摘要
第一部分 Midkine和Pleiotrophin及受体在脑膜瘤中的表达
脑膜瘤是中枢神经系统最常见的肿瘤之一。与恶性星形细胞瘤相比,大多数脑膜瘤病人生活质量良好。但是,回顾大宗病例结果不容乐观。近年来,脑膜瘤分子生物学和基因研究的发展在临床提高了对侵袭性脑膜瘤预后的预测能力,并促进发展新的靶向治疗方法以提高疗效。肝素结合性生长因子家族,由2个成员组成,即中期因子(midkine,MDK)和多效营养因子(pleiotrophin,PTN)。MDK和PTN在多种肿瘤组织中表达,具有多种生物学功能,影响肿瘤细胞的存活、增殖、分化和迁移等,已成为恶性肿瘤诊断和治疗中颇具前景的分子靶点。目前,有关MDK、PTN和脑膜瘤的关系,研究得很少。因此,本研究对MDK和PTN及受体在脑膜瘤中表达进行了探讨。
1、材料和方法
(1) 临床资料
脑膜瘤标本收集于62位患者,男性20人,女性42人,WHO Ⅰ级的良性脑膜瘤33例,WHO Ⅱ级的不典型性脑膜瘤26例,WHO Ⅲ级间变型脑膜瘤3例。5例正常脑膜作为对照。
(2) RNA分离
PART ⅠExpression of midkine and pleiotrophin and their receptors inmeningiomasMeningiomas are the second most common tumors of the central nervous system. Most patients with meningiomas have good quality of life in contrast to the patients with malignant astrocytoma. However, reviews of large patient groups reveal that this optimistic view is not always satisfying. The recent improvements in the molecular biology and genetics of meningiomas may enhance the abilities to predict prognosis and to develop targeted therapies to improve outcomes in patients with clinically aggressive meningiomas. The family of heparin-binding neurotrophic factors consists of only two members, namely heparin-binding growth factors midkine (MDK) and pleiotrophin (PTN). MDK and PTN are overexpressed in various tumors. They promote the growth, survival, and migration of tumor cells, and play roles in the cancer development. Moreover, employing MDK and PTN as molecular targets for cancer would provide a new avenue for cancer therapy. Since not much is known about the relationship between midkine, pleiotrophin, and meningiomas, the purpose of this study is to investigate the expression of midkine and pleiotrophin and their receptors in meningiomas.1. Materials and Methods1) Clinical material
The meningioma tissues were collected from 62 patients. The patients included 42 women and 20 men. Thirty-three tumors corresponded to benign meningiomas of WHO grade I, 26 tumors were atypical meningiomas of WHO grade II and 3 were anaplastic meningiomas of WHO grade III. Five normal dura tissues served as control.2) RNA IsolationRNA was isolated by TRIzoL Reagent. RNA concentrations were quantified by spectrophotometer.3) cDNA SynthesisRNase free DNase was used to destroy the contaminating genomic DNA interfering with RT-PCR experiments. For first strand synthesis, the mixture was added to random hexamer primer, 5x reaction buffer, 4 dNTP mix and RevertAid? H Minus M-MuLV Reverse Transcriptase, respectively and incubated at different temperature and duration.4) Real-time RT-PCRReal-time RT-PCR was performed in three replicates of each sample which contained 20xAssays-on-Demand? Gene Expression Assay Mix, 2> pipetted into the appropriate microtiter wells which were coated with a rabbit polyclonal anti-human midkine antibody. After incubation and washing, biotin-labelled polyclonal anti-human midkine antibody and streptavidin-horseradish peroxidase conjugate were added and incubated, respectively. After washing, the remaining conjugate was allowed to react with the substract H2O2-tetramethylbenzidine. The reaction was stopped by addition of acidic solution and absorbance of the resulting yellow product was measured at 450nm by a fluorescence microplate reader. A standard curve was constructed by plotting absorbance values against the concentrations of midkine calibrators, and values of unknown samples were determined using this standard curve.2. Results1) A higher level of midkine mRNA expression was found in meningiomas of different grades compared to the normal dura tissues. A statistically significant difference was detected between meningiomas grade II and normal dura, and between meningiomas grade III and normal dura (P<0.05, unpaired Student's t-test). However, pleiotrophin mRNA expression did not significantly differ among the meningioma samples and normal dura tissues.2) A positive immunostaining for midkine could be detected in the cytoplasm and also in the extracellular space of the meningioma cells. Normal dura tissues were not immunostained by the anti-midkine antibody. Although the proportion of positively stained versus unstained tumor cells varied among samples to sample, in meningiomas grade II, the percentage of midkine-positive cases was significantly more than that in grade I (PO.05, Chi-square test). However, a positive immunostaining for pleiotrophin could only be detected in two cases of the meningiomas. No immunoreactivity was found in other samples examined.3) Normal dura tissues showed weak expression of the midkine protein. The mean content of meningiomas of different grades was significantly higher than that of normal dura (PO.05, unpaired Student's t-test).4) Though the expression of the receptors varied strongly among different samples, in most cases a positive mRNA expression could be observed. No statistical significant differences between mRNA expression levels of ALK, RPTPzeta, and LRP in normal dura tissues and meningioma samples could be observed.
