转录共激活因子4对骨肉瘤恶性表型的影响及机制研究

发布时间：2018-06-07 02:29

  本文选题：骨肉瘤 + 肺转移　； 参考：《第三军医大学》2016年博士论文

【摘要】：目的:骨肉瘤是最常见的原发恶性骨肿瘤,预后极差,发现时常常已经发生肺转移。转录共激活因子4(PC4)在DNA转录、复制、修复、染色体构成等过程中发挥了复杂且重要的作用。目前有相关研究提示PC4可能与肿瘤有一定联系,但是其具体机制尚不明确,尤其是在骨肉瘤的研究领域中,PC4的具体作用尚无人报道。本研究目的在于探索PC4对骨肉瘤恶性表型的影响及其可能的分子机制。首先,在前期研究基础上探索PC4蛋白表达量与临床骨肉瘤分期、预后的联系。然后,在多种不同恶性程度的骨肉瘤细胞模型中检测PC4的表达情况。在高肺转移骨肉瘤细胞株143B中使用慢病毒干扰PC4的表达,检测恶性表型的变化,分析PC4可能参与哪些恶性表型的调控。通过RNA-seq的高通量测序法,分析143B细胞株在干扰PC4后基因表达的变化情况,分析可能存在的PC4下游靶基因,并与表型实验结合,分析哪些靶基因参与了PC4对骨肉瘤恶性表型的调控。针对骨肉瘤肺转移表型,通过筛选分析RNA-seq结果,剖析PC4对MMP9的具体调控机制。针对骨肉瘤骨破坏表型,通过筛选分析RNA-seq结果,剖析PC4参与骨肉瘤骨破坏的具体分子机制及可能存在的靶基因。方法:1.PC4蛋白表达量与临床骨肉瘤分期、预后的联系。1.1使用免疫组织化学法分析198例骨肉瘤临床标本和44例正常骨组织中PC4的表达量与年龄、性别、Enneking分期、肿瘤大小之间的关系,并对免疫组化结果进行量化评估。1.2使用WB法分析5例骨肉瘤临床标本和对应正常癌旁组织中PC4的表达量,分析PC4在骨肉瘤组织和正常组织中存在差异表达。1.3随访59例骨肉瘤病例,分析PC4的表达情况与患者生存时间之间的关系,绘制生存曲线。2.通过细胞模型和裸鼠荷瘤实验,在多种不同恶性程度的骨肉瘤细胞株中检测PC4的表达情况。分析PC4可能参与的骨肉瘤恶性表型调控。2.1分析PC4在7种恶性程度不同的骨肉瘤细胞株中的蛋白表达情况。2.1.1免疫荧光法检测PC4在7种恶性程度不同的骨肉瘤细胞株中的表达量及细胞定位情况。2.1.2 WB法检测PC4在7种恶性程度不同的骨肉瘤细胞株中的表达量。2.2双层软琼脂克隆实验检测7种骨肉瘤细胞的基础克隆形成能力。2.3裸鼠荷瘤实验检测多种骨肉瘤细胞的成瘤能力。2.4使用RT-PCR法在多种骨肉瘤细胞株中检测恶性表型相关基因的表达情况。2.5在143B高肺转移骨肉瘤细胞株中使用慢病毒干扰PC4的表达。2.5.1人PC4干扰慢病毒的构建。2.5.2免疫荧光法检测PC4慢病毒干扰效率,有限稀释法挑选高转染效率的亚细胞克隆。2.5.3使用WB法检测PC4慢病毒干扰效率。2.5.4使用CCK-8法检测PC4干扰前后143B细胞增殖能力的变化。2.5.5使用流式细胞仪检测PC4干扰前后143B细胞周期的变化。2.5.6使用双层软琼脂克隆法检测PC4干扰前后143B细胞集落形成能力的变化。2.5.7使用划痕实验检测PC4干扰前后143B细胞迁移能力的变化。2.5.8使用Transwell检测PC4干扰前后143B细胞侵袭能力的变化。2.5.9使用CCK-8法检测PC4干扰前后143B细胞粘附能力的变化。2.5.10裸鼠荷瘤实验检测PC4干扰前后143B细胞成瘤能力及肺转移能力的变化。3.使用RNA-seq的高通量测序法,分析143B细胞株在干扰PC4后基因表达的变化情况。3.1分析143B细胞株在干扰PC4后存在差异表达的基因。3.2使用Gene ontology分析差异表达基因在不同细胞功能、细胞组成、生物学功能中的富集程度,探寻差异表达基因可能的效应。3.3使用KEGG pathway分析差异表达基因在不同信号通路中的富集程度,探寻差异表达基因可能的参与的信号通路。4.针对骨肉瘤肺转移表型,基于RNA-seq结果,剖析PC4参与MMP9的调控进而影响骨肉瘤肺转移能力的机制。4.1使用PC4干扰慢病毒在7种骨肉瘤细胞株中验证慢病毒干扰效率;RT-PCR法检测7种骨肉瘤细胞株在PC4干扰后MMP-2、MMP-9、FN基因RNA水平的变化情况。4.2使用外源性人重组FN蛋白和FNsi RNA分别在143B和MG63两种细胞模型中验证FN对MMP-2和MMP-9的调控作用。4.3使用染色质免疫共沉淀法分析PC4与MMP-9启动子区域的结合能力。4.4使用荧光素梅报告基因和PC4si RNA及SP1si RNA,检测PC4和SP1对MMP-9基因转录激活能力。4.5免疫共沉淀法分析143B细胞中PC4蛋白与SP1蛋白的结合能力。5.针对骨肉瘤骨破坏表型,基于对RNA-seq结果的分析,剖析PC4参与骨肉瘤骨破坏的具体分子机制及可能存在的靶基因。5.1裸鼠荷瘤实验检测PC4干扰前后143B细胞对骨破坏的影响。5.1.1 Micro CT检测病理性骨折的发生率、骨小梁数量、骨小梁厚度、骨量的变化。5.1.2分别在共培养细胞模型和条件培养基细胞模型中检测PC4干扰143B细胞对RAW264.7破骨分化的影响。使用TRAP染色和骨片扫描电镜评估破骨细胞成熟程度。5.1.3分析RNA-seq结果,筛选与破骨细胞活化相关的分泌型细胞因子,并使用RT-PCR法验证RNA-seq结果。5.1.4使用WB法验证IGFBP5在PC4干扰前后的蛋白表达情况。5.1.5使用外源性IGFBP5蛋白,检测其对RAW264.7破骨分化的影响,使用TRAP染色评估破骨细胞成熟程度。5.1.6使用外源性IGFBP5蛋白,在条件培养基细胞模型中检测PC4干扰及外源性IGFBP5对RAW264.7破骨分化的影响,使用TRAP染色评估破骨细胞成熟程度。结果:1.PC4蛋白表达量与临床骨肉瘤分级、分期、预后的联系。1.1 PC4主要在细胞核表达,在高分期的骨肉瘤标本中表达较高,PC4的表达与骨肉瘤的患者的性别、年龄无关,与肿瘤的大小以及肿瘤的Ennenking分期相关。1.2 WB法检测结果提示PC4在骨肉瘤组织和配对的正常组织中存在差异表达,在骨肉瘤组织中高表达。1.3随访59例骨肉瘤病例,生存曲线分析结果提示:PC4的阳性率与患者生存时间负相关。2.通过细胞模型和裸鼠荷瘤实验,在多种不同恶性程度的骨肉瘤细胞株中检测PC4的表达情况。PC4参与多种骨肉瘤恶性表型调控。2.1 PC4在7种恶性程度不同的骨肉瘤细胞株中的蛋白表达情况。2.1.1免疫荧光法检测PC4在7种恶性程度不同的骨肉瘤细胞株中的表达量及细胞定位情况:PC4在7中骨肉瘤细胞株中均有表达,主要表达与细胞核,胞浆有少量表达。表达强度由强到弱依次为:143B、MNNG-HOS、9901、MG-63、U2OS、HOS、SAOS2。2.1.2 WB法检测PC4在7种恶性程度不同的骨肉瘤细胞株中的表达量:PC4在7中骨肉瘤细胞株中均有表达,表达强度由强到弱依次为:143B、MNNG-HOS、MG-63、U2OS、9901、HOS、SAOS2。2.2双层软琼脂克隆实验检测7种骨肉瘤细胞的基础克隆形成能力:克隆形成能力由强到弱依次为:143B、MNNG-HOS、MG-63、9901、HOS、SAOS2、U2OS。2.3裸鼠荷瘤实验检测多种骨肉瘤细胞的成瘤能力:,143B和MNNG-HOS拥有最强的成瘤能力,9901次之,MG-63较弱,HOS在观察期内未成瘤。2.4使用RT-PCR法在多种骨肉瘤细胞株中检测恶性表型相关基因的表达情况:PC4在143B、MNNG-HOS、U2OS表达较高,HOS表达最弱;而MMP9在143B中表达异常增高,其余骨肉瘤细胞株中表达相对弱。