HDGF基因在胶质瘤中调控细胞生长、侵袭、迁移及其替莫唑胺敏感性的研究

发布时间：2018-04-30 18:45

本文选题：胶质瘤 + HDGF　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景与目的胶质瘤是中枢神经系统最常见的、预后较差的原发性恶性肿瘤,占颅内肿瘤的50%-60%,即使给予传统的手术联合放、化疗的综合治疗,低级别胶质瘤病人的平均存活时间仅为3-5年,而高级别胶质瘤为1-2年。其治疗一直是神经外科领域的研究热点,胶质瘤的传统治疗策略是在手术尽可能全切除的基础上,采用多种手段杀灭残存瘤细胞或抑制其增殖。但即使是相同病理类型和相同WHO级别的胶质瘤患者,其预后也存在很大差异,治疗上的差异是一方面,但胶质瘤内在的生物学特性,特别是分子水平的不同是关键所在。肝癌衍生生长因子(hepatoma-derived growth factor, HDGF)是从人肝癌细胞系HUH-7培养液中分离出一种的肽类物质。大量研究发现HDGF在多种恶性肿瘤中高表达,具有促进细胞增殖、促进血管生成等多种生理作用。HDGF在多种恶性肿瘤中系高表达,如有研究表明HDGF在正常的肝细胞、肝癌细胞及内皮细胞中均有表达,且在胞浆及胞核中都有分布,癌组织中的HDGF表达水平明显高于癌周正常组织,肿瘤细胞核HDGF表达水平与患者的AFP水平呈正相关关系、与肿瘤的分化程度呈负相关关系；在乳腺癌中HDGF核表达程度和肿瘤的分期、分级及增殖密切相关,在细胞水平上,腺病毒转染高表达HDGF乳腺癌细胞中E钙粘蛋白表达降低而波形蛋白表达增高,从而诱导上皮细胞向间叶细胞的转化(EMT过程),使得乳腺癌的侵袭性及转移能力增强；在恶性程度较高的口腔癌中,HDGF呈现高表达,HDGF核表达的程度与口腔癌病理分期、分级及肿瘤坏死程度呈正相关。 HDGF在胶质瘤中鲜有研究,且其在胶质瘤中的具体功能尚未明确,本研究首先通过免疫组化方法明确HDGF在胶质瘤组织中的表达特性；进一步通过构建稳定低表达HDGF胶质瘤细胞系,探讨HDGF表达沉默后对于胶质瘤细胞系生物学行为的影响及其所涉及的具体信号通路,将有助于进一步揭示胶质瘤的发病机理及预后,并有可能为今后深入探讨胶质瘤的发生发展机制和今后的临床防治提供新的依据。研究内容与方法 1.胶质瘤及正常脑组织中HDGF表达鉴定 ①利用RT-PCR检测HDGF在胶质瘤及正常脑组织中mRNA表达水平。 ②利用Western blot检测HDGF在胶质瘤及正常脑组织中蛋白表达水平。 ③免疫组化检测胶质瘤及正常脑组织中HDGF的表达情况。 2.稳定低表达HDGF胶质瘤细胞株的功能研究在人胶质瘤细胞株U251、U87中,采用慢病毒载体技术将HDGF沉默表达,同时设立阴性对照组,并利用RT-PCR及Western blot检测其干扰效率。通过平板克隆实验、MTT法、EdU实验、划痕实验、Transwell小室迁移实验、Boyden小室侵袭实验等检测干扰HDGF后对胶质瘤细胞株生长、周期、侵袭、迁移等方面的影响。 3. HDGF介导的机制研究通过细胞功能实验我们发现,干扰HDGF表达后细胞生长、侵袭及迁移能力均降低。因此,采用Western blot定量检测细胞侵袭及生长相关蛋白的表达,如β-catenin、Vimentin、E-cadherin. N-cadherin及PI3K/Akt、TGF-beta信号通路相关蛋白。 4.替莫唑胺(TMZ)敏感性检测及IC50测定首先IC50定义是胶质瘤细胞株生长被抑制一半时替莫唑胺的浓度。接下来,为了研究HDGF基因是否影响药物敏感性,将干扰HDGF组和对照组分别在替莫唑胺浓度为25μM、50μM、100μM、200μM和400μM的培养基中培养48个小时,随后用MTT法对细胞生长进行检测并测定IC50。结果 1.胶质瘤及正常脑组织中HDGF表达鉴定①根据RT-PCR结果,可发现HDGF在胶质瘤组织中表达较正常脑组织明显上调(P0.0001)。 ②Western blot实验结果显示,与正常脑组织比较,HDGF在胶质瘤组织中上调,其差异有统计学意义(P0.01) ③免疫组化结果表明,与正常脑组织比较,HDGF在胶质瘤组织中显著上调(P0.001),且在大多数细胞中HDGF为核表达,少数也在细胞浆中表达。此外,根据组织标本的临床资料的统计分析,不同性别、年龄、组织学分型患者HDGF表达程度无显著差异(P值分别为0.395、0.513和0.573)。但胶质瘤组织WHO级别越高,HDGF表达越高(P0.01),即HDGF表达程度与胶质瘤的恶性程度相关。 2.稳定低表达HDGF胶质瘤细胞株的功能研究为了研究HDGF在胶质瘤细胞中的功能,我们选取了经典的U251、U87细胞株,构建稳定低表达HDGF的慢病毒载体,包装成成熟的慢病毒,转染胶质瘤细胞株。