| BackgroundCraniopharyngioma is a congenital, invasive epithelial tumor arising from the suprasellar region which is classified by histology as benign. Surgical resection is the main strategy for treating craniopharyngioma, but only 18-84% of tumors can be completely resected surgically because of internal factors, such as tumor location, calcification, range, adhesion, and complex anatomical relationships around the tumor. Additionally, severe complications are commonly seen and postoperative death rate is up to 1.7-5.4%. Moreover, the difficulty to treat recurrent craniopharyngioma is significantly increased, the surgical resection rate is decreased significantly to 0-25%, and the perioperative mortality and morbidity are increased significantly.Radiotherapy which includes fractionated radiation, interstitial radiation and gamma knife therapy is the primary therapy for craniopharyngioma, and additionally, it is also an important auxiliary and complementary therapy for surgical treatment. Stereotactic interstitial radiotherapy has been introduced for treating cystic craniopharyngioma with positive efficacy.This therapy is characterized by few complications and low mortality rate, but there are distinct individual differences.However, some patients have no satisfied outcomes because of the presence of radiation-resistant tumor cells that leads to uncertain choice of treatment methods and prognostic evaluation.Previous studies have demonstrated that activities of vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) are increased in a variety of tumors, such as lung cancer, liver cancer, breast cancer and cervical cancer, which not only causes tumor angiogenesis, but also stimulates the proliferation of vascular endothelial cells and tumor cells, thereby exerting an important role in tumor growth, metastasis and recurrence. VEGF secreted by most tumors is the most important inducing factor for tumor angiogenesis, and its receptor (VEGFR-2) is mainly expressed in the vascular endothelial cells to mediate endothelial cell division and to influence vascular permeability.Previous studies have also demonstrated that tumor angiogenesis is crucial for the occurrence and development of tumors, which is a delicate balance between stimulating and inhibiting the proliferation of endothelial cells. Vidal et al. reported that VEGFR-2 mRNA is expressed not only in interstitial capillaries, but also in the epithelium of craniopharyngioma. These findings indicate an increased possibility that VEGF produces an effect on the epithelial components, and it is also confirmed that the VEGFR-2 acts as the important regulator for the activity of VEGF in endothelial and non-endothelial cells. In recent years, VEGFR-2 is found to express not only in vascular endothelial cells, but also in some non-endothelial cells, such as melanocytes, retinal epithelial cells, hematopoietic stem cells, neuronal cells, and some tumor cells (glioblastoma, hemangioblastoma and meningioma).VEGF/VEGFR-2 active expression in tumor cells and stromal vessels is also relevant to tumor recurrence. However, these studies have focused on the relationship between VEGF/VEGFR-2 and recurrence of craniopharyngioma and angiogenesis, and there is no report on the correlation between VEGF/VEGFR-2 expression in craniopharyngioma and tumor radiosensitivity. Here, the present study aimed to investigate the relationship of imaging features of craniopharyngioma and expression of VEGF/VEGFR-2 in tumor cells with tumor radiosensitivity.Based on the above finding, the purpose of this study is to identify the possible role and mechanisms of VEGF/VEGFR-2 in craniopharyngioma rediosensitivity. We hope that this study will provide further insight into the craniopharyngioma rediosensitivity and improve the development of potential novel therapeutic targets for craniopharyngioma.Chapterl The relationship between VEGF/VEGFR-2 expression and 32P-colloid interstitial radiotherapy for recurrent craniopharyngiomaObejective:To investigate the relationship of the expression of vascular growth factor (VEGF) and its receptor-2 (VEGFR-2) and the curative effect of 32P-colloid interstitial radiotherapy in recurrent craniopharyngioma.Methods:Thirty-two patients with recurrent craniopharyngioma underwent interstitial infiltration with a radiocolloid labeled with 32P -colloid at our hospital of PLA, China from January 2006 to December 2013 were enrolled and their tumors were classified into four types according to the thickness of the cyst wall and signals of the cyst contents as shown by tumor CT and MRI images. Immunohistochemistry was used to detect the expression of VEGF and VEGFR-2 in craniopharyngioma tissues before radiotherapy. Twelve months post treatment, tumor sensitivity to the interstitial radiotherapy was determined. Tumor radiosensitivity, imaging features and expression of VEGF and VEGFR-2 were analyzed statistically using SPSS19.0.Results:After 12 months of radiotherapy,9 cases were clinically confirmed to