乳腺癌MRMT-1细胞局部骨转移大鼠模型的研究

Research of rat model of bone metastasis from rat mammary tumor MRMT-1 cells

目的 研究乳腺癌MRMT-1细胞局部骨转移大鼠模型在痛觉反应、影像学、病理学和分子生物学等方面的表现,为进一步的发病机制和药理学研究提供资料.方法 将32只雌性SD大鼠用随机数字表法分为假手术组和模型组各16只,在模型组大鼠胫骨内注射乳腺癌MRMT-1细胞,引起局部骨转移.造模后19d测定痛觉反应,21d处死大鼠取材,测定肿瘤体积,用X线对骨质损伤进行评分并测定骨密度（BMD）,HE染色观察病理形态改变,抗酒石酸酸性磷酸酶（TRAP）法染色观察破骨细胞,免疫组织化学法检测核因子κB受体活化因子配体（RANKL）和骨保护素（OPG）水平,并计算比值；实时荧光定量反转录聚合酶链反应（RT-PCR）法检测甲状旁腺激素相关蛋白（PTHrP）水平.结果 与假手术组比较,模型组存在痛觉异常状态（P＜0.01）；胫骨在X线下损伤评分较高（P＜0.01）.模型组和假手术组大鼠左侧胫骨的BMD分别为（0.11 ±0.01）g/cm2和（0.13±0.02）g/cm2（P＜ 0.05）.模型组胫骨标本上肿瘤生长明显,体积为（1082.73±679.44）mm3,而假手术组无肿瘤生长（P＜0.01）,病理可见混合性骨转移,但以溶骨性病变为主；伴有破骨细胞数量明显增多,达到（40.84±25.59）个/高倍视野,而假手术组仅为（1.88±2.92）个/高倍视野（P＜0.01）.转移灶局部RANKL水平无明显变化,OPG水平下降（P＜0.05）,OPG/RANKL比值和PTHrP下降（P＜0.05）.结论 乳腺癌MRMT-1细胞局部骨转移大鼠模型存在癌痛、骨质破坏,甚至病理性骨折等表现.其发病机制可能是MRMT-1细胞在浸润生长过程中打破了OPG-RANKL-RANK系统的平衡,从而激活破骨细胞,引起骨吸收作用亢进,引起各种表现.

Objective To study the characteristics of rat model of bone metastasis from breast cancer,which receiving intra-tibial injection of MRMT-1 rat breast cancer cells.Methods Female SD rats were divided randomly into sham operation group and model group.Sham operation group were intra-tibial injected normal saline,while model group were injected the MRMT-1 cells to develop model.On day 19 after operation,allodynia and hyperalgesia were tested.All rats were killed on day 21,tibias were collected to measure the volume of tumors,determinate bone mineral density （BMD） and bone mineral capacity （BMC）.Through HE staining,tartrate-resistant acid phosphatase （TRAP） staining and immunohistochemistry staining to observe the bone pathological changes,count osteoclasts and semi-quantitatively measure expressions of proliferating cell nuclear antigen （PCNA）,osteoprotegerin （OPG） and receptor activator of nuclear factor-κB ligand （RANKL）.By real-time quantitative RT-PCR,expression of parathyroid hormone related protein （PTHrP） was determinated.Results Model group displayed mechanical allodynia and hyperalgesia （P 〈 0.01）,bone destruction was found by image examination （P 〈 0.01）.BMD of left tibia decreased to （0.11±0.01） g/cm2 from （0.13±0.02） g/cm2.In the model tibia,visible tumor growth could be seen,and mixed bone lesion was seen through HE staining sections.Osteoclast enhanced from （1.88±2.92）/HP to （40.84±25.59）/HP （P 〈 0.01）,PTHrP and OPG reduced （P 〈 0.05）,but RANKL did not change.Conclusion The model of bone metastasis from breast cancer show characteristics of cancer pain and bone lesion.The injury mechanism is to break balance of OPG-RANKL-RANK system by inhibiting OPG,these changes would cause excessive activation of osteoclasts,which induced hyperfunction of bone resorption.