随后,采用blot实验对比干扰HDGF组及对照组细胞HDGF蛋白表达情况,在干扰组中HDGF表达明显下调(P0.001)。鉴定完干扰效率后,采用两株细胞的干扰HDGF组及阴性对照组细胞进行细胞功能实验。采用平板克隆实验和MTT法检测两组细胞的体外增殖能力的差异。两种实验的结果均显示,下调HDGF表达后,与阴性对照组相比,两株细胞的干扰组的体外增殖能力均明显减弱。统计学分析表明,两组间细胞的增殖能力差异具有统计学意义(P0.001)。接下来,为了剖析干扰组细胞增殖能力改变的原因,我们采用EdU实验检测两组细胞中处于细胞周期S期的细胞的数量。结果显示,干扰组的S期细胞较对照组减少,其差异具有统计学意义(P0.01)。这一结果也从侧面证明了干扰HDGF组细胞较对照组细胞的增殖能力减弱。采用划痕实验及Transwell小室迁移实验检测沉默表达HDGF后细胞的体外迁移能力。Transwell小室迁移实验结果显示,将HDGF沉默后,干扰组细胞穿过膜的细胞数显著减少,提示干扰组的细胞迁移能力明显减弱,差异具有显著性；同样,划痕实验也得到了一样的结果(P0.001)。为了检测干扰组细胞的侵袭能力的变化,我们采用了Boyden小室侵袭实验。结果显示,两组细胞均有穿透基质胶向下侵袭的现象,并在聚碳酸酯膜上可见肿瘤细胞形态改变。与阴性对照组相比,干扰组穿过膜的细胞明显减少,其体外侵袭能力降低(P0.001)。 3. HDGF介导的机制研究根据细胞功能结果可见,干扰HDGF表达后,胶质瘤细胞的增殖能力、迁移能力及侵袭能力均下降。为了进一步研究HDGF调控胶质瘤发生发展的机制,我们采用western blot检测了与肿瘤增殖、迁移、侵袭相关蛋白及PI3K/AKT、 TGF-beta信号通路。结果如下： ①Western blot定量分析表明,较阴性对照组相比,干扰组的侵袭、迁移相关蛋白表达除了E-cadherin无明显变化外,beta-catenin, vimentin、N-cadherin 均下降(P0.05)。 ②采用Western blot检测增殖相关的经典信号通路PI3K/AKT和TGF-beta,结果表明：下调HDGF后,PI3K、AKT总蛋白及磷酸化的PI3K、AKT亦下调(P0.05); TGF-beta通路中,TGF-beta、c-myc、CCND1、p16及p21蛋白表达均减弱(P0.05)。 4.替莫唑胺(TMZ)敏感性检测及ICso测定将干扰HDGF组和对照组分别在替莫唑胺浓度为25μM、50μM、100μM、200pM和400μM的培养基中培养48小时,随后用MTT法对细胞生长进行检测,结果显示：与正常生长的细胞相比,U251干扰组细胞在含有不同替莫唑胺浓度的培养基中分别被抑制9.4%、15.0%、35.3%、49.4%、67.2%,U251对照组细胞分别被抑制3.7%、6.5%、13.9%、28.9%、43.9%,两组间差异有统计学意义(P0.05)；U87干扰组分别被抑制11.8%、14.8%、27.5%、37.1%、49.7%,U87对照组细胞分别被抑制2.7%、4.3%、12.3%、17.5%、32.5%,两组间差异亦有统计学意义(P0.05)。随后测定的IC50为：U251干扰组202.5,对照组487.3；U87干扰组406.3,对照组808.4。讨论从本研究的结果中可知,HDGF在胶质瘤组织中系高表达,且以核表达为主,与肿瘤的恶性程度呈正相关。细胞功能实验显示,下调HDGF基因后,胶质瘤细胞株U251、U87的增殖能力、侵袭能力及迁移能力均下降。为进一步阐明HDGF调控胶质瘤发生发展的机制,我们检测了大量与细胞增殖、细胞周期、侵袭、迁移相关的蛋白,从中发现,HDGF可能通过PI3K/AKT及TGF-beta两条通路共同作用来调控胶质瘤的发生发展。此外,HDGF沉默表达后,beta-catenin、vimentin、N-cadherin等侵袭相关蛋白亦出现沉默表达,提示HDGF可能参与调控与上皮间质转化(Epithelial-to-Mesenchymal Transition, EMT)过程。最后,我们检测了HDGF对胶质瘤药物敏感性的影响,可以发现当下调HDGF基因时,胶质瘤细胞株U251、U87对于药物替莫唑胺的敏感性上升,由此可见HDGF可能与胶质瘤的药物耐药性有关。综上所述,HDGF作为一个癌基因,拥有一个复杂的调控机制,本研究发现HDGF可能通过PI3K/AKT及TGF-beta两条通路的共同作用来调控胶质瘤的发生发展,可能参与上皮间质转化过程,且与药物的耐药性相关,揭示HDGF的具体调控机制有助于阐明肿瘤的发生发展机制,并有可能成为药物治疗的一个新的靶点。
[Abstract]:Background and purpose of study

