本文選題：骨肉瘤 + Oct4�。� 參考：《吉林大學(xué)》2017年博士論文

【摘要】：骨肉瘤(Osteosarcoma;OS)又被稱作成骨肉瘤,是一種骨的惡性腫瘤,其發(fā)病年齡較早,大多常見于兒童或20歲以下的青少年,同時易于發(fā)生轉(zhuǎn)移,因此可以說其嚴(yán)重危害了廣大青少年和兒童的身體健康。此外,其發(fā)病率亦占據(jù)了原發(fā)性惡性腫瘤中的榜首。OS的惡性程度非常高,且預(yù)后相當(dāng)差,甚至與OS發(fā)現(xiàn)的時間、采取的治療措施等均無關(guān)。綜上,OS給年輕的患者、家庭乃至社會帶來了很重的負(fù)擔(dān)和打擊,故其一直是生命科學(xué)領(lǐng)域中研究的重點和熱點,但盡管相關(guān)研究很多,其發(fā)病機制尤其是其易于生長和侵襲的機制,至今為止仍未給出很好的解釋,而轉(zhuǎn)錄因子Oct4是一種重要的關(guān)鍵調(diào)控基因,密切參與多向性分化的調(diào)控,大量報道顯示其非常可能參與了成體細(xì)胞和腫瘤的發(fā)生發(fā)展過程,但與OS的相關(guān)性如何至今鮮有研究。故本研究將以分子生物學(xué)技術(shù)為手段來探討轉(zhuǎn)錄因子Oct4在OS發(fā)生發(fā)展中的具體作用機制,以便給OS發(fā)病機制的深入研究提供理論基礎(chǔ),具有重要的現(xiàn)實意義。目的檢測Oct4在骨肉瘤中的表達(dá)情況,探討Oct4對骨肉瘤的具體影響并研究這種影響產(chǎn)生的具體作用機制,為骨肉瘤的發(fā)病機制的研究奠定理論基礎(chǔ),具有重要的科學(xué)價值和研究意義。方法本論文的研究工作分為4個部分,具體如下:(1)Oct4基因在不同骨肉瘤細(xì)胞系和人類骨肉瘤組織表達(dá)分析對骨肉瘤細(xì)胞株SOSP-9607、MG63、F5M2和成骨細(xì)胞株h FOB1.19進(jìn)行培養(yǎng),同時收集吉林大中日聯(lián)誼醫(yī)院進(jìn)行骨肉瘤手術(shù)治療的10名患者于手術(shù)期間獲得的骨肉瘤組織和正常組織。對上述樣本組織進(jìn)行總RNA和蛋白質(zhì)的提取,分別用real-time RT-PCR和western blotting方式對樣本進(jìn)行Oct4基因和蛋白質(zhì)表達(dá)的檢測,檢測結(jié)果采用單向方差分析和Bonferroni檢測多重比較進(jìn)行統(tǒng)計學(xué)分析。(2)Oct4基因?qū)5M2細(xì)胞株增殖和入侵的影響研究1)對骨肉瘤細(xì)胞株F5M2進(jìn)行培養(yǎng),采用Lipofectamine 2000轉(zhuǎn)染試劑將三種Oct4 siRNAs和scramble分別轉(zhuǎn)染至F5M2細(xì)胞中。轉(zhuǎn)染三天后,分別用real-time RT-PCR和western blotting方式檢測被Oct4 siRNAs干擾的F5M2細(xì)胞株的Oct4表達(dá)情況,以評價siRNAs和scramble的轉(zhuǎn)染效果。2)對骨肉瘤細(xì)胞株F5M2分別加入Oct4 si RNA1和scramble進(jìn)行干擾培養(yǎng)三天。采用MTT試驗檢測細(xì)胞增殖情況,分別測定Oct4 si RNA1和scramble干擾后的骨肉瘤細(xì)胞的吸光度。采用膜聯(lián)蛋白V-FITC凋亡檢測試劑盒檢測,流式細(xì)胞儀監(jiān)測骨肉瘤的細(xì)胞凋亡情況。通過體外侵襲實驗對骨肉瘤細(xì)胞進(jìn)行侵襲情況的評估。檢測結(jié)果采用單向方差分析和Bonferroni檢測多重比較進(jìn)行統(tǒng)計學(xué)分析。(3)Oct4基因?qū)K055347表達(dá)影響對骨肉瘤細(xì)胞株F5M2進(jìn)行培養(yǎng),采用Lipofectamine 2000轉(zhuǎn)染試劑將Oct4si RNA-1和scramble轉(zhuǎn)染至骨肉瘤F5M2細(xì)胞株中。設(shè)計9種相關(guān)lnc RNAs引物,轉(zhuǎn)染三天后采用real-time RT-PCR檢測相關(guān)lnc RNAs的表達(dá)情況。(4)AK055347的下調(diào)表達(dá)對F5M2細(xì)胞增殖和入侵的影響研究1)對骨肉瘤細(xì)胞株F5M2進(jìn)行培養(yǎng),采用Lipofectamine 2000轉(zhuǎn)染試劑將三種AK055347 siRNAs和scramble分別轉(zhuǎn)染至F5M2細(xì)胞中。轉(zhuǎn)染三天后,分別用real-time RT-PCR方式檢測被AK055347 siRNAs干擾的F5M2細(xì)胞株的AK055347表達(dá)情況,以評價siRNAs和scramble的轉(zhuǎn)染效果。2)對骨肉瘤細(xì)胞株F5M2分別加入AK055347 si RNA3和scramble進(jìn)行干擾培養(yǎng)三天。采用MTT試驗檢測細(xì)胞增殖情況,分別測定AK055347 si RNA3和scramble干擾后的骨肉瘤細(xì)胞的吸光度。采用膜聯(lián)蛋白V-FITC凋亡檢測試劑盒檢測,流式細(xì)胞儀監(jiān)測骨肉瘤的細(xì)胞凋亡情況。通過體外侵襲實驗對骨肉瘤細(xì)胞進(jìn)行侵襲情況的評估。檢測結(jié)果采用單向方差分析和Bonferroni檢測多重比較進(jìn)行統(tǒng)計學(xué)分析。結(jié)果(1)Oct4基因在不同骨肉瘤細(xì)胞系的表達(dá)顯著增加;同時相較于癌旁周圍正常組織,Oct4基因在人類骨肉瘤組織中的表達(dá)顯著增加。(2)1)三種Oct4 siRNAs干擾骨肉瘤細(xì)胞株F5M2三天后,均顯著降低Oct4 m RNA和蛋白的表達(dá)。其中,Oct4 si RNA-1表現(xiàn)出最大抑制作用,提示Oct4si RNA-1具有最佳的Oct4基因沉默效果。2)MTT試驗檢測結(jié)果顯示Scramble組與空白組吸光度增加明顯,而Oct4 si RNA1干擾組吸光度增加不明顯,說明后者的細(xì)胞增殖情況明顯受到抑制;膜聯(lián)蛋白的血細(xì)胞計數(shù)檢測結(jié)果表明Oct4si RNA-1干擾后,癌細(xì)胞凋亡率明顯升高;侵襲小室實驗結(jié)果顯示與scramble組和空白對照組相比,Oct4下調(diào)的F5M2細(xì)胞里只有少量細(xì)胞穿越了涂有聚碳酸酯膜的人工基底膜,提示入侵減少。(3)F5M2細(xì)胞被Oct4 si RNA1和scramble干擾培養(yǎng)三天后,通過Real-time RT-PCR檢測Oct4下調(diào)對lnc RNAs表達(dá)的影響,其中只有顯著降低了AK055347的表達(dá),其它8種并沒有顯著影響。