密骨方对去卵巢大鼠骨质疏松性骨折愈合影响的实验研究
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摘要
目的:
骨质疏松症(osteoporosis,OP)是目前世界人口老龄化重点防治的病症。骨质疏松症的严重后果是骨折,骨质疏松性骨折(Osteoporotic fracture,OPF)存在不愈合或延迟愈合的危险,严重影响患者身体健康与生活质量,甚至会致残、致死。OPF以粉碎性骨折居多,骨折部位骨的质量降低,强度变弱,治疗十分困难,医疗费用非常惊人。尽管近年来在防治OP方面已取得了很大的成就,但至今仍未找到令人满意的防治OPF方法。因此,防治OPF是当今研究的热门课题。
关于促进OPF愈合的药物,现代医学主要应用雌激素替代疗法、氟化物、双膦酸盐、降钙素和钙制剂等,虽有一定疗效,但有相当大的副作用,能否长期服用一直存在争议。中医中药在促进OPF愈合方面的进展逐渐为医药学者所重视。从临床症状上看OPF可归属为中医学中“骨痿,骨疲”范畴,根据“肾藏精,精生髓,髓养骨”,认为OPF与中医的“肾”有非常密切的关系。
本研究是在建立去卵巢Wistar大鼠骨质疏松骨折模型的基础上,用中药密骨方(Migufang,MGF)进行干预,通过免疫组化、分子生物学等实验方法,探讨雌激素缺乏时产生OPF在骨折愈合过程中的相关因子在不同时期生物学特征,以阐明MGF在去卵巢OPF骨折愈合过程中的作用机制,为中医药在防治OPF的应用开发提供实验依据。
方法:
1.骨质疏松骨折动物模型的建立
6月龄成年雌性Wistar大鼠90只,260-300克,随机分为3组,假去卵巢对照组(Sham)30只,模型组(OVX)30只,密骨方治疗组(MGF)30只。
MGF组与OVX组完整摘除双侧卵巢,Sham组只切除同量的脂肪。饲养3个月后全部在右后侧股骨中上1/3段建立闭合性骨折模型。
2.给药方法:骨折固定术后,MGF组按10 mg/(kg·d)密骨方汤剂(华中科技大学同济医学院老年医药学研究所提供)灌胃,OVX组与Sham组给予同量的生理盐水,疗程为2周、4周、6周。
3.观察指标及测定方法
3.1血液生化的变化:术后2周、4周及6周在每组随机选取5只大鼠摘眼球取血,离心5min取血清,用放射免疫法检测血清E_2、血清BGP,用生化分析仪检测血清ALP。
3.2骨折愈合过程骨折端IL-6、TGF-β_1的动态变化:采用SABC染色法,并用HPIAS-1000高清晰度彩色病理图像分析系统作图像分析。检测术后2周、4周及6周骨折端IL-6、TGF-β_1的表达。
3.3骨折愈合过程骨折端BMP-3、OPG的动态变化:采用荧光定量逆转录聚合酶链反应(RT-PCR)法,检测术后2周、4周及6周骨折端BMP-3mRNA、OPGmRNA的表达。
结果:
1.血液生化的变化:
1.1 MGF对去卵巢大鼠血清E2的影响((?)±s)
OVX组在各时间段均显著低于Sham组与MGF组(P＜0.05);MGF组在第2周和第4周的血清E_2均显著低于Sham组(P＜0.05);MGF组在第6周的血清E_2均显著高于Sham组(P＜0.05);OVX组与MGF组血清E_2在时间上有显著升高均势(P＜0.05)。Sham组在时间上无统计学差异(P＞0.05)。
1.2 MGF对去卵巢大鼠血清BGP的影响((?)±s)
Sham组在三个时间段内无显著性差异(P＞0.05)。MFG组在三个时间段有降低趋势且有显著性差异(P＜0.05)。MFG组血清BGP值在第6周显著低于Sham组(P＜0.05)。OVX组血清BGP值在三个时间段内有升高趋势,且各组间有显著性差异(P＜0.05)。
1.3 MGF对去卵巢大鼠血清ALP的影响((?)±s)
Sham组在三个时间段内无显著性差异(P＞0.05)。OVX组在第2周显著高于Sham组(P＜0.05)。MFG组血清ALP值总体比较有显著降低趋势(P＜0.05)。OVX组血清ALP值总体比较有显著升高的趋势(P＜0.05)。各组在第4周时血清ALP值最低(P＜0.05)。
2.组织学观察:
2.1 Sham组与OVX组的比较
2.1.1骨折后2周,OVX组软骨性骨痂形成以取代纤维骨痂,早期纤维骨痂内软骨岛出现,不断扩展,血管周围软骨细胞多体积较小的圆形幼稚软骨细胞,随着时间的推移,软骨痂周边部软骨细胞大部分变性坏死,并出现少量原始(幼稚)小梁状骨,原始小梁状骨边成骨细胞数较少,原始骨小梁间为结缔组织(包括血细胞)。Sham组原始小梁状骨表面可见大量排列整齐的成骨细胞。
2.1.2骨折后4周,OVX组骨痂量少,纤维结缔组织逐渐减少,软骨性骨痂逐渐增多。Sham组骨折端出现大部分骨性愈合,且有连续性骨痂包绕。
2.1.3骨折后6周,OWN组软骨痂周边部继续不断为原始小梁状骨(编织骨)替代,但软骨性骨痂仍未吸收融合,仍有软骨岛出现;早期形成的原始小梁状骨内软骨细胞或变性坏死或分化、转化为骨细胞;同时,表面破骨细胞出现,并与成骨细胞相偶联,使原始骨小梁不改建成为成熟骨小梁。OVX组与Sham组相比,小梁状骨吸收快且被吸收数多,小梁间距增宽,其间充满大量脂肪细胞。Sham组此时骨折端已大部分形成骨性骨痂。
2.2 MGF组与OVX组的比较
2.2.1骨折后2周,MGF组膜内成骨与OVX组基本相似,在纤维性骨痂中出现软骨岛,并逐渐扩大,并在软骨痂周边出现新生骨小梁。OVX组大量纤维组织及少数纤维性骨痂形成。OWN组的软骨骨痂多于MGF组。MGF组纤维性骨痂较OVX组多见。
2.2.2骨折后4周,MGF组骨痂中骨性骨痂较多,软骨细胞较少,未见到软骨岛存在,骨小梁粗细均匀一致,间距无明显增宽,板层骨形成,周围结缔组织和骨髓腔可见较多毛细血管植入。OWN组四周纤维结缔组织逐渐减少,软骨性骨痂逐渐增多。MGF已有少量成熟的骨性骨痂出现。
2.2.3骨折后6周,MGF组软骨骨痂周边部新生小梁状骨不断增加,在中早期形成的原始小梁状骨内软骨细胞此时逐渐转化为骨细胞,原始骨小梁不断改建成为成熟骨小梁,骨小梁融合、粗大,软骨成分减少,软骨性骨痂已逐渐转化为成熟的骨性骨痂。OVX组软骨骨痂向骨性骨痂转化缓慢,软骨性骨痂仍未吸收融合,仍有软骨岛出现,透明样软骨仍然存在,软骨痂仍然较多。
3.免疫组化结果
3.1 SABC染色组织学观察:MGF组和OVX组在骨折愈合过程中TGF-β_1、IL-6细胞因子的细胞定位无差别。TGF-β_1主要定位于间充质细胞、吞噬细胞、成骨细胞、软骨细胞等。IL-6主要定位于成骨细胞、软骨细胞等。
3.2 TGF-β_1在骨折端的表达图像分析结果:Sham组TGF-β_1在骨折端的表达在时间的变化上无显著性差异(P＞0.05);OVX组TGF-β_1在骨折端的表达在时间的变化上呈显著减弱趋势(P＜0.05);MGF组TGF-β_1在骨折端的表达在时间的变化上呈显著升高趋势(P＜0.05);MGF组在4周与6周的TGF-β_1在骨折端的表达结果显著高于OVN组和Sham组。
3.3 IL-6在骨折端的表达图像分析结果:Sham组IL-6在骨折端的表达在时间的变化上有显著性减弱趋势(P＞0.05);OVX组IL-6在骨折端的表达在时间的变化上呈显著升高趋势(P＜0.05);MGF组IL-6在骨折端的表达在时间的变化上呈显著减弱趋势(P＜0.05);MGF组在4周与6周的IL-6在骨折端的表达结果显著低于OVX组和Sham组。
4.荧光定量逆转录聚合酶链反应(RT-PCR)法的检测结果:
4.1 BMP-3 mRNA在骨折端的表达
大鼠骨折端OPG mRNA表达的循环数值:在时间上的总体比较呈递减的趋势并有显著性差异(F=153.19、P＜0.05);在组别的总体比较是OVX组＜MGF组＜Sham组并有显著性差异(F=262.23、P＜0.05);在时间与组别的交互比较也存在显著性差异(F=81.50、P＜0.05);任两组间都有统计学差异(P＜0.05)。
4.2 OPG mRNA在骨折端的表达
大鼠骨折端OPG mRNA表达的循环数值:在时间上的总体比较呈递减的趋势并有显著性差异(F=124.51、P＜0.05);在组别的总体比较是OVX组＜MGF组＜Sham组并有显著性差异(F=118.42、P＜0.05);在时间与组别的交互比较也存在显著性差异(F=16.23、P＜0.05);任两组间都有统计学差异(P＜0.05)。
结论:
MGF对去卵巢大鼠OPF有促进骨折愈合的作用。其作用机制是:有可能提高E水平促进骨形成,抑制骨吸收来调节骨代谢平衡;降低骨折断端BMP-3的基因表达及提高TGF-β_1的合成分泌,刺激成骨细胞生成和分化,从而促进骨形成;降低骨折断端OPG的基因表达及降低IL-6的合成分泌,降低破骨细胞的活性,从而抑制骨吸收。总之,MGF是从促进骨形成和抑制骨吸收两方面促进OPF的骨折愈合。
Objective:Osteoporosis(osteoporosis,OP)is the world focus controlling disease of the ageing of the population.Fractures are the serious consequences of OP.And not healing or delayed healing osteoporotic fracture(Osteoporotic fracture,OPF)will seriously affect the health of patients and their quality of life,or even make them maimed or killed.Most of Osteoporotic fracture are comminuted fractures,the bone quality of the fractural site reduces,the strength weakens,and the treatment is quite difficult,so the medical expenses are very alarming.Although in recent years the research of the osteoporosis prevention and treatment has made great achievements, the effective ways of treating the osteoporotic fractures have not been found yet. Therefore,osteoporotic fracture combating has become the hot topic of research nowadays.
On the promotion drugs of osteoporotic fracture healing,the estrogen replacement therapy,fluoride,bisphosphonates,calcitonin and calcium preparations are mainly applicated.Although it has made a certain effect,it is still not satisfied,and there is considerable side-effects and whether can be long-term used has been controversial. Chinese medical academics are paying great attention to Chinese medicine promotion of osteoporotic fracture healing progress gradually.The Chinese traditional medicine classified osteoporotic fracture as "bone contaction,bone fratigue" on the vision of clinical symptoms.It is also considered that the bone growth and metabolism had very close relationship with "kidney" in Chinese medicine,depending on the saying that "kidney of fine,fine Health and pulp,pulp Custody Bone".
