【作者】 李燕；

【导师】 王惠惠；

【作者基本信息】 中国医科大学 ， 公共卫生（专业学位）， 2023， 硕士

【摘要】 目的:癌症已成为严重的全球性公共卫生问题,其病理机制复杂,血管生成在癌症发生发展过程中起重要作用。核转录因子Nfe2l1(Nuclear factor erythroid-2-related factor 1)是调节多种应激反应的核心转录因子,在调节机体氧化还原反应和蛋白酶体稳态等多种生物学作用中发挥重要作用。肿瘤生长需要新血管形成来获得氧气及营养物质,血管内皮细胞的增殖和迁移会促进血管形成。血管内皮细胞中Nfe2l1在肿瘤血管生成中所发挥的作用尚未见报道,本研究通过对血管内皮细胞Nfe2l1敲除小鼠进行皮下荷瘤肺癌细胞建立体内肿瘤生长模型,利用Nfe2l1沉默的小鼠血管内皮细胞系(SVEC4-10)与肿瘤细胞(3LL)建立体外共培养模型,研究在肿瘤生长过程中血管内皮细胞Nfe2l1对肿瘤血管生成的影响,并探索血管内皮细胞Nfe2l1调控血管生成的可能机制,为临床肿瘤治疗提供实验依据。研究方法:1.构建血管内皮细胞Nfe2l1基因敲除小鼠模型:利用C57BL/6遗传背景的小鼠建立Cdh5-Cre以及Nfe2l1-Flox和Nfe2l1(E)-KO转基因小鼠,通过PCR扩增法进行基因型鉴定。2.建立小鼠肿瘤生长模型:应用6～8周的Cdh5-Cre、Nfe2l1-Flox和Nfe2l1(E)-KO的小鼠,皮下注射小鼠肺癌细胞(3LL)8×10~5个/只,隔天量取一次小鼠体重和肿瘤大小,两周后收取肿瘤组织。3.荷瘤小鼠肿瘤生长的评价:收取小鼠肿瘤组织后拍摄瘤体照片并测量其重量;通过冰冻切片免疫荧光染色血小板-内皮细胞粘附分子(Platelet endothelial cell adhesion molecule-1,PECAM-1/CD31),观察肿瘤血管密度;通过石蜡切片免疫组化染色Ki-67,观察肿瘤细胞增殖情况;检测血管内皮细胞生长因子受体2(Vascular endothelial growth factor receptor 2,VEGFR2)及p-VEGFR2的表达水平,评价血管生成相关信号通路激活情况。4.小鼠皮下肿瘤血管通透性检测:通过尾静脉注射Evans blue(30 mg/kg)至荷瘤小鼠,半小时后,同时收取同窝实验小鼠,测量肿瘤组织重量,提取并检测组织中Evans blue含量,评估血管通透性。5.建立Nfe2l1沉默的血管内皮细胞:血管内皮细胞转入携带靶向Nfe2l1 sh RNA的慢病毒(Nfe2l1-KD)与其阴性对照(Scramble,Scr),利用1μg/ml嘌呤霉素(puromycin)筛选稳转的细胞,通过提取m RNA及蛋白来鉴定Nfe2l1沉默效果。6.细胞共培养:以7.5×10~5个/孔的细胞数,将Scramble和Nfe2l1-KD组分别接种于6孔板,以1×10~6个/孔的细胞数,将3LL细胞接种于0.4μm孔径的共培养插件中,共培养体系中细胞均使用含10%FBS的DMEM培养液。共培养两天后收集Scramble和Nfe2l1-KD进行实验。7.细胞迁移实验:将共培养后的血管内皮细胞Scramble和Nfe2l1-KD接种至Transwell小室(8μm微孔)中,上室加DMEM,下层加含10%FBS的DMEM作为诱导细胞迁移的因素,诱导24小时后,采用结晶紫进行染色,正倒置一体显微镜观察细胞穿至小室下层的数目。8.划痕愈合实验:将共培养后的血管内皮细胞Scramble和Nfe2l1-KD重新接种于6孔板,并将共培养插件中接种3LL细胞处理上述两种细胞,观察血管内皮细胞6、12、18、24、30 h划痕愈合的百分比。9.3LL细胞条件培养液处理SVEC4-10细胞系:血管内皮细胞Scramble和Nfe2l1-KD同时采用3LL细胞条件培养液处理5、10、20 min,检测VEGFR2及p-VEGFR2的表达水平。结果:1.成功构建血管内皮细胞Nfe2l1基因敲除小鼠模型:通过PCR基因型鉴定,确认了Cdh5-Cre、Nfe2l1-Flox和Nfe2l1(E)-KO小鼠模型构建成功。2.血管内皮细胞Nfe2l1敲除促进荷瘤小鼠瘤体生长:3LL细胞接种到三组小鼠的背部皮下,持续监测肿瘤生长两周后收取瘤体。与其他两组相比,Nfe2l1(E)-KO小鼠体内移植瘤生长更快(P<0.05)、肿瘤重量更大(P<0.05)。3.血管内皮细胞Nfe2l1敲除小鼠瘤体内肿瘤细胞增殖更快:通过对肿瘤组织石蜡切片,并进行了肿瘤细胞增殖标记物Ki-67免疫组化染色,来评估Nfe2l1缺失是否影响肿瘤细胞增殖,通过定量分析表明,与Nfe2l1-Flox相比Nfe2l1(E)-KO小鼠肿瘤组织中Ki-67表达量更高(P<0.05)。4.血管内皮细胞Nfe2l1敲除促进荷瘤小鼠瘤体血管生成:通过对肿瘤组织冰冻切片,血管内皮细胞标记物CD31免疫荧光染色,观察肿瘤血管密度进而评价肿瘤血管的生成情况。对血管阳染区域进行定量分析显示,与Nfe2l1-Flox组相比Nfe2l1(E)-KO组肿瘤组织中的血管密度更高(P<0.05)。5.血管内皮细胞Nfe2l1敲除小鼠瘤体内微血管通透性升高:为了检测肿瘤血管通透性,我们将Evans blue通过尾静脉注射到小鼠体内,然后从肿瘤组织中提取Evans blue,并用分光光度法进行定量,发现Nfe2l1(E)-KO小鼠肿瘤组织的Evans blue含量高于其对照小鼠(P<0.05)。6.血管内皮细胞Nfe2l1敲除小鼠瘤体VEGFR2及p-VEGFR2表达水平升高:通过提取肿瘤组织蛋白,检测肿瘤血管生成相关蛋白VEGFR2及p-VEGFR2的蛋白表达水平,来评估Nfe2l1缺失对肿瘤血管生成的影响,发现小鼠体内移植瘤中Nfe2l1(E)-KO组VEGFR2及p-VEGFR2的蛋白表达水平高于其对照组(P<0.05)。7.成功构建血管内皮细胞Nfe2l1沉默细胞:通过建立Nfe2l1稳转沉默的SVEC4-10细胞系,来研究血管内皮细胞Nfe2l1参与肿瘤血管生成的可能机制,提取m RNA和蛋白,Nfe2l1稳转沉默的血管内皮细胞(Nfe2l1-KD)Nfe2l1的m RNA和蛋白水平均低于Scramble组。8.与肿瘤细胞共培养后血管内皮细胞Nfe2l1沉默促进血管内皮细胞迁移:建立肿瘤细胞与血管内皮细胞共培养体系,上室接种3LL细胞,下室分别接种Scramble和Nfe2l1-KD血管内皮细胞,并评估血管内皮细胞的迁移能力。结果表明与3LL细胞共培养两天后的Nfe2l1-KD组内皮细胞迁出小室的数量,明显多于Scramble组(P<0.05)。9.与肿瘤细胞共培养后血管内皮细胞Nfe2l1沉默促进内皮细胞划痕愈合:经共培养处理两天的SVECs,重新接种于6孔板中,观察6、12、18、24、30 h划痕愈合能力。结果显示,共培养处理的Nfe2l1-KD组较Scramble组划痕愈合能力更强。10.血管内皮细胞Nfe2l1沉默促进肿瘤细胞条件培养液诱导的VEGFR2磷酸化:3LL细胞条件培养液处理Scramble组和Nfe2l1-KD组血管内皮细胞5、10、20 min,评估在肿瘤环境中血管内皮细胞VEGFR2的激活水平,可以发现与Scramble组相比,Nfe2l1-KD组VEGFR2的磷酸化水平较高(P<0.05)。结论:血管内皮细胞特异性Nfe2l1缺失可能通过上调VEGFR2的磷酸化水平,进而促进血管内皮细胞迁移加速肿瘤血管生成,加速肿瘤生长。更多还原

