本文選題：骨肉瘤 + 肺轉(zhuǎn)移��； 參考：《第三軍醫(yī)大學(xué)》2016年博士論文

【摘要】：目的:骨肉瘤是最常見的原發(fā)惡性骨腫瘤,預(yù)后極差,發(fā)現(xiàn)時常常已經(jīng)發(fā)生肺轉(zhuǎn)移。轉(zhuǎn)錄共激活因子4(PC4)在DNA轉(zhuǎn)錄、復(fù)制、修復(fù)、染色體構(gòu)成等過程中發(fā)揮了復(fù)雜且重要的作用。目前有相關(guān)研究提示PC4可能與腫瘤有一定聯(lián)系,但是其具體機(jī)制尚不明確,尤其是在骨肉瘤的研究領(lǐng)域中,PC4的具體作用尚無人報(bào)道。本研究目的在于探索PC4對骨肉瘤惡性表型的影響及其可能的分子機(jī)制。首先,在前期研究基礎(chǔ)上探索PC4蛋白表達(dá)量與臨床骨肉瘤分期、預(yù)后的聯(lián)系。然后,在多種不同惡性程度的骨肉瘤細(xì)胞模型中檢測PC4的表達(dá)情況。在高肺轉(zhuǎn)移骨肉瘤細(xì)胞株143B中使用慢病毒干擾PC4的表達(dá),檢測惡性表型的變化,分析PC4可能參與哪些惡性表型的調(diào)控。通過RNA-seq的高通量測序法,分析143B細(xì)胞株在干擾PC4后基因表達(dá)的變化情況,分析可能存在的PC4下游靶基因,并與表型實(shí)驗(yàn)結(jié)合,分析哪些靶基因參與了PC4對骨肉瘤惡性表型的調(diào)控。針對骨肉瘤肺轉(zhuǎn)移表型,通過篩選分析RNA-seq結(jié)果,剖析PC4對MMP9的具體調(diào)控機(jī)制。針對骨肉瘤骨破壞表型,通過篩選分析RNA-seq結(jié)果,剖析PC4參與骨肉瘤骨破壞的具體分子機(jī)制及可能存在的靶基因。方法:1.PC4蛋白表達(dá)量與臨床骨肉瘤分期、預(yù)后的聯(lián)系。1.1使用免疫組織化學(xué)法分析198例骨肉瘤臨床標(biāo)本和44例正常骨組織中PC4的表達(dá)量與年齡、性別、Enneking分期、腫瘤大小之間的關(guān)系,并對免疫組化結(jié)果進(jìn)行量化評估。1.2使用WB法分析5例骨肉瘤臨床標(biāo)本和對應(yīng)正常癌旁組織中PC4的表達(dá)量,分析PC4在骨肉瘤組織和正常組織中存在差異表達(dá)。1.3隨訪59例骨肉瘤病例,分析PC4的表達(dá)情況與患者生存時間之間的關(guān)系,繪制生存曲線。2.通過細(xì)胞模型和裸鼠荷瘤實(shí)驗(yàn),在多種不同惡性程度的骨肉瘤細(xì)胞株中檢測PC4的表達(dá)情況。分析PC4可能參與的骨肉瘤惡性表型調(diào)控。2.1分析PC4在7種惡性程度不同的骨肉瘤細(xì)胞株中的蛋白表達(dá)情況。2.1.1免疫熒光法檢測PC4在7種惡性程度不同的骨肉瘤細(xì)胞株中的表達(dá)量及細(xì)胞定位情況。2.1.2 WB法檢測PC4在7種惡性程度不同的骨肉瘤細(xì)胞株中的表達(dá)量。2.2雙層軟瓊脂克隆實(shí)驗(yàn)檢測7種骨肉瘤細(xì)胞的基礎(chǔ)克隆形成能力。2.3裸鼠荷瘤實(shí)驗(yàn)檢測多種骨肉瘤細(xì)胞的成瘤能力。2.4使用RT-PCR法在多種骨肉瘤細(xì)胞株中檢測惡性表型相關(guān)基因的表達(dá)情況。2.5在143B高肺轉(zhuǎn)移骨肉瘤細(xì)胞株中使用慢病毒干擾PC4的表達(dá)。2.5.1人PC4干擾慢病毒的構(gòu)建。2.5.2免疫熒光法檢測PC4慢病毒干擾效率,有限稀釋法挑選高轉(zhuǎn)染效率的亞細(xì)胞克隆。2.5.3使用WB法檢測PC4慢病毒干擾效率。2.5.4使用CCK-8法檢測PC4干擾前后143B細(xì)胞增殖能力的變化。2.5.5使用流式細(xì)胞儀檢測PC4干擾前后143B細(xì)胞周期的變化。