摘要
研究背景
脑胶质瘤是中枢神经系统最为好发的、恶性程度很高的肿瘤,目前临床给予手术加放疗、化疗的综合治疗方案后,高级别的胶质瘤患者其中位生存期仅能达到16.7个月,其在很大程度上是由于复发并形成转移灶所致。传统的化疗药物是通过促进细胞的凋亡,达到抑制肿瘤的生长,但耐药性出现较快。为了逆转这种现象,我们急需寻找新的细胞蛋白靶点,改进目前的化疗方案,延长患者的生存期。细胞死亡目前认为包括3种类型：坏死、凋亡和自噬性细胞死亡。在肿瘤细胞中,凋亡蛋白都有不同程度的缺陷(限制药物对凋亡通路的激活),自噬(现在被认为是Ⅱ型细胞死亡,有别于细胞凋亡)是指细胞通过单层或双层膜包裹待降解物形成自噬体,然后形成自噬溶酶体并进行消化及降解,其作为一种细胞的保护机制,其蛋白往往是被优先保留的。mTOR信号通路是细胞生长、增殖及细胞周期的中心调控器,其也是PI3K/AKT信号通路(调控细胞自噬)的中心环节。依维莫司作为mTOR信号通路特异性的阻断剂,近年来发现其对肝癌、胰腺癌等肿瘤有明显的抗癌作用。但是目前关于mTOR通路及其抑制剂对胶质瘤的研究较少,有以下不足之处：①mTOR相关通路蛋白在人脑胶质瘤的表达及其机制仍未完全明确;②mTOR抑制剂对不同遗传背景胶质瘤细胞株中对凋亡和自噬的影响及其之间的关系仍不明确;③mTOR抑制剂诱导对胶质瘤裸鼠皮下移植瘤的疗效仍不明确。本研究在既往研究的基础上,试图通过体外培养来自不同遗传背景的胶质瘤细胞株U-87MG、 U251、SHG44,给予不同浓度的依维莫司,来检测胶质瘤细胞株给药前后细胞生长、细胞周期变化及细胞的自噬；并通过建立三种胶质瘤细胞株裸鼠皮下移植瘤模型,给与依维莫司腹腔注射,采用免疫组化、细胞周期、肿瘤血管密度等方法进行测定,来检测依维莫司对不同细胞株抑制情况。试图证明mTOR信号通路能够作为胶质瘤治疗的一个新的靶点,为脑胶质瘤的治疗提供新的实验依据。
研究目的
探讨依维莫司作用于人U87-MG、 U251-MG、SHG-44胶质瘤细胞的体内外作用及其可能的机制。
1.培养胶质瘤细胞株U87、U251、SHG44,并用不同浓度的药物干预细胞的生长,明确其凋亡和自噬情况。
2.建立胶质瘤细胞株U87、U251、SHG44裸鼠移植瘤模型,并用不同浓度的药物干预移植瘤的生长,明确依维莫司对不同细胞株抑制情况及其机制。
研究方法：
1.免疫组织化学染色检测32例人脑胶质瘤标本中mTOR信号通路相关蛋白表达情况,并用Western Blot法检测人脑胶质瘤标本的mTOR蛋白的表达情况。
2.培养稳定生长的胶质瘤细胞株U87、U251、SHG44,采用MTT法观察不同剂量mTOR抑制剂依维莫司对人脑胶质瘤细胞抑制作用；细胞凋亡荧光Hoechst33342/PI双染发测定细胞细胞凋亡；单丹磺酰尸胺(MDC)荧光染色检测自噬囊泡；流式细胞法定量观察细胞周期；Western blot法进一步分析验证依维莫司对胶质瘤细胞作用的通路及其机理。
3.通过建立胶质瘤细胞株U87、U251、SHG44裸鼠皮下移植瘤模型,并给与依维莫司腹腔注射,采用免疫组化、细胞周期及凋亡、肿瘤血管密度等方法进行测定,了解依维莫司对不同胶质瘤细胞株移植瘤的抑制作用,并探讨其可能引起的机制。
研究结果：
1. mTOR蛋白在Ⅲ、Ⅳ级胶质瘤较Ⅰ、Ⅱ级胶质瘤的表达有统计学意义(P<0.01),Ⅰ级胶质瘤与对照组表达无统计学意义(P>0.05)。
2.体外培养的胶质瘤细胞株中,不同细胞株受抑制程度均随着依维莫司浓度的增加而增加。其中U87-MG细胞株是最敏感的；U251细胞株有中度敏感性；SHG-44细胞株是最不敏感的。
3.细胞凋亡荧光Hoechst33342/PI双染结果显示各株胶质瘤细胞均发生了一种不同于凋亡的死亡方式,可能为细胞自噬。行MDC活细胞染色显示,依维莫司作用于胶质瘤细胞增加了酸性囊泡样细胞器(自噬泡)的形成,可认为依维莫司可促进胶质瘤细胞自噬的形成。三种细胞株均给与IC50剂量的依维莫司,在48小时后进行流式细胞周期分析。我们发现三种细胞株均有特异性的G0/G1期细胞阻滞。用AnnexinV检测没有发现明显的细胞凋亡。
4.用Western blot分析依维莫司作用于胶质瘤细胞株后对mTOR, p70S6K,4E-BP-1和p70S6K的非磷酸化和磷酸化水平的影响。在所有三个细胞株中,随着依维莫司剂量的增加,mTOR、p70S6K、4E-BP-1的磷酸化表达水平均明显减少,非磷酸化的p70S6K并没有发现具有明显的变化,只有4E-BP-1的非磷酸化表达水平亦随依维莫司浓度的升高而有所减少。
5.建立胶质瘤细胞株U87、U251、SHG44裸鼠皮下移植瘤模型,绘制移植瘤生长曲线。在第12天末开始给与裸鼠依维莫司后发现,各细胞株实验组裸鼠移植瘤体积较对照组明显减小,并通过公式测量不同细胞株的抑瘤率,U-87MG, SHG-44, U251细胞株的抑瘤率分别为30.6%,42.1%,29.7%,依维莫司对胶质瘤有明显的抑制作用。
6.对不同胶质瘤细胞株行细胞凋亡测定,各细胞株中实验组较对照组的凋亡并没有明显的统计学差异(P>0.05)。行细胞周期测量,各细胞株中实验组较对照组均有明显的G0/G1期停滞(P<0.05)。其中U-87MG细胞株的G0/G1期停滞较对照组差异最大,SHG-44次之, U251最小。
7.行肿瘤血管密度测定,细胞株U-87MG、U-251中实验组肿瘤血管密度值较对照组没有明显差异(P>0.05),细胞株SHG-44实验组肿瘤血管MVD值为31.1(25.0-37.2),较对照组20.9(14.8-27.0)有明显差异(P<0.05)。进一步测量不同细胞株VEGF的表达水平,在SHG-44细胞株实验组的VEGF表达水平较对照组明显下调,其余两种细胞株间没有发现明显差异。