Gliomas are the most common and poor prognosis primary malignant tumors of the central nervous system , accounting for 50 - 60 % of the intracranial tumors , and the average survival time of the patients with low grade glioma is only 3 - 5 years , and the high - grade glioma is 1 - 2 years .

The expression level of HDGF in normal liver cells , liver cancer cells and endothelial cells was significantly higher than that in normal tissues , and the expression level of HDGF in cancer tissues was significantly higher than that in normal tissues .
The expression of E - cadherin and the expression of E - cadherin in human breast cancer cells were closely related to the level of expression of E - cadherin in human breast cancer . The expression of E - cadherin in human breast cancer cells was decreased , and the expression of P - cadherin increased , thus inducing the transformation of epithelial cells to mesenchymal cells ( EMT process ) , which resulted in increased invasion and metastasis ability of breast cancer .
HDGF was highly expressed in the higher malignant degree of oral cancer , and the level of HDGF was positively correlated with pathological stage , grade and tumor necrosis degree of oral cancer .

The specific function of HDGF in glioma was not clear . First , the expression of HDGF in glioma tissues was determined by immunohistochemistry .
Furthermore , by constructing the stable low - expression HDGF glioma cell line , the effects of HDGF on the biological behavior of glioma cell lines and the specific signal pathways involved will be discussed , which will help to further reveal the pathogenesis and prognosis of glioma .

Study contents and methods

Expression of HDGF in glioma and normal brain tissue

( 1 ) The mRNA expression level of HDGF in human glioma and normal brain tissue was detected by RT - PCR .

2 . Western blot was used to detect the expression level of HDGF in human glioma and normal brain tissue .

( 3 ) The expression of HDGF in glioma and normal brain tissues was detected by immunohistochemistry .

2 . The function of stable low - expression HDGF glioma cell line was studied in human glioma cell lines U251 and U87 , and the interference efficiency was detected by RT - PCR and Western blot . The effects of HDGF on growth , cycle , invasion and migration of glioma cell line were studied by means of plate cloning , MTT assay , EdU experiment , scratch test , Transwell chamber migration experiment , Boyden chamber invasion experiment and so on .