This study is on the basis of the establishment of castration Wistar rat model of osteoporotic fracture,intervents the taking of the Chinese medicine administration of the decoction,uses the immunohistochemistry,molecular biology,and other experimental methods,discusses the biological characteristics of the relevant factor producing Osteoporotic fracture in the fracture healing process in different periods with the lack of estrogen,to clarify the function organization of the MGF in postmenopausal osteoporotic fractures healing process,and provide the experimental basis for the application and the development in the Chinese medical prevention and treatment of osteoporotic fracture healing process.
Methods:
1.The establishment of an animal model of osteoporotic fracture
Devide the 90 6-month female Wistar adult big rats of 260 to 300 grams into 3 groups randomly:30 rats into a castrated contrasting group(Sham),30 into a model group(OVX)and the other 30 into the treatment study group of taking the MGF.
A MGF group and an OVX group extract an ovary on the double sides perfectly, and a Sham group only removes the same amount of fat.After raising for 3 months, establish a fracture model on a thighbone in one side by an oppening operation.
2.The medicine given way:after the firming operation of the fracture,10mg,/(kg d) of the decoction(offered by the research center of old medicine science of Douser's medical academy of Huazhong University of Science and Technoiogy)is pushed into the stomach towards a MGF group.An OVX group and a Sham group are given the same amount of physiological saline and the course of treatment is 2 weeks,4 weeks and 6 weeks.
3.Observation targets and determination ways:
3.1 The blood biochemical changes:select five big rats randomly in each group to pick the eyeball and draw blood in the second,forth and sixth week after operation and extract serum from centrifugal 5 min,test the serum E2 and serum BGP with RIA, and test the serum ALP using the Biochemical Analyzer.
3.2 The dynamic changes of the IL-6,TGF-β1 in the callus bone in the fracture healing process:make image analysis by using the SABC staining,and the HP IAS-1000 high-resolution color pathological image analysis system.Test the expression of the IL-6,TGF-β1 in the bone callus in the second,forth and sixth week after operation.
3.3 The dynamic changes of the BMP-3 and OPG in the callus bone in the fracture healing process:test the expression of the BMP-3mRNA,OPGmRNA in the bone callus in the second,forth and sixth week after operation using the fluorescence quantitative reverse transcription polymerized chain reaction(RT-PCR)method.
Results:
1.The blood biochemical changes:
1.1 The MGF influence on the serum E2 of ovary extracted rats((?)±s)
The OVX group is significantly lower than the Sham group and the MGF group in various time periods(P＜0.05);the serum E2 in the MGF group in the second and the forth week is significantly lower than the Sham group(P＞0.05);the serum E2 in the MGF group in the sixth week is significantly higher than the Sham group(P＜0.05); the serum E2 in the OVX group and the MGF group have a significant increasing trend in terms of time(P＜0.05).The Sham group has no statistic differences in terms of time(P＞0.05).
1.2 The MGF influence on the serum E2 and the plasma BGP of ovary extracted rats ((?)±s)
The Sham group has no significant difference in the three time periods(P＞0.05). The MFG group has discending trend and significant differences in the three time periods(P＜0.05).The serum BGP value in the MFG group is significantly lower than the Sham group in the sixth week(P＜0.05).The serum BGP value in the OVX group has increasing trend and also significant differences in the three time periods(P＜0.05).
1.3 The MGF influence on the serum E2 and the plasma ALP of ovary extracted rats ((?)±s)
The Sham group in the three time periods has no significant differences(P＞0.05). The OVX group is significantly higher than the Sham group in the second week(P＜0.05).The serum ALP value in the MFG group has a significant descending trend comparatively(P＜0.05).The serum ALP value in the OVX group has a significant increasing trend comparatively(P＜0.05).The serum ALP has the minimum value of each group in the forth week(P＜0.05).
2.Histological observation:
2.1 The comparison between the Sham group and the OVX group
2.1.1 In the second week after fracture,the cartilage bone callus in the OVX group form to take the place of fiber bone callus.The cartilage island appears in the early fiber bone callus and expanses constantly.Most of the cartilage cells surrounding the blood vessels are small circular naive cartilage cells,and as time goes by,most of the cartilage cells surrounding the cartilage callus degenerate and necros,and a small amount of the original(naive)trabecular bones come out.The osteoblast quantity around the original trabecular bones is small and the organization among the trabecular bones is the connective tissues(including the blood cells).There is a large number of neatly arranged osteoblast on the surface of the origina trabecular bone in the Sham group.
2.1.2 In the forth week after fracture,the bone callus number decreases in the OVX group,the liber connective tissues decrease gradually and the cartilage bone callus increase gradually.There is mostly bone healing in the fracture side,as well as constant bone callus wrapping in the Sham group.
2.1.3 In the sixth week after fracture,the cartilage bone callus peripheral areas are constantly filled with the original trabecular bone(bone knitting)instead,but the cartilage bone callus have not absorbed and mixed yet and there are still some carlitage islands;the early formed original trabecular inside bone cartilage cells degenerate and necros or differentiate,or turn into bone cells;the surface osteoclasts appear at the same time and couple with the osteoblasts,so that the original trabecular bones do not become the mature ones.Compared with the Sham group,the trabecular bone absorbed fast and the number is large in the OVX group,and the distant among the trabecular bones are widened,inside which are full of a large amount of fat cells. At the same time,the fracture sides mostly form bone callus
2.2 The comparison between the MGF group and OVX group
2.2.1 Two weeks after fracture,the membrane bones are basically similar between the MGF group and the OVX group.The cartilage islands appear in the fiber bone callus and expand gradually,and newly-born trabecular bones appear around the cartilage bone callus.A big amount of liber organiazation,as well as a small amount of liber bone callus form in the OVX group.The cartilage bone callus in the OVX group are more than those of the MGF group.The liber bone callus in the MGF group are more than those of the OVX group.
2.2.2 Four weeks after fracture,the bone callus are more and the cartilage cells are less in the MGF Group.No cartilage bone islands exsist.The trabecular bones are uniformed in size and there is no significant broadening in distance.The lamellar bones form,and there are many visible capillaries implantating in the surrounding connective tissues and the bone marrow cavity.The surrounding liber connective tissues decrease gradually in the OVX group and the carlitage bone callus increase gradually.A small amount of mature bone callus appear in the MGF group.
2.2.3 Six weeks after fracture,the newly-born trabecular bones around the cartilage bone callus increase gradually in the MGF group.The early formed original trabecular cartilage bone callus turn into bone cells gradually at that time.The original trabecular bones turn into mature trabecular bones constantly.The trabecular bones become mixed and windened,the portion of carlitage bones becomes less, and the cartilage bone callus transmit into mature bone callus gradually.The cartilage bone callus transform into bone cells slowly in the OVX group,and the cartilage bone callus haven't absorbed or mixed yet.Cartilage bone islands still come out,tansparent cartilage bones still exist,and the amount of the cartilage bone callus is still very large.
3.Immunohistochemical results
3.1 SABC stained histological observation:there is no difference of the cytokine localization of TGF-β1,IL-6 in the fracture healing process in the MGF group and the OVX group.TGF-β1 mainly locats in mesenchymal cells,phagocytes,bone cells, cartilage cells,and so on.IL-6 mainly locats in osteoblast cells,cartilage cells and so on.
3.2 The fracture side TGF-β1 expression image analysis results:the TGF-β1 in the fracture side expression has no significant difference in the Sham group in terms of time(P＞0.05);TGF-β1 in the fracture side expression has a significant descending trend in the OVX group in terms of time(P＜0.05);TGF-β1 in the fracture side expression has a significant increasing trend in the MGF group in terms of time(P＜0.05);TGF-β1 in the fracture side expression result is significantly higher than that of the OVX group and the Sham group in the forth and the sixth week.
3.3 IL-6 in the fracture side in the expression image analysis results:IL-6 in the fracture side expression has a significant descending trend in the Sham group in terms of time(P＞0.05);IL-6 in the fracture side expression has a significant increasing trend in the OVX group in terms of time(P＜0.05);IL-6 in the fracture side expression has a significant descending trend in the MGF group in terms of time(P＜0.05);IL-6 in the fracture side expression in the MGF group is significantly lower than that of the OVX group and the Sham group in the forth and the sixth week.
4.The test results of the Fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR):
4.1 The expression of BMP-3mRNA in the fracture side
Rat fracture side BMP-3mRNA expression levels:there is a descending trend in the overall comparison in terms of time and there are significant differences(F=153.19, P＜0.05);the overall comparison in groups is the model group＜experimental group＜control group and there are significant differences(F=262.23,P＜0.05);the interactive comparison in terms of time and groups also has significant differences(F =81.50,P＜0.05);there are significant statistic differences between any two groups (P＜0.05)
4.2 The expression of OPGmRNA in the fracture side
Rat fracture side OPG mRNA expression levels:there is a descending trend in the overall comparison in terms of time and there are significant differences(F=124.51, P＜0.05);the overall comparison in groups is the model group＜experimental group＜control group and there are significant differences(F=118.42,P＜0.05);the interactive comparison in terms of time and groups also has significant differences(F =16.23,P＜0.05);there are significant statistic differences between any two groups (P＜0.05)
Conclusion:
MGF has a fracture healing promoting function towards the OPF of the castration rats. The function organizations:maybe improve the E level to promote the bone formation and inhibit the bone resorption to regulate the bone metabolism balance; reduce the gene expression of the fracture side BMP-3 and promote the TGF-β1 synthesis and secretion,stimulate the osteoblasts' generation and differentation to promote the bone formation;reduce the gene expression of the fracture side OPG and reduce the synthesis and secretion of IL-6,inhibit the activity of the osteoclasts to inhibit the bone resorption.Anyway,the MGF promotes the OPF fracture healing in both promoting the bone generation and inhibiting the bone resorption ways.
