TREX1对人骨肉瘤细胞生物学特性影响及机制研究

发布时间：2018-06-11 20:20

本文选题：TREX1 + 骨肉瘤　；参考：《福建医科大学》2015年博士论文

【摘要】：目的1通过免疫组化比较(3年内经临床证实无转移或转移)骨肉瘤患者肿瘤组织中TREX1(Three Prime Repair Exonuclease 1)表达的差异,探讨TREX1与骨肉瘤患者远处转移及预后相关性。2比较人骨肉瘤细胞系CD133~+细胞亚群与CD133-细胞亚群中TREX1基因的表达差异,探讨TREX1基因与骨肉瘤细胞生物学特性的相关性。3通过调节TREX1基因的表达水平,比较调节前后骨肉瘤细胞生物学特性的改变,研究其对骨肉瘤细胞生物特性的影响。4通过调节TREX1基因的表达水平,检测TREX1基因下游相关基因的变化,寻找TREX1基因相关的信号通路,为了解TREX1基因功能提供实验依据。方法1收集临床病例标本:根据3年内是否发生转移分为转移组与无转移组,免疫组化检测骨肉瘤组织细胞中TREX1蛋白表达差异。2将人骨肉瘤细胞经CD133抗体标记后,经由流式细胞仪分选出CD133~+细胞亚群和CD133-细胞亚群。通过qPCR及Western Blot检测两细胞亚群中TREX1及干细胞相关基因Nanog、Oct4表达情况;以骨肉瘤MNNG/HOS细胞为代表,通过肿瘤球成球培养、克隆形成、生长曲线(MTT法)、化疗药物敏感性、成骨及成脂方向诱导分化和裸鼠体内成瘤能力等实验,比较MNNG/HOS细胞CD133~+细胞亚群与CD133-细胞亚群细胞生物学特性差异。3下调人骨肉瘤MNNG/HOS细胞CD133-细胞亚群中TREX1基因,观察下调前后生长曲线、化疗药物敏感性、成骨及成脂方向诱导分化、迁移及侵袭和裸鼠体内成瘤能力的变化情况。4下调人骨肉瘤MNNG/HOS细胞CD133-细胞亚群中TREX1基因,通过qPCR检测干细胞相关基因Nanog和Oct4等基因表达变化情况。结果1免疫组化检测结果表明:TREX1蛋白在3年内无转移组患者骨肉瘤组织中高表达,转移组患者骨肉瘤组织中低表达,两组差异有统计学意义(P0.05),随访发现无转移组骨肉瘤患者平均存活时间长截至最后一次随访,平均存活时间达52.6个月,而转移组平均存活时间仅为24.4个月,两者差异有统计学意义(P0.05)。2人骨肉瘤MNNG/HOS细胞中CD133~+细胞亚群高表达干细胞相关基因Nanog和Oct4,但TREX1基因低表达;在生物学特性鉴定实验中可见其形成肿瘤球、克隆形成集落数多、细胞增殖能力强、对化疗药物顺铂敏感性低、向成骨及成脂分化的潜能及裸鼠体内成瘤性强;而CD133-细胞亚群低表达干细胞相关基因Nanog和Oct4,但TREX1基因表达水平较高;在生物学特性鉴定实验中无法形成肿瘤球、克隆形成集落数少、细胞增殖慢、对化疗药物顺铂敏感性高、缺乏向成骨及成脂方向分化的潜能、裸鼠体内成瘤不明显。3下调骨肉瘤MNNG/HOS细胞CD133-细胞亚群中TREX1基因表达后,其增殖能力未见明显改变,但其对化疗药物顺铂的敏感性降低、成骨及成脂相关基因表达水平升高(mRNA水平)、迁移及侵袭能力和裸鼠体内成瘤性增强。4下调骨肉瘤MNNG/HOS细胞CD133-细胞亚群中TREX1基因表达后,Oct4基因表达水平约为干扰前的3倍(P0.05);CD133 mRNA表达水平约为干扰前的2倍(P0.05);而Nanog基因表达略有增加,差异均无统计学意义(P0.05)。结论1在25例有转移患者中有21例的骨肉瘤组织TREX1蛋白低表达,而20例无转移患者中有17例的骨肉瘤组织高表达TREX1蛋白。即TREX1蛋白表达量与骨肉瘤患者是否发生转移密切相关,可作为患者预后判断的有益指标。2人骨肉瘤MNNG/HOS细胞CD133~+细胞亚群中TREX1低表达,具有自我更新、多向分化潜能、化疗抵抗、裸鼠体内成瘤等肿瘤干细胞生物学特性,而CD133-细胞亚群中TREX1高表达,无明显肿瘤干细胞生物学特性。即TREX1在骨肉瘤干细胞中低表达。3下调人骨肉瘤MNNG/HOS细胞CD133-细胞亚群中TREX1的表达水平后,该亚群细胞多项肿瘤干细胞特性增强,如向成骨及成脂分化、化疗抵抗、迁移、侵袭及体内成瘤能力等;但增殖能力无明显改变。4下调人骨肉瘤MNNG/HOS细胞中CD133-细胞亚群中TREX1的表达水平后,干细胞相关基因Oct4表达明显升高,表明TREX1介导的信号通路可能通过调节Oct4的表达水平,进而影响骨肉瘤相应的生物学特性。
[Abstract]:Objective 1 to investigate the difference in the expression of TREX1 (Three Prime Repair Exonuclease 1) in the tumor tissues of patients with osteosarcoma in 3 years, and to explore the correlation of distant metastasis and prognosis in patients with TREX1 and osteosarcoma.2 comparison of CD133~+ cell subsets and TREX1 based CD133- cell subsets in human osteosarcoma cell line The correlation between the expression of TREX1 gene and the biological characteristics of osteosarcoma cells.3 by regulating the expression level of TREX1 gene and comparing the changes of biological characteristics of osteosarcoma cells before and after regulating the biological characteristics of osteosarcoma cells, and studying the effect of.4 on the expression level of TREX1 gene to detect the downstream correlation of the TREX1 gene. The change of gene, finding the signal pathway related to TREX1 gene and providing experimental basis for understanding the function of TREX1 gene. Method 1 collection of clinical case specimens: according to whether metastasis in 3 years is divided into transfer group and non metastasis group, the differential expression of TREX1 protein in the tissue cells of osteosarcoma by immunohistochemical method,.2 can be used as the CD133 antibody for human osteosarcoma cells. CD133~+ cell subsets and CD133- cell subsets were selected by flow cytometry. The expression of TREX1 and stem cell related genes Nanog and Oct4 in two cell subsets were detected by qPCR and Western Blot, and the expression of Oct4 in osteosarcoma MNNG/HOS cells was cloned, growth curve (MTT method), chemotherapeutic drug sensitivity, The differentiation of osteogenic and fat induced differentiation and the ability of tumorigenesis in nude mice, the difference of biological characteristics of CD133~+ cell subsets and CD133- cell subsets in MNNG/HOS cells.3 downregulated the TREX1 gene in the CD133- cell subgroup of human osteosarcoma MNNG/HOS cells, observed the growth curve, chemosensitivity, osteogenesis and lipid orientation. Induction of differentiation, migration, invasion and tumorigenesis in nude mice.4 down regulated the TREX1 gene in the CD133- cell subgroup of human osteosarcoma cell MNNG/HOS