(4)1)三種AK055347 siRNAs干擾骨肉瘤細(xì)胞株F5M2三天后,均顯著降低AK055347 m RNA的表達(dá)。其中,AK055347 si RNA-3表現(xiàn)出最大抑制作用,提示AK055347 si RNA-3具有最佳的AK055347基因沉默效果。2)等量F5M2細(xì)胞被AK055347 si RNA3和scramble轉(zhuǎn)染并培養(yǎng)3天,AK055347的表達(dá)下調(diào)與scramble組相比顯著降低了MTT的吸光度值,表明了AK055347的表達(dá)下調(diào)降低了F5M2細(xì)胞的增殖;采用流式細(xì)胞儀檢測膜聯(lián)蛋白V的細(xì)胞表面表達(dá),結(jié)果顯示,AK055347的表達(dá)下降顯著增加了膜聯(lián)蛋白V表面表達(dá),所以同時增加了癌細(xì)胞的凋亡;F5M2細(xì)胞分別加入AK055347 si RNA-3和scramble干擾培養(yǎng)三天后,通過侵襲小室實驗檢測細(xì)胞入侵能力,結(jié)果表明與scramble組相比,AK055347下調(diào)的F5M2細(xì)胞里只有少量細(xì)胞穿越了涂有聚碳酸酯膜的人工基底膜,即入侵減少,提示了AK055347下調(diào)降低了F5M2細(xì)胞的入侵能力。結(jié)論(1)Oct4參與骨肉瘤的發(fā)生發(fā)展過程,與其細(xì)胞增殖密切相關(guān)。(2)靶向Oct4的RNAi導(dǎo)致F5M2細(xì)胞增殖抑制,其原因與細(xì)胞死亡相關(guān)聯(lián),且死亡形式以凋亡為主而非壞死。(3)Oct4可能參與骨肉瘤細(xì)胞的侵襲和轉(zhuǎn)移。(4)轉(zhuǎn)錄因子Oct4通過調(diào)節(jié)lnc RNA AK055347的表達(dá)促進(jìn)骨肉瘤的發(fā)生發(fā)展創(chuàng)新率先發(fā)現(xiàn)Oct4基因在骨肉瘤組織及細(xì)胞系中表達(dá),且呈現(xiàn)增高表達(dá)。提出Oct4可能是骨肉瘤中的重要轉(zhuǎn)錄因子之一,對其發(fā)生、發(fā)展起到巨大的推動作用。
[Abstract]:Osteosarcoma (OS), also known as osteosarcoma, is a malignant tumor of bone. The age of the osteosarcoma is early, most common in children or young people under 20 years of age, and it is easy to metastasize. Therefore, it can be said that it seriously endangers the body health of young people and children. In addition, the incidence of the disease also occupies the primary malignant tumor. .OS, at the top of the list, has a high degree of malignancy, and the prognosis is very poor, even with the time that OS has been found and the treatment measures are taken. To sum up, OS has brought heavy burden and shock to young patients, family and society, so it has been the focus and hot spot in the field of life science. Mechanism, especially its mechanism that is easy to grow and invasion, has still not been well explained, and the transcription factor Oct4 is an important key regulatory gene, closely involved in the regulation of multidirectional differentiation. A large number of reports show that it is very likely to be involved in the development of adult cells and tumors, but how the correlation with OS is so far It is rarely studied. Therefore, this study will study the specific mechanism of the transcription factor Oct4 in the development of OS by means of molecular biology technology, so as to provide theoretical basis for the in-depth study of the pathogenesis of OS. The purpose of this study is to detect the appearance of Oct4 in osteosarcoma and to explore the specific effects of Oct4 on osteosarcoma. And study the specific mechanism of this effect to lay a theoretical foundation for the study of the pathogenesis of osteosarcoma. It has important scientific value and research significance. The research work of this paper is divided into 4 parts: (1) the expression analysis of Oct4 gene in different osteosarcoma cell lines and human osteosarcoma tissue is fine for osteosarcoma The cells SOSP-9607, MG63, F5M2 and osteoblast h FOB1.19 were cultured, and the osteosarcoma tissues and normal tissues obtained during the operation were