骨质疏松症(osteoporosis,OP)是目前世界人口老龄化重点防治的病症。骨质疏松症的严重后果是骨折,骨质疏松性骨折(Osteoporotic fracture,OPF)存在不愈合或延迟愈合的危险,严重影响患者身体健康与生活质量,甚至会致残、致死。OPF以粉碎性骨折居多,骨折部位骨的质量降低,强度变弱,治疗十分困难,医疗费用非常惊人。尽管近年来在防治OP方面已取得了很大的成就,但至今仍未找到令人满意的防治OPF方法。因此,防治OPF是当今研究的热门课题。
关于促进OPF愈合的药物,现代医学主要应用雌激素替代疗法、氟化物、双膦酸盐、降钙素和钙制剂等,虽有一定疗效,但有相当大的副作用,能否长期服用一直存在争议。中医中药在促进OPF愈合方面的进展逐渐为医药学者所重视。从临床症状上看OPF可归属为中医学中“骨痿,骨疲”范畴,根据“肾藏精,精生髓,髓养骨”,认为OPF与中医的“肾”有非常密切的关系。
本研究是在建立去卵巢Wistar大鼠骨质疏松骨折模型的基础上,用中药密骨方(Migufang,MGF)进行干预,通过免疫组化、分子生物学等实验方法,探讨雌激素缺乏时产生OPF在骨折愈合过程中的相关因子在不同时期生物学特征,以阐明MGF在去卵巢OPF骨折愈合过程中的作用机制,为中医药在防治OPF的应用开发提供实验依据。
方法:
1.骨质疏松骨折动物模型的建立
6月龄成年雌性Wistar大鼠90只,260-300克,随机分为3组,假去卵巢对照组(Sham)30只,模型组(OVX)30只,密骨方治疗组(MGF)30只。
MGF组与OVX组完整摘除双侧卵巢,Sham组只切除同量的脂肪。饲养3个月后全部在右后侧股骨中上1/3段建立闭合性骨折模型。
2.给药方法:骨折固定术后,MGF组按10 mg/(kg·d)密骨方汤剂(华中科技大学同济医学院老年医药学研究所提供)灌胃,OVX组与Sham组给予同量的生理盐水,疗程为2周、4周、6周。
3.观察指标及测定方法
3.1血液生化的变化:术后2周、4周及6周在每组随机选取5只大鼠摘眼球取血,离心5min取血清,用放射免疫法检测血清E_2、血清BGP,用生化分析仪检测血清ALP。
3.2骨折愈合过程骨折端IL-6、TGF-β_1的动态变化:采用SABC染色法,并用HPIAS-1000高清晰度彩色病理图像分析系统作图像分析。检测术后2周、4周及6周骨折端IL-6、TGF-β_1的表达。
3.3骨折愈合过程骨折端BMP-3、OPG的动态变化:采用荧光定量逆转录聚合酶链反应(RT-PCR)法,检测术后2周、4周及6周骨折端BMP-3mRNA、OPGmRNA的表达。
结果:
1.血液生化的变化:
1.1 MGF对去卵巢大鼠血清E2的影响((?)±s)
OVX组在各时间段均显著低于Sham组与MGF组(P＜0.05);MGF组在第2周和第4周的血清E_2均显著低于Sham组(P＜0.05);MGF组在第6周的血清E_2均显著高于Sham组(P＜0.05);OVX组与MGF组血清E_2在时间上有显著升高均势(P＜0.05)。Sham组在时间上无统计学差异(P＞0.05)。
1.2 MGF对去卵巢大鼠血清BGP的影响((?)±s)
Sham组在三个时间段内无显著性差异(P＞0.05)。MFG组在三个时间段有降低趋势且有显著性差异(P＜0.05)。MFG组血清BGP值在第6周显著低于Sham组(P＜0.05)。OVX组血清BGP值在三个时间段内有升高趋势,且各组间有显著性差异(P＜0.05)。
1.3 MGF对去卵巢大鼠血清ALP的影响((?)±s)
Sham组在三个时间段内无显著性差异(P＞0.05)。OVX组在第2周显著高于Sham组(P＜0.05)。MFG组血清ALP值总体比较有显著降低趋势(P＜0.05)。OVX组血清ALP值总体比较有显著升高的趋势(P＜0.05)。各组在第4周时血清ALP值最低(P＜0.05)。
2.组织学观察:
2.1 Sham组与OVX组的比较
2.1.1骨折后2周,OVX组软骨性骨痂形成以取代纤维骨痂,早期纤维骨痂内软骨岛出现,不断扩展,血管周围软骨细胞多体积较小的圆形幼稚软骨细胞,随着时间的推移,软骨痂周边部软骨细胞大部分变性坏死,并出现少量原始(幼稚)小梁状骨,原始小梁状骨边成骨细胞数较少,原始骨小梁间为结缔组织(包括血细胞)。Sham组原始小梁状骨表面可见大量排列整齐的成骨细胞。
2.1.2骨折后4周,OVX组骨痂量少,纤维结缔组织逐渐减少,软骨性骨痂逐渐增多。Sham组骨折端出现大部分骨性愈合,且有连续性骨痂包绕。
2.1.3骨折后6周,OWN组软骨痂周边部继续不断为原始小梁状骨(编织骨)替代,但软骨性骨痂仍未吸收融合,仍有软骨岛出现;早期形成的原始小梁状骨内软骨细胞或变性坏死或分化、转化为骨细胞;同时,表面破骨细胞出现,并与成骨细胞相偶联,使原始骨小梁不改建成为成熟骨小梁。OVX组与Sham组相比,小梁状骨吸收快且被吸收数多,小梁间距增宽,其间充满大量脂肪细胞。Sham组此时骨折端已大部分形成骨性骨痂。
2.2 MGF组与OVX组的比较
2.2.1骨折后2周,MGF组膜内成骨与OVX组基本相似,在纤维性骨痂中出现软骨岛,并逐渐扩大,并在软骨痂周边出现新生骨小梁。OVX组大量纤维组织及少数纤维性骨痂形成。OWN组的软骨骨痂多于MGF组。MGF组纤维性骨痂较OVX组多见。
2.2.2骨折后4周,MGF组骨痂中骨性骨痂较多,软骨细胞较少,未见到软骨岛存在,骨小梁粗细均匀一致,间距无明显增宽,板层骨形成,周围结缔组织和骨髓腔可见较多毛细血管植入。OWN组四周纤维结缔组织逐渐减少,软骨性骨痂逐渐增多。MGF已有少量成熟的骨性骨痂出现。
2.2.3骨折后6周,MGF组软骨骨痂周边部新生小梁状骨不断增加,在中早期形成的原始小梁状骨内软骨细胞此时逐渐转化为骨细胞,原始骨小梁不断改建成为成熟骨小梁,骨小梁融合、粗大,软骨成分减少,软骨性骨痂已逐渐转化为成熟的骨性骨痂。OVX组软骨骨痂向骨性骨痂转化缓慢,软骨性骨痂仍未吸收融合,仍有软骨岛出现,透明样软骨仍然存在,软骨痂仍然较多。
3.免疫组化结果
3.1 SABC染色组织学观察:MGF组和OVX组在骨折愈合过程中TGF-β_1、IL-6细胞因子的细胞定位无差别。TGF-β_1主要定位于间充质细胞、吞噬细胞、成骨细胞、软骨细胞等。IL-6主要定位于成骨细胞、软骨细胞等。
3.2 TGF-β_1在骨折端的表达图像分析结果:Sham组TGF-β_1在骨折端的表达在时间的变化上无显著性差异(P＞0.05);OVX组TGF-β_1在骨折端的表达在时间的变化上呈显著减弱趋势(P＜0.05);MGF组TGF-β_1在骨折端的表达在时间的变化上呈显著升高趋势(P＜0.05);MGF组在4周与6周的TGF-β_1在骨折端的表达结果显著高于OVN组和Sham组。
3.3 IL-6在骨折端的表达图像分析结果:Sham组IL-6在骨折端的表达在时间的变化上有显著性减弱趋势(P＞0.05);OVX组IL-6在骨折端的表达在时间的变化上呈显著升高趋势(P＜0.05);MGF组IL-6在骨折端的表达在时间的变化上呈显著减弱趋势(P＜0.05);MGF组在4周与6周的IL-6在骨折端的表达结果显著低于OVX组和Sham组。
4.荧光定量逆转录聚合酶链反应(RT-PCR)法的检测结果:
4.1 BMP-3 mRNA在骨折端的表达
大鼠骨折端OPG mRNA表达的循环数值:在时间上的总体比较呈递减的趋势并有显著性差异(F=153.19、P＜0.05);在组别的总体比较是OVX组＜MGF组＜Sham组并有显著性差异(F=262.23、P＜0.05);在时间与组别的交互比较也存在显著性差异(F=81.50、P＜0.05);任两组间都有统计学差异(P＜0.05)。
4.2 OPG mRNA在骨折端的表达
大鼠骨折端OPG mRNA表达的循环数值:在时间上的总体比较呈递减的趋势并有显著性差异(F=124.51、P＜0.05);在组别的总体比较是OVX组＜MGF组＜Sham组并有显著性差异(F=118.42、P＜0.05);在时间与组别的交互比较也存在显著性差异(F=16.23、P＜0.05);任两组间都有统计学差异(P＜0.05)。
结论:
MGF对去卵巢大鼠OPF有促进骨折愈合的作用。其作用机制是:有可能提高E水平促进骨形成,抑制骨吸收来调节骨代谢平衡;降低骨折断端BMP-3的基因表达及提高TGF-β_1的合成分泌,刺激成骨细胞生成和分化,从而促进骨形成;降低骨折断端OPG的基因表达及降低IL-6的合成分泌,降低破骨细胞的活性,从而抑制骨吸收。总之,MGF是从促进骨形成和抑制骨吸收两方面促进OPF的骨折愈合。
Objective:Osteoporosis(osteoporosis,OP)is the world focus controlling disease of the ageing of the population.Fractures are the serious consequences of OP.And not healing or delayed healing osteoporotic fracture(Osteoporotic fracture,OPF)will seriously affect the health of patients and their quality of life,or even make them maimed or killed.Most of Osteoporotic fracture are comminuted fractures,the bone quality of the fractural site reduces,the strength weakens,and the treatment is quite difficult,so the medical expenses are very alarming.Although in recent years the research of the osteoporosis prevention and treatment has made great achievements, the effective ways of treating the osteoporotic fractures have not been found yet. Therefore,osteoporotic fracture combating has become the hot topic of research nowadays.
On the promotion drugs of osteoporotic fracture healing,the estrogen replacement therapy,fluoride,bisphosphonates,calcitonin and calcium preparations are mainly applicated.Although it has made a certain effect,it is still not satisfied,and there is considerable side-effects and whether can be long-term used has been controversial. Chinese medical academics are paying great attention to Chinese medicine promotion of osteoporotic fracture healing progress gradually.The Chinese traditional medicine classified osteoporotic fracture as "bone contaction,bone fratigue" on the vision of clinical symptoms.It is also considered that the bone growth and metabolism had very close relationship with "kidney" in Chinese medicine,depending on the saying that "kidney of fine,fine Health and pulp,pulp Custody Bone".
This study is on the basis of the establishment of castration Wistar rat model of osteoporotic fracture,intervents the taking of the Chinese medicine administration of the decoction,uses the immunohistochemistry,molecular biology,and other experimental methods,discusses the biological characteristics of the relevant factor producing Osteoporotic fracture in the fracture healing process in different periods with the lack of estrogen,to clarify the function organization of the MGF in postmenopausal osteoporotic fractures healing process,and provide the experimental basis for the application and the development in the Chinese medical prevention and treatment of osteoporotic fracture healing process.
Methods:
1.The establishment of an animal model of osteoporotic fracture
Devide the 90 6-month female Wistar adult big rats of 260 to 300 grams into 3 groups randomly:30 rats into a castrated contrasting group(Sham),30 into a model group(OVX)and the other 30 into the treatment study group of taking the MGF.
A MGF group and an OVX group extract an ovary on the double sides perfectly, and a Sham group only removes the same amount of fat.After raising for 3 months, establish a fracture model on a thighbone in one side by an oppening operation.