【Abstract】 Objective:The complex pathological mechanism of cancer has become a serious global public health problem.The occurrence and development of cancer are mainly affected by angiogenesis.Nuclear factor erythroid-2-related factor 1(Nfe2l1)is a core transcription factor that regulates a variety of stress response and plays an important role in regulating a variety of biological functions such as redox reactions and proteasome homeostasis.Tumors need to form blood vessels to get the oxygen and nutrients they need.The proliferation and migration of endothelium promote angiogenesis.However,little is known about the role of Nfe2l1 in vascular endothelial cells.Therefore,in the present study,we established an in vivo tumor growth model of subcutaneous tumor-bearing lung cancer cells in Nfe2l1 knockout mice and an in vitro co-culture model of Nfe2l1silenced mouse vascular endothelial cell line(SVEC4-10)and tumor cells(3LL).To study the possible mechanism of Nfe2l1 regulating vascular endothelial cells affecting tumor growth,and to provide reference for clinical tumor treatment.Methods:1.Construction of Nfe2l1 gene knockout mouse model of vascular endothelial cells:Cdh5-Cre,Nfe2l1-Flox and Nfe2l1(E)-KO transgenic mice were established from C57BL/6 genetic background,and genotype identification was conducted by PCR amplification.2.A mouse tumor growth model was established:Cdh5-Cre,Nfe2l1-Flox and Nfe2l1(E)-KO mice aged 6-8 weeks were injected subcutaneously with mouse lung cancer cells(3LL),and each mouse was injected with 8×10~5cells,the weight and tumor size of mice were measured every other day,and tumor tissues were collected two weeks later.3.Evaluation and characterization of mouse tumor growth model:After collecting tumor tissues from mice,we took gross photographs and measured their weights;observed tumor vascular density by immunofluorescence staining of platelet endothelial cell adhesion molecule-1(PECAM-1/CD31)in frozen sections.The expression levels of Vascular endothelial growth factor receptor 2(VEGFR2)and p-VEGFR2 were measured to evaluate the activation of angiogenesis-related signaling pathways.4.Detection of vascular permeability of mouse subcutaneous tumor:Evans blue(30mg/kg)was injected into the blood circulation of tumor-bearing mice for half an hour through the tail vein,and mice in the same litter were collected at the same time to measure the weight of tumor tissue,extract and detect the content of Evans blue in the tissue and evaluate the vascular permeability.5.Establishment and detection of Nfe2l1 silenced vascular endothelial cells:Vascular endothelial cells were transfected with lentivirus