cells, and detected the changes in the gene expression of the stem cell related genes Nanog and Oct4 by qPCR. Results 1 immunohistochemistry showed that TREX1 protein had no metastasis in 3 years. The high expression in the tumor tissue and the low expression of the osteosarcoma tissue in the metastasis group were statistically significant (P0.05). The average survival time of the patients with osteosarcoma in the non metastatic group was long up to the last follow-up, the average survival time was 52.6 months, and the average survival time of the metastatic group was only 24.4 months, and the difference was statistically significant (P 0.05) the CD133~+ cell subsets in the.2 human osteosarcoma MNNG/HOS cells express the stem cell related genes Nanog and Oct4, but the TREX1 gene is low expressed. In the biological characterization experiments, the tumor cells are formed, the colony forming colony is more, the cell proliferation ability is strong, the chemosensitivity of cisplatin is low, and the potential of osteogenesis and lipid differentiation is found. The tumorigenicity of the nude mice was strong, while the CD133- cell subsets showed low expression of stem cell related genes Nanog and Oct4, but the expression level of TREX1 gene was high, and the tumor cells were not formed in the identification experiments of biological characteristics, the number of colony forming colony was less, the cell proliferation was slow, the chemosensitivity of cisplatin was high, and the potential of differentiation to osteogenesis and fat was lack. The tumor formation in nude mice was not obvious.3 down regulation of the TREX1 gene expression in the CD133- cell subgroup of osteosarcoma, the proliferation ability of MNNG/HOS cells was not significantly changed, but its sensitivity to chemotherapy cisplatin decreased, the expression level of osteogenic and adipogenic genes increased (mRNA level), migration and invasion ability and tumorigenicity of nude mice enhanced.4 down regulation After the expression of TREX1 gene in the CD133- cell subgroup of osteosarcoma MNNG/HOS cells, the expression level of Oct4 gene was about 3 times as much as before interference (P0.05), and the expression level of CD133 mRNA was about 2 times as much as before interference (P0.05), but the expression of the Nanog gene was slightly increased, and the difference was not statistically significant (P0.05). Conclusion 1 in 25 cases with metastatic patients, 21 osteosarcoma tissues TRE The expression of X1 protein was low, while 17 cases of osteosarcoma were highly expressed in 20 cases without metastasis. That is, the expression of TREX1 protein is closely related to the metastasis of osteosarcoma patients. It can be used as a useful indicator of prognosis in patients with.2 human osteosarcoma MNNG/HOS cell CD133~+ cell subsets of human osteosarcoma, which has a low expression of TREX1 and is self renewing and multidirectional. The biological characteristics of the tumor stem cells, such as chemical potential, chemotherapeutic resistance and tumor formation in nude mice, and the high expression of TREX1 in CD133- cell subsets, and no obvious biological characteristics of tumor stem cells. That is, the low expression of TREX1 in osteosarcoma stem cells reduces the expression level of TREX1 in the CD133- cell subgroup of human osteosarcoma MNNG/HOS cells, and the subgroup is a number of swollen cells. The tumor stem cell characteristics are enhanced, such as osteogenesis and lipid differentiation, chemotherapeutic resistance, migration, invasion and tumorigenicity in the body, but the proliferation ability does not significantly alter the expression level of TREX1 in the CD133- cell subsets of human osteosarcoma cells of.4, and the expression of Oct4 in the stem cell related genes is significantly elevated, indicating that the TREX1 mediated signaling pathway is possible. By regulating the expression level of Oct4, the corresponding biological characteristics of osteosarcoma will be affected.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R738

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