143B的MTA1表达最高,MNNG-HOS次之,其余细胞株表达较弱。143B中P53几乎测不到表达,而HOS和U2OS相对表达最高。2.5在143B高肺转移骨肉瘤细胞株中使用慢病毒干扰PC4的表达。2.5.1人PC4干扰慢病毒的构建。2.5.1免疫荧光法结果提示PC4慢病毒转染率约90%;有限稀释法挑选高转染效率的亚细胞克隆并成功扩增。2.5.2 WB法检测PC4慢病毒干扰效率约为70%,且多次传代后干扰效率无明显下降。2.5.3 CCK-8法检测结果提示:PC4干扰后143B细胞增殖能力有一定程度的下降。2.5.4流式细胞仪检测结果提示:PC4干扰后143B细胞周期出现G1期阻滞。2.5.5双层软琼脂克隆法检测结果提示:PC4干扰后143B细胞集落形成能力的明显下降。2.5.6划痕实验检测结果提示:PC4干扰后143B细胞迁移能力下调。2.5.7 Transwell检测结果提示:PC4干扰后143B细胞侵袭能力下调。2.5.8 CCK-8法检测结果提示:PC4干扰后143B细胞粘附能力明显下降。2.5.9裸鼠荷瘤实验检测结果提示:PC4干扰后143B细胞成瘤能力及肺转移能力均出现明显下降。3.RNA-seq的高通量测序法,分析143B细胞株在干扰PC4后基因表达的变化情况。3.1在143B细胞株中干扰PC4后,513个基因出现下调,571个基因上调。3.2 Gene ontology分析结果提示:PC4干扰后的差异表达基因可能参与了蛋白粘附、细胞连接、细胞外基质的分泌、细胞运动、细胞应激等多种细胞生物学功能的调节。3.3 KEGG pathway分析结果提示:PC4干扰后的差异表达基因在p53等20条信号通路中呈现富集状态。4.PC4参与MMP9的调控进而影响骨肉瘤肺转移能力。4.1 PC4干扰慢病毒在7种骨肉瘤细胞株中均有干扰效果;PC4干扰后MMP-2、MMP-9、FN基因RNA水平除个别细胞株外均有不同程度的下调。4.2外源性人重组FN蛋白和FNsi RNA对MG63细胞的MMP-2和MMP-9均有明显调控作用,但是对143B和143BPC4-4.3染色质免疫共沉淀法结果提示:PC4蛋白可以与MMP-9启动子区域结合。细胞的MMP-2和MMP-9没有明显的调控作用。4.4荧光素梅报告基因结果提示:PC4si RNA及SP1si RNA均可降低MMP-9的转录活性,二者存在协同效应。4.5免疫共沉淀法结果提示:143B细胞中PC4蛋白与SP1蛋白可以结合。5.PC4参与骨肉瘤骨破坏的具体分子机制及可能存在的靶基因。5.1裸鼠荷瘤实验检测PC4干扰前后143B细胞对骨破坏的影响。5.1.1 Micro CT检测结果提示:PC4干扰组的裸鼠病理性骨折的发生率明显降低、骨小梁数量增加、骨小梁厚度增厚、骨量增加。5.1.2共培养细胞模型和条件培养基细胞模型均提示:骨肉瘤细胞可以促进RAW264.7的破骨分化,PC4干扰可抑制该效应。5.1.3 RNA-seq结果提示:在PC4干扰后CXCL2、IGFBP5、MCP1、IL1A、IL1B明显下调,RT-PCR法验证结果与RNA-seq结果一致。5.1.4 WB法检测IGFBP5在PC4干扰后的蛋白表达明显下调。5.1.5外源性IGFBP5蛋白,可增加TRAP阳性细胞的数量,且该效应呈浓度依赖性。5.1.6外源性IGFBP5蛋白在条件培养基细胞模型中,可部分抵消PC4干扰对RAW264.7破骨分化的抑制作用。结论:1.PC4在骨肉瘤中高表达;2.PC4与骨肉瘤的分期及预后存在相关性;3.PC4参与调控骨肉瘤的增殖、侵袭、迁移、粘附、集落形成、成瘤、转移等恶性表型;4.PC4通过与MMP9的转移因子SP1形成转录复合体,参与MMP9的转录调控;5.PC4的表达量与143B细胞引起的骨吸收正相关;6.干扰在143B骨肉瘤细胞中PC4的表达,可抑制143B分泌CXCL2、CCL2、IGFBP5、IL1A、IL1B,从而抑制破骨细胞的激活,降低骨肉瘤引起的骨破坏。
[Abstract]:Objective: osteosarcoma is the most common primary malignant bone tumor. The prognosis is very poor and pulmonary metastasis is often found. Transcription co activating factor 4 (PC4) plays a complex and important role in the process of DNA transcription, replication, repair, and chromosome composition. There are relevant studies suggesting that PC4 may be associated with tumor, but its specific mechanism The purpose of this study is to explore the effect of PC4 on the malignant phenotype of osteosarcoma and the possible molecular mechanism of PC4. First, the relationship between the expression of PC4 protein and the prognosis of osteosarcoma was explored on the basis of previous studies. The expression of PC4 was detected in a malignant osteosarcoma cell model. The expression of lentivirus interference with PC4 was used in the osteosarcoma cell line 143B of high lung metastases, the changes of malignant phenotype were detected, and the regulation of which malignant phenotype may be involved in PC4 was analyzed. The expression of 143B cell lines after PC4 interference was analyzed by high throughput sequencing of RNA-seq. Change, analyze the possible downstream target genes of PC4 and combine with phenotypic experiments to analyze which target genes are involved in the regulation of the malignant phenotype of osteosarcoma by PC4. According to the phenotype of osteosarcoma lung metastasis, the specific regulation mechanism of PC4 on MMP9 is analyzed by screening and analyzing the results of