【作者】 周斌；

【导师】 王芳；

【作者基本信息】 华中科技大学 ， 药理学， 2021， 硕士

【摘要】 目的:Twist1是一种基本的转录因子,属于碱性螺旋-环-螺旋蛋白家族中的一员,参与多种癌症(包括乳腺癌)的发生和转移。研究发现多种癌症会诱导患者产生抑郁症,被称为癌症诱导的抑郁症(cancer-induced depression,CID)。但CID的发病机制还尚不清楚。外泌体是一种由不同类型的细胞分泌的囊泡,可以携带各种生物活性分子,如蛋白质、RNA、DNA和脂质,可以作为细胞间通讯物质传递到其他靶细胞,参与多种神经系统退行性疾病的发生和预后,同时也在癌症发生发展中扮演重要角色。前期工作已证明Twist1在慢性应激诱导的抑郁小鼠的内侧前额叶皮层(medial prefrontal cortex,mPFC)中显著增高并增加小鼠对应激的易感性,但在CID中的作用尚未见报道。本课题主要采用乳腺癌4T1细胞诱导的小鼠抑郁症模型,研究Twist1在CID中的作用及其机制。方法:利用慢病毒表达短发夹RNA(short hairpin RNA,sh RNA)以及嘌呤霉素筛选系统构建Twist1稳定低表达的4T1细胞系(后面简称稳转sh Twist1的4T1细胞),将正常4T1细胞以及稳转sh Twist1的4T1细胞经皮下接种至小鼠,构建荷瘤小鼠模型。采用悬尾实验(tail suspension test,TST)、强迫游泳实验(forced swimming test,FST)和糖水偏好实验(sucrose preference test,SPT)检测小鼠的抑郁样行为。使用超高速离心法得到细胞培养液或者荷瘤小鼠血清中的外泌体,使用定量聚合酶链式反应(quantitative polymerase chain reaction,q PCR)和蛋白质免疫印迹(western blot,WB)来检测细胞、外泌体和小鼠mPFC脑区中Twist1的m RNA和蛋白表达水平。通过向小鼠mPFC脑区注射慢病毒敲减Twist1,研究Twist1在小鼠抑郁样行为中的作用。将外泌体经鼻腔或尾静脉给予小鼠,或者利用外泌体抑制剂处理小鼠,研究外泌体Twist1在小鼠抑郁样行为中的作用。采用共聚焦显微镜研究外泌体在小鼠脑区中的分布以及小鼠mPFC脑区中锥体神经元的形态变化。结果:(1)小鼠接种4T1细胞悬浮液后表现出显著的抑郁样行为,包括TST和FST中不动时间增加以及糖水偏好率降低。(2)WB和透射电子显微镜(transmission electron microscope,TEM)证明超高速离心法成功提取外泌体。(3)提取4T1细胞培养液中的外泌体(cell-derived exosomes,CDE),小鼠分别经鼻腔吸收以及尾静脉注射外泌体后,表现出显著的抑郁样行为,包括TST和FST中不动时间增加以及糖水偏好率降低。(4)小鼠经鼻腔或者尾静脉给予经PKH67标记过的外泌体后,共聚焦显微镜检测外泌体在大脑具有广泛的分布。(5)荷瘤小鼠mPFC中Twist1 m RNA和蛋白表达水平显著上升。(6)慢病毒敲减荷瘤小鼠mPFC脑区的Twist1改善荷瘤小鼠抑郁样行为,包括TST和FST中不动时间降低以及糖水偏好率增加。(7)利用慢病毒表达sh RNA以及嘌呤霉素筛选系统构建稳转sh Twist1的4T1细胞,该细胞系Twist1 m RNA和蛋白表达水平显著下降。提取该细胞系中外泌体,检测发现Twist1 m RNA表达水平显著下降。(8)与接种4T1细胞的荷瘤小鼠相比,接种稳转sh Twist1的4T1细胞的荷瘤小鼠抑郁样行为明显改善,包括TST和FST中不动时间降低以及糖水偏好率增加,其mPFC脑区Twist1 m RNA和蛋白水平显著降低。(9)GW4869显著抑制外泌体的分泌和释放,同时改善荷瘤小鼠的抑郁样行为,包括TST中不动时间降低以及糖水偏好率增加。(10)小鼠分别接种正常4T1细胞以及稳转sh Twist1的4T1细胞,提取荷瘤小鼠血清中的外泌体(serum-derived exosomes,SDE),将SDE分别经鼻腔或者尾静脉给予至正常小鼠,接种正常4T1细胞的荷瘤小鼠来源的SDE可诱导小鼠抑郁样行为,包括TST和FST中不动时间增加以及糖水偏好率降低,而接种稳转sh Twist1的4T1细胞的荷瘤小鼠来源的SDE无此作用。接种正常4T1细胞的荷瘤小鼠来源的SDE上调小鼠mPFC脑区Twist1 m RNA和蛋白表达水平,而接种稳转sh Twist1的4T1细胞的荷瘤小鼠来源的SDE对小鼠mPFC脑区Twist1 m RNA和蛋白表达水平无明显影响。(11)小鼠分别接种正常4T1细胞以及稳转sh Twist1的4T1细胞悬浮液,提取荷瘤小鼠的SDE,经尾静脉分别给予正常小鼠,检测小鼠mPFC脑区锥体神经元形态。接种正常4T1细胞的荷瘤小鼠来源的SDE降低小鼠mPFC脑区神经元树突长度、树突分支节点数以及树突复杂性,而接种稳转sh Twist1的4T1细胞的荷瘤小鼠来源的SDE无此作用。结论:接种乳腺癌4T1细胞的荷瘤小鼠表现出抑郁样行为,肿瘤细胞源性的Twist1参与该过程。乳腺癌4T1细胞通过分泌外泌体Twist1释放入外周循环系统,作用于小鼠mPFC脑区,降低锥体神经元树突长度、树突分支节点数以及树突复杂性,进而诱导小鼠抑郁样行为。更多还原