8.分离肿瘤组织后,免疫组化分析各标本,其中U251细胞株实验组较对照组的pmTOR表达增加,p70S6K1、pAKT表达减少,VEGF表达没有明显差异；U-87MG细胞株实验组较对照组的表达p70S6K1表达减少,pAKT、pmTOR、VEGF表达均没有明显差异；SHG-44细胞株实验组较对照组的pmTOR、p70S6K1表达均没有明显差异、pAKT表达减少,VEGF表达明显减少。
结论：
1. mTOR抑制剂依维莫司通过阻滞细胞周期,诱导胶质瘤细胞的自噬,并抑制肿瘤血管的生成,从而发挥抗胶质瘤的作用。
2. mTOR信号通路能够作为治疗胶质瘤的一个新靶点,为脑胶质瘤的治疗提供新的思路。
BACKGROUND
Glioma is a central nervous system is the most good, highly malignant tumor, the present study to give the surgery plus radiotherapy, and chemotherapy treatment programs, senior other glioma patients with a median survival of only16.7its due in large part because of the recurrence and the formation of metastases. Traditional chemotherapy drugs to inhibit tumor growth by promoting cell apoptosis, but the rapid emergence of resistance. In order to reverse this phenomenon, we need to find a new cellular protein targets to improve the current chemotherapy regimens prolong survival of patients. Cell death is considered to include three types:necrosis, apoptosis and autophagic cell death. Apoptotic proteins in cancer cells, have varying degrees of impairment (restricted drugs on the activation of the apoptotic pathway), autophagy (now considered to be type Ⅱ cell death, unlike apoptosis) refers to the cells by single or bilayers wrapped to be degradation products to the formation of autophagosomes, and the formation of autophagic lysosomes and the digestion and degradation, as a cellular protection mechanism, and its protein are preferentially retained. mTOR signaling pathway of cell growth, proliferation and cell cycle regulation, the PI3K/AKT signaling pathway (regulation of autophagy) is the central link. Everolimus mTOR signaling pathway-specific blocker, in recent years significant role in cancer of the liver, pancreas and other tumors. On the mTOR pathway and its inhibitors on glioma less, the following shortcomings:①of mTOR-related pathway protein expression in human gliomas and its mechanism is still not entirely clear;②mTOR inhibitor on different genetic background glioma cell lines and the relationship between apoptosis and autophagy remains unclear;(3) mTOR inhibitor-induced gliomas subcutaneously into nude mice transplanted tumor efficacy remains unclear. In this study, on the basis of previous research, trying in vitro from different