【作者】 钟小文；

【导师】 庄洪；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2011， 博士

【摘要】 绝经后骨质疏松症(postmenopausal osteoporosis,PMOP)是一种多因素相关的高龄常见病。随着世界人口老龄化时代的到来,PMOP将成为前所未有的公共卫生问题,对其防治的研究愈来愈重要。尽管近年来对PMOP研究取得了很大进展,但仍存在不足,如何将基因水平和中医药方面的研究有机结合起来,是我们一直在思考和探索的问题。中国传统医学并无对骨质疏松症的记载,从其病因病机与临床症状分析,将其归属于骨痿、骨枯、骨痹等范畴。近年来中医学者多认为肾虚、脾虚是骨质疏松症的主要原因,同时也认为因虚致瘀、瘀阻血络,血瘀在本病发生发展过程中扮演重要角色,血瘀是骨质疏松症必经病理过程,瘀血是病理产物。骨组织血供丰富,PMOP骨组织显微结构改变,毛细血管数量减少,血供下降,使骨细胞处于低氧状态,而低氧时无氧代谢增加,酸性代谢产物积聚,可引起局部微环境不同程度的酸化,长时间的缺血缺氧无疑会造成微环境血瘀的形成,进一步加剧局部的缺氧和酸化,其相互循环作用之下,从而引起一系列病理反应。由此可以推测缺氧、酸化和血瘀之间是密切相关的。目前普遍认为酸中毒对骨的负面影响是由于理化作用溶解骨密度,骨骼以被动方式缓冲全身性酸中毒。但近期的研究资料表明,多种因素影响了骨细胞的骨吸收功能,而非理化作用。缺氧与酸化伴随着PMOP的发展过程,两者是通过什么途径参与PMOP的病理过程,具体机制是什么?两者关系如何?有关报道尚罕见。国内外研究发现缺氧诱导因子-1α(hypoxia inducible factors-1α, HIF-1α)是普遍存在于人和哺乳动物细胞内的缺氧应答调控因子,其与缺血/缺氧状态下机体众多炎症因子表达、免疫反应的各种应答机制有密切的关系,是反映组织缺氧的重要指标。酸敏感离子通道(ASICs)是一类由细胞外酸化所激活的阳离子通道,H+是目前唯一已知的通道激动剂,在病理情况下如炎症、缺血等伴随着剧烈的组织酸化,细胞外的H+浓度升高,从而激活ASICs途径,导致胞外Na+、Ca2+内流,激发各种生理病理效应。其在局部酸化的组织和细胞中扮演重要角色。目的本研究临床实验拟通过观察绝经后骨质疏松患者,对其骨密度、血清HIF-1α、血清ASICs含量进行检测,了解各指标间的相关性。动物实验拟通过建立稳定的去势大鼠动物模型,探讨微环境的改变与PMOP的关系,揭示HIF-1α、ASICs在实验性绝经后骨质疏松症中的作用机理和相互关系,并通过中药骨康干预,在细胞分子水平研究中药骨康对HIF-1α、ASICs调控的影响。从缺氧和酸化的角度探讨微环境的改变与骨质疏松症血瘀的关系,有助于阐明其病机,进一步认识“从瘀论治”骨质疏松症,为中医药防治PMOP提供新的策略及实验依据,拓展其基因预防和治疗的新领域。方法临床实验：自2009年3月始至2011年2月份期间,在广州中医药大学第一附属医院门诊就诊或住院部住院治疗的女性患者,选择经骨密度测定及相关临床检查,且自愿加入临床实验研究,符合纳入标准者。对纳入研究对象的患者根据骨密度测定结果分为三组：绝经后骨质疏松症患者20例(PMOP组),绝经后无骨质疏松症患者20例(骨量减少组),绝经前妇女20例(对照组)。进行腰椎第2、3、4椎体侧位骨密度BMD测试；酶联免疫法测定血清缺氧诱导因子1α(HIF-1α)及酸敏感离子通道(ASICs)水平。动物实验：选择健康雌性SD大鼠52只,随机结果分为对照组16只和手术组36只。术后3个月后从两组随机各选出6只大鼠行活体骨密度检查,以确定造模成功。在确定造模成功后,将手术组中剩下的30只大鼠再随机分为3组,每组10只,分别为手术模型组、雌激素组、中药组。空白组中剩下的10只大鼠为空白对照组。各组进行灌胃给药治疗,灌胃给药容积为10ml/kg,空白组、模型组灌服蒸馏水；中药组给予中药按4.8g/kg灌胃(相当于临床剂量的10倍),药物以蒸馏水定容至所需浓度,即10ml/kg灌服给药,1次/日；尼尔雌醇组给予尼尔雌醇1mg/kg灌胃,使用前配成混悬液(浓度为0.167mg/ml),1次/周,其余时间给予蒸馏水10ml/kg灌胃。所有大鼠在5个月后处死,动物处死前1天行活体骨密度测定,处死前采用内眦取血法收集血液,提取血清,采用酶联免疫检测缺氧诱导因子-1α(HIF-1α),酸敏感离子通道(ASICs).结果临床实验部分通过对60例绝经前和绝经后患者的观察,对其骨密度、血清HIF-1α、血清ASICs含量进行检测,发现绝经后妇女血清HIF-1α、ASICs含量高于对照组妇女,PMOP组的HIF-1α含量高于对照组(P<0.01),但与骨量减少组的HIF-1α含量无统计学差异(P>0.05)。PMOP组的ASICs含量最高,高于对照组和骨量减少组(P<0.01),骨量减少组的ASICs含量也高于对照组(P<0.01)。三组HIF-1α、ASICs及BMD之间具有相关性关系,BMD与HIF-1α含量呈现负相关性(r=-0.681,P<0.01),BMD值与ASICs含量呈现负相关性(r=-0.840,P<0.01),而HIF-1α和ASICs含量之间呈现正相关性(1=0.783,P<0.01)。动物实验部分通过建立去势大鼠动物模型,以中药骨康为实验药物,以尼尔雌醇为阳性对照药物,观察中药骨康对骨密度、血清HIF-1α、血清ASICs含量的影响。手术模型组大鼠的BMD均较其他组低；手术模型组的治疗前、治疗后BMD均小于空白组(P<0.05)。雌激素组和中药组治疗后BMD均高于治疗前BMD(P<0.01)。手术模型组、雌激素和中药组的HIF-1α含量均明显高于空白组(P<0.05),而中药组和雌激素组的HIF-1α含量低于手术模型组(P<0.05)。手术模型组的ASICs含量高于空白组(P<0.05),而其他组间的ASICs含量比较则无统计学差异(P>0.05)。治疗后BMD与HIF-1α含量呈现负相关性(r=-0.869,P<0.01),治疗后BMD与ASICs含量呈现负相关性(r=-0.706,P<0.01),而HIF-1α和ASICs之间呈现正相关性(r=0.846,P<0.01)。结论本临床实验发现绝经后妇女血清HIF-1α、ASICs含量高于绝经前妇女。动物实验表明中药骨康在降低大鼠HIF-1α、ASICs含量方面,具有一定作用。临床和动物实验均表明HIF-1α、ASICs及BMD之间具有相关性关系。初步从缺氧和酸化角度上说明PMOP存在微环境的循环障碍,导致“血瘀”的病理基础改变。实验结果表明PMOP导致机体微环境改变,存在不同程度的缺氧和酸化,两者与BMD密切相关,提示HIF-1α、ASICs可能直接参与了骨代谢的过程,亦有可能通过雌激素或其他某种中介因子间接地参与骨代谢的过程。其确切价值有待进一步研究。血清HIF-1α、ASICs水平的异常改变对于PMOP血瘀的形成机制具有重要的参考价值。HIF-1α、ASICs作为缺氧和酸化的特异性指标之一,其变化对机体微环境影响有着重要生理病理意义。两者在PMOP发生发展过程中相互关系如何,是否参与对PMOP其他相关因素的调控,具体作用机制尚不清楚。因而深入探讨HIF-1α、ASICs在血瘀形成过程中的作用机理及其与PMOP的内在相关联系,能完善中西医结合治疗PMOP病因病理机制研究,进一步认识“从瘀论治”骨质疏松症。更多还原