be sensitive to the 32P-colloid interstitial radiotherapy, but 23 cases were non-sensitive to the radiotherapy. VEGF mainly expressed in the tumor cytoplasm, and VEGFR-2 expressed either in vascular endothelial cells or in tumor endothelial cells. VEGF/VEGFR-2 expressions varied significantly in cases sensitive or insensitive to the radiotherapy (VEGF:z=-2.194, p= 0.028; VEGFR-2:z=-2.382, p= 0.017).Conclusion:VEGF/VEGFR-2 expressions of craniopharyngioma are correlated with tumor radiosensitivity.VEGFR-2 is expressed lowly or not expressed in tumor endothelial cells, the tumors are mostly sensitive to the 32P-colloid interstitial radiotherapy; otherwise, VEGFR-2 is highly expressed in tumor endothelial cells, the tumors are mostly insensitive to the 32P-colloid interstitial radiotherapy.Chapter 2 Cell culture and morphology of craniopharyngioma cellObejective:To explore an effective primary cell culture of craniopharyngioma and observe the biological characteristics of craniopharyngioma cell in vitro.Methods:The fresh specimen of adamantiomatous craniopharygioma were treated with trypsin and cultured in vitro. Craniopharyngioma cell was obtained from fresh craniopharyngioma tissue specimens used by primary culture with tissue explants method,and was identified by immunofluorescence. The cell morphology was observed by inverted microscope.Results:The craniopharyngioma cell line were cultured and expanded effectively in DMEM/f12 medium containing 10% fetal bovine serum. Tumor cells from 10 patients were successfully cultured. Microscopically, the cells had a typical epithelial morphology with a higher nucleus to cytoplasm ratio than nontumorous epithelial cells. CP cells were trigonal, tetragonal or pentagonal.All CP cells adhered to the flask tightly and stopped growing at confluence.Their doubling time was approximately 3 days.The craniopharyngioma cells demonstrated the Pan-CK positive phenotype.Conclusion:We had successfully established an the cell line of primary craniopharyngiomas in vitro by the way of enzymtic digestion through the fresh tumor tissue in DMEM/f12 medium.The craniopharyngioma cells demonstrated the Pan-CK positive phenotype,which confirmed that the cultured cells were craniopharyngioma cells.Chapter3 VEGFR-2 overexpression as a promoter in craniopharyngioma cells growth and radiation-resistance2.1 Literature search and selectionObejective:To explore the effects of VEGFR-2 overexpression mediated by Aadenovirus-mediated VEGFR-2 on radiosensitivity of craniopharyngioma cells on the basis of the primary craniopaharyngioma cells culture previously.Methods:Craniopharyngioma cells were transfected with Aadenovirus-mediated VEGFR-2.The mRNA and protein expression of VEGFR-2 gene in craniopharyngioma were investigated after transfection by RT-PCR and western blot analysis, respectively. The proliferation of craniopharyngioma cells were detected with MTT after transfection.After transfection with the recombinant adenovirus vector VEGFR-2, the craniopharyngioma cells were treated with X-ray irradiation. The cell apoptosis were detected by flow cytometry.Results:(1)The transfection efficiency at 24h,48h,72h were: 30.54±2.15%,80.45±3.37%,85.87±4.32% respectively. Among them, the transfection efficiency at 48h was significantly higher than transfected at 24h (P<0.01), so we selected 48h as the best point to do a follow-up experiment analysis in the rest part of the experiment.(2) There were no difference in VEGFR-2 mRNA level between normal and Ad-GFP group (P>0.05).Compared with the Ad-GFP group, VEGFR-2 mRNA level in Ad-VEGFR-2 overexpression group was significantly elevated (P<0.05).These results suggested that all the process including viral transfection and VEGFR-2 gene expression is successful.(3) There were no difference in VEGFR-2 expression between normal and Ad-GFP group (P>0.05). Compared with Ad-GFP group,VEGFR-2 expression increased significantly in VEGFR-2 overexpression group (P<0.01), which further proved that VEGFR-2 transfected and expressed successfully.(4)The proliferation of craniopharyngioma cells after being transfected with VEGFR-2 was faster than that of the other two control groups (P< 0.05).(5)The results revealed that apoptosis was significantly decreased in cells overexpressing VEGFR-2 compared with control group:2gy(15.8%±0.5%, vs. 19.4%±0.3%),4 gy (21.7%±0.6% vs.15.6%±0.3%),6 gy (24.4%±0.5%, vs. 17.5%±0.2%),8 gy (25.6%±0.4% vs.19.2%±0.6%). It showed that normal group had a higher apoptosis rate compared with VEGFR-2 overexpression groupConclusion:Gene transfer technology mediated by adenoviral vector can transfect VEGFR-2 gene into craniopharyngioma cells with high efficiency. Craniopharyngioma cells transfected by VEGFR-2 gene could expressVEGFR-2.VEGFR-2 overexpression in craniopharyngioma could promote tumor cells growth.It confirmed VEGFR-2 overexpression in craniopharyngioma cells were related to radioresistance through the experiment in vitro. |
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