2.5.6使用雙層軟瓊脂克隆法檢測PC4干擾前后143B細(xì)胞集落形成能力的變化。2.5.7使用劃痕實(shí)驗(yàn)檢測PC4干擾前后143B細(xì)胞遷移能力的變化。2.5.8使用Transwell檢測PC4干擾前后143B細(xì)胞侵襲能力的變化。2.5.9使用CCK-8法檢測PC4干擾前后143B細(xì)胞粘附能力的變化。2.5.10裸鼠荷瘤實(shí)驗(yàn)檢測PC4干擾前后143B細(xì)胞成瘤能力及肺轉(zhuǎn)移能力的變化。3.使用RNA-seq的高通量測序法,分析143B細(xì)胞株在干擾PC4后基因表達(dá)的變化情況。3.1分析143B細(xì)胞株在干擾PC4后存在差異表達(dá)的基因。3.2使用Gene ontology分析差異表達(dá)基因在不同細(xì)胞功能、細(xì)胞組成、生物學(xué)功能中的富集程度,探尋差異表達(dá)基因可能的效應(yīng)。3.3使用KEGG pathway分析差異表達(dá)基因在不同信號通路中的富集程度,探尋差異表達(dá)基因可能的參與的信號通路。4.針對骨肉瘤肺轉(zhuǎn)移表型,基于RNA-seq結(jié)果,剖析PC4參與MMP9的調(diào)控進(jìn)而影響骨肉瘤肺轉(zhuǎn)移能力的機(jī)制。4.1使用PC4干擾慢病毒在7種骨肉瘤細(xì)胞株中驗(yàn)證慢病毒干擾效率;RT-PCR法檢測7種骨肉瘤細(xì)胞株在PC4干擾后MMP-2、MMP-9、FN基因RNA水平的變化情況。4.2使用外源性人重組FN蛋白和FNsi RNA分別在143B和MG63兩種細(xì)胞模型中驗(yàn)證FN對MMP-2和MMP-9的調(diào)控作用。4.3使用染色質(zhì)免疫共沉淀法分析PC4與MMP-9啟動子區(qū)域的結(jié)合能力。4.4使用熒光素梅報(bào)告基因和PC4si RNA及SP1si RNA,檢測PC4和SP1對MMP-9基因轉(zhuǎn)錄激活能力。4.5免疫共沉淀法分析143B細(xì)胞中PC4蛋白與SP1蛋白的結(jié)合能力。5.針對骨肉瘤骨破壞表型,基于對RNA-seq結(jié)果的分析,剖析PC4參與骨肉瘤骨破壞的具體分子機(jī)制及可能存在的靶基因。5.1裸鼠荷瘤實(shí)驗(yàn)檢測PC4干擾前后143B細(xì)胞對骨破壞的影響。5.1.1 Micro CT檢測病理性骨折的發(fā)生率、骨小梁數(shù)量、骨小梁厚度、骨量的變化。5.1.2分別在共培養(yǎng)細(xì)胞模型和條件培養(yǎng)基細(xì)胞模型中檢測PC4干擾143B細(xì)胞對RAW264.7破骨分化的影響。使用TRAP染色和骨片掃描電鏡評估破骨細(xì)胞成熟程度。5.1.3分析RNA-seq結(jié)果,篩選與破骨細(xì)胞活化相關(guān)的分泌型細(xì)胞因子,并使用RT-PCR法驗(yàn)證RNA-seq結(jié)果。5.1.4使用WB法驗(yàn)證IGFBP5在PC4干擾前后的蛋白表達(dá)情況。5.1.5使用外源性IGFBP5蛋白,檢測其對RAW264.7破骨分化的影響,使用TRAP染色評估破骨細(xì)胞成熟程度。5.1.6使用外源性IGFBP5蛋白,在條件培養(yǎng)基細(xì)胞模型中檢測PC4干擾及外源性IGFBP5對RAW264.7破骨分化的影響,使用TRAP染色評估破骨細(xì)胞成熟程度。結(jié)果:1.PC4蛋白表達(dá)量與臨床骨肉瘤分級、分期、預(yù)后的聯(lián)系。1.1 PC4主要在細(xì)胞核表達(dá),在高分期的骨肉瘤標(biāo)本中表達(dá)較高,PC4的表達(dá)與骨肉瘤的患者的性別、年齡無關(guān),與腫瘤的大小以及腫瘤的Ennenking分期相關(guān)。