carrying sh RNA targeting Nfe2l1(Nfe2l1-KD)and its negative control Scramble(Scr).The stable transfected cells were screened with 1μg/ml puromycin,and the silencing effect of Nfe2l1 was identified by extracting m RNA and protein.6.Cell co-culture:Scramble and Nfe2l1-KD groups were seeded in 6-well plates at a cell number of 7.5×10~5 cells/well,and 3LL cells were seeded in 0.4μm pore diameter co-culture inserts at a cell number of 1×10~6 cells/well.The cells in the co-culture system were cultured in DMEM medium containing 10%fetal bovine serum.Scramble and Nfe2l1-KD were collected for experiments after two days of co-culture.7.Cell migration assay:Scramble and Nfe2l1-KD co-cultured vascular endothelial cells were seeded into Transwell chambers(8μm microwells).The upper chamber was added with DMEM,and the lower layer was added with DMEM containing 10%FBS as a factor to induce cell migration.The number of cells penetrating the lower layer of the chamber was observed under an inverted microscope.8.Scratch healing experiment:Scramble and Nfe2l1-KD of co-cultured vascular endothelial cells were re-inoculated into the 6-well plate,and 3LL cells were inoculated into the co-culture plug-in to treat the above two kinds of cells,and the percentage of scratch healing of vascular endothelial cells at 6,12,18,24 and 30 h was observed.9.SVEC4-10 cell line treated with 3LL cell conditioned medium:vascular endothelial cell Scramble and Nfe2l1-KD were treated with 3LL cell conditioned medium at the same time,and the expression levels of VEGFR2 and p-VEGFR2 were detected after 5,10 and 20 min of treatment.Results:1.The successful construction of Nfe2l1 gene knockout mouse models of vascular endothelial cells:Cdh5-Cre,Nfe2l1-Flox and Nfe2l1(E)-KO mouse models were confirmed by genotype identification.2.Accelerated tumor growth in vascular endothelial Nfe2l1 knockout mice:3LL cells were inoculated into the back of three groups of mice,the tumor growth volume was recorded every other day,and the tumor was harvested two weeks later.Compared with the other two groups,Nfe2l1(E)-KO mice had faster tumor growth(P<0.05)and larger tumor weight(P<0.05).3.Vascular endothelial Nfe2l1 knockout mice had faster tumor cell proliferation:To assess whether Nfe2l1 loss affects tumor cell proliferation,paraffin sections of tumor tissue were subjected to immunohistochemical staining for the tumor cell proliferation marker Ki-67.The quantitative results showed that Ki-67 expression in transplanted tumors of Nfe2l1(E)-KO mice was higher than that of Nfe2l1-Flox mice(P<0.05).4.Vascular endothelial Nfe2l1 knockout mice showed increased tumor angiogenesis:Frozen sections of tumor tissues were stained with immunofluorescence for vascular endothelial cell marker CD31 to evaluate tumor angiogenesis.Compared with the Nfe2l1-Flox group,the Nfe2l1(E)-KO group had a significantly higher vascular density in tumor