【Abstract】 Objective:Investigate the relationship between micro-environment changes and PMOP from the perspeetive of hypoxia and acidification,which to provide the objective reasonable quantification and seientific basis for Chinese medicine theory of "blood stasis" in the treatment of osteoporosis.Methods:Clinical Research:60 female patients were selected from the first hospital affiliated to Guang zhou university of Chinese medicine between March 2009 and February 2011, who were diagnosed for bone mineral density. The patients were divided into three groups:patients with postmenopausal osteoporosis in 20 cases (PMOP group), postmenopausal women without osteoporosis and 20 (bone loss group), pre-menopausal women 20 patients (control group). Lateral lumbar vertebral bone mineral density test; serum enzyme-linked immunosorbent assay of hypoxia inducible factor 1α(HIF-1α) and acid-sensing ion channels (ASICs) level.Animal experiments:52 healthy female SD rats were randomly divided into control group for 16 rats and the surgery group for 36 rats.3 months after surgery from two randomly selected 6 of the rats in vivo bone density examination to determine the successful model. After modeling in determining the success, the rest of the surgery group,30 rats were randomly divided into 3 groups,10 in each group were surgical model group, estrogen group, Chinese group. Oral administration further treatment, Chinese medicine group was given by gavage GuKang; estrogen group was given orally nilestriol;blank control group, operation model group were given distilled water. All rats were sacrificed at 5 months, animals were killed 1 day before the line of bone mineral density in vivo, were killed after the collection of blood, preparation of serum for the ELISA (HIF-1αand ASICs)Results:The clinical experimentClinical studies on 60 patients with premenopausal and postmenopausal patients with observation of their bone mineral density, serum HIF-1αand ASICs were detected in serum of postmenopausal women found that HIF-1 a, ASICs content higher than women, PMOP group of HIF-1αlevel was higher (P<0.01), but with the bone loss of HIF-1αgroup showed no significant difference (P> 0.05). PMOP ASICs group was the highest,higher than the control group, and bone loss (P<0.01), bone loss is also higher than the control group ASICs content (P<0.01). Three