2.The medicine given way:after the firming operation of the fracture,10mg,/(kg d) of the decoction(offered by the research center of old medicine science of Douser's medical academy of Huazhong University of Science and Technoiogy)is pushed into the stomach towards a MGF group.An OVX group and a Sham group are given the same amount of physiological saline and the course of treatment is 2 weeks,4 weeks and 6 weeks.
3.Observation targets and determination ways:
3.1 The blood biochemical changes:select five big rats randomly in each group to pick the eyeball and draw blood in the second,forth and sixth week after operation and extract serum from centrifugal 5 min,test the serum E2 and serum BGP with RIA, and test the serum ALP using the Biochemical Analyzer.
3.2 The dynamic changes of the IL-6,TGF-β1 in the callus bone in the fracture healing process:make image analysis by using the SABC staining,and the HP IAS-1000 high-resolution color pathological image analysis system.Test the expression of the IL-6,TGF-β1 in the bone callus in the second,forth and sixth week after operation.
3.3 The dynamic changes of the BMP-3 and OPG in the callus bone in the fracture healing process:test the expression of the BMP-3mRNA,OPGmRNA in the bone callus in the second,forth and sixth week after operation using the fluorescence quantitative reverse transcription polymerized chain reaction(RT-PCR)method.
Results:
1.The blood biochemical changes:
1.1 The MGF influence on the serum E2 of ovary extracted rats((?)±s)
The OVX group is significantly lower than the Sham group and the MGF group in various time periods(P＜0.05);the serum E2 in the MGF group in the second and the forth week is significantly lower than the Sham group(P＞0.05);the serum E2 in the MGF group in the sixth week is significantly higher than the Sham group(P＜0.05); the serum E2 in the OVX group and the MGF group have a significant increasing trend in terms of time(P＜0.05).The Sham group has no statistic differences in terms of time(P＞0.05).
1.2 The MGF influence on the serum E2 and the plasma BGP of ovary extracted rats ((?)±s)
The Sham group has no significant difference in the three time periods(P＞0.05). The MFG group has discending trend and significant differences in the three time periods(P＜0.05).The serum BGP value in the MFG group is significantly lower than the Sham group in the sixth week(P＜0.05).The serum BGP value in the OVX group has increasing trend and also significant differences in the three time periods(P＜0.05).
1.3 The MGF influence on the serum E2 and the plasma ALP of ovary extracted rats ((?)±s)
The Sham group in the three time periods has no significant differences(P＞0.05). The OVX group is significantly higher than the Sham group in the second week(P＜0.05).The serum ALP value in the MFG group has a significant descending trend comparatively(P＜0.05).The serum ALP value in the OVX group has a significant increasing trend comparatively(P＜0.05).The serum ALP has the minimum value of each group in the forth week(P＜0.05).
2.Histological observation:
2.1 The comparison between the Sham group and the OVX group
2.1.1 In the second week after fracture,the cartilage bone callus in the OVX group form to take the place of fiber bone callus.The cartilage island appears in the early fiber bone callus and expanses constantly.Most of the cartilage cells surrounding the blood vessels are small circular naive cartilage cells,and as time goes by,most of the cartilage cells surrounding the cartilage callus degenerate and necros,and a small amount of the original(naive)trabecular bones come out.The osteoblast quantity around the original trabecular bones is small and the organization among the trabecular bones is the connective tissues(including the blood cells).There is a large number of neatly arranged osteoblast on the surface of the origina trabecular bone in the Sham group.
2.1.2 In the forth week after fracture,the bone callus number decreases in the OVX group,the liber connective tissues decrease gradually and the cartilage bone callus increase gradually.There is mostly bone healing in the fracture side,as well as constant bone callus wrapping in the Sham group.
2.1.3 In the sixth week after fracture,the cartilage bone callus peripheral areas are constantly filled with the original trabecular bone(bone knitting)instead,but the cartilage bone callus have not absorbed and mixed yet and there are still some carlitage islands;the early formed original trabecular inside bone cartilage cells degenerate and necros or differentiate,or turn into bone cells;the surface osteoclasts appear at the same time and couple with the osteoblasts,so that the original trabecular bones do not become the mature ones.Compared with the Sham group,the trabecular bone absorbed fast and the number is large in the OVX group,and the distant among the trabecular bones are widened,inside which are full of a large amount of fat cells. At the same time,the fracture sides mostly form bone callus
2.2 The comparison between the MGF group and OVX group
2.2.1 Two weeks after fracture,the membrane bones are basically similar between the MGF group and the OVX group.The cartilage islands appear in the fiber bone callus and expand gradually,and newly-born trabecular bones appear around the cartilage bone callus.A big amount of liber organiazation,as well as a small amount of liber bone callus form in the OVX group.The cartilage bone callus in the OVX group are more than those of the MGF group.The liber bone callus in the MGF group are more than those of the OVX group.
2.2.2 Four weeks after fracture,the bone callus are more and the cartilage cells are less in the MGF Group.No cartilage bone islands exsist.The trabecular bones are uniformed in size and there is no significant broadening in distance.The lamellar bones form,and there are many visible capillaries implantating in the surrounding connective tissues and the bone marrow cavity.The surrounding liber connective tissues decrease gradually in the OVX group and the carlitage bone callus increase gradually.A small amount of mature bone callus appear in the MGF group.
2.2.3 Six weeks after fracture,the newly-born trabecular bones around the cartilage bone callus increase gradually in the MGF group.The early formed original trabecular cartilage bone callus turn into bone cells gradually at that time.The original trabecular bones turn into mature trabecular bones constantly.The trabecular bones become mixed and windened,the portion of carlitage bones becomes less, and the cartilage bone callus transmit into mature bone callus gradually.The cartilage bone callus transform into bone cells slowly in the OVX group,and the cartilage bone callus haven't absorbed or mixed yet.Cartilage bone islands still come out,tansparent cartilage bones still exist,and the amount of the cartilage bone callus is still very large.
3.Immunohistochemical results
3.1 SABC stained histological observation:there is no difference of the cytokine localization of TGF-β1,IL-6 in the fracture healing process in the MGF group and the OVX group.TGF-β1 mainly locats in mesenchymal cells,phagocytes,bone cells, cartilage cells,and so on.IL-6 mainly locats in osteoblast cells,cartilage cells and so on.
3.2 The fracture side TGF-β1 expression image analysis results:the TGF-β1 in the fracture side expression has no significant difference in the Sham group in terms of time(P＞0.05);TGF-β1 in the fracture side expression has a significant descending trend in the OVX group in terms of time(P＜0.05);TGF-β1 in the fracture side expression has a significant increasing trend in the MGF group in terms of time(P＜0.05);TGF-β1 in the fracture side expression result is significantly higher than that of the OVX group and the Sham group in the forth and the sixth week.
3.3 IL-6 in the fracture side in the expression image analysis results:IL-6 in the fracture side expression has a significant descending trend in the Sham group in terms of time(P＞0.05);IL-6 in the fracture side expression has a significant increasing trend in the OVX group in terms of time(P＜0.05);IL-6 in the fracture side expression has a significant descending trend in the MGF group in terms of time(P＜0.05);IL-6 in the fracture side expression in the MGF group is significantly lower than that of the OVX group and the Sham group in the forth and the sixth week.
4.The test results of the Fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR):
4.1 The expression of BMP-3mRNA in the fracture side
Rat fracture side BMP-3mRNA expression levels:there is a descending trend in the overall comparison in terms of time and there are significant differences(F=153.19, P＜0.05);the overall comparison in groups is the model group＜experimental group＜control group and there are significant differences(F=262.23,P＜0.05);the interactive comparison in terms of time and groups also has significant differences(F =81.50,P＜0.05);there are significant statistic differences between any two groups (P＜0.05)
4.2 The expression of OPGmRNA in the fracture side
Rat fracture side OPG mRNA expression levels:there is a descending trend in the overall comparison in terms of time and there are significant differences(F=124.51, P＜0.05);the overall comparison in groups is the model group＜experimental group＜control group and there are significant differences(F=118.42,P＜0.05);the interactive comparison in terms of time and groups also has significant differences(F =16.23,P＜0.05);there are significant statistic differences between any two groups (P＜0.05)
Conclusion:
MGF has a fracture healing promoting function towards the OPF of the castration rats. The function organizations:maybe improve the E level to promote the bone formation and inhibit the bone resorption to regulate the bone metabolism balance; reduce the gene expression of the fracture side BMP-3 and promote the TGF-β1 synthesis and secretion,stimulate the osteoblasts' generation and differentation to promote the bone formation;reduce the gene expression of the fracture side OPG and reduce the synthesis and secretion of IL-6,inhibit the activity of the osteoclasts to inhibit the bone resorption.Anyway,the MGF promotes the OPF fracture healing in both promoting the bone generation and inhibiting the bone resorption ways.
引文
1.张月锋,韩景献.骨质疏松动物模型研究进展[J]动物医学进展,2005;26(3):8-11
2.刘康,季晖,李昭平,等.三种大鼠骨质疏松模型的比较研究[J].中国骨质疏松杂志,1998,4(4):13-18
3.Edwards MW,Bain SD,Bailey MC,Lantry MM,Howard GA.17 beta estradiol stimulation of endosteal bone formation in the ovariectomized mouse:an animal model for the evaluation of bone-targeted estrogens[J].Bone. 1992;13(1):29□34.
4.Kalu DN,Liu CC,Salerno E,et al.Skeletal response of ovariectomized rats to low and high doses of 17 β-estradiol[J].Bone and Miner,1991,14:175-179
5.Smith RL,Thomas KD,Schurman DJ.Rabbit knee immobilization:Bone remodeling precedes cartilage degradation[J].Orthop Res,1992,10(1)88-95
6.郭世绂,罗先正,邱贵兴.骨质疏松基础与临床[M].天津:天津科学技术出版社,2001:330-428
7.Young,DR,Niklowitz,WJ,Brown,JR,et al.WSS immobilization associated osteoporosis in primates[J].Bone,1986,7:109-117
8.FERBER D.Cholesterol drugs show promise as bone builders[J]_Science,2000,288(5475):2297-2298.
9.吴波.骨质疏松动物模型研究的现状与展望[J].药学学报,1996;31(4):316-320
10.史之桢,沈士良.崔伟等.刺五加对模拟失重大鼠骨丧失的改善作用[J].航天医学与医学工程,1990,3(1):1-4
11.涂生平,冯英,许杰,等.植物雌激素对去卵巢大鼠骨质疏松性骨折愈合的影响[J].解剖学研究,2005;27(3):173-175
12.李明,刘植珊,高建章等.中频电流预防废用性骨质疏松的实验观察[J].中华理疗杂志.1991,4:195-197
13.冼汉昭,卢宝廉.体力载荷对废用性兔胫骨超微结构和力量性能的影响[J].中国临床解剖学杂志,1991,9(2):114-117
14.王素伟,李海平.废用性骨质疏松及三苯氧胺治疗作用的实验研究[J].湖南医科大学学报.1992,17(2):187-189
15.曹丽梅,董培智,李波.糖皮质激素诱导的骨质疏松大鼠模型的综合评价[J].中国药事,2005,19(10):589-591,594
16.赵和平,李宁,陈列.骨质疏松症动物实验模型的建立和评价[J].中国中医骨伤科杂志,2001,9(3):55-57
17.费琴明,陈统一,陈中伟等.大鼠胫骨标准骨折模型的制作[J].上海实验动物科学,2002;22:10-12
18.Wang S-J,Lewallen DG,Bolander ME,et al.Low intensity ultrasound treatment increases strength in a rat femoral fracture model[J].J Orthop Res,1994,12:40-47
19.周晓中,董启榕,张健。大鼠股骨闭合骨折模型的制作[J].东南大学学报(医学版),2007,26(1):60-62
20.Walsh WR,Sherman P,Howlett CR,et al.Fracture healing in a rat osteopenia model[J].Clin Orthop,1997,342:218-227.