RNA-seq. In view of the bone destruction phenotype of osteosarcoma, the analysis of RNA- by screening and analyzing the phenotype of osteosarcoma bone. Seq results, the specific molecular mechanism of PC4 participation in osteosarcoma bone destruction and the possible target genes. Methods: the relationship between the expression of 1.PC4 protein and clinical osteosarcoma staging and the prognosis of.1.1, the expression of PC4 in 198 cases of osteosarcoma and 44 cases of normal bone tissue was analyzed by immunohistochemistry and the age, sex, Enneking staging, and swelling were observed. The relationship between the size of the tumor and the quantitative evaluation of the immunohistochemical results.1.2 was used to analyze the expression of PC4 in 5 cases of osteosarcoma and normal para cancerous tissues by WB method. The difference expression of PC4 in osteosarcoma tissues and normal tissues was analyzed with.1.3, and the expression of PC4 and the survival time of the patients were analyzed. The relationship between the.2. and the tumor bearing experiments in nude mice, the expression of PC4 in a variety of malignant osteosarcoma cell lines was detected by the cell model and nude mice. The.2.1 analysis of the malignant phenotype of osteosarcoma that may be involved in PC4 was analyzed by PC4, and the protein expression of PC4 in the osteosarcoma cell lines with different levels of malignancy was immune to.2.1.1 immunity The expression of PC4 in 7 different malignant osteosarcoma cell lines and cell localization by fluorescence method.2.1.2 WB method was used to detect the expression of PC4 in osteosarcoma cell lines with different levels of malignancy.2.2 double layer soft agar cloning test for the detection of the basic clone formation ability of 7 osteosarcoma cells in.2.3 nude mice The tumorigenicity of osteosarcoma cells.2.4 using RT-PCR to detect the expression of malignant phenotype related genes in a variety of osteosarcoma cell lines.2.5 in 143B high lung metastatic osteosarcoma cell lines using lentivirus interference PC4,.2.5.1 human PC4 interfering with lentivirus, the construction of.2.5.2 immunofluorescence for the detection of the interference efficiency of the PC4 lentivirus, limited dilute Detection of high transfection efficiency subcellular clone.2.5.3 using WB method to detect PC4 lentivirus interference efficiency.2.5.4 use CCK-8 method to detect the changes of 143B cell proliferation ability before and after PC4 interference.2.5.5 using flow cytometry to detect the changes of 143B cell cycle before and after PC4 interference,.2.5.6 using double layer soft agar cloning method to detect PC4 interference before and after interference Changes in cell colony formation ability.2.5.7 use scratch test to detect changes in migration ability of 143B cells before and after interference of PC4 interference.2.5.8 use Transwell to detect the changes of 143B cell invasiveness before and after PC4 interference.2.5.9 use CCK-8 method to detect the changes of adherence of 143B cells before and after PC4 interference.2.5.10 nude mice bearing tumor test before interference .3. using high throughput sequencing of RNA-seq to analyze the changes of gene expression after