 周检测一次全身骨密度(BMD).8 周后安乐死所有大鼠,检测股骨和椎骨离体 BMD、进行 micro CT 扫描和骨生物力学实验.结果 各组大鼠实验过程中体质量未出现统计学意义,8 周时 CT-9 组和 CT-18 组的全身 BMD 及离体股骨、椎骨BMD均显著高于 Control组,CT-18 组的股骨弹性模量和最大载荷显著高于 Control组,CT-9 组和 CT-18 组的相对骨体积、骨小梁数和体积骨密度均显著高于 Control组,骨小梁分离度显著低于 Control 组.结论 CT 能提高生长期大鼠的骨质量,具有开发预防和治疗骨质疏松药物的潜力.

目的 研究仙灵骨葆胶囊对生长期大鼠骨强度的影响.方法 将 20 只 1 月龄 SPF级 SD雌性大鼠按随机数字表法分为对照组和仙灵骨葆组,每组 10 只.仙灵骨葆组每天灌服 378 mg/kg仙灵骨葆胶囊,对照组每天给予等体积蒸馏水.实验期间每两周测一次全身骨密度,待第 6 周仙灵骨葆组显著高于对照组后安乐死取材.取脏器、股骨、胫骨和椎骨进行病理学分析、骨密度检测、生物力学检测、micro CT 分析以及血清生化指标检测.结果 实验期间各组大鼠体质量呈上升趋势,两组间比较并无统计学意义,脏器无明显病理学改变;给药 6 周后仙灵骨葆组全身骨密度、股骨骨密度和椎骨骨密度都显著高于对照组(P<0.05);仙灵骨葆组股骨最大载荷和屈服强度显著高于对照组(P<0.05),椎骨最大载荷也显著高于对照组(P<0.05);仙灵骨葆组胫骨骨体积分数、骨小梁厚度和骨小梁数显著高于对照组(P<0.05),骨小梁分离度显著低于对照组(P<0.05);与对照组相比,仙灵骨葆组血清 OCN 含量上升、TRACP-5b含量下降,差异具有统计学意义(P<0.05).结论 仙灵骨葆胶囊可能通过抑制骨吸收与促进骨形成来提高生长期大鼠骨强度,具有通过提高生长期大鼠峰值骨量来预防后期骨质疏松的潜力.

OBJECTIVE:To explore effects of isopsoralen (ISO) with different doses on fracture and vascular healing in mice.METHODS:Sixty 2-month-old male C57BL/6 mices with body mass of (20±2) g were selected and divided into 4 groups by random number table method:model group (model), low dose group (isopsoralen-low dose, ISO-L), medium dose group (isopsoralen-medium dose, ISO-M) and high dose group (isopsoralen-high dose, ISO-H), with 15 animals in each group. The right tibial fracture model was established. After operation, ISO-L group, ISO-M group and ISO-H group were given ISO concentration of 10 mg·kg-1, 20 mg·kg-1 and 40 mg·kg-1, respectively. Model group was given same volume of normal saline once a day for 28 days. Weighed once a week. X-ray was performed on 7, 14, 21 and 28 days, respectively, and modified I.R. Garrett scoring method was used to evaluate callus growth. After 28 days, the main organs were stripped and weighed, and organ coefficients were calculated. Hematoxylin eosin staining (HE staining) was performed on the organs to observe whether there were pathological structural changes. Micro-computed tomography (Micro-CT) was used to scan fracture area and conduct three-dimensional reconstruction to obtain the effect map, and quantify bone volume fraction (bone volume/total volume, BV/TV). After decalcification, the tibia was embedded in paraffin wax and sectioned. The healing and shape of fracture end were observed by HE staining and ferruxin solid green staining. The right tibia was removed and decalcified after intravascular infusion of Microfil contrast agent. Micro-CT was used to scan the callus microvessels in the fracture area, and the vascular volume fraction and vessel diameter were quantified.RESULTS:After 28 days of administration, there was no significant difference in body mass and organ coefficient among all groups (P>0.05), and no significant pathological changes were found in HE staining of organs. The results of X-ray and improved I.R. Garrett score showed that ISO-M group was higher than that of Model group at 28 days (P<0.05). Scores of ISO-H group at 14, 21 and 28 days were higher than those of the other 3 groups (P<0.05). Micro-CT results showed intracavitary callus in ISO-M group was significantly reduced, which was lower than that in Model group (P<0.05), most of the callus in ISO-H group were subsided, and BV/TV in ISO-H group was lower than that in the other 3 groups (P<0.05). The results of HE staining and ferrubens solid green staining showed fracture area of ISO-H group was closed, continuous laminar bone had appeared, and the fracture healing process was higher than that of other groups. Angiographic results showed vascular volume fraction in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05), and the vascular diameter in ISO-H and ISO-M groups was higher than that in Model and ISO-L groups (P<0.05).CONCLUSION:In the concentration range of 10-40 mg·kg-1, ISO has no obvious toxic and side effects, and could improve bone microstructure, promote formation of callus microvessels, and accelerate healing of fracture ends in a concentration-dependent manner.