21.Walsh WR,Sherman P,Howlett CR,et al.Fracture healing in a rat osteopenia model[J].Clin Orthop,1997,342:218-227.
22.郝永强,戴戎.骨质疏松性骨折实验模型的设计与建立.中国矫形外科杂志,2002,9:569-572.
23.刘忠厚.原发性骨质疏松症诊断标准的探讨[J].中国骨质疏松杂志,1997,3:1-4
24.周永昌,郭万学,主编.超声医学[M].第二版,北京:科技文献出版社,1997,23-25
1.Garnero P,Delmas PD.Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metablic bone disease.J Clin Endocrinol Metab,1993,77:1046-1053
2.Davey RA,Morris HA.Effects of dihydrotestrone on osteoblast gene expression in osteopenic ovariectomized rats.Endocr Res,2004,30:361-368
3.Erem C,Tanakol R,Alagol F,et al.Relationship of bone truenover parameters endogenous hormones and VitD deficiency to hip fracture in elderly postmenopausal women.Int J Clin Pract,2002,56:333-337
4. Lee YH ,Rho YH , Choi SJ ,et al . Predictors of bone mineral density and osteoporosis in patients attending a rheumatology outpatient clinic. Rheumatol Int, 2003, 23: 67-69
5. Ivaska KK, Kakonen SM, Gerdhem P , et al. Urinary Osteocalcin as a Marker of Bone Metabolism. Clin Chem, 2005,51:618-628
6. Akesson K, Kakonen SM, Josefsson PO , et al . Fracture2induced changes in bone turnover : a potential confounder in the use of biochemical markers in osteoporosis. J Bone Miner Metab, 2005, 23: 30-35
7. Liu B , Wang C , Yang M, et al . Clinical value of urinary deoxypyridionllne excretion in postmenopausal osteoporosis. Chinese Journal Osteoporosis ,2002,8:43-46
8. Xiao Y, Zhang J , Hua B , et al . Experimental Study of QCT in predicting the Strength of Vertebra. Chinese Journal of Medical Imaging Technology ,2002,18:625-627
1.EINHOM TA.The cell and molecular biology of fracture healing[J].Clln Orthop,1998,355:7-21.
2.Hughes D E,Dai A,Tiffee J C,et al.Estrogen promote apoptosis of murine osteoclasts mediated by TGF-beta[J].Nat Med,1996,2:1132-1136.
3.Shevde N K,Pike J W.Estrogen modulates the recruitment of myelopoietic cell progenitors in rat through a stromal cellindependent mechanism involving apoptosis[J].Blood,1996,87:2683-2692.
4.Majeska R J,Ryaby J T,Einhorm T A.Direct modulation ofosteoblastic activity with estrogen[J].J Bone Joint Surg(Am),1994,76(5):713-721.
5.郝永强,戴克戎.骨质疏松性骨折实验模型的设计与建立[J].中国矫形外科杂志,2002,9(6):569-572.
6.李昕,王集锷.雌激素对去卵巢SD雌性大鼠骨折愈合的早期骨痂形成影响的实验研究[J].中国矫形外科杂志,1999,6(7):524-525.
1.李颖,薛存宽,何学彬等.红车轴草异黄酮对去卵巢大鼠骨质疏松防治作用的实验研究[J].中国骨质疏松杂志,2005;11(4)509-511,436
2.Wildemann B,Schmidmaier G,Brenner N,et al.Quantification,localization,and expression Of IGF-1 and TGF-beta1 during growth factor—stimulated fracture healing.CalcifTissue Int,2004,4:388-397
3.Massague J.The transforming growth factor-β family.Ann Rev cell Biol,1990,6:597
4.李伟,赵光锋,喻任.转移生长因子碱性成纤维细胞生长因子,骨形态发生蛋白-2在大鼠骨质疏松骨折愈合骨痂中的表达[J].中华急诊医学杂志,2003;12(8):530-532
5.Makhijani NS,Bischoff DS,Yamaguchi DT.Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 Cells by TGF-beta isoforms.J Cell Physiol,2005,202:304-313
6.Xu SW,Yu R,Zhao GF,et al.Early period of fracture healing in ovariectomized rats.Chin J Traumatol.2003,6:.160-166
7.Tatsuyama K,Maezawa Y,Baba H,et al.Expression of various growth factors for cell proliferation and cytodiferentiation during fractor repair Of bone.EurJ nistochem,2000,44:269-278
8.Schmidmaier G,Wildemann B,Gabelein T,et al.Synergistic effect of IGF—1 and TGF—beta1 on fracture healing in rats:single versus combined application of IGF—1 and TGF—betal.Seand,2003,74:4-10
9.Schmidmaier G,WildemannB,Lubbertedt M,et al.IGF-1 and TGF-betal incorporated in a poly(D,L-lactide)implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture.J BiomedMater Res B hppl Biomater,2003,65:157-162
10.WildemannB,Sehmidmaier G,Ordel S,et al.Cell proliferation and differentiation during fracture healing are influenced by locally applied IGF-1 and TGF—betal:comparison of two proliferation markers,PCNA and BrdU.J Biomed Mater Res B hppl Biomater,2003,65:150-156
11.Park SH,0Connor KM,McKellop H.Interaction between active motion and exogenous transforming growth factor Beta during tibial fracture repair.J Orthop Trauma,2003,17:2-10
12.刑渊,倪安民.激素及相关因子与骨质疏松的研究进展[J].实用诊断与治疗杂志,2005;19(11):816-818,824
13.赵刚,胡侦明,罗先正.骨质疏松性骨折愈合与细胞因子的作用[J].中国骨肿瘤骨病,2005;4(6):364-367,369
14.孙玉鹏,陆裕补,胡蕴玉,等.牛血小板TGF-β提取,纯化及其诱导小鼠股骨成骨作用.中华骨科杂志,1995,15:610
15.曾敬,徐栋梁,张惠忠,等.成骨细胞移植促进骨质疏松性骨折愈合的机制研究.中华实验外科杂志,2003,20:439-441
16.Cho TJ,Gerstenfeld LC,Einhom TA.Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing(J].J Bone Miner Res,2002;17(3):513-20
17.马文辉,时述山,李亚非,等.骨修复中骨形态发生蛋白(BMP)成骨机理的研究及其应用现状[J].中国矫形外科杂志,2001,8(2):167
18.Croteau S,Rauch F,Silvestri A,et al.Bone morphogenetic proteins in orthopedics:from basic scirece to clinical practice[J].Orthopedics,1999,22(7):686-689
19.Einbrom TA,Majeska RJ,Oloumi RJ,et al.Enhancement of experimental fracture healing with a local percutaneous injection of recombinant human bone morphogenetic protein- 2[J].Trans Am Acad Or thop Surg,1997,64:216.
20.Stone CA.A molecular approach to bone regeneration[J].British J Plastic Surgery,1997,50(5):369.
21.Reddi AH.Morphogenesis and tissue engineering of bone and cartilage:inductive signals stem cells and biomimetic biomaterials[J].Tissue Eng,2000,6(4):351.
22.宋兴贤.骨形态发生蛋白的基础研究及临床应用[J].现代医药卫生;2007,23(7):1003-1004
23.Chen D,Feng J Q,Feng M,et al.Sequence and expression of bone morphotenetic protein 3 mRNA in prolonged cultures of fetal rat calvarial osteoblasts and in rat prostate adenocarcinoma PA Ⅲcells[J]DNA and cell Biology,1995,14(3):235-239.
24.崔有宏,陈苏明,刘新平,等.重组人骨形成蛋白23羧基端肽在大肠杆菌中表达及其诱骨活性[J].生物化学志,1997,13(5):513-516.
25.何焕玲,李星星,赖天霞,等.BMP-2、-3、-6和-12在骨肉瘤细胞株中的表达[J].中国组织化学与细胞化学杂志;2007,16(2):134-137
1.罗南萍,杨道理,赵玉堂,等,骨代谢指标与老年男性骨质疏松症关系的研究[J].中华核医学杂志,2001,21(3):223.
2.VARGAS SJ,NAPRETA A,LEE SK,et al.Lack of evidence for an increase in interleukin-6 expression in adult murine bone,bone marrow,and marrow stromal cell cultures after ovariectomy[J].J Bone Miner Res,1996,11(12):1926-1934.
3.MARITZ FJ,CONRADIE MM,HULLY PA,et al.Efect of statins on bone mineral density and bone histomorphometry in rodents[J].ArteriosclerThromb Vasc Biol,2001,21(10):1636-1641.
4.MUNDY G,GREET R,HARRIS S,et al.Stimulation of hone formation in vitro and in rodents by statins[J].Science,1999,286(3):1946-1949.
5.杨茂伟,王占友,屠冠军,等.骨质疏松大鼠椎间盘的超微结构研究[J].中国医科大学学报,2004,33(2):107-109.
6.WALSH WR,SHERMAN P,HOWLETF CR,et al.Fracture healing in a rat osteopeniamodel[J].ClinOrthop RelatRes,1997,(342):218-227.