1.2 WB法檢測結(jié)果提示PC4在骨肉瘤組織和配對的正常組織中存在差異表達(dá),在骨肉瘤組織中高表達(dá)。1.3隨訪59例骨肉瘤病例,生存曲線分析結(jié)果提示:PC4的陽性率與患者生存時間負(fù)相關(guān)。2.通過細(xì)胞模型和裸鼠荷瘤實(shí)驗(yàn),在多種不同惡性程度的骨肉瘤細(xì)胞株中檢測PC4的表達(dá)情況。PC4參與多種骨肉瘤惡性表型調(diào)控。2.1 PC4在7種惡性程度不同的骨肉瘤細(xì)胞株中的蛋白表達(dá)情況。2.1.1免疫熒光法檢測PC4在7種惡性程度不同的骨肉瘤細(xì)胞株中的表達(dá)量及細(xì)胞定位情況:PC4在7中骨肉瘤細(xì)胞株中均有表達(dá),主要表達(dá)與細(xì)胞核,胞漿有少量表達(dá)。表達(dá)強(qiáng)度由強(qiáng)到弱依次為:143B、MNNG-HOS、9901、MG-63、U2OS、HOS、SAOS2。2.1.2 WB法檢測PC4在7種惡性程度不同的骨肉瘤細(xì)胞株中的表達(dá)量:PC4在7中骨肉瘤細(xì)胞株中均有表達(dá),表達(dá)強(qiáng)度由強(qiáng)到弱依次為:143B、MNNG-HOS、MG-63、U2OS、9901、HOS、SAOS2。2.2雙層軟瓊脂克隆實(shí)驗(yàn)檢測7種骨肉瘤細(xì)胞的基礎(chǔ)克隆形成能力:克隆形成能力由強(qiáng)到弱依次為:143B、MNNG-HOS、MG-63、9901、HOS、SAOS2、U2OS。2.3裸鼠荷瘤實(shí)驗(yàn)檢測多種骨肉瘤細(xì)胞的成瘤能力:,143B和MNNG-HOS擁有最強(qiáng)的成瘤能力,9901次之,MG-63較弱,HOS在觀察期內(nèi)未成瘤。2.4使用RT-PCR法在多種骨肉瘤細(xì)胞株中檢測惡性表型相關(guān)基因的表達(dá)情況:PC4在143B、MNNG-HOS、U2OS表達(dá)較高,HOS表達(dá)最弱;而MMP9在143B中表達(dá)異常增高,其余骨肉瘤細(xì)胞株中表達(dá)相對弱。143B的MTA1表達(dá)最高,MNNG-HOS次之,其余細(xì)胞株表達(dá)較弱。143B中P53幾乎測不到表達(dá),而HOS和U2OS相對表達(dá)最高。2.5在143B高肺轉(zhuǎn)移骨肉瘤細(xì)胞株中使用慢病毒干擾PC4的表達(dá)。2.5.1人PC4干擾慢病毒的構(gòu)建。2.5.1免疫熒光法結(jié)果提示PC4慢病毒轉(zhuǎn)染率約90%;有限稀釋法挑選高轉(zhuǎn)染效率的亞細(xì)胞克隆并成功擴(kuò)增。2.5.2 WB法檢測PC4慢病毒干擾效率約為70%,且多次傳代后干擾效率無明顯下降。2.5.3 CCK-8法檢測結(jié)果提示:PC4干擾后143B細(xì)胞增殖能力有一定程度的下降。2.5.4流式細(xì)胞儀檢測結(jié)果提示:PC4干擾后143B細(xì)胞周期出現(xiàn)G1期阻滯。2.5.5雙層軟瓊脂克隆法檢測結(jié)果提示:PC4干擾后143B細(xì)胞集落形成能力的明顯下降。2.5.6劃痕實(shí)驗(yàn)檢測結(jié)果提示:PC4干擾后143B細(xì)胞遷移能力下調(diào)。2.5.7 Transwell檢測結(jié)果提示:PC4干擾后143B細(xì)胞侵襲能力下調(diào)。2.5.8 CCK-8法檢測結(jié)果提示:PC4干擾后143B細(xì)胞粘附能力明顯下降。2.5.9裸鼠荷瘤實(shí)驗(yàn)檢測結(jié)果提示:PC4干擾后143B細(xì)胞成瘤能力及肺轉(zhuǎn)移能力均出現(xiàn)明顯下降。3.RNA-seq的高通量測序法,分析143B細(xì)胞株在干擾PC4后基因表達(dá)的變化情況。