目的 本研究旨在通过综合比较本课题组既往研究中建立大鼠骨折模型的不同方法之间的差异性及模型的稳定性,并评价改良后大鼠截骨模型建立方法的科学性及可靠性,为骨折研究提供可靠的动物模型建立方法.方法 根据不同造模方式将大鼠分为闭合性骨折模型组以及开放性骨折模型组,闭合性骨折模型组通过自制闭合性骨折造模仪建立股骨中段骨折,砝码 500 g,下落高度分别为 22、26、30 cm.开放性骨折模型组通过手术制造大鼠股骨中段横形或短斜形骨折,其中单纯克氏针固定组采用不同型号克氏针进行髓内固定,克氏针加线固定组在克氏针进行髓内固定的基础上,使用可吸收线将两骨折断端进行牵拉固定.结果 闭合性骨折模型组中砝码下落高度为 22、26、30 cm,造模成功率依次为 33%、50%、17%.开放性骨折模型组中使用 1.2 mm 克氏针内固定其骨折断端的稳定性明显优于 0.8 和 1.0 mm克氏针内固定.克氏针加线固定组相比于单纯克氏针固定组骨折断端更稳定.HE染色结果显示克氏针加线固定组其固定稳定,骨折愈合良好且未见可吸收线对其产生不良影响.结论 开放性骨折模型较闭合性骨折模型更容易控制骨折类型,保证实验对象的同质性.克氏针加线固定组较单纯克氏针固定组固定效果更稳定,可以避免因骨折断端固定不牢造成骨折不愈合甚至延迟愈合,进而导致样本脱落以及因固定不牢造成的实验结果偏差,确保了实验对象的同质性并降低了随机误差.

目的 探讨淫羊藿苷对模拟微重力环境大鼠骨丢失的防治作用.方法 取2 月龄Wistar雄性大鼠30 只,采用随机数字表法分为对照组、尾吊组、淫羊藿苷组,每组 10 只.尾吊组、淫羊藿苷组均建立尾吊模型,淫羊藿苷组给予淫羊藿苷 50 mg/kg灌胃,对照组和尾吊组则给予等体积蒸馏水,每周称体质量1 次.给药 28d后麻醉下腹主动脉采血处死大鼠,检测股骨骨密度并进行生物力学检测,观察并分析胫骨的HE染色结果,进行胫骨Mirco-CT检测,测定血清骨代谢生化指标.结果 尾吊组和淫羊藿苷组大鼠体质量下降,但差异无统计学意义;淫羊藿苷组骨密度、骨生物力学参数较尾吊组均有所改善;淫羊藿苷组骨小梁形态、数量与厚度较尾吊组均有显著改善;淫羊藿苷组骨钙素(osteocalcin,OCN)水平较尾吊组明显增加,抗酒石酸酸性磷酸酶 5b(tartrate-resistant acid phosphatase-5b,TRACP-5b)水平较尾吊组明显降低.结论 淫羊藿苷对模拟微重力环境大鼠骨丢失具有良好的防治作用.

