【作者】 眭承志；

【导师】 王和鸣；

【作者基本信息】 福建中医学院 ， 中医骨伤科学， 2008， 博士

【摘要】 目的:从理论角度分析总结老年性骨质疏松症的发病原因,并通过实验研究探讨骨碎补总黄酮对骨髓基质细胞脂质化的影响,以及对成骨细胞衰变和凋亡的影响,从临床角度观察其治疗老年性骨质疏松症的效果,为临床应用提供科学理论依据。方法:临床研究:选择股骨近端BMD较同性别年轻成人均值低2.5SD以上,诊断为老年性骨质疏松症者70例。采用随机对照的方法,分为治疗组（口服强骨胶囊）36例,对照组（口服钙尔奇D）34例,依据对腰背痛及腰痛综合评分进行动态观察;依据“疼痛VAS线段分级法”对腰背痛（静止痛、翻身痛、久坐痛、起身痛、弯腰痛、步行痛、睡眠痛、叩击痛、压痛）进行量化动态观察;依据“Barthel指数评定法”对老年性骨质疏松症日常生活活动能力（ADL）进行观察;采用双能X线骨密度仪对腰椎（L₂-L₄）、股骨近端各部位:股骨颈（Neck）、股骨三角区（Ward）、股骨粗隆（Troch）进行骨密度（BMD）定量检测;应用放射免疫法对骨转换指标进行测定,骨形成指标:骨钙素（BGP）、Ⅰ型前胶原氨基端前肽（PINP）;骨吸收指标:Ⅰ型胶原交联羧基末端肽（ICTP）等,评价骨碎补总黄酮治疗老年性骨质疏松症的临床效果。实验研究:通过WST-8分析骨碎补总黄酮含药血清对大鼠骨髓基质细胞活性的影响;以地塞米松诱导骨髓基质细胞脂质化后,建立脂肪细胞与骨髓基质细胞的共培养体系,用骨碎补总黄酮含药血清干预后,以TUNEL法检测骨髓基质细胞的凋亡情况;采用三丁基过氧化氢（t-BHP）体外诱导大鼠成骨细胞衰老,用骨碎补总黄酮含药血清干预后,以SA-β-半乳糖苷酶（Se-nescence associatedβ-galactosidase）化学染色,观察细胞衰老情况;将大鼠成骨细胞培养于含有肿瘤坏死因子-α（TNF-α）或骨碎补总黄酮加TNF-α培养基中后,通过荧光染色细胞核DNA及电镜下超微结构观察,检测成骨细胞凋亡情况。结果:1.“疼痛六点行为评分法（BRS-6）”观察结果显示,治疗前老年性骨质疏松症患者的腰背痛及腰痛多在Ⅲ～Ⅴ级之间,疼痛无法忽视,对日常生活有一定的影响。运用强骨胶囊（骨碎补总黄酮）治疗3个月后疼痛分级多降至到Ⅰ～Ⅱ级,自觉疼痛基本消失。说明骨碎补总黄酮在一定程度上能够改善老年性骨质疏松症引起的腰背痛及腰痛,缓解疼痛的幅度以治疗的前3个月最为明显。2.“疼痛VAS线段分级法”观察结果显示,治疗前静止时腰背痛、久坐时腰背痛、步行时腰背痛均呈现中度疼痛,翻身时腰背痛、起身时腰背痛、弯腰时腰背痛、腰背压痛与叩击痛多表现重度疼痛,而以弯腰时腰背痛、腰背叩击痛尤为显著,部分病人腰背痛对睡眠会有一定影响。运用骨碎补总黄酮治疗3个月后,各种行为方式引起的腰背痛均有不同程度的改善,其中治疗1个月后腰背痛对睡眠的影响较小,治疗2个月后多数静止痛、久坐痛基本消失,治疗3个月多数翻身痛、起身痛、步行痛、压痛可缓解,但部分病人仍然呈现出一定程度的弯腰痛和腰背叩击痛。由此可见,骨碎补总黄酮对老年性骨质疏松症病人静态生活方式出现的腰背痛改善迅速且明显,对动态生活方式引起腰背痛加重,以及外界干预加重的腰背痛也有一定的作用,但相对较慢。3.“Barthel指数评定法”对日常生活活动能力（ADL）进行观察结果显示:治疗前多数病人Barthel指数评分为60～40分,表现中度残疾,有功能障碍,生活需要帮助;治疗3个月后积分可达到90分以上,生活基本自理,生活独立性有一定程度的改善,生活质量有很大提高。4.双能X线骨密度仪对老年性骨质疏松症腰椎（L₂-L₄）、股骨近端各部位:股骨颈（Neck）、股骨三角区（Ward）、股骨粗隆（Troch）进行应用骨碎补总黄酮治疗前后骨密度（BMD）定量检测结果表明,应用骨碎补总黄酮治疗6个月后各部位BMD均有一定程度的升高;与对照组（钙尔奇D）相比,除外腰椎（L₂-L₄）的骨密度改善程度无差异,骨碎补总黄酮的治疗效果更为明显,二者对比,有显著性差异。说明骨碎补总黄酮对老年性骨质疏松症具有提高整体骨量的作用,同时能够提高股骨近端骨强度,预防髋部骨折。5.骨代谢标志物检测结果表明,老年性骨质疏松症BGP、PINP比较正常成年人均有一定程度降低,说明老年性骨质疏松症成骨细胞功能衰退,合成胶原减少,成骨能力较弱;运用骨碎补总黄酮治疗6个月后,BGP、PINP均有一定程度上升,说明骨碎补总黄酮具有促进低转换型骨质疏松症骨胶原的合成,提高骨转换,增加骨形成等作用。老年性骨质疏松症ICTP比较正常成年人有一定程度升高,说明老年性骨质疏松症骨溶解增加,骨吸收相对较快。运用骨碎补总黄酮治疗6个月后,ICTP有一定程度上降低,说明骨碎补总黄酮具有减缓低转换型骨质疏松症骨吸收状况的功效。6.骨碎补总黄酮含药血清对大鼠骨髓基质细胞活性的影响研究结果表明,经骨碎补总黄酮干预后大鼠骨髓基质细胞数量显著增加,说明骨碎补总黄酮能够提高大鼠骨髓基质细胞的活性,并具有促进其增殖分裂的作用。7.骨碎补总黄酮对TNF-α诱导的大鼠成骨细胞凋亡的抑制作用研究结果表明,在用空白血清培养的成骨细胞中加入TNF-α,可见到成骨细胞出现凋亡征象,凋亡的成骨细胞显著增多;而用骨碎补总黄酮含药血清培养的成骨细胞,凋亡细胞明显减少,虽然仍有细胞凋亡的形态改变,但可部分对抗TNF-α所致的成骨细胞凋亡。8.骨碎补总黄酮含药血清对脂质化大鼠骨髓基质细胞凋亡的保护作用研究结果表明,脂肪细胞能够诱导骨髓基质细胞的凋亡,用骨碎补总黄酮含药血清干预后,凋亡细胞明显减少,说明骨碎补总黄酮含药血清对脂质化大鼠骨髓基质细胞凋亡具有保护作用。9.骨碎补总黄酮对大鼠成骨细胞衰老的影响研究表明,经t-BHP处理后的成骨细胞SA-β-半乳糖苷酶的细胞化学染色阳性的细胞数明显增多,而用骨碎补总黄酮干预的衰老细胞明显减少,说明骨碎补总黄酮含药血清对t-BHP诱导的大鼠成骨细胞衰老具有保护作用。结论:1.随着人类寿命的延长和人口老龄化,老年性骨质疏松症的发病率逐渐增加,严重危害着老年人的健康和生命。2.老年性骨质疏松症是机体自然衰退、老化过程的组成部分,骨髓基质细胞分化方向改变和成骨细胞的生物学功能衰退是其发病的细胞病理学基础,而五脏虚损和气血功能紊乱与其发病密切相关,其中肾虚为其核心病机。3.临床研究表明:骨碎补总黄酮具有促进老年性骨质疏松症骨胶原的合成,提高骨转换,增加骨形成,减缓骨吸收状况等作用,进而提高老年性骨质疏松的整体骨量,缓解腰背痛,提高股骨近端骨强度,预防髋部骨折,提升独立性生活水准,全面提高老年性骨质疏松症患者的生命质量。4.实验研究表明:骨碎补总黄酮能够提高大鼠骨髓基质细胞的活性,促进其增殖分裂,并对脂质化大鼠骨髓基质细胞凋亡具有保护作用,而且骨碎补总黄酮含药血清对TNF-α诱导的大鼠成骨细胞凋亡和t-BHP诱导的大鼠成骨细胞衰老均具有一定的保护作用。实验研究结果说明,骨碎补总黄酮对增强老年性骨质疏松症成骨作用具有重要的意义。更多还原

