【作者】 王旭；

【导师】 闫志勇；

【作者基本信息】 青岛大学 ， 外科学（专业学位）， 2023， 硕士

【摘要】 研究背景:一直以来,肿瘤作为影响全世界人口死亡的主要原因,已成为威胁人类健康的重大问题之一,也是医学难以解决的难题之一,作为神经系统恶性肿瘤,脑胶质母细胞瘤（GBM）占比率较高,且中位存活率较低,目前治疗尚未有有效的治疗方案,由于胶质母细胞瘤（GBM）肿瘤细胞具备可渗透到正常脑组织的固有能力,阻碍了手术根除性治疗,会导致较高的早期复发率。为了更好地了解GBM肿瘤进化和治疗耐药性的决定因素,癌症基因组图谱联盟（TCGA）对近600种GBM肿瘤进行了高维分析和分子分类,TCGA发现了三个与基因组异常密切相关的簇,称为经典（CL）、间充质（MES）和原神经（PN）^[1],前期的研究发现三种类型中PN型预后最好,分子层面的差异可能导致了表达亚型的不一致,最终决定预后的不同,近年来,研究发现组蛋白脱乙酰酶（HDAC）的乙酰化修行为与体内多种肿瘤的发生、发展及预后存在着紧密关联^[2]。有研究表明,引起HDAC表达失衡的相关调节因子同时也调节着相关肿瘤细胞的发生,发展与转归,现如今,许多与HDAC相关的调节因子被认为是肿瘤治疗的新靶点,组蛋白脱乙酰酶1（HDAC1）是HDAC家族中的重要一员^[3]。近些年来,很多研究证明了,HDAC1影响着身体多种肿瘤的发生和发展,在这些肿瘤中可能发挥促癌或抑癌作用。同时组蛋白脱乙酰酶1的乙酰转移酶活性也有可能影响这些肿瘤细胞的增殖和侵袭能力,对其生物学行为造成影响,进一步导致了恶性肿瘤的演变专归及预后^[4],我们课题组前期研究发现HDAC1的抑制剂RG2833在GBM细胞生长与亚型表达方面发挥着重要作用,然而抑制剂RG2833发挥何种作用及其机制尚不明确。本研究先利用生物信息学的方法筛选影响肿瘤生存及预后的关键因素,然后观察抑制剂RG2833对脑GBM细胞的生长,侵袭,转移功能的影响,旨在探讨抑制剂RG2833在脑GBM细胞生长生存中的影响作用,研究目的:通过公共数据库筛选不同亚型的GBM,并分析其预后差异,通过细胞培养研究人脑GBM细胞培养的方法,明确HDAC1在不同亚型的GBM中的表达,以及在HDAC1的抑制剂RG2833的作用下对GBM细胞系生长、增殖及侵袭能力的影响。观察在RG2833影响下培养肿瘤细胞的生存生长活性与肿瘤级别之间的关系,探究RG2833与临床药物合用的可能性。研究方法:选取U251细胞系和LN229细胞系,通过基因组表达的不同,将二者划分为GBM不同类型中预后较好的PN型和预后较差的MES型,明确HDAC1在不同亚型的GBM中的表达,我们通过细胞培养,细胞传代,cck8实验,划痕实验,transwell侵袭实验探索在不同浓度RG2833的影响下两种分型的肿瘤细胞系的迁徙和侵袭能力的差别,对照不同亚型,我们比较影响肿瘤细胞生长的最佳抑制浓度。针对MES型胶质母细胞我们通过功能试验来验证RG2833对其的影响。结合临床用药替莫唑胺,研究与RG2833联合用药对GBM细胞的生长侵袭影响。研究结论:一、胶质母细胞瘤的分子亚型影响了肿瘤的转归和病人的预后。二、抑制剂RG2833可以影响胶质母细胞瘤细胞生长侵袭迁移能力且对MES型胶质母细胞瘤有明显的抑制作用。三、RG2833是通过组蛋白去乙酰化对MES型胶质母细胞瘤细胞发挥抑制作用。四、RG2833与替莫唑胺（TMZ）联合用药,抑制MES型胶质母细胞瘤细胞生长更明显。更多还原