Objective:To study the effects of a simulated plateau environment on fracture healing in rats.Methods:A rat model of mid-femoral fracture was established by hacksaw truncation and intramedullary fixation with Kirschner wires in 60 male Wistar rats which were divide into 2 groups ( n=30) by the random number table method. The rats in the control group were raised in the animal experiment center of The 940 Hospital of Joint Logistic Support Force of Chinese PLA at an altitude of 1,400 m, while the rats in the plateau group were placed in an animal experimental cabin in a simulated plateau environment at a simulated altitude of 5,000 m. The body weight was weighed once a week and X-ray films were taken every 2 weeks. Blood samples were collected after 4 weeks for detection of biochemical indicators of bone metabolism. After 8 weeks, the femurs of the surgical side were taken for bone biomechanical detection and the bone mineral density of the healthy side was detected. After 4 and 8 weeks, the femurs of the surgical side were taken for in vitro Micro-CT scanning and angiography detection. After 1, 2, 4 and 8 weeks, the femurs of the surgical side were taken for bone histopathologic detection. Results:During the entire experiment, no rats in the control group died while the mortality rate of the rats in the plateau group was as high as 26.7% (8/30). In the plateau group, some organs were pathologically damaged in the rats, fracture union was delayed, and the callus differentiated and matured slowly with the chondrocytes still dominant at the 8th week. The bone mineral density and the maximum load of the femur in the plateau group were significantly lower than those in the control group ( P< 0.05). Angiography showed that the rats in the plateau group had microvascular proliferation which did not penetrate the fracture end at the 8th week. The bone formation indexes like osteocalcin, procollagen type Ⅰ N-terminal propeptide (PⅠNP), and osteoprotegerin of the rats in the plateau group were significantly lower than those in the control group at the 4th week ( P<0.05). The bone resorption indexes like tartrate resistant acid phosphatase 5b (TRACP-5b) and receptor activator for nuclear factor-κB ligand (RANKL) in the plateau group were significantly higher than those in the control group ( P<0.05). Conclusion:A simulated plateau environment at an altitude of 5,000 m may lead to delayed fracture healing in rats.

Objective To determine the effect of pinoresinol diglucoside (PDG) on bone quality in growing rats and evaluate its potential as an anti-osteoporosis drug. Methods A total of 30 1-month-old female SD rats (body weight 107±7 g) were randomly divided into 3 groups: control group (Control), 25 mg/(kg·d) PDG treatment group (PDG-25) and 50 mg/(kg·d) PDG treatment group (PDG-50), with 10 rats in each group. The rats in the PDG-25 group and PDG-50 group were given corresponding doses of PDG intragastrically every day, and those of the control group was given equal volume distilled water. Their body mineral density (BMD) of the whole body was measured in 2, 4 and 6 weeks after treatment. In the end of 6 weeks, all rats were sacrificed, and organ coefficients of important organs were calculated. Micro-CT scanning, bone biomechanical test and serum biochemical tests of bone metabolism were performed. Results There were no significant differences in body weight and main organ coefficients among the groups. At 6 weeks, the BMD value was significantly higher in the PDG-25 group and PDG-50 group than the Control group (P < 0.05). The PDG-50 group had significantly higher relative bone volume, trabecular number and volumetric bone mineral density (P < 0.05), lower trabecular separation (P < 0.05) than the Control group, and the bone microstructure of PDG-25 group and PDG-50 group was significantly better than that of Control group. The PDG-50 group had elevated elastic modulus of femur (P < 0.05), larger maximum load of vertebrae (P < 0.05). The PDG-50 group had increased content of serum osteocalcin (P < 0.01), but obviously lower content of serum tartrate resistant acid phosphatase 5b (P < 0.01), when compared with the Control group. The content of serum osteocalcin in the PDG-25 group was significantly higher than that in the Control group (P < 0.05). Conclusion PDG can effectively improve bone mineral density, optimize bone microstructure, enhance bone strength, promote bone formation, inhibit bone resorption and improve bone quality.

目的 研究淫羊藿苷对模拟高原环境下雄性大鼠生殖系统损伤的保护作用,并初步探讨相关作用机制.方法 选取30只12周龄雄性Wistar大鼠,随机分为平原对照组、高原模型组以及淫羊藿苷实验组,每组10只.平原对照组饲养于本院动物实验中心(海拔1 400 m),高原模型组以及淫羊藿苷实验组置人模拟海拔高度6 000 m高原环境动物实验舱;淫羊藿苷实验组给予100 mg/kg淫羊藿苷悬浊液于每日上午9时舱内灌胃,其余两组给予等体积生理盐水灌胃.模拟高原环境干预满30 d后麻醉取材,睾丸组织称重后一侧多聚甲醛固定用于进行苏木素-伊红(HE)染色观察大鼠睾丸组织形态学改变,一侧破碎匀浆处理用于检测超氧化物歧化酶(SOD)、丙二醛(MDA)氧化应激指标检测;附睾尾制备成精子悬液,检测精子质量.取大鼠血清检测睾酮(T)、黄体生成素(LH)、卵泡刺激素(FSH)等激素水平.结果 (1)高原模型组与淫羊藿苷实验组的体重增长显著低于平原对照组(P＜0.05),且高原模型组睾丸指数显著低于平原对照组与淫羊藿苷实验组(P＜0.05).(2)HE染色结果示,高原模型组睾丸组织生精小管内各级生精细胞排列不规则,层数减少,管腔内精子数量稀少,支持细胞空泡化,间质内红细胞较多;淫羊藿苷实验组较高原模型组的睾丸组织形态有明显改善.(3)精子质量检测结果显示,与高原模型组相比,平原对照组和淫羊藿苷实验组的精子数量、精子存活率和精子总活动率均显著上升(P＜0.01),精子畸形率显著下降(P＜0.05);而平原对照组和淫羊藿苷实验组间精子质量均无显著性差异(P＞0.05).(4)性激素检测结果显示,与高原模型组相比,平原对照组和淫羊藿苷实验组的大鼠血清T、FSH和LH水平均显著上升(P＜0.01);而平原对照组和淫羊藿苷实验组间的血清性激素水平无显著性差异(P＞0.05).(5)氧化应激检测结果显示,与高原模型组相比,平原对照组和淫羊藿苷实验组的睾丸组织内SOD活性显著上升(P＜0.01)、MDA水平显著下降(P＜0.01),而平原对照组和淫羊藿苷实验组间的氧化应激指标水平无显著性差异(P＞0.05).结论 淫羊藿苷对模拟高原环境下雄性大鼠生殖损伤具有保护作用.