interference with PC4 by high throughput sequencing of RNA-seq..3.1 analysis of the differential expression of gene.3.2 using Gene ontology after the interference of PC4 in the 143B cell line analysis of the differentially expressed genes of the 143B cell lines using Gene ontology analysis of the differential expression genes in different cell functions and cell composition, The enrichment degree in biological function, exploring the possible effect of differentially expressed genes,.3.3 uses KEGG pathway to analyze the concentration of differentially expressed genes in different signaling pathways, and explores the possible signaling pathway.4. for differentially expressed genes in the lung metastasis phenotype of osteosarcoma. Based on the RNA-seq results, the regulation of PC4 to participate in the regulation of MMP9 is analyzed. The mechanism.4.1 affected the lung metastasis of osteosarcoma using PC4 interfering with lentivirus in 7 osteosarcoma cell lines to verify the efficiency of lentivirus interference; RT-PCR assay was used to detect the changes of MMP-2, MMP-9, FN gene RNA levels in 7 osteosarcoma cell lines after PC4 interference..4.2 used exogenous human weight group FN protein and FNsi RNA, respectively. The cellular model was used to verify the regulatory role of FN on MMP-2 and MMP-9.4.3 using chromatin immunoprecipitation method to analyze the binding ability of PC4 and MMP-9 promoter region.4.4 using the fluorescent primer reporter gene and PC4si RNA and SP1si RNA. The binding ability of protein.5. against osteosarcoma bone destruction phenotype, based on the analysis of RNA-seq results, the specific molecular mechanism of PC4 involvement in osteosarcoma bone destruction and the possible target gene of.5.1 nude mice were tested to detect the effect of 143B cells on bone destruction before and after PC4 interference, the incidence of pathological fracture in.5.1.1 Micro CT detection, and small bone marrow The number of beams, the thickness of the trabecular bone, and the changes in bone mass were detected by.5.1.2 in the co culture cell model and the conditioned medium cell model. The effects of PC4 interfering 143B cells on the osteoclast differentiation were detected by TRAP staining and bone slice scanning electron microscopy to evaluate the extent of osteoclast maturation.5.1.3 analysis of RNA-seq results, and to screen the activation of osteoclast activation. Secretory cytokines, and using the RT-PCR method to verify the RNA-seq result,.5.1.4 used WB to verify the protein expression of IGFBP5 before and after PC4 interference..5.1.5 used exogenous IGFBP5 protein to detect its effect on RAW264.7 osteoclast differentiation, and TRAP staining was used to evaluate the maturation of osteoclasts by using exogenous IGFBP5 protein. The effects of PC4 interference and exogenous IGFBP5 on RAW264.7 osteoclast differentiation were detected in the base cell model. TRAP staining was used to evaluate the degree of osteoclast maturation. Results: the relationship between the expression of 1.PC4 protein and the classification, stage and prognosis of the clinical osteosarcoma was mainly expressed in the nucleus, and the expression of PC4 in the high staging osteosarcoma