【Abstract】 Research Background: For a long time,tumor,as the main cause of death in the world,has become one of the major problems threatening human health and one of the difficult problems to be solved by medicine.As a malignant tumor of the nervous system,glioma accounts for a high proportion and the median survival rate is low.Currently,there is no effective treatment plan.Due to the innate ability of glioblastoma multiforme(GBM)tumor cells to penetrate into normal brain tissue,surgical eradication is prevented and leads to a high rate of early recurrence.In order to better understand The determinants of GBM tumor evolution and treatment resistance,The Cancer Genome Atlas(TCGA)conducted a high-dimensional analysis and molecular classification of nearly 600 GBM tumors,and TCGA identified three clusters that were closely related to genomic abnormalities.They are called classical(CL),mesenchymal(MES),and neuronal(PN).Previous studies have found that PN has the best prognosis among the three types.Differences in the molecular level may lead to inconsistencies in the expression of subtypes,ultimately determining the different prognosis.Acetylation modification of HDAC is closely related to the occurrence,development and prognosis of various tumors in vivo.Studies have shown that the regulatory factors that cause the imbalance of HDAC expression also regulate the occurrence,development and outcome of related tumor cells.Now,many regulatory factors related to HDAC are considered as new targets for tumor therapy.histone deacetylase1(HDAC1)is an important member of the HDAC family.In recent years,many studies have proved that HDAC-1 affects the occurrence and development of various tumors in the body,and may play a role in promoting or suppressing cancer in these tumors.At the same time,the acetyltransferase activity of histone deacetylase 1 May also affect the proliferation and invasion ability of these tumor cells,affecting their biological behavior,and further leading to the evolution and prognosis of malignant tumors.Our previous study found that the inhibitor of HDAC-1,RG2833,plays an important role in the growth of glioma cells and expression of subtypes.However,the role and mechanism of the inhibitor RG2833 remain unclear.This study first used bioinformatics methods to screen the key factors affecting tumor survival and prognosis,and then observed the effect of inhibitor RG2833 on the growth,invasion and metastasis of glioma cells,aiming to explore the effect of inhibitor RG2833 on the growth and survival of glioma cells.Objective: To screen different subtypes of GBM in public database and analyze their prognostic differences,and to study human glioma cell culture by cell culture method,so as to clarify the expression of HDAC1 in different subtypes of glioma and the effect of HDAC1 inhibitor RG2833 on the growth,proliferation and invasion ability of glioma cell lines.To observe the relationship between survival and growth activity of cultured tumor cells and tumor grade under the influence of RG2833,and explore the possibility of combining RG2833 with clinical drugs.Research methods: Different subtypes of GBM were screened in the public database and their prognostic differences were analyzed.U251 cell lines and LN229 cell lines were selected and divided into PN type with good prognosis and MES type with poor prognosis among different types of glioma based on their different genomic expressions,so as to clarify the expression of HDAC1 in different subtypes of glioblastoma.We used cell culture,cell passage,CCK8 assay,scratch assay and transwell invasion assay to explore the differences in migration and proliferation ability of the two tumor cell lines under the influence of different concentrations of RG2833.Compared with different subtypes,we compared the optimal inhibitory concentration affecting the growth of tumor cells.Functional tests were conducted to verify the effect of RG2833 on the function of MES glioblasts.Combined with clinical drug Temozolomide,to study the effect of combined drug with RG2833 on the growth and invasion of glioblastoma cells.Research conclusion:First:Molecular subtypes of glioblastoma influence tumor specificity and patient prognosis.Second:inhibitor RG2833 can affect the growth,invasion and migration ability of glioblastoma cells and has obvious inhibitory effect on MES type glioblastoma.Three : RG2833 exerts an inhibitory effect on glioma cells through histone deacetylation.Four : The combination of RG2833 and temozolomide(TMZ)can significantly inhibit the growth of MES glioma cells.更多还原