7.Seidel C,Hjertner O,Abildgaard N,et al.Serumosteoprotegerin levels are reduced in patients with multiple myeloma with lytic bone disease.BIood,2001,98:2269-2271
8.Seidel C,Hjertner O,Abildgaard N,et al.Serumosteoprotegerin levels are reduced in patients with multiple myeloma with lytic bone disease.Blood,2001,98:2269-2271
9.Midori N,Nobuyuki U,Sachiko M,et al.Osteoprotegerin regulate bone formation through a coupling mechanism bone resorption[J]Endocrinology,2003,144(12):5441
10.Bucay N,Sarosi I,Dunstan CR,et al.Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification[J].Genes Dev,1998,12:1260
11.张晓明,陈孙裕,肖德明等.骨质疏松性骨折愈合过程中骨保护素的表达[J].咸宁学院学报(医学版),2006,20(4):35-37
1.刘忠厚.骨质疏松学[M].第2版.北京:北京科学技术出版社,1998;246-248
2.刘忠厚,潘子昂,王石鹏,等.骨骼生长衰老规律和原发性骨质疏松症预诊的研究[J].中国骨质疏松杂志,1995;1(1):1-4
3.关怀.骨质疏松的流行病学与危险度评价[J].国外医学(卫生学分册),2002;28(3):140-142
4.崔彦红节译.骨质疏松防治展望[J].国外医学(卫生学分册),1999;22(1):32-35
5.Lane JM,Riley,Wirganowiez.Osteoporosis:diagnosis and treatment [J].J Bone Joint Surg Am 1996;78:618-620
6.朱建民.骨质疏松症的发病原因及防治[J].中华内科杂志,1999;11(31):714-716
7.杨定焯.骨质疏松与骨折[J].实用妇产科学杂志,1999;11(5):232-235
8.闫丽娅.中国沈阳髋部骨折的流行病学研究[J].中国骨质疏松杂志,1996;2(2):69-71
9.Lindsay R.The pharmacoiogy of estrogens,progressinbasic and clinical[J].pharmacology.1990.4:55-72
10.Kiel DP,Felson DT,hnderson JJ,et al.Hip fracture and the use of estrogen in postmenopausal women;the Framingham Study[J].N Engl Med,1987,3(17):1169
11.Maxim P,Etting B,Spitalny GM.Fracture protection provided by longterm estrogen treatment[J].Osteoporosis Int,1995,5:23-29
12.Lufkin EG,Riggs BL.Three-year Follow-up of effects of transdermal estrogen[J],Ann Intern Me.1996,12(5):77
13.Mallmin H,LjunghallS,Persson I,et al.Risk factors for fracture of the distal forearm;a polulation-bassed case-control study[J].Osteoporosis Int. 1994,4:298-304
14.Kanis JA.Treatment of osteoporosis in elderly women[J].Am J Med,1995,98(supp1 2A):60-66
15.Komm BS.estrogen binding receptor mRNA and biologic reponse in osteoblast-like osteosarcoma cell[J].Science,1998,241:81-84
16.Fiorelli G,Gori F,Petilli M,et al.Functional estrogen receptors in a human preosteoclastic cell line[J].Proc Natl Acad Sei USA,1995,92:2672-2676
17.Hughes DE,Dai A,Tiffee JC,et al.Estrogen promote apotosis of murine osteoclasts mediated by TGF-beta[J].Nat Med,1996,2:1132-1136
18.Shevde NK,Pike JW.Estrogen modulates the recruimtment of myelopoietic cell progenitors in rat Through a stromal cell-independent mechanism involving apoptosis[J].Blood,1996,87:2683-2692
19.赵刚,胡侦明,罗先正.骨质疏松性骨折愈合与细胞因子的作用[J].中国骨肿瘤骨病,2005;4(6):364-367,369
20.Chelauine D,Mizmno S,ClowackiJ.In vitro of cytokines by immune bone marrow;effects of age and estrogen status[J].J Clin Endoecinol Metab.1998,83:2043-2051
21.Teielbaum SL.Bone resorption by osteoclasts[J].Science,2000;289(5484):1504-1508
22.李青南,廖进民,吴铁等.淫羊蕾提取液防治激素所致大鼠骨质疏松的实验研究[J].中国药学杂志,1996,31(8):467-469
23.刘舫,刘和娣,李恩.肾虚与骨质疏松及补肾中药的作用[J].中国骨质疏松杂志,2000,6(4):83-85
24.谭清武.补肾健脾法治疗老年性骨质疏松症47例[J].湖北中医杂志,2000,22)(11):52-55
25.吕建元,王根福,周建国,等.论骨质疏松从中药补肾论治[J].河北医学,2004,4(10):375-376
26.夏东胜,郭盛君,郭志强,金哲等.绝经后妇女骨质疏松程度与肾虚证型的 关系[J].新中医,2001,:33(9):14-17
27.吴官保,杨军辉,谢心军.中药促进骨折愈合研究进展.湖南中医学院学报,1997;17(2):72-74
28.周琦.补钙防治骨质疏松症的卫生经济价值[J].中国临床营养杂志,2003,11(4):309-311.
29.杜建层,冯艳萍,庞义存.他汀类药物防治骨质疏松症[J].中国骨质疏松杂志,2004,10(1):109-111.
30.SchoofMW,ScurkeenboomMC,VanderKM,etal.HMG-CoAreductaseinhibitorsan dtheriskofvertebral fracture[J].BoneMinerRes,2004,19(9):1525-1530.
31.Imanishiy,Nishizaway.Strontiumranelateasnewtherapeuticagentforoste oporosis[J].ClinCalcium,2005,1(1):25-33.
32.张洪,魏之玉,董建之,等.骨宝胶囊治疗原发性骨质疏松症疗效观察[J].中医正骨,1999,11(10):579-581.
33.李双蕾,范冠杰,唐爱华,等.壮骨胶囊治疗绝经后骨质疏松症的临床研究[J].中国骨质疏松杂志,2004,10(3):330-332.
34.张荣华,陈可冀,陆大祥,等.益骨胶囊治疗绝经后骨质疏松症的临床研究[J].中国中西医结合杂志,2004,24(8):680-684.
35.Schmidmaier G,Wildemann B,Gabelein T,et al.Synergistic effect of IGF-1 and TCF-betal on fracture healing in rats:single versus combined application of IGF-1 and TGF-betal.hcta Orthop Scand,2003,74:604-610
36.杨丕斌,胡侦明,劳汉昌,等.雌激素对骨折愈合过程中BMP—4mRNA表达的影响.中国矫形外科杂志,2003,11:620-621
37.杨庆秋,胡侦明,劳汉昌,等.雌激素对骨质疏松性骨折愈合过程中基质mRNA 表达的影响.中华创伤杂志,2001,2:96-98
38.Zeng jing Xu Dongliang,Zhang Huizhong,et al.Dynamic expression of several grow factors in process of osteoblast grafts to promote healing of osteoporotic fracture.Chinese Journal of Clinical Rehabilitation,2003,7:662-664
[1]沙漠,郭征.骨质疏松骨折外科内固定的研究进展[J].中国骨与关节损伤杂志,2007,22(2):170-172
[2]冯坤,刘月桂,张灵菊,等.高转换型骨质疏松模型的生化特点[J].中国骨质疏松杂志,1997,3(2):25-27.
[3]徐栋梁,李佛保,陈维清,等.广州地区老年人群骨质疏松性骨折和骨关节病的流行病学调查和分析[J].中国骨质疏松杂志,2000,3(6):73-76.
[4]吴波,徐冰,黄添友,等.维A酸致大鼠骨质疏松模型与机理研究[J].药学学报,1996,31(4):241-243
[5]中华医学会骨质疏松和骨矿盐疾病分会.骨质疏松性骨折诊疗指南(讨论稿)[J].中华全科医师杂志,2006,5(8):458-459.
[6]国家中医药管理局.中医病证诊断疗效标准[M].南京:南京大学出版社,1998:176.
[7]张耀光,席战现,等.原发性骨质疏松骨折治疗探讨[J].右江医学,2003,31(3):236-237
[8]李建新.接骨合剂用治老年骨质疏松骨折疗效观察[J].临床和实验医学杂志,2007,6(9):46-47
[9]钟润泉,潘刚明,邓伟民,等.固肾益精汤治疗骨质疏松性胸腰椎压缩性骨折的临床观察[J].南方医科大学学报,2007,27(11):1724-1725
[10]周忠民,安建武,张根印,等.双骨汤治疗骨质疏松性骨折48例[J].陕西中医,2006,27(12):1053-1055
[11]张俐,移平,张安桢,等.针灸对骨折家兔血液流变学的影响[J].中国中医骨伤科杂志,1997,5(6):1-2
[12]苏恩亮,梅铁成,李广琪,等.针刺对骨质疏松性骨折愈合的影响[J].中国骨质疏松杂志,2003,9(3):250-252,272
[13]程志安 吴燕峰 黄智清,等.续断对成骨细胞增殖、分化、凋亡和细胞周 期的影响[J].中医正骨,2004,16(12):1-3
[14]徐栋梁 李佛保 黄芝胜等.伤科接骨片对骨质疏松性骨折疗效和机制的研究[J].中华创伤杂志,1998,14(1):25-27
[15]李新建,闻辉,刘钟华,等.灵鹿接骨胶囊促进大鼠实验性骨质疏松性骨折愈合的研究[J].中国中医骨伤科杂志,2004,12(4):8-11
[16]张柳,刘锋,梁春雨,等.麝香乌龙丸对卵巢切除大鼠股骨骨折愈合的实验研究[J].中国骨质疏松杂志,2007,,13(1):33-36
[17]李平泽,李元校,译.刘献祥,吴明霞,吴炳煌,等针灸对实验性骨质疏松性骨折的影响[J].中国骨伤,2001,14(2):81-82·
[18]刘献祥,吴明霞,吴炳煌,等.针灸对实验性骨质疏松性骨折愈合影响的研究.中国骨伤,2001,14(2):81-82
[19]罗秀夏.骨折迟缓愈合的中医辨证论治体会[J].中华中医药杂志(原中国医药学报),2005,20(10):610-611
[20]吴明霞,刘献祥,吴炳煌等.艾灸对去卵巢大鼠骨质疏松性骨折愈合的影响[J].现代康复,2001,5(6):46-47
[21]韩冰,杨峻山.淫羊藿药理作用研究概况[J].中草药,2000,31(11):873-875
[22]李也白,温宏,徐华辛,等.复合中药促进骨折愈合的实验研究[J].骨与关节损伤杂志,1999,14(2):108-109.
[23]陈之喜,李志强,陈津岩,等.驳骨煎剂对骨折愈合中32P沉积量的影响[J].中医研究,1999,12(1):12-14.
[24]王维佳,许林薇,余杭平.补肾活血中药促进骨折愈合的实验研究[J].中国中医基础医学杂志,1998,4(9):35-38.
[25]刘献祥,余希杰,许书亮.无名异冲剂在骨折愈合过程中对DNA和BMP含量的实验研究[J].中国骨伤,1999,12(3):21-23.
[26]柴本甫.绝经期后骨质疏松症的病理生理及治疗[J].中华骨科杂志,1984,4(1):58-59.
2.刘康,季晖,李昭平,等.三种大鼠骨质疏松模型的比较研究[J].中国骨质疏松杂志,1998,4(4):13-18
3.Edwards MW,Bain SD,Bailey MC,Lantry MM,Howard GA.17 beta estradiol stimulation of endosteal bone formation in the ovariectomized mouse:an animal model for the evaluation of bone-targeted estrogens[J].Bone. 1992;13(1):29□34.
4.Kalu DN,Liu CC,Salerno E,et al.Skeletal response of ovariectomized rats to low and high doses of 17 β-estradiol[J].Bone and Miner,1991,14:175-179
5.Smith RL,Thomas KD,Schurman DJ.Rabbit knee immobilization:Bone remodeling precedes cartilage degradation[J].Orthop Res,1992,10(1)88-95
6.郭世绂,罗先正,邱贵兴.骨质疏松基础与临床[M].天津:天津科学技术出版社,2001:330-428
7.Young,DR,Niklowitz,WJ,Brown,JR,et al.WSS immobilization associated osteoporosis in primates[J].Bone,1986,7:109-117
8.FERBER D.Cholesterol drugs show promise as bone builders[J]_Science,2000,288(5475):2297-2298.