collected by 10 patients undergoing osteosarcoma operation in Jilin Dazhong friendship hospital. The total RNA and protein were extracted from the above samples with real-time RT-PCR and Western blotting respectively. Methods to detect the expression of Oct4 gene and protein in the sample, the results were analyzed statistically by one-way ANOVA and Bonferroni detection. (2) the effect of Oct4 gene on the proliferation and invasion of F5M2 cell line 1) culture of osteosarcoma cell line F5M2, and three Oct4 Si using Lipofectamine 2000 transfection reagent. RNAs and scramble were transfected into F5M2 cells respectively. After three days, real-time RT-PCR and Western blotting were used to detect Oct4 expression of Oct4 siRNAs interfering F5M2 cell lines. The cell proliferation was detected by MTT test, and the absorbance of osteosarcoma cells after Oct4 Si RNA1 and scramble interference were measured respectively. The apoptosis of osteosarcoma cells was detected by the membrane associated protein V-FITC apoptosis detection kit and the flow cytometry was used to evaluate the invasion of osteosarcoma cells through the invasion test in vitro. The results were statistically analyzed by one-way ANOVA and Bonferroni detection. (3) the effect of Oct4 gene on osteosarcoma cell line F5M2 was cultured on AK055347 expression, and Oct4si RNA-1 and scramble were transfected to F5M2 cell line of osteosarcoma by Lipofectamine 2000 transfection reagent. 9 kinds of LNC RNAs primers were designed and transfected for three days. Then real-time RT-PCR was used to detect the expression of related LNC RNAs. (4) the effect of AK055347 down expression on the proliferation and invasion of F5M2 cells. 1) the osteosarcoma cell line F5M2 was cultured and three AK055347 siRNAs and scramble were transfected into F5M2 cells respectively with Lipofectamine 2000 transfection reagent. The transfection for three days was used respectively. The expression of AK055347 in F5M2 cell lines interfered by AK055347 siRNAs was detected by -time RT-PCR, and the transfection effect of siRNAs and scramble was evaluated for three days. The absorbance of osteosarcoma cells after e interference. The apoptosis of osteosarcoma was detected by the flow cytometry with V-FITC apoptosis detection kit. The invasion of osteosarcoma cells was evaluated by invasive test in vitro. The detection results were statistically analyzed by one-way ANOVA and Bonferroni detection. Results (1) the expression of Oct4 gene in different osteosarcoma cell lines increased significantly, and the expression of Oct4 gene in human osteosarcoma tissue was significantly increased. (2) 1) three Oct4 siRNAs interfered osteosarcoma cell line F5M2 for three days, and the expression of Oct4 m RNA and protein was significantly reduced. Oct4 Si RNA-1 The maximum inhibitory effect showed that Oct4si RNA-1 had the best Oct4 gene silencing effect.2) MTT test results showed that the absorbance of the Scramble group and the blank group increased obviously, while the increase in the