补肾壮骨中药对大鼠乳腺癌骨转移模型的影响

【作者】 姚暄；

【导师】 贾立群；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 骨是乳腺癌最常见的远处转移部位,大约有65%-80%的晚期乳腺癌患者会发生骨转移。乳腺癌骨转移严重影响病人的生活质量,甚至会危及生命。因此,控制骨转移的发生发展是当今肿瘤学研究的重要课题,需要通过综合治疗的方法解决。中医补肾壮骨法是临床治疗骨转移常用的治法之一,其作用机理有待深入探讨。目的:根据中医学“肾主骨”理论选取淫羊藿、骨碎补和补骨脂三味补肾壮骨中药,以破骨细胞活性作为切入点,研究补肾壮骨中药对乳腺癌骨转移大鼠模型在溶骨性骨质破坏和癌性骨痛等方面的影响,为中医药防治骨转移肿瘤提供实验依据。方法:实验使用雌性SD大鼠,随机分为6组,即假手术组、模型对照组、淫羊藿组、骨碎补组、补骨脂组和唑来膦酸组,每组16只。在假手术组大鼠左后肢胫骨的骨髓腔内注射生理盐水,其余各组则在相同部位接种乳腺癌MRMT-1细胞以制成乳腺癌骨转移模型。次日开始对模型对照组和假手术组灌胃去离子水,淫羊藿组、骨碎补组和补骨脂组灌胃相应中药水煎剂（相当于生药0.5g/100g体重·天）,唑来膦酸组皮下注射唑来膦酸。在造模第19天时观察von Frey纤维刺激的50%缩足阈（50%PWT）、双足负重差值和热刺激缩足潜伏期（TWL）。第21天取材,测定子宫系数、肿瘤体积,收集血清检测雌二醇（E₂）和骨特异性碱性磷酸酶（BALP）水平;对一部分胫骨标本用荧光实时定量RT-PCR法测定甲状旁腺激素相关蛋白（PTHrP）的mRNA水平;另一部分胫骨标本用影像学方法进行评分、测定骨密度（BMD）和骨矿物质含量（BMC）后脱钙,包埋切片,通过HE染色观察形态,抗酒石酸酸性磷酸酶（TRAP）染色计数破骨细胞,用免疫组化法对增殖细胞核抗原（PCNA）、骨保护素（OPG）、核因子κB受体活化因子配体（RANKL）和雌激素受体（ER）染色并进行半定量分析。结果:本项研究中乳腺癌骨转移大鼠模型的特点是以溶骨性病变为主的混合性骨转移,出现破骨细胞过度激活、溶骨性为主的骨质破坏、骨转移性肿瘤生长和癌性骨痛等表现。与模型对照组比较,淫羊藿组和唑来膦酸组BMD升高,唑来膦酸组影像学评分改善,其余各组的BMD和影像学评分亦有改善趋势,另外,各治疗组的BMC均有升高趋势,提示各种药物能够不同程度地减轻骨转移引发的溶骨性骨质破坏。各治疗组的TRAP（+）破骨细胞数量均较模型对照组减少,而OPG表达有增加趋势,从而使RANKL/OPG比值呈现下降趋势,各组PTHrP mRNA和RANKL表达无统计学差异,提示补肾壮骨中药和唑来膦酸能有效抑制具有骨吸收活性的成熟破骨细胞激活,这是其减轻溶骨性病变的中心环节,这一作用是通过提高OPG降低了RANKL/OPG比值而实现的。各中药组血清E₂和子宫系数均与模型对照组无统计学差异,提示补肾壮骨中药的雌激素样作用不明显,补肾壮骨中药在乳腺癌骨转移时的骨保护作用并非通过这一途径。与模型对照组比较,补骨脂组肿瘤体积显著减小,其余各治疗组也表现出此趋势,且与PCNA表达的下降趋势相一致,提示各组药物存在不同程度的抑瘤作用。各治疗组的50%PWT均较模型对照组提高,淫羊藿组、补骨脂组的双足负重差值与淫羊藿组的TWL均有降低,其余各治疗组也有改善趋势,提示补肾壮骨中药能够提高痛阈,减轻乳腺癌骨转移所引发的疼痛。结论:补肾壮骨中药淫羊藿、骨碎补和补骨脂具有减轻乳腺癌骨转移引发的溶骨性骨质破坏和癌性骨痛的效果,其作用的中心环节是抑制了破骨细胞的过度激活,这是通过提高组织中OPG的水平以降低RANKL/OPG比值和抑制骨转移性肿瘤的生长而实现的。本项实验的结果证明补肾壮骨法是治疗乳腺癌骨转移的有效方法。更多还原