【Abstract】 Objective: Twist1 is a basic transcription factor,a member of the basic helix-loop-helix protein family,and is involved in the occurrence and metastasis of a variety of cancers(including breast cancer).Studies have found that a wide variety of cancers induce depression in patients,which is called cancer-induced depression(CID).However,the mechanism of CID is still unclear.Exosomes are vesicles secreted by different types of cells and package various biologically active molecules,such as protein,RNA,DNA and lipids.They can act as intercellular communication materials transmitted to other target cells and are involved in occurrence and prognosis of systemic degenerative diseases and cancers.Previous work has proved that Twist1 is significantly increased in the medial prefrontal cortex(mPFC)of chronic stress induced depressive mice and increases susceptibility to stress.This study mainly explored whether breast cancer 4T1 cells could secrete exosomes Twist1 to promote the depressive-like behaviors in mice.Methods: A 4T1 cell line with stable and low expression of Twist1(referred to as stable expressing sh Twist1 4T1 cells)were obtained via lentivirus and puromycin.4T1 cells and stable expressing sh Twist1 4T1 cells were subcutaneously inoculated into mice to construct a tumorbearing mice model.Tail suspension test(TST),forced swimming test(FST)and sucrose preference test(SPT)were used to detect depressive-like behaviors in mice.Ultra-high-speed centrifugation were used to extract exosomes in cell medium or serum.Quantitative polymerase chain reaction(q PCR)and western blot(WB)were used to detect m RNA and protein expression levels of Twist1 in the cells,exosomes and mPFC.The role of Twist1 in depressive-like behavior in mice was studied by injecting lentivirus into the mPFC to knock down Twist1.The exosomes were administered to mice through the intranasal administration or intravenous injection,or mice were treated with exosomal inhibitors to study the role of exosomal Twist1 in depressive-like behaviors in mice.Confocal microscope was used to study the distribution of exosomes in the brain regions and the morphological changes of pyramidal neurons in the mPFC.Results:(1)Mice inoculated with 4T1 cell suspension showed significant depressive-like behaviors,including increased immobility time in TST and FST and decreased sucrose preference in SPT.(2)WB and transmission electron microscope(TEM)were used to prove the purity of exosomes.(3)The mice showed significant depressive-like behavior after absorbing the exosomes extracting from the 4T1 cell culture supernatant through the intranasal administration or intravenous injection,including increased immobility time in TST and FST and decreased sucrose preference in SPT.(4)After the mice absorbed the exosomes labeled with PKH67 through the intranasal administration or intravenous injection,the exosomes were detected by the confocal microscope and showed extensive expression in the brain.(5)The m RNA and protein expression levels of Twist1 in mPFC of tumor-bearing mice were increased.(6)Knockdown of Twist1 via lentivirus in the mPFC reversed depressive-like behavior in tumor-bearing mice,including reduced immobility time in TST and FST and increased sucrose preference in SPT.(7)Stable expressing sh Twist1 4T1 cells were obtained via lentivirus and puromycin,the m RNA and protein expression levels of Twist1 in stable expressing sh Twist1 4T1 cells were decreased significantly.The exosomes from this cell line were extracted and the m RNA expression level of Twist1 in the exosomes was decreased significantly.(8)Tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells reversed depressive-like behaviors,including decreased immobility time in TST and FST and increased sucrose preference in SPT.The m RNA and protein expression levels of Twist1 in the mPFC of tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells were decreased significantly compared with tumor-bearing mice inoculated with 4T1 cells.(9)GW4869 inhibited the secretion and release of exosomes significantly,and reversed the depressive-like behavior of tumor-bearing mice,including decreased immobility time in TST and increased sucrose preference in SPT.(10)Mice were respectively inoculated with normal 4T1 cells or stable expressing sh Twist1 4T1 cells.The exosomes(serum-derived exosomes,SDE)in the serum of tumor-bearing mice were extracted,and were given to new normal mice via intranasal administration or intravenous injection.SDE derived from tumor-bearing mice inoculated with 4T1 cells induced significant depressive-like behaviors,including increased immobility time in TST and FST and decreased sucrose preference in SPT,while SDE derived from tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells had no effects.SDE derived from tumor-bearing mice inoculated with 4T1 cells increased the m RNA and protein expression level of Twist1 in the mPFC of normal mice,while the SDE derived from tumorbearing mice inoculated with stable expressing sh Twist1 4T1 cells had no effect.(11)SDE were extracted from mice inoculated with normal 4T1 cells or stable expressing sh Twist1 4T1 cells,and were given to new normal mice via intravenous injection.SDE derived from tumor-bearing mice inoculated with 4T1 cells reduced total dendrite length and branching and dendritic complexity of pyramidal neurons in the mPFC,while SDE derived from tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells had no effect.Conclusion: Tumor-bearing mice inoculated with breast cancer 4T1 cells showed depressive-like behavior,and tumor cell-derived Twist1 was involved in this process.4T1 cells secreted exosomal Twist1 into the peripheral circulatory system and targeted mPFC of mice,reducing the dendritic complexity,the number of branch and length of dendrites to induce depressive-like behavior.更多还原