【Abstract】 Objective:From the theoretical angle,to analyze and summarize the morbility reason of Senile Osteoporosis,to approach the effect of the flavone of rhizome drynaria on the lipidision of marrow stroma cell,as well as the senescence and apoptosis of osteoblast through the experimental study;from the clinical angle,to observe the clinical effect of the flavone of rhizome drynania on Senile Osteoporosis,to supply scientific theory basis for clinical application.Methods:Clinical study:70 patients with Senile Osteoporosis who were depended the diagnostic criteria,were divided into two groups randomly,36 patients in treat group (treated with the Qianggu capsule);34 patients in control group(treated with Caltrate D 600 chew tablet).Appraised the clinical effect of the flavone of rhizome drynania on Senile Osteoporosis,according to the dynamic observation on integrative scores of lumbodorsal pain and lumbodynia,"VAS section grading method" on lumbodynia, "Barthel index rating method" on ADL,detected BMD on anterior-posterior position lumbar2～4 vertebra body and proximate of femur including neck/ward/troch,and determined the index of bone transition including BGP/PINP/ICTP through the radioimmunity method before and after treated half a year,then contrast intragroup and selfgroup.Depending on the standard of curative effect,contrast the difference of curative effect between two groups.experimental study:The effect of flavone of rhizome drynaria’s serum in blood on cytoactive of marrow stroma cell of rats from analyse of WST-8.After dexamethasone induced lipidsion of marrow stroma cell,based the co-culture system of cell and marrow stroma cell.we use TUNEL standard detect the marrow stroma cells’ apotosis,after intervened of flavone of rhizome drynaria’s serum.Osteoblast senescence of rats by we uesd t-BHP induce in vitro,after we used the flavone of rhizome drynaria’s serum intervene,Se-nescence associatedβ-galactosidase chemi-staining, observe the cell senescence.To detect the osteoblast apoptosis,after cultured rats’ osteoblast in the medium including TNF-a or the the flavone of rhizome drynaria and TNF-a,by fluorescence staining DNA of nucleus,and observing ultrastructure under electron microscope.Results:1.The observation results of 6-points Behavioural Rating Scale(BRS-6) displayed,the lumbodosal pain and lumbodynia with pretherapy SOP patients nearly were three or four degree,ignored impossible.There is several impact to daily living.The lumbodosal pain and lumbodynia lowered to one or tow degree after 3 months treated by the flavone of rhizome drynaria,the aware pain vanished.To illustrate the flavone of rhizome drynaria can extently amend the lumbodosal pain and lumbodynia with SOP,the pain obviously released after 3 months.2.The observation results of "VAS section grading method" displayed,the lumbodynia showed midrange pain while