genetic backgrounds glioma cell line U-87MG, of U251, of SHG44treated with different concentrations of everolimus to detect the administration of glioma cell lines before and after cell growth, cell cycle and cell autophagy; and through the establishment of the three kinds of glioma cell lines subcutaneously into nude mice xenograft model, given by intraperitoneal injection of everolimus, immunohistochemistry, cell cycle, tumor vascular density and other methods of measurement, to detect everolimus inhibition of different cell lines. Trying to prove that the mTOR signaling pathway can be used as a new target for glioma therapy, a new experimental basis for the treatment of glioma.
OBJECTIVE
Explore the role of everolimus people in U87-MG, of U251-the MG, the SHG-44glioma cells in vivo and in vitro and its possible mechanism.
1. cultured glioma cell lines in U87, of U251, of SHG44with different concentrations of drug intervention in cell growth, clear apoptosis and autophagy.
2.the establishment of strains in U87glioma cells of U251, of SHG44nude mouse xenograft tumor model, tumor growth with different concentrations of drug intervention, clear and its mechanism of inhibition of different cell lines according to Weimo Secretary.
METHODS
1. The mTOR signaling pathway related protein expression and mTOR protein expression by Western Blot Detection of human glioma specimens, an immunohistochemical staining detected32cases of human glioma specimens.
2. Fostering stable growth in glioma cell lines in U87, of U251, of SHG44, MTT assay was used to observe different doses of the mTOR inhibitor everolimus inhibition of human glioma cells; apoptosis fluorescent Hoechst33342/PI hair Determination of cell apoptosis; single dansyl cadaverine (MDC), the fluorescence staining of autophagic vesicles; flow cytometry for quantitative cell cycle; Western blot for further analysis to verify the pathways of everolimus on the role of glioma cells and its mechanism.
3. Through the establishment of the glioma cell lines in U87, of U251, of SHG44nude mouse subcutaneous xenograft model, and given intraperitoneal injection of everolimus, immunohistochemistry, cell cycle and apoptosis, tumor vascular density were measured, understanding of everolimus inhibition of transplanted tumors of different glioma cell lines and to explore its possible mechanism.
