【作者】 汪学红；

【导师】 熊昌源；

【作者基本信息】 湖北中医学院 ， 中医骨伤科学， 2008， 博士

【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E₂、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-β₁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-β₁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E₂均显著低于Sham组（P＜0.05）;MGF组在第6周的血清E₂均显著高于Sham组（P＜0.05）;OVX组与MGF组血清E₂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-β₁、IL-6细胞因子的细胞定位无差别。TGF-β₁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-β₁在骨折端的表达图像分析结果:Sham组TGF-β₁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-β₁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-β₁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-β₁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-β₁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。更多还原

【Abstract】 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 fractureDevide 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 group2.1.1 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 callus2.2 The comparison between the MGF group and OVX group2.2.1 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 results3.1 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 sideRat 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.更多还原