目的 研究松脂醇二葡萄糖苷(pinoresinol diglucoside,PDG)对青年大鼠骨代谢的影响.方法 将实验大鼠按随机数字表达法分为Control组、PDG-25组和PDG-50组.PDG-25组和PDG-50组每天分别按25 mg·kg-1和50 mg·kg-1的剂量灌服PDG,Control组每天灌服等体积蒸馏水.6周后处死所有大鼠,取脏器、血清、股骨、胫骨以及椎骨,用于组织病理学检测、离体骨密度检测、Micro CT成像分析、血清生化指标检测、双荧光标记观察以及股骨骨组织蛋白表达分析.结果 各组大鼠主要脏器的组织病理学结果均未见明显异常;与Control组相比,PDG-25组和PDG-50组的离体骨密度均明显增高;Micro CT结果显示,PDG-50组的骨体积分数、骨小梁数以及骨小梁厚度均明显升高,但骨小梁分离度明显下降;PDG-25组和PDG-50组荧光标记间距明显增大;PDG-25组和PDG-50组血清OPG含量明显提高,但RANKL含量明显降低;PDG-25组和PDG-50组BMP-2、Runx-2、OSX和OPG表达量明显增多,RANKL表达量明显减少.结论 25 mg·kg-1和50 mg·kg-1的PDG可能通过促进骨形成和抑制骨吸收来提高青年大鼠骨质量与骨强度.

目的 研究LED多波段光谱治疗对大鼠皮肤创伤愈合的影响.方法 选取50只雄性Wistar大鼠,在大鼠背部制备小面积全层皮肤缺损创面模型,随机分为模型对照(MC)组、LED治疗(LEDT)组、红外线治疗(IR)组、红光治疗(RL)组以及紫外线治疗(UV)组,每组10只.各治疗组大鼠每天暴露于对应光源下照射40 min,模型对照组放入治疗仪但不通电.每组分别于第3、7、14天处死3只大鼠,计算创面愈合率,观察HE染色结果,并检测相关蛋白表达量.结果 LEDT组、IR组、RL组和UV组创面愈合率治疗第3、7、14天愈合率均高于MC组(P<0.05);创面HE染色显示,LED多波段光谱治疗能够降低炎症反应,促进皮肤组织增殖分化,加速创伤愈合进程;LEDT组、IR组、RL组和UV组治疗第14天后EGF、VEGF、TGF-β1表达量均高于MC组(P<0.05),但LEDT与IR组TGF-β1比较,差异无统计学意义(P>0.05).结论 LED多波段光谱治疗能够促进大鼠皮肤创伤愈合,促进皮肤再上皮化进程,提高创面中EGF、TGF-β1及VEGF蛋白表达水平.

In recent years, international and domestic academics have made a series of studies on themechanism of icariin which is the main effective component of Herba Epimedii, to accelerate fracture healing.The current research results show that the mechanism of icariin accelerating fracture healing is to promote themigration and homing of BMSCs and ASCs by activating Wnts, BMP, MAPK, ER and other related signalpathways, so as to carry out osteogenic differentiation into osteoblasts. At the same time, it can promotethe maturation and mineralization of osteoblasts to form callus, and inhibit the bone resorption activity ofosteoclasts. This article reviews the mechanism of icariin in accelerating fracture healing from the aspectsof its effects on related cells and vascular remodeling, so as to provide reference for the follow-up research.

