【作者】 周红；

【导师】 罗庆；

【作者基本信息】 重庆医科大学 ， 儿科学， 2022， 硕士

【摘要】 第一部分靶向抑制PHGDH表达对骨肉瘤细胞生物学行为、成骨分化的影响目的:靶向抑制3-磷酸甘油酸脱氢酶(PHGDH)探讨其对人骨肉瘤细胞生物学行为及成骨分化的影响。方法:(1)运用qRT-PCR及Western blot法验证在正常人成骨细胞h FOB1.19和不同恶性程度人骨肉瘤细胞TE85、MG63、143B中PHGDH基因的表达;(2)感染PHGDH干扰RNA慢病毒的人骨肉瘤细胞作为实验组(sh PHGDH组),感染空载慢病毒的人骨肉瘤细胞作为阴性对照组(NC组),未感染病毒的人骨肉瘤细胞作为空白对照组(Control组),运用qRTPCR检测PHGDH基因的m RNA水平的表达,Westernblot法检测PHGDH蛋白水平的表达,验证PHGDH在骨肉瘤细胞中的抑制效果;(3)运用CCK-8、Ed U染色法和Western blot法(检测PCNA的表达),检测骨肉瘤细胞增殖能力;在体内实验中,运用异位成瘤法,运用体外活体成像技术观察肿瘤大小;(4)运用DAPI染色法检测骨肉瘤细胞凋亡,运用Western blot法检测抗凋亡基因Bcl-2的表达;(5)运用流式细胞术检测敲低PHGDH对骨肉瘤细胞周期分布的影响;(6)运用划痕实验、Transwell实验检测敲低PHGDH对骨肉瘤细胞的迁移能力的影响;(7)运用碱性磷酸酶(ALP)染色和茜素红S染色检测敲低PHGDH对骨肉瘤细胞的早期及晚期成骨分化的影响,Western blot法和免疫组化法检测成骨分化相关基因的变化。结果:(1)与成骨细胞h FOB 1.19相比,在人骨肉瘤细胞TE85、MG63、143B中PHGDH高表达,PHGDH在143B细胞中的表达水平高于TE85、MG63细胞(P<0.01);(2)在骨肉瘤143B、MG63及143B-luc细胞中靶向抑制PHGDH进行抗性筛选后,PHGDH在m RNA及蛋白水平的表达较Control组和NC组均明显降低(P<0.01),表明敲低PHGDH的人骨肉瘤细胞稳转株构建成功;(3)敲低PHGDH后,骨肉瘤细胞增殖能力明显降低,增殖相关基因PCNA表达明显降低(P<0.01);体内实验中,NC组和sh PHGDH组裸鼠均成瘤,sh PHGDH组裸鼠皮下移植瘤体积明显小于NC组;(4)敲低PHGDH后,骨肉瘤细胞凋亡率明显增高(P<0.01),抗凋亡蛋白Bcl-2的表达降低(P<0.05);(5)敲低PHGDH后,骨肉瘤细胞G0/G1群体的增加和S期、G2/M期细胞群体减少(P<0.01);(6)敲低PHGDH后,划痕愈合率明显降低(P<0.01),且Transwell实验细胞穿膜数也明显减少(P<0.01);(7)诱导骨肉瘤细胞成骨分化,敲低PHGDH后,ALP染色阳性增加,茜素红染色示钙盐结节明显增多;成骨分化相关基因Runx2、OC、OPN的表达增高。结论:PHGDH的表达水平与骨肉瘤细胞的恶性程度呈正相关;PHGDH促进骨肉瘤细胞的体外增殖和体内生长;PHGDH抑制骨肉瘤细胞的凋亡,促进骨肉瘤细胞的迁移和周期进程;PHGDH降低骨肉瘤细胞的成骨分化能力。第二部分靶向抑制PHGDH对骨肉瘤能量代谢的影响及其机制研究目的:靶向能量代谢探讨抑制3-磷酸甘油酸脱氢酶(PHGDH)对人骨肉瘤细胞的影响。方法:(1)运用乳酸检测试剂盒测定乳酸;ATP检测试剂盒测定ATP;(2)运用TMRE试剂、Mito Traker-red试剂、Mito SOX试剂检测骨肉瘤细胞线粒体膜电位、线粒体结构及线粒体ROS水平;(3)运用qRT-PCR检测骨肉瘤细胞中能量代谢相关基因表达。结果:(1)敲低PHGDH后,乳酸降低(P<0.01),ATP增多(P<0.01);(2)敲低PHGDH后,线粒体膜电位降低(P<0.01),线粒体形态发生变化,线粒体ROS荧光强度增强;(3)敲低PHGDH后,能量代谢相关基因(GLUT1、PFK1、PKM2、LDHA)的表达下调(P<0.01)。结论:PHGDH促进骨肉瘤细胞的糖酵解,抑制骨肉瘤细胞通过氧化磷酸化产生能量,可改变骨肉瘤的能量代谢模式。更多还原