was higher. The expression of PC4 in high staging osteosarcoma and the expression of PC4 The sex of the patients with osteosarcoma was independent of age, the size of the tumor and the Ennenking staging of the tumor were related to the.1.2 WB method. The results suggested that PC4 was expressed differently in the osteosarcoma tissue and the matched normal tissues. The high expression of.1.3 in the osteosarcoma tissue was followed up in 59 cases of osteosarcoma, and the survival curve analysis showed that the positive rate of PC4 was positive. Negative correlation with patient survival time.2. detection of PC4 expression in osteosarcoma cell lines with various malignant degrees by cell model and nude mice bearing tumor cell strain.PC4 participation in multiple osteosarcoma phenotype regulation of.2.1 PC4 protein expression in 7 different malignant osteosarcoma cell lines.2.1.1 immunofluorescence method for PC4 detection of PC4 Expression and cell location in 7 different malignant osteosarcoma cell lines: PC4 was expressed in 7 osteosarcoma cell lines, mainly expressed in the cell nucleus and a small amount of cytoplasm. The expression intensity from strong to weak was 143B, MNNG-HOS, 9901, MG-63, U2OS, HOS, and SAOS2.2.1.2 WB method for detecting PC4 in 7 different malignant bones. The expression in sarcomas cell line: PC4 was expressed in 7 osteosarcoma cell lines. The expression intensity from strong to weak was 143B, MNNG-HOS, MG-63, U2OS, 9901, HOS, SAOS2.2.2 double layer soft agar cloning test to detect the basic clone formation ability of 7 osteosarcoma cells: the ability of clonogenic formation from strong to weak was 143B, MNNG-HOS, MG-639901, HO. S, SAOS2, U2OS.2.3 nude mice were used to detect the tumorigenicity of a variety of osteosarcoma cells: 143B and MNNG-HOS had the strongest tumorigenicity, 9901 times, and MG-63 weak. HOS in the observation period was not a tumor.2.4 using RT-PCR method to detect the expression of the malignant phenotypic correlation gene in a variety of osteosarcoma cell lines: PC4 in 143B, MNNG-HOS, expression. The expression of HOS was the weakest, and the expression of MMP9 was abnormal in 143B. The expression of MTA1 in the other osteosarcoma cell lines was highest, MNNG-HOS was the highest, and the other cell lines were weakly expressed in.143B and the P53 was almost undetected, while HOS and U2OS relative expression was the highest in the 143B high lung metastatic osteosarcoma cell lines using lentivirus interference. The expression of.2.5.1 human PC4 interfering lentivirus showed that the transfection rate of PC4 lentivirus was about 90%, the limited dilution method selected the high transfection efficiency subcell clone and the.2.5.2 WB method to detect PC4 lentivirus interference efficiency was about 70%, and the interference efficiency after multiple passages did not decrease.2.5.3 CCK-8 detection results. The results suggested that the proliferation ability of 143B cells decreased to a certain extent after PC4 interference. The results of.2.5.4 flow cytometry showed that after PC4 interference, 143B cell cycle appeared G1 phase block.2.5.5 double layer soft agar cloning method, and the result suggested that the 143B