9.吴波.骨质疏松动物模型研究的现状与展望[J].药学学报,1996;31(4):316-320
10.史之桢,沈士良.崔伟等.刺五加对模拟失重大鼠骨丧失的改善作用[J].航天医学与医学工程,1990,3(1):1-4
11.涂生平,冯英,许杰,等.植物雌激素对去卵巢大鼠骨质疏松性骨折愈合的影响[J].解剖学研究,2005;27(3):173-175
12.李明,刘植珊,高建章等.中频电流预防废用性骨质疏松的实验观察[J].中华理疗杂志.1991,4:195-197
13.冼汉昭,卢宝廉.体力载荷对废用性兔胫骨超微结构和力量性能的影响[J].中国临床解剖学杂志,1991,9(2):114-117
14.王素伟,李海平.废用性骨质疏松及三苯氧胺治疗作用的实验研究[J].湖南医科大学学报.1992,17(2):187-189
15.曹丽梅,董培智,李波.糖皮质激素诱导的骨质疏松大鼠模型的综合评价[J].中国药事,2005,19(10):589-591,594
16.赵和平,李宁,陈列.骨质疏松症动物实验模型的建立和评价[J].中国中医骨伤科杂志,2001,9(3):55-57
17.费琴明,陈统一,陈中伟等.大鼠胫骨标准骨折模型的制作[J].上海实验动物科学,2002;22:10-12
18.Wang S-J,Lewallen DG,Bolander ME,et al.Low intensity ultrasound treatment increases strength in a rat femoral fracture model[J].J Orthop Res,1994,12:40-47
19.周晓中,董启榕,张健。大鼠股骨闭合骨折模型的制作[J].东南大学学报(医学版),2007,26(1):60-62
20.Walsh WR,Sherman P,Howlett CR,et al.Fracture healing in a rat osteopenia model[J].Clin Orthop,1997,342:218-227.
21.Walsh WR,Sherman P,Howlett CR,et al.Fracture healing in a rat osteopenia model[J].Clin Orthop,1997,342:218-227.
22.郝永强,戴戎.骨质疏松性骨折实验模型的设计与建立.中国矫形外科杂志,2002,9:569-572.
23.刘忠厚.原发性骨质疏松症诊断标准的探讨[J].中国骨质疏松杂志,1997,3:1-4
24.周永昌,郭万学,主编.超声医学[M].第二版,北京:科技文献出版社,1997,23-25
1.Garnero P,Delmas PD.Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metablic bone disease.J Clin Endocrinol Metab,1993,77:1046-1053
2.Davey RA,Morris HA.Effects of dihydrotestrone on osteoblast gene expression in osteopenic ovariectomized rats.Endocr Res,2004,30:361-368
3.Erem C,Tanakol R,Alagol F,et al.Relationship of bone truenover parameters endogenous hormones and VitD deficiency to hip fracture in elderly postmenopausal women.Int J Clin Pract,2002,56:333-337
4. Lee YH ,Rho YH , Choi SJ ,et al . Predictors of bone mineral density and osteoporosis in patients attending a rheumatology outpatient clinic. Rheumatol Int, 2003, 23: 67-69
5. Ivaska KK, Kakonen SM, Gerdhem P , et al. Urinary Osteocalcin as a Marker of Bone Metabolism. Clin Chem, 2005,51:618-628
6. Akesson K, Kakonen SM, Josefsson PO , et al . Fracture2induced changes in bone turnover : a potential confounder in the use of biochemical markers in osteoporosis. J Bone Miner Metab, 2005, 23: 30-35
7. Liu B , Wang C , Yang M, et al . Clinical value of urinary deoxypyridionllne excretion in postmenopausal osteoporosis. Chinese Journal Osteoporosis ,2002,8:43-46
8. Xiao Y, Zhang J , Hua B , et al . Experimental Study of QCT in predicting the Strength of Vertebra. Chinese Journal of Medical Imaging Technology ,2002,18:625-627
1.EINHOM TA.The cell and molecular biology of fracture healing[J].Clln Orthop,1998,355:7-21.
2.Hughes D E,Dai A,Tiffee J C,et al.Estrogen promote apoptosis of murine osteoclasts mediated by TGF-beta[J].Nat Med,1996,2:1132-1136.
3.Shevde N K,Pike J W.Estrogen modulates the recruitment of myelopoietic cell progenitors in rat through a stromal cellindependent mechanism involving apoptosis[J].Blood,1996,87:2683-2692.
4.Majeska R J,Ryaby J T,Einhorm T A.Direct modulation ofosteoblastic activity with estrogen[J].J Bone Joint Surg(Am),1994,76(5):713-721.
5.郝永强,戴克戎.骨质疏松性骨折实验模型的设计与建立[J].中国矫形外科杂志,2002,9(6):569-572.
6.李昕,王集锷.雌激素对去卵巢SD雌性大鼠骨折愈合的早期骨痂形成影响的实验研究[J].中国矫形外科杂志,1999,6(7):524-525.
1.李颖,薛存宽,何学彬等.红车轴草异黄酮对去卵巢大鼠骨质疏松防治作用的实验研究[J].中国骨质疏松杂志,2005;11(4)509-511,436
2.Wildemann B,Schmidmaier G,Brenner N,et al.Quantification,localization,and expression Of IGF-1 and TGF-beta1 during growth factor—stimulated fracture healing.CalcifTissue Int,2004,4:388-397
3.Massague J.The transforming growth factor-β family.Ann Rev cell Biol,1990,6:597
4.李伟,赵光锋,喻任.转移生长因子碱性成纤维细胞生长因子,骨形态发生蛋白-2在大鼠骨质疏松骨折愈合骨痂中的表达[J].中华急诊医学杂志,2003;12(8):530-532
5.Makhijani NS,Bischoff DS,Yamaguchi DT.Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 Cells by TGF-beta isoforms.J Cell Physiol,2005,202:304-313
6.Xu SW,Yu R,Zhao GF,et al.Early period of fracture healing in ovariectomized rats.Chin J Traumatol.2003,6:.160-166
7.Tatsuyama K,Maezawa Y,Baba H,et al.Expression of various growth factors for cell proliferation and cytodiferentiation during fractor repair Of bone.EurJ nistochem,2000,44:269-278
8.Schmidmaier G,Wildemann B,Gabelein T,et al.Synergistic effect of IGF—1 and TGF—beta1 on fracture healing in rats:single versus combined application of IGF—1 and TGF—betal.Seand,2003,74:4-10
9.Schmidmaier G,WildemannB,Lubbertedt M,et al.IGF-1 and TGF-betal incorporated in a poly(D,L-lactide)implant coating stimulates osteoblast differentiation and collagen-1 production but reduces osteoblast proliferation in cell culture.J BiomedMater Res B hppl Biomater,2003,65:157-162
10.WildemannB,Sehmidmaier G,Ordel S,et al.Cell proliferation and differentiation during fracture healing are influenced by locally applied IGF-1 and TGF—betal:comparison of two proliferation markers,PCNA and BrdU.J Biomed Mater Res B hppl Biomater,2003,65:150-156
11.Park SH,0Connor KM,McKellop H.Interaction between active motion and exogenous transforming growth factor Beta during tibial fracture repair.J Orthop Trauma,2003,17:2-10
12.刑渊,倪安民.激素及相关因子与骨质疏松的研究进展[J].实用诊断与治疗杂志,2005;19(11):816-818,824
13.赵刚,胡侦明,罗先正.骨质疏松性骨折愈合与细胞因子的作用[J].中国骨肿瘤骨病,2005;4(6):364-367,369
14.孙玉鹏,陆裕补,胡蕴玉,等.牛血小板TGF-β提取,纯化及其诱导小鼠股骨成骨作用.中华骨科杂志,1995,15:610
15.曾敬,徐栋梁,张惠忠,等.成骨细胞移植促进骨质疏松性骨折愈合的机制研究.中华实验外科杂志,2003,20:439-441
16.Cho TJ,Gerstenfeld LC,Einhom TA.Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing(J].J Bone Miner Res,2002;17(3):513-20
17.马文辉,时述山,李亚非,等.骨修复中骨形态发生蛋白(BMP)成骨机理的研究及其应用现状[J].中国矫形外科杂志,2001,8(2):167
18.Croteau S,Rauch F,Silvestri A,et al.Bone morphogenetic proteins in orthopedics:from basic scirece to clinical practice[J].Orthopedics,1999,22(7):686-689
19.Einbrom TA,Majeska RJ,Oloumi RJ,et al.Enhancement of experimental fracture healing with a local percutaneous injection of recombinant human bone morphogenetic protein- 2[J].Trans Am Acad Or thop Surg,1997,64:216.
20.Stone CA.A molecular approach to bone regeneration[J].British J Plastic Surgery,1997,50(5):369.
21.Reddi AH.Morphogenesis and tissue engineering of bone and cartilage:inductive signals stem cells and biomimetic biomaterials[J].Tissue Eng,2000,6(4):351.
22.宋兴贤.骨形态发生蛋白的基础研究及临床应用[J].现代医药卫生;2007,23(7):1003-1004
23.Chen D,Feng J Q,Feng M,et al.Sequence and expression of bone morphotenetic protein 3 mRNA in prolonged cultures of fetal rat calvarial osteoblasts and in rat prostate adenocarcinoma PA Ⅲcells[J]DNA and cell Biology,1995,14(3):235-239.
24.崔有宏,陈苏明,刘新平,等.重组人骨形成蛋白23羧基端肽在大肠杆菌中表达及其诱骨活性[J].生物化学志,1997,13(5):513-516.
25.何焕玲,李星星,赖天霞,等.BMP-2、-3、-6和-12在骨肉瘤细胞株中的表达[J].中国组织化学与细胞化学杂志;2007,16(2):134-137
1.罗南萍,杨道理,赵玉堂,等,骨代谢指标与老年男性骨质疏松症关系的研究[J].中华核医学杂志,2001,21(3):223.
2.VARGAS SJ,NAPRETA A,LEE SK,et al.Lack of evidence for an increase in interleukin-6 expression in adult murine bone,bone marrow,and marrow stromal cell cultures after ovariectomy[J].J Bone Miner Res,1996,11(12):1926-1934.
3.MARITZ FJ,CONRADIE MM,HULLY PA,et al.Efect of statins on bone mineral density and bone histomorphometry in rodents[J].ArteriosclerThromb Vasc Biol,2001,21(10):1636-1641.
4.MUNDY G,GREET R,HARRIS S,et al.Stimulation of hone formation in vitro and in rodents by statins[J].Science,1999,286(3):1946-1949.
5.杨茂伟,王占友,屠冠军,等.骨质疏松大鼠椎间盘的超微结构研究[J].中国医科大学学报,2004,33(2):107-109.
6.WALSH WR,SHERMAN P,HOWLETF CR,et al.Fracture healing in a rat osteopeniamodel[J].ClinOrthop RelatRes,1997,(342):218-227.