【Abstract】 PART Ⅰ EFFECT OF TARGETED INHIBITION OF PHGDH EXPRESSION ON BIOLOGICAL BEHAVIOR AND OSTEOGENIC DIFFERENTIATION OF OSTEOSARCOMA CELLSObjective: To investigate the effect of targeted inhibition of 3-phosphate dehydrogenase(PHGDH)on biological behavior and osteogenic differentiation of human osteosarcoma cells.Methods:(1)qRT-PCR and Western blot were used to verify the expression of PHGDH gene in normal osteoblasts FOB1.19 and human osteosarcoma cells TE85,MG63 and 143 B with different malignant degrees.(2)Human osteosarcoma cells infected with PHGDH interference RNA lentivirus were used as experimental group(sh PHGDH group),human osteosarcoma cells infected with empty lentivirus were used as negative control group(NC group),and human osteosarcoma cells without lentivirus were used as blank control group(Control group).qRT-PCR was used to detect the expression of PHGDH gene m RNA level,and Western blot was used to detect the expression of PHGDH protein level to verify the inhibitory effect of PHGDH in osteosarcoma cells.(3)CCK-8,Ed U staining and Western blot(detection of PCNA expression)were used to detect proliferation of osteosarcoma cells;in vivo experiments,ectopic tumor formation method and in vitro in vivo imaging technology were used to observe the tumor size.(4)The apoptosis rate of osteosarcoma cells was detected by DAPI staining,and the expression of anti-apoptotic gene Bcl-2 was detected by Western blot.(5)The effect of PHGDH knockdown on cell cycle of osteosarcoma was detected by flow cytometry;(6)The effect of PHGDH knockdown on the migration ability of osteosarcoma cells was detected by cell scratch test and Transwell test.(7)Alkaline phosphatase(ALP)staining and alizarin red S staining were used to detect the effect of PHGDH knockdown on early and late osteogenic differentiation of osteosarcoma cells.Western blot and immunohistochemistry were used to detect the changes of osteogenic differentiation-related genes.Results:(1)Compared with osteoblast h FOB 1.19,PHGDH was highly expressed in human osteosarcoma cells TE85,MG63 and 143B(P <0.05),and the expression level of PHGDH in 143 B cells was higher than that in TE85 and MG63 cells.(2)After targeted inhibition of PHGDH in osteosarcoma 143 B,MG63and 143B-luc cells for resistance screening,the expression levels of PHGDH in m RNA and protein were significantly lower than those in the control group and the NC group(P < 0.01),indicating that the stable transfer strain of human osteosarcoma cells with low PHGDH was successfully constructed.(3)After knocking down PHGDH,the proliferation ability of osteosarcoma cells was significantly decreased,and the expression of PCNA was significantly decreased(P < 0.01).In the in vivo experiment,the nude mice in the NC group and sh PHGDH group were all tumors,and the subcutaneous transplanted tumor volume in the sh PHGDH group was significantly smaller than that in the NC group.(4)After knocking down PHGDH,the apoptosis rate of osteosarcoma cells was significantly increased(P < 0.01),and the expression of antiapoptotic protein Bcl-2 was decreased(P < 0.05).(5)After knocking down PHGDH,the cell population of G0 / G1 of osteosarcoma cells increased and the cell population of S phase and G2 / M phase decreased(P < 0.01);(6)After knocking down PHGDH,the healing rate of scratches was significantly reduced(P < 0.01),and the number of cells penetrating membrane in Transwell experiment was also significantly reduced(P <0.01).(7)Osteogenic differentiation of osteosarcoma cells was induced.After knocking down PHGDH,the positive rate of ALP staining increased,and alizarin red staining showed that calcium salt nodules increased significantly;the expression of osteogenic differentiation-related genes Runx2,OC,OPN increased.Conclusion: The expression level of PHGDH was positively correlated with the malignant degree of osteosarcoma cells;PHGDH promotes the proliferation of osteosarcoma cells in vitro and growth in vivo;PHGDH inhibits the apoptosis of osteosarcoma cells and promotes the migration and cycle process of osteosarcoma cells;PHGDH reduces the osteogenic differentiation of osteosarcoma cells.PART Ⅱ EFFECT OF TARGETED INHIBITION OF PHGDH ON ENERGY METABOLISM IN OSTEOSARCOMA AND ITS MECHANISMObjective: To investigate the effect of inhibiting 3-phosphate dehydrogenase(PHGDH)on human osteosarcoma cells by targeting energy metabolism.Methods:(1)Using lactic acid detection kit to determine lactic acid;ATP determination kit.(2)TMRE reagent,Mito Traker-red reagent and Mito SOX reagent were used to detect mitochondrial membrane potential,mitochondrial structure and mitochondrial ROS level in osteosarcoma cells.(3)qRT-PCR was used to detect the expression of energy metabolism related genes in osteosarcoma cells.Results:(1)After knocking down PHGDH,lactic acid decreased(P <0.01)and ATP increased(P < 0.01).(2)After knocking down PHGDH,mitochondrial membrane potential decreased(P < 0.01),mitochondrial morphology changed and mitochondrial ROS fluorescence intensity increased.(3)After knocking down PHGDH,the expression of energy metabolism-related genes(GLUT1,PFK1,PKM2,LDHA)were downregulated(P < 0.01).Conclusion:PHGDH can promote the glycolysis of osteosarcoma cells and inhibit the energy production of osteosarcoma cells by oxidative phosphorylation,which can change the energy metabolism mode of osteosarcoma.更多还原