3. ConclusionsThe midkine mRNA and protein expression level were elevated in meningiomas compared to normal dura tissues. The expression of midkine mRNA and protein in poorly differentiated atypical and anaplastic meningiomas was more prominent than that in the well differentiated low-grade meningiomas, and, this enhanced midkine expression indicated the possibility that midkine was also involved in malignant transformation from low-grade meningiomas to high-grade meningiomas. In contrast, the expression of pleiotrophin mRNA was not found to increase in meningiomas compared to normal dura. In most of the investigated samples, the expression of the different midkine receptors could be observed by real-time RT-PCR. High expression of LRP could be detected, whereas ALK and RPTPzeta were expressed only at low levels in tumor samples and normal dura tissues.
PART II Expression of truncated form of midkine in meningiomasRecently, a midkine was detected in some specimens of human tumors. This variant lacks exon 3, which encodes a N-terminally located domain, but contains the intact C-terminal half which is responsible for midkine activity. The purpose of this study is to investigate the expression of the truncated form of midkine in meningiomas and normal tissues.1. Materials and Methods1) Clinical materialThe meningioma tissues were collected from 25 patients. The patients included 17 women and 8 men. Eleven tumors corresponded to benign meningiomas of WHO grade I, 11 tumors were atypical meningiomas of WHO grade II and 3 were anaplastic meningiomas of WHO grade III. Two normal dura tissues served as control.2) RNA IsolationRNA was isolated by TRIzoL Reagent. RNA concentrations were quantified by spectrophotometer.3) RT-PCRThe RNA was first digested by DNase and RT-PCR was performed using the Qiagen OneStep RT-PCR Kit. After cDNA synthesis followed by activation of HotStarTaq DNA Polymerase, 40 cycles of 30 seconds at 94°C (denaturation), 1 minute at 58°C (annealing) and 90 seconds at 72°C (extension). The RT-PCR products were identified by 2% agarose gel electrophoresis following standard procedures with ethidium bromide staining.2. ResultsThe intact form of midkine, a PCR fragment of 448 base pair (bp), was observed in all the samples including meningiomas of grade I, II, III and normal dura. The truncated form of midkine, a PCR fragment of 280bp, was expressed in all normal dura tissues and the meningiomas samples except one (grade II). It seemed that higher expression
levels of the 448 bp were detected in all samples while various intensities of the 280 bp fragment could be observed.3. ConclusionsThe intact form of midkine, a PCR fragment of 448 bp, and the truncated form of midkine, a PCR fragment of 280bp, were observed not only in meningiomas of different grades but also in normal dura. The 448 bp band intensities were always strong whereas the 280 bp band intensities differed from low to moderate among different samples.