7.Seidel C,Hjertner O,Abildgaard N,et al.Serumosteoprotegerin levels are reduced in patients with multiple myeloma with lytic bone disease.BIood,2001,98:2269-2271
8.Seidel C,Hjertner O,Abildgaard N,et al.Serumosteoprotegerin levels are reduced in patients with multiple myeloma with lytic bone disease.Blood,2001,98:2269-2271
9.Midori N,Nobuyuki U,Sachiko M,et al.Osteoprotegerin regulate bone formation through a coupling mechanism bone resorption[J]Endocrinology,2003,144(12):5441
10.Bucay N,Sarosi I,Dunstan CR,et al.Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification[J].Genes Dev,1998,12:1260
11.张晓明,陈孙裕,肖德明等.骨质疏松性骨折愈合过程中骨保护素的表达[J].咸宁学院学报(医学版),2006,20(4):35-37
1.刘忠厚.骨质疏松学[M].第2版.北京:北京科学技术出版社,1998;246-248
2.刘忠厚,潘子昂,王石鹏,等.骨骼生长衰老规律和原发性骨质疏松症预诊的研究[J].中国骨质疏松杂志,1995;1(1):1-4
3.关怀.骨质疏松的流行病学与危险度评价[J].国外医学(卫生学分册),2002;28(3):140-142
4.崔彦红节译.骨质疏松防治展望[J].国外医学(卫生学分册),1999;22(1):32-35
5.Lane JM,Riley,Wirganowiez.Osteoporosis:diagnosis and treatment [J].J Bone Joint Surg Am 1996;78:618-620
6.朱建民.骨质疏松症的发病原因及防治[J].中华内科杂志,1999;11(31):714-716
7.杨定焯.骨质疏松与骨折[J].实用妇产科学杂志,1999;11(5):232-235
8.闫丽娅.中国沈阳髋部骨折的流行病学研究[J].中国骨质疏松杂志,1996;2(2):69-71
9.Lindsay R.The pharmacoiogy of estrogens,progressinbasic and clinical[J].pharmacology.1990.4:55-72
10.Kiel DP,Felson DT,hnderson JJ,et al.Hip fracture and the use of estrogen in postmenopausal women;the Framingham Study[J].N Engl Med,1987,3(17):1169
11.Maxim P,Etting B,Spitalny GM.Fracture protection provided by longterm estrogen treatment[J].Osteoporosis Int,1995,5:23-29
12.Lufkin EG,Riggs BL.Three-year Follow-up of effects of transdermal estrogen[J],Ann Intern Me.1996,12(5):77
13.Mallmin H,LjunghallS,Persson I,et al.Risk factors for fracture of the distal forearm;a polulation-bassed case-control study[J].Osteoporosis Int. 1994,4:298-304
14.Kanis JA.Treatment of osteoporosis in elderly women[J].Am J Med,1995,98(supp1 2A):60-66
15.Komm BS.estrogen binding receptor mRNA and biologic reponse in osteoblast-like osteosarcoma cell[J].Science,1998,241:81-84
16.Fiorelli G,Gori F,Petilli M,et al.Functional estrogen receptors in a human preosteoclastic cell line[J].Proc Natl Acad Sei USA,1995,92:2672-2676
17.Hughes DE,Dai A,Tiffee JC,et al.Estrogen promote apotosis of murine osteoclasts mediated by TGF-beta[J].Nat Med,1996,2:1132-1136
18.Shevde NK,Pike JW.Estrogen modulates the recruimtment of myelopoietic cell progenitors in rat Through a stromal cell-independent mechanism involving apoptosis[J].Blood,1996,87:2683-2692
19.赵刚,胡侦明,罗先正.骨质疏松性骨折愈合与细胞因子的作用[J].中国骨肿瘤骨病,2005;4(6):364-367,369
20.Chelauine D,Mizmno S,ClowackiJ.In vitro of cytokines by immune bone marrow;effects of age and estrogen status[J].J Clin Endoecinol Metab.1998,83:2043-2051
21.Teielbaum SL.Bone resorption by osteoclasts[J].Science,2000;289(5484):1504-1508
22.李青南,廖进民,吴铁等.淫羊蕾提取液防治激素所致大鼠骨质疏松的实验研究[J].中国药学杂志,1996,31(8):467-469
23.刘舫,刘和娣,李恩.肾虚与骨质疏松及补肾中药的作用[J].中国骨质疏松杂志,2000,6(4):83-85
24.谭清武.补肾健脾法治疗老年性骨质疏松症47例[J].湖北中医杂志,2000,22)(11):52-55
25.吕建元,王根福,周建国,等.论骨质疏松从中药补肾论治[J].河北医学,2004,4(10):375-376
26.夏东胜,郭盛君,郭志强,金哲等.绝经后妇女骨质疏松程度与肾虚证型的 关系[J].新中医,2001,:33(9):14-17
27.吴官保,杨军辉,谢心军.中药促进骨折愈合研究进展.湖南中医学院学报,1997;17(2):72-74
28.周琦.补钙防治骨质疏松症的卫生经济价值[J].中国临床营养杂志,2003,11(4):309-311.
29.杜建层,冯艳萍,庞义存.他汀类药物防治骨质疏松症[J].中国骨质疏松杂志,2004,10(1):109-111.
30.SchoofMW,ScurkeenboomMC,VanderKM,etal.HMG-CoAreductaseinhibitorsan dtheriskofvertebral fracture[J].BoneMinerRes,2004,19(9):1525-1530.
31.Imanishiy,Nishizaway.Strontiumranelateasnewtherapeuticagentforoste oporosis[J].ClinCalcium,2005,1(1):25-33.
32.张洪,魏之玉,董建之,等.骨宝胶囊治疗原发性骨质疏松症疗效观察[J].中医正骨,1999,11(10):579-581.
33.李双蕾,范冠杰,唐爱华,等.壮骨胶囊治疗绝经后骨质疏松症的临床研究[J].中国骨质疏松杂志,2004,10(3):330-332.
34.张荣华,陈可冀,陆大祥,等.益骨胶囊治疗绝经后骨质疏松症的临床研究[J].中国中西医结合杂志,2004,24(8):680-684.
35.Schmidmaier G,Wildemann B,Gabelein T,et al.Synergistic effect of IGF-1 and TCF-betal on fracture healing in rats:single versus combined application of IGF-1 and TGF-betal.hcta Orthop Scand,2003,74:604-610
36.杨丕斌,胡侦明,劳汉昌,等.雌激素对骨折愈合过程中BMP—4mRNA表达的影响.中国矫形外科杂志,2003,11:620-621
37.杨庆秋,胡侦明,劳汉昌,等.雌激素对骨质疏松性骨折愈合过程中基质mRNA 表达的影响.中华创伤杂志,2001,2:96-98
38.Zeng jing Xu Dongliang,Zhang Huizhong,et al.Dynamic expression of several grow factors in process of osteoblast grafts to promote healing of osteoporotic fracture.Chinese Journal of Clinical Rehabilitation,2003,7:662-664
[1]沙漠,郭征.骨质疏松骨折外科内固定的研究进展[J].中国骨与关节损伤杂志,2007,22(2):170-172
[2]冯坤,刘月桂,张灵菊,等.高转换型骨质疏松模型的生化特点[J].中国骨质疏松杂志,1997,3(2):25-27.
[3]徐栋梁,李佛保,陈维清,等.广州地区老年人群骨质疏松性骨折和骨关节病的流行病学调查和分析[J].中国骨质疏松杂志,2000,3(6):73-76.
[4]吴波,徐冰,黄添友,等.维A酸致大鼠骨质疏松模型与机理研究[J].药学学报,1996,31(4):241-243
[5]中华医学会骨质疏松和骨矿盐疾病分会.骨质疏松性骨折诊疗指南(讨论稿)[J].中华全科医师杂志,2006,5(8):458-459.
[6]国家中医药管理局.中医病证诊断疗效标准[M].南京:南京大学出版社,1998:176.
[7]张耀光,席战现,等.原发性骨质疏松骨折治疗探讨[J].右江医学,2003,31(3):236-237
[8]李建新.接骨合剂用治老年骨质疏松骨折疗效观察[J].临床和实验医学杂志,2007,6(9):46-47
[9]钟润泉,潘刚明,邓伟民,等.固肾益精汤治疗骨质疏松性胸腰椎压缩性骨折的临床观察[J].南方医科大学学报,2007,27(11):1724-1725
[10]周忠民,安建武,张根印,等.双骨汤治疗骨质疏松性骨折48例[J].陕西中医,2006,27(12):1053-1055
[11]张俐,移平,张安桢,等.针灸对骨折家兔血液流变学的影响[J].中国中医骨伤科杂志,1997,5(6):1-2
[12]苏恩亮,梅铁成,李广琪,等.针刺对骨质疏松性骨折愈合的影响[J].中国骨质疏松杂志,2003,9(3):250-252,272
[13]程志安 吴燕峰 黄智清,等.续断对成骨细胞增殖、分化、凋亡和细胞周 期的影响[J].中医正骨,2004,16(12):1-3
[14]徐栋梁 李佛保 黄芝胜等.伤科接骨片对骨质疏松性骨折疗效和机制的研究[J].中华创伤杂志,1998,14(1):25-27
[15]李新建,闻辉,刘钟华,等.灵鹿接骨胶囊促进大鼠实验性骨质疏松性骨折愈合的研究[J].中国中医骨伤科杂志,2004,12(4):8-11
[16]张柳,刘锋,梁春雨,等.麝香乌龙丸对卵巢切除大鼠股骨骨折愈合的实验研究[J].中国骨质疏松杂志,2007,,13(1):33-36
[17]李平泽,李元校,译.刘献祥,吴明霞,吴炳煌,等针灸对实验性骨质疏松性骨折的影响[J].中国骨伤,2001,14(2):81-82·
[18]刘献祥,吴明霞,吴炳煌,等.针灸对实验性骨质疏松性骨折愈合影响的研究.中国骨伤,2001,14(2):81-82
[19]罗秀夏.骨折迟缓愈合的中医辨证论治体会[J].中华中医药杂志(原中国医药学报),2005,20(10):610-611
[20]吴明霞,刘献祥,吴炳煌等.艾灸对去卵巢大鼠骨质疏松性骨折愈合的影响[J].现代康复,2001,5(6):46-47
[21]韩冰,杨峻山.淫羊藿药理作用研究概况[J].中草药,2000,31(11):873-875
[22]李也白,温宏,徐华辛,等.复合中药促进骨折愈合的实验研究[J].骨与关节损伤杂志,1999,14(2):108-109.
[23]陈之喜,李志强,陈津岩,等.驳骨煎剂对骨折愈合中32P沉积量的影响[J].中医研究,1999,12(1):12-14.
[24]王维佳,许林薇,余杭平.补肾活血中药促进骨折愈合的实验研究[J].中国中医基础医学杂志,1998,4(9):35-38.
[25]刘献祥,余希杰,许书亮.无名异冲剂在骨折愈合过程中对DNA和BMP含量的实验研究[J].中国骨伤,1999,12(3):21-23.
[26]柴本甫.绝经期后骨质疏松症的病理生理及治疗[J].中华骨科杂志,1984,4(1):58-59.