本文選題：腎臟透明細胞癌 + HPPCn��； 參考：《濟南大學(xué)》2014年碩士論文

【摘要】：目的意義： HPPCn(Hepatopoietin Cn)是在我們實驗室一種新發(fā)現(xiàn)的再生肝臟的細胞生長因子，哺乳動物為其主要來源，在多種組織中廣泛表達。作為一類多功能的核蛋白，它可以表達于腫瘤組織中，同時也表達于自我具有更新能力的細胞中和正常組織中，同時在很多方面發(fā)揮了重要的作用，其中包括在蛋白質(zhì)的降解、細胞骨架動力學(xué)、細胞之間的信號轉(zhuǎn)導(dǎo)、和細胞的形態(tài)變化方面和其他許多生物過程。 在CRCC細胞中HIF-1α和HIF-2α的轉(zhuǎn)錄靶基因并不完全相同，并清楚定義了HIF-1α和HIF-2α的特異靶基因。多種癌基因如CyclinD1、TGF-α、VEGF等是HIF-2α的特異靶基因，而促凋亡基因BNIP3是HIF-1α的特異靶基因。而且HIF-1α和HIF-2α與不同的信號轉(zhuǎn)導(dǎo)通路交聯(lián)。激活的HIF-1α能夠降低c-Myc依賴的細胞增殖，而HIF-2α則與之相反。除此以外，HIF-1α比HIF-2α對VHL缺失CRCC細胞中蛋白酶體的降解更敏感。CRCC的多血管特征常與HIF-2α上調(diào)密切相關(guān)，而CRCC中高表達HIF-1α患者的生存期要長于其他類型患者。因此，HIF，特別是HIF-2α可能在CRCC發(fā)展中發(fā)揮了更為重要的作用 近年來的研究發(fā)現(xiàn)HPPCn的表達和活性與腫瘤的生長與預(yù)后密切相關(guān)。HPPCn能夠抑制多種化療藥物（Sorafinib，ADM，CDD，TSA）誘導(dǎo)的肝癌細胞凋亡，并經(jīng)過調(diào)控mRNA的表達促進腫瘤細胞上皮-間質(zhì)轉(zhuǎn)化（EMT）和血管內(nèi)皮細胞遷移，促進腫瘤的轉(zhuǎn)移和浸潤。轉(zhuǎn)基因小鼠的肝癌誘發(fā)實驗顯示HPPCn能夠在誘導(dǎo)早期增加原癌基因c-myc的表達，并顯著增加腫瘤的肺轉(zhuǎn)移率。肝癌細胞尾靜脈注射實驗顯示HPPCn高表達能夠增加肝癌細胞的肺轉(zhuǎn)移和腫瘤中血管的數(shù)量。同時，初步的研究還發(fā)現(xiàn)HPPCn在腎臟透明細胞癌中的表達明顯高于癌旁組織和正常腎臟組織。并且發(fā)現(xiàn)HPPCn可促進HIF-2的表達。那么，腎臟透明細胞癌的發(fā)生、發(fā)展過程中與HPPCn之間會有著什么樣的關(guān)系呢？HIF-2又在這之中是否有作用？ 圍繞上述問題，本實驗?zāi)康氖茄芯縃PPCn調(diào)控HIF-2a促進腎癌細胞生長轉(zhuǎn)移的作用和機制。為此做了如下的研究工作。 方法: 構(gòu)建干涉HIF-2的慢病毒載體，應(yīng)用Western blot和Q-PCR檢測HIF-2的干涉效率。再通過干涉腫瘤細胞的HIF-2表達，應(yīng)用MTS，劃痕實驗、Transwell實驗驗證HIF-2在HPPCn調(diào)控腎癌細胞發(fā)生發(fā)展中的作用。 結(jié)果: 我們通過慢病毒載體構(gòu)建技術(shù)構(gòu)建了PLKO.1-shHIF-2，并將其包裝成慢病毒，它可以有效的干涉細胞內(nèi)的HIF-2的表達，干涉效率在蛋白質(zhì)水平和mRNA水平上均大于80%，病毒感染細胞后經(jīng)過克隆擴大培養(yǎng)，獲得了穩(wěn)定干涉HIF-2的786-0細胞，慢病毒的獲取和穩(wěn)定細胞的篩選為下一步闡述HPPCn在腎臟透明細胞癌中的作用奠定了基礎(chǔ)。 隨后，我們驗證了HPPCn蛋白通過對腎臟透明細胞癌中HIF-2的調(diào)控進而影響腎透明細胞癌細胞生物特性。我們用穩(wěn)定干涉HIF-2的786-0細胞來進行實驗，證明干涉786-0細胞內(nèi)HIF-2表達后，HPPCn蛋白對細胞的增殖能力，遷移能力，細胞的凋亡率都消失。 基于以上的研究，，我們得出結(jié)論HPPCn在腎臟透明細胞癌的生物特性方面有重要的作用，HPPCn是通過對HIF-2的調(diào)節(jié)來促進腫瘤細胞的增殖，遷移、活化和轉(zhuǎn)移等生物過程，該研究將為腎臟透明細胞癌的分子靶向治療提供新的理論支持。
[Abstract]:Objective significance:
HPPCn (Hepatopoietin Cn) is a newly discovered cell growth factor of the regenerated liver in our laboratory, which is widely expressed in many tissues as its main source. As a class of multifunctional nucleoprotein, it can be expressed in tumor tissues, and also in cells and normal tissues that have the ability to renew themselves. At the same time, it plays an important role in many aspects, including protein degradation, cytoskeleton dynamics, signal transduction between cells, and cell morphological changes and many other biological processes.
The transcriptional target genes of HIF-1 alpha and HIF-2 a are not exactly the same in CRCC cells, and the specific target genes of HIF-1 A and HIF-2 alpha are clearly defined. A variety of oncogenes, such as CyclinD1, TGF- a, VEGF and so on, are the specific target genes of HIF-2 alpha, while BNIP3 is the specific target gene of HIF-1 alpha, and HIF-1 A and alpha are different signal transduction pathways. Cross linking. Activated HIF-1 alpha can reduce the proliferation of c-Myc dependent cells, while HIF-2 alpha is the opposite. In addition to this, HIF-1 alpha is more sensitive than HIF-2 a to the degradation of proteasome in VHL missing CRCC cells. The multiple vascular features of.CRCC are closely related to the up regulation of HIF-2 alpha, while the survival period of the high expression HIF-1 alpha patient in CRCC is longer than that of other types of patients. Therefore, HIF, especially HIF-2 alpha, may play a more important role in the development of CRCC.