absorbance of the Oct4 Si RNA1 interference group was not obvious, indicating that the cell proliferation of the latter was obviously inhibited; the blood cell count detection junction of the annexin was found. The results showed that the apoptosis rate of cancer cells increased significantly after Oct4si RNA-1 interference, and the results of invasive chamber experiment showed that only a few cells in Oct4 down regulated F5M2 cells passed the artificial basement membrane coated with polycarbonate membrane compared with the scramble group and the blank control group, suggesting that the invasion was reduced. (3) F5M2 cells were disturbed by Oct4 Si RNA1 and scramble three. Real-time RT-PCR was used to detect the effect of down regulation of Oct4 on the expression of LNC RNAs, which only significantly reduced the expression of AK055347, and the other 8 had no significant influence. (4) 1) three AK055347 siRNAs interfered osteosarcoma cell lines, which significantly decreased the expression of AK055347 m RNA. It was suggested that AK055347 Si RNA-3 had the best AK055347 gene silencing effect.2) and the F5M2 cells were transfected and cultured for 3 days by AK055347 Si RNA3 and scramble, and the downregulation of AK055347 expression decreased significantly compared with the scramble group, indicating that the expression lowered the proliferation of the cells; flow cytometry was used. The expression of the cell surface of annexin V was detected. The results showed that the decrease of AK055347 expression significantly increased the expression of the membrane associated protein V, so the apoptosis of cancer cells was increased at the same time. F5M2 cells were added to AK055347 Si RNA-3 and scramble for three days, and the invasion ability of the cells was detected by invasive chamber experiment, and the results showed that the cells were with scram. Compared with the ble group, only a few cells in the AK055347 down F5M2 cells passed through the artificial basement membrane coated with polycarbonate membrane, that is, the invasion decreased, suggesting that the AK055347 downregulation reduced the invasion ability of F5M2 cells. Conclusion (1) Oct4 is involved in the development process of osteosarcoma and is closely related to its fine cell proliferation. (2) RNAi of the target to Oct4 leads to F5M2 fine. The inhibition of cell proliferation is associated with cell death, and the death form is mainly apoptosis and not necrotic. (3) Oct4 may be involved in the invasion and metastasis of osteosarcoma cells. (4) the transcription factor Oct4 first found the expression of Oct4 gene in osteosarcoma tissue and cell lines by regulating the expression of LNC RNA AK055347. It is suggested that Oct4 may be one of the important transcription factors in osteosarcoma, which may play an important role in the development of osteosarcoma.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R738.1

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9 朱U，

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