RESULTS
1. There were significant differences in the expression of mTOR protein between the control and grade Ⅱ, grade Ⅲ or grade Ⅳ (all P<0.01). There was no significant difference in the expression of those protein between the control and grade Ⅰ (P>0.05)..
2. Glioma cell lines in vitro in different cell lines by the degree of inhibition increased with the in accordance with the increase in the concentration of Weimo Secretary. In U87-MG cell line is most sensitive; U251cell lines with moderate sensitivity; the SHG-44cell line is the least sensitive.
3. Apoptosis fluorescent Hoechst33342/PI double staining results showed that the glioma cell a different apoptotic forms of death, may be autophagy. OK MDC live cell staining showed that the glioma cells to increase the role of everolimus in the acidic vesicle-like organelles (from macrophage foam) formation that everolimus can promote the formation of glioma cell autophagy. The three cell lines are given to the IC50dose of everolimus, flow cytometry cycle analysis after48hours. We found that the three cell lines are specific cells in G0/G1phase arrest. Using the AnnexinV detection did not find significant apoptosis.
4. Western blot analysis of the role of everolimus in glioma cell lines after the non-phosphorylation of mTOR, of p70S6K,4E-BP-1and p70S6K and phosphorylation levels. In all three cell lines, with the increase in everolimus dose of mTOR, of p70S6K,4E-BP-1phosphorylation expression levels were significantly reduced phosphorylation of p70S6K and did not find significant changes, only The phosphorylation of4E-BP-1expression levels tandem according Weimo Secretary concentration increased and decreased.
5. Established in U87glioma cell lines of U251, of SHG44nude mice subcutaneous xenograft model, to draw the tumor growth curve. Give nude everolimus found at the end of12days, the transplanted tumor volume of each cell line the experimental group compared with the control group was significantly reduced, and by a formula measuring the inhibition rate of the different cell lines, U-87MG the SHG-44and U251cells lines, tumor inhibition rates were30.6%,42.1%,29.7%, everolimus significantly inhibited glioma.
6. Determination of different glioma cell lines line apoptosis, each cell line the experimental group and control group, apoptosis, and there is no statistically significant difference (P>0.05). OK cell cycle measurement, each cell line in the experimental group than in the control group were significantly G0/G1phase arrest (P<0.05). U-87MG cell lines of the G0/G1phase of stagnation than the control group, the biggest difference, the SHG-44, followed by the smallest of U251.
7. Determination of tumor vascular density, cell line U-87MG, U-251in tumor vascular density values of the experimental group than the control group did not differ significantly (P>0.05), tumor blood vessels of the cell line SHG-44experimental group MVD value of31.1(25.0-37.2) compared with the control group20.9(14.8-27.0) were significantly different (P <0.05). Further measurement of the different cell lines the expression of VEGF in the SHG-44cell line the experimental group, VEGF expression level compared with the control group was significantly lowered, and found no significant difference in the remaining two cell lines.
8. Separation of tumor tissue, the immunohistochemical analysis of various specimens, which the U251cell line the experimental group than in the control group pmTOR expression increase in p70S6K1, pAKT expression was decreased, VEGF expression did not differ significantly; U-87MG cell line the experimental group than the control group expression of p70S6K1expression to reduce pAKT pmTOR, VEGF expression did not differ significantly; SHG-44cell line the experimental group than the control group pmTOR, p70S6K1expression did not differ significantly, pAKT expression was decreased, VEGF expression significantly reduced.
CONCLUSION
1. mTOR inhibitor everolimus by blocking the cell cycle, thereby inducing autophagy of glioma cells, and inhibit tumor angiogenesis, which play the role of anti-glioma.
2. mTOR signaling pathway can be used as a new target in the treatment of gliomas, and to provide new ideas for the treatment of glioma.
引文
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