3 . The mechanism of HDGF - mediated mechanism was found to decrease the cell growth , invasion and migration ability after interfering with HDGF expression . Therefore , Western blot was used to quantitatively detect the invasion and growth - related protein expression , such as 尾 - catenin , Vimentin , E - cadherin . N - cadherin and PI3 , TGF - beta signaling pathway related proteins .

4 . The IC50 determination of temozolomide ( TMZ ) was first defined as the concentration of temozolomide when the growth of glioma cell line was inhibited by half . Next , in order to study whether the HDGF gene affected drug sensitivity , the interference HDGF group and the control group were cultured for 48 hours in the medium of temozolomide concentration of 25 . m u.M , 50 . m u.M , 100 . m u.M , 200 . m u.M and 400 . m u.M , respectively , and then the cell growth was detected and the IC50 was determined by MTT assay .

Results

1 . The expression of HDGF in human glioma and normal brain tissue was determined ( 1 ) According to the results of RT - PCR , it was found that the expression of HDGF in glioma tissues was significantly higher than that in normal brain tissue ( P < 0 . 0001 ) .

The results of Western blot showed that HDGF was up - regulated in glioma tissues compared with normal brain tissue ( P0.01 ) .

In addition , the higher the WHO grade of glioma , the higher the level of HDGF , the higher the expression of HDGF ( P0.01 ) , that is , the degree of HDGF was correlated with the degree of malignancy of glioma .

2 . In order to study the function of HDGF in glioma cells , we selected classical U251 and U87 cell lines , constructed a slow virus vector stably expressing HDGF , packaged into mature lentivirus and transfected glioma cell line .

The results showed that the S phase cells of the interfering group decreased significantly compared with the control group ( P0.01 ) . The results also showed that the proliferation ability of the cells from the interfering HDGF group was weakened compared with the control group .

Transwell small cell migration experiment showed that the number of cells passing through the membrane decreased significantly after the silencing of HDGF , suggesting that the cell migration ability of the interfering group was significantly decreased , and the difference was significant .
Similarly , the same results were obtained in the scratch experiment ( P0.001 ) .

In order to detect the invasion ability of interfering group cells , we used Boyden ' s cell invasion experiment . The results showed that both groups had the phenomenon of penetrating matrix glue and the morphological changes of tumor cells were seen on the polycarbonate membrane . Compared with the negative control group , the cells of the interference group decreased significantly , and the invasion ability in vitro was decreased ( P0.001 ) .

3 . Mechanism of HDGF - mediated Mechanism

In order to further study the mechanism of HDGF in the development of glioma , Western blot was used to detect the proliferation , migration , invasion - related protein and the 3 - 3 / 3 - 1 , TGF - beta signaling pathway .

Western blot analysis showed that compared with the negative control group , the invasion and migration of the interference group were related .

In addition to E - cadherin , the expression of 尾 - catenin , vimentin , N - cadherin

Both decreased ( P0.05 ) .

2 . Western blot was used to detect the proliferation - related classical signal pathway of PI3 - 3 and TGF - beta . The results showed that , after downregulating the HDGF , the total protein and the phosphorylation of PI3 - 3 were down - regulated ( P0.05 ) , and the expression of TGF - beta , c - myc , CCND1 , p16 and p21 protein in TGF - beta pathway decreased ( P0.05 ) .

4 . Temozolomide ( TMZ ) Sensitivity Detection and Determination of ICso

Cell growth was detected by MTT assay . The results showed that U251 cells were inhibited by 9 . 4 % , 15.0 % , 35 . 3 % , 49.4 % , 67.2 % in culture medium containing different temozolomide concentration , and that of U251 control group were inhibited by 3 . 7 % , 6 . 5 % , 13.9 % , 28 . 9 % and 43.9 % , respectively .
U87 interfered group was inhibited 11.8 % , 14.8 % , 27.5 % , 37.5 % , 49.7 % , U87 control group cells were inhibited 2.7 % , 4.3 % , 12.3 % , 17.5 % , 32.5 % , respectively . The IC50 of U251 interference group was 202.5 and 487.3 % in U251 interference group .
U87 interfered group 406.3 , control group 808.4 .

discuss

In conclusion , HDGF may be involved in regulating the development of glioma . In conclusion , HDGF may be involved in regulating and regulating the development of glioma . In conclusion , HDGF may be involved in regulating and regulating the development of glioma . In conclusion , HDGF may be involved in regulating and regulating the development of glioma .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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