PART IIIBiological activities and signal transduction pathways of midkine in meningiomasMidkine is one kind of heparin-binding growth factors and is detected in human carcinoma specimens from pre-cancerous stages to advanced stages. MDK shows biological activities that include fibrinolysis, proliferation, mitogenisis, transformation, anti-apoptosis, angiogenesis and migration. These activities explain how this molecule is involved in carcinogenesis. The purpose of this study is to study the signal transduction mechanisms underlying the biological activities of midkine in meningioma cells.1. Materials and Methods1) Clinical materialThe fresh meningioma tissues were collected from 7 patients. The patients included 4 women and 3 men. Six tumors corresponded to benign meningiomas of WHO grade I and 1 tumor was atypical meningiomas of WHO grade II. One normal dura tissue served as control.2) Cell cultureThe freshly meningioma tissue, taken from operation, was separated by chemical methods. The meningiomas cells were incubated at 37°C and 5% CO2 in the culture flask. All cell cultures used showed 95-100% purity by fibronectin immunohistochemistry staining.3) Western blotCultivated meningioma cells were stimulated for 10 minutes, 20 minutes, and 160minutes in DMEM+0.5%FCS with 20ng/ml midkine. Cells were harvested with lysis buffer and the supernatant was collected for further research. Protein samples were loaded on 10% SDS-polyacrylamide gels for electrophoresis and were then transferred to a polyvinylidene difluoride membrane. After blocking with 5% ECL Advance Blocking Agent, the membrane was incubated with the primary antibody and secondary antibodies respectively. Horseradish peroxidase activity was detected by
applying an ECL Advance Western Blotting Detection Kit followed by exposure of the membrane to a sheet of autoradiography film. Equal protein loading was confirmed by reprobing following antibody stripping and the duplicated gel stained by Coomassie solution.4) Proliferation assayCultivated meningioma cells were incubated 48 to 72 hours in DMEM+0.5%FCS with 20ng/ml midkine or with 20ng/ml bFGF. After lysis, samples were added to the wells of a microplate. A standard curve was constructed by plotting DNA concentrations of ADNA versus fluorescence of each sample, corrected for the background fluorescence determined for the no-DNA control. The concentrations of unknown samples were determined using this standard curve.5) Caspase-3 Activity AssayCultivated meningioma cells were incubated for 6 hours in DMEM+0.5%FCS with 4(Ag/ml camptothecin, 20ng/ml midkine or both. After lysis and following centrifugation, the supernatant was collected for further detection. The activation of the caspase-3 was measured by using the Caspase 3 Activity Assay kit. The samples were transferred to the anti-caspase 3-coated well of a microplate. After washing, the samples were incubated with caspase substrate (Ac-DEVD-AFC) and the generated AFC was measured by a fluorescence microplate reader. For calibration, a calibration curve was set up with different dilutions of AFC-standards. The values of unknown samples were determined using this standard curve6) Bcl-2 mRNACultivated meningioma cells were incubated for 6 or 16 hours in DMEM+0.5%FCS with 4ug/ml camptothecin, 20ng/ml midkine or both. After washing and following centrifugation, the supernatant was discarded. After RNA isolation and cDNA synthesis, the samples were detected by real-time RT-PCR.2. Results1) No increase in phosphorylation and thereby in activation of p42/44 MAPK (Thr202/Tyr204) and p38 MAPK (Thrl80/Tyrl82) after treatment with midkine could be observed. In contrast to this, Akt phosphorylation was induced after 10 minutes of stimulation with midkine and increased significantly after 20 minutes compared to the control. After 160 minutes, the phosphorylation signal decreased to about former level.
These results indicated that PBK/Akt pathway might play a predominant role in signal transduction of midkine action on meningiomas.2) There was no significant increase in proliferation after stimulation with midkine while the positive control, bFGF promoted the meningioma cells proliferation obviously.3) The meningioma cells treated with camptothecin showed high levels of caspase-3 activity compared with controls. When incubation with camptothecin combined with midkine was performed, an inhibition of activated caspase-3 in meningioma cells could be observed. The meningioma cells treated with camptothecin showed low levels of Bcl-2 mRNA expression compared with controls. When incubation with camptothecin combined with midkine was performed, up-regulated Bcl-2 mRNA expression in meningioma cells could be observed.3. ConclusionsThe activation of the PI3K/Akt pathway might substantially contribute to the biological activity of midkine. The anti-apoptotic effect is an important activity of midkine on meningiomas and may transduce the anti-apoptotic signal via PI3K/Akt pathway.
引文
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