HIF-1α,ASICs and relevant relationships between BMD, BMD and HIF-1 a levels showed negative correlation(r=-0.681,P<0.01), BMD values and ASICs content showed negative correlation (r=-0.840, P<0.01), while HIF-1αand ASICs showed a positive correlation between levels (r=0.783, P<0.01).The animal experimentAnimal experiments through the establishment of ovariectomized rat model to the experimental drugs GuKang to Nilestriol as a positive control drug, observed GuKang on bone mineral density, serum HIF-1α, the serum levels of ASICs. Operation model group, low BMD than other groups;operation model group before treatment,after treatment, BMD was less than the control group (P <0.05).Estrogen treatment group and TCM group was higher than before treatment BMD BMD (P<0.01). Operation model group, estrogen and Medicines HIF-1αwere significantly higher than the control group (P<0.05),while the Chinese group and the estrogen group were lower than HIF-1αoperation model group (P<0.05). ASICs operation model group was higher than the control group (P<0.05), while the other comparison between groups was no significant difference between ASICs content (P>0.05).BMD after treatment with the HIF-1αlevels showed negative correlation (r=-0.869, P<0.01),BMD after treatment with ASICs content showed negative correlation(r=-0.706, P<0.01), while HIF-1αand ASICs showed a positive correlation between (r=0.846, P<0.01).Conclusion:Animal experiments and clinical research shows GuKang to reduce HIF-1α, ASICs content.Clinical study found that postmenopausal women with serum HIF-1α, ASICs were significantly higher in women. Groups HIF-1α, ASICs, and BMD related to sexual relations between, HIF-1α,ASICs, and BMD positive and statistically significant correlation (P<0.01).Confirmed from the microscopic objective of molecular biology in postmenopausal osteoporosis patients have micro-environment of circulation, there is "Blood stasis" the pathological basis of change. That HIF-1α,ASICs may be directly involved in the process of bone metabolism, may also be through the estrogen or some other intermediary factor indirectly involved in the process of bone metabolism. The exact value needs further study.Serum HIF-1α,ASICs abnormal levels of blood stasis in the formation mechanism for PMOP has important physiological and pathological significance. HIF-1α,ASICs as a specific marker for hypoxia and acidification, one of the development process in PMOP how the relationship between the specific mechanism remains uncertain, pending further study.Thus depth HIF-1α, ASICs in the role of blood stasis in the formation of the internal mechanism and the relevant contact with PMOP can further improve the Integrative Medicine PMOP mechanism of etiology and pathology.更多还原