cell colony formation ability after PC4 interference was obviously reduced by.2.5.6 scratch test results: PC4 dry test results suggested: PC4 dry The.2.5.7 Transwell detection results of 143B cell migration ability after disturbance showed that the invasion ability of 143B cells was down regulated by.2.5.8 CCK-8 method after PC4 interference: the adhesion ability of 143B cells decreased obviously after PC4 interference, and the detection results of the.2.5.9 nude mice bearing tumor experiment showed that the tumor formation ability and lung metastasis ability of 143B cells were obvious after PC4 interference. A high throughput sequencing method of decreasing.3.RNA-seq was used to analyze the changes of gene expression in 143B cell lines after interference with PC4. After interference of PC4 in.3.1, 513 genes were downregulated and 571 genes up-regulated.3.2 Gene ontology analysis results suggested that the differential expression basis after PC4 interference may be involved in protein adhesion, cell connection, and extracellular matrix. Regulation of various cellular biological functions, such as matrix secretion, cell movement, and cell stress,.3.3 KEGG pathway analysis showed that the differentially expressed genes after PC4 interference were enriched in the 20 signal pathways such as p53,.4.PC4 participated in the regulation of MMP9 and the pulmonary metastasis of osteosarcoma,.4.1 PC4 interfering lentivirus in 7 kinds of osteosarcoma There were interference effects in the cell lines. After PC4 interference, the RNA level of MMP-2, MMP-9, FN gene, except for a few cell lines, had different degrees of downregulation of.4.2, the recombinant FN protein and FNsi RNA had obvious regulation effect on MMP-2 and MMP-9 of MG63 cells. P-9 promoter region binding. MMP-2 and MMP-9 of cells have no obvious regulatory effect on.4.4 fluorescent primer report gene results suggest that PC4si RNA and SP1si RNA can reduce the transcriptional activity of MMP-9, and the synergistic effect of.4.5 immunoprecipitation in the two is suggested that PC4 egg white and protein can participate in osteosarcoma bone in 143B cells. The specific molecular mechanism of the destruction and the possible target gene.5.1 nude mice test the effect of 143B cells on bone destruction before and after PC4 interference.5.1.1 Micro CT detection results suggested that the incidence of pathological fracture in the PC4 interference group was significantly decreased, the number of bone trabecula increased, the thickness of bone trabecula increased, and the bone mass increased.5.1.2 co culture. Cell model and conditioned medium cell model suggest that osteosarcoma cells can promote osteoclast differentiation of RAW264.7, PC4 interference can inhibit this effect,.5.1.3 RNA-seq results suggest that after PC4 interference, CXCL2, IGFBP5, MCP1, IL1A, IL1B obviously down, RT-PCR method verification results are consistent with RNA-seq results to detect the eggs after interference. The expression of exogenous.5.1.5 significantly decreased the number of TRAP positive cells in IGFBP5, and the effect was.5.1.6 dependent.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

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