rest/prolonged sitting/walking,and heavy-range pain while to turn the body over/to get up/bend from the waist/tenderness/percussion pain before treated,is especial significance while /to turn the body over/ percussion pain.A piece of patients’ lumbodynia impacted their sleep.Various kinds of lumbodynia amended diversity extently through the flavone of rhizome drynaria treated after 3 months.After 1 month,the effect of lumbodynia on sleep is extremely trifling;after 2 months,the majority of rest and prolonged sitting pains nearly vanished;after 3 months,the majority of pains while to turn the body over/to get up/bend from the waist/tenderness/percussion pain can amend,but a piece of patients still showed the pain that bend from the waist and the percussion pain.From that to illustrate the flavone of rhizome drynaria can quickly and obviously amend the lumbodynia that SOP patients showed in static life style,but amend slowly in dynamic life style and surrounding intervention.3.The observation results of "Barthel index rating method" on ADL displayed,the majority of patients’ scores were 60～40 before treated,their life with moderate disability and dysfunction need help.After 3 months,the scores can achieve above 90,life nearly self cared,living independence amended extently,quality of life improved quietly.4.The results that detected BMD on anterior-posterior position lumbar2～4 vertebra body and proximate of femur including neck/ward/troch displayed,all positions’ BMD improved extently through treated by the flavone of rhizome drynaria after 6 months.The treat group has more conspicuous therapeutic efficacy than control group,there is a significant difference between the two groups,except the BMD on anterior-posterior position lumbar2～4 vertebra body.To illustrate that the flavone of rhizome drynaria can improve proximate of femur bone strength,and prevent fracture of hip. 5.The results that determined bone metabolism maker displayed,the SOP patients’ BGP and PINP lower than normal adults,to illustrate that the SOP patients’ osteoblast function decayed,synthesis collagen decreased,and bone formation capability weaken.After 6 months treated,BGP and PINP all upgraded extently,to illustrate that the flavone of rhizome drynaria can promote collagen synthesis, improve bone transition,and increase bone formation.The SOP patients’ ICTP more advanced than normal adults,to illustrate that the SOP patients’ osteolysis increased and bone resorption was more quickly.After 6 months treated,the SOP patients’ ICTP lowered extently,to illustrate that the flavone of rhizome drynaria can delay the bone resorption.6.The study results that the effect of flavone rhizome drynaria’s serum on cytoactive of marrow stroma cell of rats indicated,the marrow stroma cell’s quantity significant increased after intervened of flavone of rhizome drynaria’s serum in,to illustrate that the flavone of