【作者】 刘畅；

【导师】 余希杰；

【作者基本信息】 四川大学 ， 内科学（内分泌代谢）（专业学位）， 2021， 博士

【摘要】 第一章动物体内骨髓脂肪细胞对乳腺癌骨转移的影响目的:在进展期乳腺癌患者当中,骨骼位居远距离转移的首选器官。骨髓中的细胞成分在骨转移进程中发挥不可低估的影响。有研究已经发现成骨细胞等对乳腺癌骨转移有加速效应。骨髓脂肪细胞是骨髓中数量最多的细胞组分,但其对乳腺癌骨转移的影响和作用机制尚不清楚,有必要对此做进一步研究。材料和方法:将骨髓间充质干细胞OP9诱导为骨髓脂肪细胞OP9A,然后把OP9A细胞分别与4T1和MDA-MB-231乳腺癌细胞混合注射到小鼠的后肢胫骨旁软组织内。使用动物活体生物发光检查小鼠肿瘤负荷,micro-CT扫描检查骨侵袭及骨破坏情况。结果:骨髓脂肪细胞OP9A使4T1和MDA-MB-231乳腺癌细胞在小鼠体内产生了更重的肿瘤负荷,并且乳腺癌细胞的骨侵袭性更高而且骨质破坏更严重。结论:骨髓脂肪细胞对乳腺癌骨转移具有潜在的促进效应。第二章骨髓脂肪细胞对乳腺癌细胞演进作用的研究目的:探究骨髓脂肪细胞对乳腺癌细胞肿瘤生物学行为的影响,为进一步研究骨髓脂肪细胞影响乳腺骨转移的机制提供线索。材料和方法:收集骨髓脂肪细胞OP9A的培养上清,制备OP9A条件培养基。采用OP9A条件培养基分别对4T1和MDA-MB-231乳腺癌细胞进行处理。利用CCK-8和克隆形成实验比较乳腺癌细胞的增殖能力变化,划痕实验和Transwell实验比较乳腺癌细胞迁移能力的变化,成球实验比较乳腺癌细胞形成肿瘤干细胞的能力变化。结果:骨髓脂肪细胞的条件培养基使4T1和MDA-MB-231乳腺癌细胞的增殖能力、迁移能力、干细胞形成能力增强,从而使乳腺癌细胞肿瘤生物学行为恶性度增强,增加了骨转移的潜能。结论:骨髓脂肪细胞对乳腺癌细胞的恶性演进具有促进效应。第三章骨髓脂肪细胞促进乳腺癌细胞演进及骨转移作用的机制研究目的:探究骨髓脂肪细胞来源的脂肪因子resistin对乳腺癌细胞肿瘤生物学行为和小鼠体内乳腺癌骨侵袭的影响,以及resistin在乳腺癌细胞内作用的下游靶点,明确骨髓脂肪细胞影响乳腺癌细胞骨转移潜力的机理。材料和方法:用ELISA试剂盒检测骨髓脂肪细胞上清中resistin的含量。添加外源性resistin,或用抗体中和resistin的骨髓脂肪细胞条件培养基分别对4T1和MDAMB-231乳腺癌细胞进行处理。利用CCK-8和克隆形成实验比较乳腺癌细胞的增殖能力变化,划痕实验和Transwell实验比较乳腺癌细胞迁移能力的变化,成球实验比较乳腺癌细胞形成肿瘤干细胞的能力变化。在小鼠体内注射外源性resistin,或者在OP9A细胞处理的同时注射resistin抗体中和。使用动物活体生物发光检查小鼠肿瘤负荷,micro-CT扫描检查骨侵袭及骨破坏情况。用q PCR检测resistin作用于乳腺癌细胞的下游靶点。在乳腺癌骨转移患者取样,验证resistin、IL-11蛋白的表达水平,及resistin作用于乳腺癌细胞的下游靶点。结果:骨髓脂肪细胞分泌较多的resistin,而乳腺癌细胞不分泌resistin。Resistin使4T1和MDA-MB-231乳腺癌细胞的增殖能力、迁移能力、干细胞形成能力增强,这增加了乳腺癌细胞骨转移的潜力。中和resistin削弱了骨髓脂肪细胞条件培养基增强的4T1和MDA-MB-231乳腺癌细胞增殖能力、迁移能力、干细胞形成能力。Resistin使4T1和MDA-MB-231乳腺癌细胞在小鼠体内产生了更重的肿瘤负荷,并且骨侵袭及骨质破坏更严重。中和resistin削弱了骨髓脂肪细胞造成的4T1和MDA-MB-231乳腺癌细胞在小鼠体内更重的肿瘤负荷、更严重的骨侵袭及骨质破坏。骨髓脂肪细胞的条件培养基上调乳腺癌细胞内NF-κB、IL-11的转录水平;resistin能够增加乳腺癌细胞的IL-11表达、细胞增殖和迁移,但抑制NF-κB使resistin这种效应减弱。最后在乳腺癌骨转移患者骨髓脂肪细胞验证了resistin蛋白的表达以及骨转移患者更高的血清resistin、IL-11含量,同时验证了骨转移灶肿瘤组织中NF-κB蛋白水平更高。结论:骨髓脂肪细胞来源的resistin介导了骨髓脂肪细胞对乳腺癌细胞骨转移潜力的促进效应。具有促破骨作用的IL-11是resistin发挥这种作用的介质。Resistin可以上调调控IL-11表达的转录因子NF-κB。因此,resistin可能通过增加转录因子NF-κB及IL-11的表达,发挥促乳腺癌骨转移的作用(resistin/NF-κB/IL-11信号通路)。更多还原