健康状态下骨的生成与吸收过程维持着稳定的动态平衡,其主要通过细胞增殖、分化和凋亡完成骨组织代谢过程.受某些因素影响,骨吸收与骨重建之间稳定状态失衡,骨组织代谢性疾病随之发生.研究发现,正弦交变电磁场(sinusoidal electromagnetic fields,SEMFs)作为一种极低频电磁场通过与生物体内的电场磁场相互耦合共振,产生非热效应,在骨代谢平衡的调控中具有显著的影响.相关研究报道SEMFs通过促进骨髓间充质干细胞、成骨细胞的成骨分化、抑制破骨细胞的骨吸收活性,从而调控骨组织的代谢过程.本文综述近年来国内外针对SEMFs调控骨代谢的相关文献,阐述SEMFs调控骨代谢的窗效应以及调控的作用机制.

目的 基于影像学客观评价正弦电磁场对大鼠股骨骨折愈合的影响.方法 选取SPF级Wistar雄性大鼠40只,股骨骨折模型建立成功后随机分为模型对照组和正弦电磁场治疗组,模型对照组不采取任何治疗手段,正弦电磁场治疗组每天给予50 Hz、1.8 mT电磁场治疗1.5 h,每2周对所有大鼠行X线检查观察骨折愈合情况.开始实验4周和(或)8周后,处死大鼠,取出主要脏器行器官指数计算和病理学观察,取术侧股骨行显微CT扫描、生物力学测定、骨组织病理学检测.结果 开始实验8周后,2组大鼠主要脏器的器官指数比较差异无统计学意义(P>0.05),组织病理学表现也未见明显异常改变.开始实验2、4、6周后,正弦电磁场治疗组X线骨痂评分显著高于模型对照组(P<0.01);开始实验4、8周后显微CT显示正弦电磁场治疗组骨痂生长情况及骨折愈合情况明显优于模型对照组.开始实验8周后,与模型对照组比较,正弦电磁场治疗组弹性模量显著升高(P<0.01),而2组最大载荷与断裂点载荷比较差异均无统计学意义(P>0.05).骨组织病理学观察显示,正弦电磁场治疗组骨痂组织中可见大量成熟的骨组织,排列相对整齐,而模型对照组骨折断端主要以纤维软骨细胞为主,钙化不明显.结论 正弦电磁场对股骨骨折大鼠骨折愈合具有良好的促进作用,可明显促进骨痂生长,改善骨组织形态学.

骨缺损的修复一直是临床中的治疗难点,随着骨组织工程技术与再生医学的发展,负载中药活性成分以及种子细胞和细胞因子的复合支架材料成为研究热点.近年来,负载淫羊藿苷(icariin,ICA)的骨缺损修复支架材料在修复骨缺损方面取得了重大进展,为临床治疗骨缺损提供了新的治疗方法.本文对近年来负载淫羊藿苷的骨缺损修复支架材料的相关研究作一归纳总结,为后续研究提供一定的参考.

目的 研究脉冲电磁场干预对模拟高原环境下大鼠骨折愈合的影响.方法 选取36只6～8周龄、体质量(220±10)g的雄性Wistar大鼠,采用圆锯截断加克氏针髓内固定法建立大鼠股骨中段骨折模型.造模成功后大鼠随机分为平原对照组、高原对照组、高原电磁场治疗组,每组12只.将高原对照组及高原电磁场治疗组放入低压氧舱,在模拟高原海拔5000 m环境中饲养,平原对照组饲养于本院SPF级动物实验中心(海拔1400 m).高原电磁场治疗组采用50 Hz、0.6 mT、50％占空比的脉冲电磁场处理,90 min/d,其余2组在各自饲养环境下做相同处理但设备不通电.每2周对3组大鼠行X线检查并予影像学评分,开始实验8周后采用三点弯曲试验测量骨痂处弹性模量及最大载荷值,开始实验4、8周后分别使用显微CT对标本扫描分析.结果 实验期间平原对照组及高原电磁场治疗组骨折X线骨痂评分均高于高原对照组(P<0.05),平原对照组与高原电磁场治疗组相比差异无统计学意义(P>0.05).开始实验8周后,高原对照组股骨弹性模量、最大载荷均低于平原对照组与高原电磁场治疗组(P<0.05),平原对照组与高原电磁场治疗组比较差异无统计学意义(P>0.05).显微CT扫描结果显示,高原对照组骨折愈合较其他2组缓慢.结论 脉冲电磁场干预可以提高模拟高原环境下骨折大鼠骨形成能力,促进骨痂形成,提高股骨强度,使骨折愈合情况与平原低海拔地区相近.