tissues(P<0.05).5.Increased permeability of tumor microvessels in Nfe2l1 knockout mice:To detect tumor vascular permeability,Evans blue was injected into mice through the tail vein,and then Evans blue was extracted from tumor tissue and quantized by spectrophotometry.It was found that the content of Evans blue in tumor tissue of Nfe2l1(E)-KO mice was higher than control mice(P<0.05).Thus,tumors transplanted into Nfe2l1(E)-KO mice were characterized by increased microvascular permeability.6.Elevated VEGFR2 and p-VEGFR2 expression in endothelial Nfe2l1 knockout mice:The effect of Nfe2l1 deficiency on tumor angiogenesis was evaluated by extracting tumor tissue protein and detecting the protein expression levels of tumor angiogenesis-related proteins VEGFR2 and p-VEGFR2.The protein expression levels of VEGFR2 and p-VEGFR2 in the transplanted tumor of Nfe2l1(E)-KO group were higher than those in the control group(P<0.05).7.Nfe2l1 silencing cells of vascular endothelial cells were successfully constructed:The Nfe2l1 silencing SVEC4-10 cell line was established to study the possible mechanism of Nfe2l1 involved in tumor angiogenesis.The m RNA and protein were extracted to verify the successful construction of Nfe2l1 silencing vascular endothelial cell line Scramble and Nfe2l1-KD cells.8.Nfe2l1 silencing of vascular endothelial cells promotes cell migration in a tumor cell co-culture environment:A co-culture system of tumor cells and vascular endothelial cells was established,in which 3LL cells were inoculated in the upper chamber and Scramble and Nfe2l1-KD cells were inoculated in the lower chamber,respectively,to evaluate the migration ability of vascular endothelial cells in the tumor environment.The results showed that after two days of co-culture with 3LL cells,the number of endothelial cells migrated out of the chamber in Nfe2l1-KD group was significantly higher than that in the Scramble group(P<0.05).9.Nfe2l1 silencing of vascular endothelial cells promotes cell proliferation and migration in tumor cell co-culture environment:SVECs treated with co-culture for two days were re-inoculated into 6-well plates and the scratch healing ability was observed at6,12,18,24,and 30 h.The results showed that the scratch healing ability of the co-culture Nfe2l1-KD group was stronger than that of the Scramble group.10.Nfe2l1 silencing promotes VEGFR2 phosphorylation in vascular endothelial cells in tumor cell co-culture:3LL cell conditioned medium treated Scramble and Nfe2l1-KD vascular endothelial cells for the following time.The phosphorylation level of VEGFR2was significantly higher in the Nfe2l1-KD group than in the Scramble group at 5,10,and20 min(P<0.05).Conclusions:Vascular endothelial cell-specific Nfe2l1 deletion may promote vascular endothelial cell migration to accelerate tumor angiogenesis by up-regulating VEGFR2phosphorylation,and ultimately leading to accelerated tumor growth.更多还原