【Abstract】 Bone metastasis is the most common distant metastasis from breast cancer.In advanced breast cancer,about 65%-80%patients are suffering from bone metastasis,which decreased their quality of life.Thus, comprehensive treatment was adopted to control bone metastasis, reinforcing kidney and strengthening bone of Traditional Chinese medicine（TCM） is a effective therapy,the mechanism should be researched.Objective:According to the TCM theory,kidney controlling bone,TCM herbs including Herba Epimedii,Rhizoma Drynariae and Fructus Psoraleae,which has the function of reinforcing kidney and strengthening bone were given to rats with bone metastasis.Osteoclast activity was determinated as breakthrough point,effects on bone lesion and bone pain of the rats was observed.Methods:Female Sprague-Dawley rats were divided into 6 groups,rats of sham operation group were Intra-tibial injected normal saline,while others were inoculated the MRMT-1 cells to develop model of bone metastasis from breast cancer.Herba Epimedii,Rhizoma Drynariae, Fructus Psoraleae,pure water and Zoledronic acid was given to different group respectively to observe the effects.On day 19 after the injection of MRMT-1,behavior of von Frey hair stimulated 50%Paw withdrawal threshold（50%PWT）,difference of weight bearing between hind paws and thermal withdrawal latency（TWL） were tested in all groups. All rats were killed on day 21,volume of tumors were measured,the uterus coefficients were calculated,serum and tibias were collected: level of estradiol（E₂） and bone alkaline phosphatase（BALP） in serum was detected by ELISA method.Real-time RT-PCR technique was used to examine mRNA expressions of parathyroid hormone related protein（PTHrP） in half of the tibias,the others were used to evaluate the bone lesions by radiological analysis,quantitative measurement of bone mineral density（BMD） and bone mineral capacity（BMC）,then the tissues were made into paraffin sections,through HE stain,tartrate-resistant acid phosphatase（TRAP） stain and immunohistochemistry to observe the bone pathological changes,count TRAP stained osteoclasts and semi-quantitatively measure expressions of proliferating cell nuclear antigen（PCNA）,osteoprotegerin（OPG）,receptor activator of nuclear factor-κB ligand（RANKL） and estrogen receptor（ER）. Results:The model showed feature of mixed metastasis,dominated by osteolytic bone lesion,manifested as over activation of osteoclasts, excessive bone resorption and bone pain.Compared to model group,BMD was enhanced by Herba Epimedii and Zoledronic acid,radiological analysis was improved by Zoledronic acid, BMC of all treatment groups showed tendency to increase,but no statistical difference.Above results indicated that all treatment can relieve the osteolytic bone lesion induced by bone metastasis.Osteoclast activity were decreased in all treatment groups,and increased expression of OPG made RANKL/OPG ratio drop,PTHrP mRNA was no significance between treatment groups and model group.So,TCM herbs of reinforcing kidney and strengthening bone and Zoledronic acid showed effect of blocking the excessive activation of osteoclasts,and it’s the core link in osteolytic bone defect.There was no change in the level of uterus coefficient,serum E₂ level, didn’t show estrogenic effect significantly,it’s not a operating pathway of bone protection.Tumor volumes were decreased by Fructus Psoraleae,and other treatment groups showed tendencies to reduce the volumes than model group,but no significance,so did PCNA level,revealed that all the TCM herbs had the effect of tumor inhibiting in different degree.As regards behavior tests,50%PWT of treatment groups were higher than model group,differential values between hind paws weight bearing of Herba Epimedii and Fructus Psoraleae group were lower than models. Herba Epimedii also prolonged TWL,showed that the TCM herbs can improve the threshold of pain,and relieve pain caused from bone metastasis. Conclusion:TCM herbs of reinforcing kidney and strengthening bone showed effect of alleviating bone destruction and bone pain induced by bone metastasis,the core link is to block the excessive activation of osteoclasts.The major mechanism can be derived from increased OPG level to decrease the ratio between RANKL and OPG,the function of inhibiting tumor growth maybe another reason.In a word,reinforcing kidney and strengthening bone of TCM is a effective therapy to bone metastasis from breast cancer.更多还原