rhizome drynaria can improve the cytoactive of marrow stroma cell of rats and promote its multiplicative division.7.The study results that the depressant action of flavone rhizome drynaria’s serum to osteoblast apoptosis of rats induced by TNF-a indicated,osteoblast cultured in vacant serum adding TNF-a showed apoptotic signs,and the quantity of apoptotic osteoblast significant increased.Osteoblast cultured in flavone rhizome drynaria’s serum that its apoptotic significant decreased,although has the chang of apoptotic morphous,also can partially antagnoise osteoblast apoptosis with TNF-a.8.The study results that the protection of flavone rhizome drynaria’s serum to apoptosis of lipidsion marrow stroma cell of rats indicated,adipocyte can induce apoptosis of marrow stroma cell,and it significant decreased after intervened of flavone of rhizome drynaria’s serum,to illustrate that the flavone of rhizome drynaria can protect the apoptosis of lipidsion marrow stroma cell of rats.9.The study results that the effect of flavone rhizome drynaria’s serum on osteoblast senescence of rats indicated,quantity of osteoblast Se-nescence associatedβ-galactosidase which cytochmical staining positive increased obviously after induced by t-BHP,but the quantity of osteoblast senescence decreased obviously,after intervened of flavone of rhizome drynaria’s serum,to illustrate that the flavone of rhizome drynaria can protect the osteoblast senescence of rats induced by t-BHP.Conclusions:1.The attack rate of SOP is gradually increasing with the mankind life extending and population aging,seriously jeopardizing the aged health and life.2.SOP is ingredient of the organism natural retrogression and aging process.Its pathogenetic cell pathology is marrow stroma cells’ different direction changing and osteoblastics’ biological function decaying,as well as,is intimate correlated with five viscera consumptive disease and Qi-blood functional disorder,renal deficiency is the core pathogenesis of that.3.Clinical study indicated:the flavone of rhizome drynaria can promote collagen synthesis,improve bone transition,increase bone formation,delay bone resorption, furthermore,improve SOP patients’ integer bone mass and proximate of femur bone strength,and prevent fracture of hip.Eventually,the level of living independence amended and the quality of life improved overall.4.Experimental study indicated:the flavone of rhizome drynaria can improve the cytoactive of marrow stroma cell of rats,promote its multiplicative division,and protect the marrow stroma cells’ apotosis with lipidsion.The flavone of rhizome drynaria’s serum can extently protect the osteoblast apoptosis of rats induced by TNF-a and the osteoblast senescence of rats induced by t-BHP.The results of empirical study to illustrate that the flavone of rhizome drynaria has considerable significance to improve the bone formation action of SOP.更多还原