Recent studies have found that the expression and activity of HPPCn are closely related to the growth and prognosis of tumors..HPPCn can inhibit the apoptosis of hepatoma cells induced by various chemotherapeutic drugs (Sorafinib, ADM, CDD, TSA), and promote the metastasis and immersion of tumor cells by regulating the expression of mRNA and promoting the epithelial mesenchymal transformation (EMT) and vascular endothelial cell migration. The induction of liver cancer in transgenic mice showed that HPPCn could increase the expression of proto oncogene c-myc in the early induction and significantly increase the lung metastasis rate of the tumor. The tail vein injection experiment of hepatoma cells showed that the high expression of HPPCn could increase the lung metastasis of hepatoma cells and the number of blood vessels in the tumor. At the same time, the preliminary study also found that HPPCn was in the tumor. The expression of the renal clear cell carcinoma is significantly higher than that of the paracancerous and normal renal tissues. And it is found that HPPCn can promote the expression of HIF-2. Then, what is the relationship between the occurrence of clear cell carcinoma of the kidney and the development of the kidney with HPPCn? Is there any effect of HIF-2 in this process?
The aim of this study is to study the role and mechanism of HPPCn regulating HIF-2a in promoting the growth and metastasis of renal cell carcinoma.
Method:
The interference efficiency of HIF-2 was detected by Western blot and Q-PCR. By interfering with the expression of HIF-2 in tumor cells, MTS, scratch test and Transwell experiment were used to verify the role of HIF-2 in the regulation of the development of renal cell carcinoma cells by HPPCn blot and Q-PCR.
Result:
We constructed PLKO.1-shHIF-2 through the construction of lentivirus vector and packaged it into a lentivirus, which could effectively interfere with the expression of HIF-2 in the cell. The interference efficiency was more than 80% at the protein level and the level of mRNA. After the virus infected cells, the cells were cloned and expanded, and the 786-0 cells, which were stable interfering with HIF-2, were obtained. Obtaining and stabilizing cell screening laid the foundation for the next step to explain the role of HPPCn in renal clear cell carcinoma.
Then we verify that HPPCn protein affects the biological characteristics of renal clear cell carcinoma cells through the regulation of HIF-2 in the renal clear cell carcinoma. We use 786-0 cells that interfere with HIF-2 to carry out the experiment. It is proved that after the expression of HIF-2 in the 786-0 cells, the proliferation ability, migration ability and apoptosis rate of HPPCn protein are eliminated. Lost.
Based on the above study, we conclude that HPPCn plays an important role in the biological characteristics of renal clear cell carcinoma. HPPCn is a biological process that promotes the proliferation, migration, activation and metastasis of tumor cells through the regulation of HIF-2, which will provide new theoretical support for the molecular targeting therapy of renal cell carcinoma.

【學(xué)位授予單位】：濟南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.11

【參考文獻】

相關(guān)期刊論文 前1條

1 丁震宇,李澤桂;低氧誘導(dǎo)因子-1的生物學(xué)特性與胚胎發(fā)育[J];中國臨床康復(fù);2004年36期

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