3.1在143B細(xì)胞株中干擾PC4后,513個基因出現(xiàn)下調(diào),571個基因上調(diào)。3.2 Gene ontology分析結(jié)果提示:PC4干擾后的差異表達(dá)基因可能參與了蛋白粘附、細(xì)胞連接、細(xì)胞外基質(zhì)的分泌、細(xì)胞運(yùn)動、細(xì)胞應(yīng)激等多種細(xì)胞生物學(xué)功能的調(diào)節(jié)。3.3 KEGG pathway分析結(jié)果提示:PC4干擾后的差異表達(dá)基因在p53等20條信號通路中呈現(xiàn)富集狀態(tài)。4.PC4參與MMP9的調(diào)控進(jìn)而影響骨肉瘤肺轉(zhuǎn)移能力。4.1 PC4干擾慢病毒在7種骨肉瘤細(xì)胞株中均有干擾效果;PC4干擾后MMP-2、MMP-9、FN基因RNA水平除個別細(xì)胞株外均有不同程度的下調(diào)。4.2外源性人重組FN蛋白和FNsi RNA對MG63細(xì)胞的MMP-2和MMP-9均有明顯調(diào)控作用,但是對143B和143BPC4-4.3染色質(zhì)免疫共沉淀法結(jié)果提示:PC4蛋白可以與MMP-9啟動子區(qū)域結(jié)合。細(xì)胞的MMP-2和MMP-9沒有明顯的調(diào)控作用。4.4熒光素梅報(bào)告基因結(jié)果提示:PC4si RNA及SP1si RNA均可降低MMP-9的轉(zhuǎn)錄活性,二者存在協(xié)同效應(yīng)。4.5免疫共沉淀法結(jié)果提示:143B細(xì)胞中PC4蛋白與SP1蛋白可以結(jié)合。5.PC4參與骨肉瘤骨破壞的具體分子機(jī)制及可能存在的靶基因。5.1裸鼠荷瘤實(shí)驗(yàn)檢測PC4干擾前后143B細(xì)胞對骨破壞的影響。5.1.1 Micro CT檢測結(jié)果提示:PC4干擾組的裸鼠病理性骨折的發(fā)生率明顯降低、骨小梁數(shù)量增加、骨小梁厚度增厚、骨量增加。5.1.2共培養(yǎng)細(xì)胞模型和條件培養(yǎng)基細(xì)胞模型均提示:骨肉瘤細(xì)胞可以促進(jìn)RAW264.7的破骨分化,PC4干擾可抑制該效應(yīng)。5.1.3 RNA-seq結(jié)果提示:在PC4干擾后CXCL2、IGFBP5、MCP1、IL1A、IL1B明顯下調(diào),RT-PCR法驗(yàn)證結(jié)果與RNA-seq結(jié)果一致。5.1.4 WB法檢測IGFBP5在PC4干擾后的蛋白表達(dá)明顯下調(diào)。5.1.5外源性IGFBP5蛋白,可增加TRAP陽性細(xì)胞的數(shù)量,且該效應(yīng)呈濃度依賴性。5.1.6外源性IGFBP5蛋白在條件培養(yǎng)基細(xì)胞模型中,可部分抵消PC4干擾對RAW264.7破骨分化的抑制作用。結(jié)論:1.PC4在骨肉瘤中高表達(dá);2.PC4與骨肉瘤的分期及預(yù)后存在相關(guān)性;3.PC4參與調(diào)控骨肉瘤的增殖、侵襲、遷移、粘附、集落形成、成瘤、轉(zhuǎn)移等惡性表型;4.PC4通過與MMP9的轉(zhuǎn)移因子SP1形成轉(zhuǎn)錄復(fù)合體,參與MMP9的轉(zhuǎn)錄調(diào)控;5.PC4的表達(dá)量與143B細(xì)胞引起的骨吸收正相關(guān);6.干擾在143B骨肉瘤細(xì)胞中PC4的表達(dá),可抑制143B分泌CXCL2、CCL2、IGFBP5、IL1A、IL1B,從而抑制破骨細(xì)胞的激活,降低骨肉瘤引起的骨破壞。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R738.1

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