本文選題：TREX1 + 骨肉瘤��； 參考：《福建醫(yī)科大學(xué)》2015年博士論文

【摘要】：目的1通過(guò)免疫組化比較(3年內(nèi)經(jīng)臨床證實(shí)無(wú)轉(zhuǎn)移或轉(zhuǎn)移)骨肉瘤患者腫瘤組織中TREX1(Three Prime Repair Exonuclease 1)表達(dá)的差異,探討TREX1與骨肉瘤患者遠(yuǎn)處轉(zhuǎn)移及預(yù)后相關(guān)性。2比較人骨肉瘤細(xì)胞系CD133~+細(xì)胞亞群與CD133-細(xì)胞亞群中TREX1基因的表達(dá)差異,探討TREX1基因與骨肉瘤細(xì)胞生物學(xué)特性的相關(guān)性。3通過(guò)調(diào)節(jié)TREX1基因的表達(dá)水平,比較調(diào)節(jié)前后骨肉瘤細(xì)胞生物學(xué)特性的改變,研究其對(duì)骨肉瘤細(xì)胞生物特性的影響。4通過(guò)調(diào)節(jié)TREX1基因的表達(dá)水平,檢測(cè)TREX1基因下游相關(guān)基因的變化,尋找TREX1基因相關(guān)的信號(hào)通路,為了解TREX1基因功能提供實(shí)驗(yàn)依據(jù)。方法1收集臨床病例標(biāo)本:根據(jù)3年內(nèi)是否發(fā)生轉(zhuǎn)移分為轉(zhuǎn)移組與無(wú)轉(zhuǎn)移組,免疫組化檢測(cè)骨肉瘤組織細(xì)胞中TREX1蛋白表達(dá)差異。2將人骨肉瘤細(xì)胞經(jīng)CD133抗體標(biāo)記后,經(jīng)由流式細(xì)胞儀分選出CD133~+細(xì)胞亞群和CD133-細(xì)胞亞群。通過(guò)qPCR及Western Blot檢測(cè)兩細(xì)胞亞群中TREX1及干細(xì)胞相關(guān)基因Nanog、Oct4表達(dá)情況;以骨肉瘤MNNG/HOS細(xì)胞為代表,通過(guò)腫瘤球成球培養(yǎng)、克隆形成、生長(zhǎng)曲線(MTT法)、化療藥物敏感性、成骨及成脂方向誘導(dǎo)分化和裸鼠體內(nèi)成瘤能力等實(shí)驗(yàn),比較MNNG/HOS細(xì)胞CD133~+細(xì)胞亞群與CD133-細(xì)胞亞群細(xì)胞生物學(xué)特性差異。3下調(diào)人骨肉瘤MNNG/HOS細(xì)胞CD133-細(xì)胞亞群中TREX1基因,觀察下調(diào)前后生長(zhǎng)曲線、化療藥物敏感性、成骨及成脂方向誘導(dǎo)分化、遷移及侵襲和裸鼠體內(nèi)成瘤能力的變化情況。4下調(diào)人骨肉瘤MNNG/HOS細(xì)胞CD133-細(xì)胞亞群中TREX1基因,通過(guò)qPCR檢測(cè)干細(xì)胞相關(guān)基因Nanog和Oct4等基因表達(dá)變化情況。結(jié)果1免疫組化檢測(cè)結(jié)果表明:TREX1蛋白在3年內(nèi)無(wú)轉(zhuǎn)移組患者骨肉瘤組織中高表達(dá),轉(zhuǎn)移組患者骨肉瘤組織中低表達(dá),兩組差異有統(tǒng)計(jì)學(xué)意義(P0.05),隨訪發(fā)現(xiàn)無(wú)轉(zhuǎn)移組骨肉瘤患者平均存活時(shí)間長(zhǎng)截至最后一次隨訪,平均存活時(shí)間達(dá)52.6個(gè)月,而轉(zhuǎn)移組平均存活時(shí)間僅為24.4個(gè)月,兩者差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2人骨肉瘤MNNG/HOS細(xì)胞中CD133~+細(xì)胞亞群高表達(dá)干細(xì)胞相關(guān)基因Nanog和Oct4,但TREX1基因低表達(dá);在生物學(xué)特性鑒定實(shí)驗(yàn)中可見(jiàn)其形成腫瘤球、克隆形成集落數(shù)多、細(xì)胞增殖能力強(qiáng)、對(duì)化療藥物順鉑敏感性低、向成骨及成脂分化的潛能及裸鼠體內(nèi)成瘤性強(qiáng);而CD133-細(xì)胞亞群低表達(dá)干細(xì)胞相關(guān)基因Nanog和Oct4,但TREX1基因表達(dá)水平較高;在生物學(xué)特性鑒定實(shí)驗(yàn)中無(wú)法形成腫瘤球、克隆形成集落數(shù)少、細(xì)胞增殖慢、對(duì)化療藥物順鉑敏感性高、缺乏向成骨及成脂方向分化的潛能、裸鼠體內(nèi)成瘤不明顯。3下調(diào)骨肉瘤MNNG/HOS細(xì)胞CD133-細(xì)胞亞群中TREX1基因表達(dá)后,其增殖能力未見(jiàn)明顯改變,但其對(duì)化療藥物順鉑的敏感性降低、成骨及成脂相關(guān)基因表達(dá)水平升高(mRNA水平)、遷移及侵襲能力和裸鼠體內(nèi)成瘤性增強(qiáng)。4下調(diào)骨肉瘤MNNG/HOS細(xì)胞CD133-細(xì)胞亞群中TREX1基因表達(dá)后,Oct4基因表達(dá)水平約為干擾前的3倍(P0.05);CD133 mRNA表達(dá)水平約為干擾前的2倍(P0.05);而Nanog基因表達(dá)略有增加,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1在25例有轉(zhuǎn)移患者中有21例的骨肉瘤組織TREX1蛋白低表達(dá),而20例無(wú)轉(zhuǎn)移患者中有17例的骨肉瘤組織高表達(dá)TREX1蛋白。即TREX1蛋白表達(dá)量與骨肉瘤患者是否發(fā)生轉(zhuǎn)移密切相關(guān),可作為患者預(yù)后判斷的有益指標(biāo)。2人骨肉瘤MNNG/HOS細(xì)胞CD133~+細(xì)胞亞群中TREX1低表達(dá),具有自我更新、多向分化潛能、化療抵抗、裸鼠體內(nèi)成瘤等腫瘤干細(xì)胞生物學(xué)特性,而CD133-細(xì)胞亞群中TREX1高表達(dá),無(wú)明顯腫瘤干細(xì)胞生物學(xué)特性。即TREX1在骨肉瘤干細(xì)胞中低表達(dá)。3下調(diào)人骨肉瘤MNNG/HOS細(xì)胞CD133-細(xì)胞亞群中TREX1的表達(dá)水平后,該亞群細(xì)胞多項(xiàng)腫瘤干細(xì)胞特性增強(qiáng),如向成骨及成脂分化、化療抵抗、遷移、侵襲及體內(nèi)成瘤能力等;但增殖能力無(wú)明顯改變。4下調(diào)人骨肉瘤MNNG/HOS細(xì)胞中CD133-細(xì)胞亞群中TREX1的表達(dá)水平后,干細(xì)胞相關(guān)基因Oct4表達(dá)明顯升高,表明TREX1介導(dǎo)的信號(hào)通路可能通過(guò)調(diào)節(jié)Oct4的表達(dá)水平,進(jìn)而影響骨肉瘤相應(yīng)的生物學(xué)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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R738

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本文編號(hào)：2006570