【Abstract】 Part Ⅰ.The effect of bone marrow adipocytes on bone metastasis of breast cancer in vivo Objective:Bone is the most common distant metastasis site of advanced breast cancer.The cellular components in the bone marrow play an important role in the bone metastasis of breast cancer.Previous studies have shown that osteoclasts and osteoblasts have a promoting effect on bone metastasis of breast cancer.Bone marrow adipocytes are the most abundant cell component in the bone marrow,but their effect on bone metastasis of breast cancer and the mechanism of this effect are still unclear,so further research is necessary.Materials and Methods:Bone marrow mesenchymal stem cells OP9 were induced to bone marrow adipocytes OP9 A.OP9A cells were mixed with 4T1 or MDA-MB-231 breast cancer cells respectively.They were injected into the soft tissues of the hind limbs adjacent to the tibia of mice.Animal bioluminescence was used to detect tumor burden in mice.Micro-CT scan was used to check bone invasion and bone destruction.Results:Bone marrow adipocyte OP9 A caused 4T1 and MDA-MB-231 breast cancer cells to produce a heavier tumor burden in mice,more bone invasion,and more severe bone destruction.Conclusion:Bone marrow adipocytes have the potential to promote bone metastasis of breast cancer.Part Ⅱ.The preliminary study on the effect of bone marrow adipocytes on breast cancer cells progressionObjective:To explore the effect of bone marrow adipocytes on the biological behavior of breast cancer cells,and provide clues for further research on the mechanism of bone marrow adipocytes influencing bone metastasis of breast cancer.Materials and Methods:The culture supernatant of bone marrow adipocyte OP9 A was collected to prepare OP9 A conditioned medium.OP9 A conditioned medium was used to treat 4T1 and MDA-MB-231 breast cancer cells respectively.CCK-8 and colony-forming assay were used to compare the proliferation ability of breast cancer cells,the wound scratch assay and the transwell assay were used to compare the migration ability of breast cancer cells,and the sphere formation assay was used to compare the ability of breast cancer cells to form tumor stem cells.Results:The conditioned medium of bone marrow adipocytes promoted the proliferation,migration,and stem cell formation capabilities of 4T1 and MDA-MB-231 breast cancer cells,thereby enhancing the malignancy of the tumor biological behavior of breast cancer cells and increasing the potential for bone metastasis.Conclusion:Bone marrow adipocytes promote the malignant progression of breast cancer cells.Part Ⅲ.The exploration of the mechanism of bone marrow adipocytes in promoting progression and bone metastasis of breast cancer cellsObjective:To explore the effects of adipokines resistin derived from bone marrow adipocytes on tumor biological behavior and bone invasion of breast cancer cells in mice,as well as the downstream targets of resistin in breast cancer cells,and clarify the mechanism that bone marrow adipocytes affect bone metastasis potential of breast cancer cells.Materials and Methods:ELISA kit was used to detect the content of resistin in the supernatant of bone marrow adipocytes.Add exogenous resistin or use antibody to neutralize resistin in the bone marrow adipocyte conditioned medium to treat 4T1 or MDA-MB-231 breast cancer cells respectively.CCK-8 and clone formation assay were used to compare the proliferation ability of breast cancer cells,the wound scratch assay and the transwell assay were used to compare the migration ability of breast cancer cells,and the sphere formation assay was used to compare the ability of breast cancer cells to form tumor stem cells.Inject exogenous resistin into mice,or inject resistin antibody to neutralize resistin from OP9 A cells in vivo.Then animal bioluminescence was uesed to detect tumor burden in mice,and micro-CT scan was used to check bone invasion and bone destruction.QPCR was used to test the downstream target of resistin on breast cancer cells.Take samples from breast cancer patients with bone metastasis to verify the expression level of resistin protein and the effect of resistin on the downstream targets of breast cancer cells.Results:Bone marrow adipocytes secreted more resistin,while breast cancer cells did not secrete resistin.Resistin enhanced the proliferation,migration,and stem cell formation of 4T1 and MDA-MB-231 breast cancer cells.These increased the potential for bone metastasis of breast cancer cells.Neutralizing resistin weakened the 4T1 and MDAMB-231 breast cancer cell proliferation,migration,and stem cell formation enhanced by the bone marrow adipocyte conditioned medium.Resistin caused 4T1 and MDAMB-231 breast cancer cells to produce heavier tumor burden in mice,higher bone invasion,and more bone destruction.Neutralizing resistin weakened the tumor burden,bone invasion,and bone destruction of breast cancer cells in mice enhanced by bone marrow adipocytes.The conditioned medium of bone marrow adipocytes up-regulated the transcription levels of NF-κB,IL-11 in breast cancer cells.The expression of resistin protein was verified in bone marrow adipocytes of breast cancer patients with bone metastasis.And these patients with bone metastasis presented higher concentration of resistin and IL-11 in their serum.Besides the higher level of NF-κB protein in tumor tissues of bone metastasis was confirmed.Conclusion:The resistin derived from bone marrow adipocytes mediates the promoting effect of bone marrow adipocytes on the bone metastasis potential of breast cancer cells.IL-11 with osteolytic effect is the medium for resistin to exert this effect.Resistin can upregulate the transcription factor NF-κB that increases the expression of IL-11.Therefore,resistin may up-regulate the expression of NF-κB and IL-11 in breast cancer cells to promote bone metastasis of breast cancer(resistin / NF-κB / IL-11 signaling pathway).更多还原