| Objective:It is well known that gliomas are the most common primary central nervous tumors, which account for 40-60% of intracranial tumors. Extensive research devoted to improving treatment of malignant gliomas over the past three decades has yielded little progress, the prognosis of the patients who harbored the malignant gliomas remains grim, even though treated with multiple approaches including surgical resection, radiotherapy and chemotherapy. It is reported that the median survival of the patients with glioblastomas is only fifty-two weeks. Therefore, malignant gliomas is an intractable disease and it is still a problem for urgently being solved.Gamma knife radiosurgery with high precise and safety characteristic is widely applied to the clinic of Neurosurgery. Moreover, it has better curative effects than the conventional radiotherapy. Especially for small lesion which diameter is less than 3 cm, its therapeutic result is equal to the surgical resection. Evidence indicates that gamma knife radiosurgery is an favorable modality for treatment of malignant gliomas. However its effect on prolonging the survival of patients is poor, due to the intrinsic radioresistance and infiltrative growth of malignant gliomas. How to improve the radiosensitivity of such tumors is the hotspot in radiosurgery and radiotherapy research. In current status, most radiosensible drugs are toxic for normal tissue, then, it is important to find out some approaches to enhance the radiosensitity of gliomas.As the knowledge of tumor biology and molecular genetics increased, it has been shown that the development of gliomas, just the same as the rumors in the other site of the body, result from the activation of proto-oncogene and inactivation of tumor suppressor genes. Of this genes, the p53 gene is one of the most importantsuppressor gene. It , and its encoded protein, has emerged as the most frequent somatic genetic event in human cancer. It is estimated that more than 50% of human cancers lack a wild-type p53 allele and normal p53 protein, or retain a wild-type allele yet have dysfunction of the p53 pathway. Clinical trial indicated that the tumors may keep silence or grow slowly when they were treated with exogenous wild-type p53, moreover, the p53 gene can improve the radiosensitity of tumors. We will evaluate the effects of therapy combined gamma knife radiosurgery and wild-type p53 for malignant gliomas in vivo, for the aim of finding out a new and effective approach to cure this kind of tumors.Meanwhile, from molecular biology basis, p53 is responsible for DNA repair, the second part of this study aims to reduce the expression of Ku which is the important factor for DNA repair with RNA interfering technology.Materials and Methods:Part I Parental C6 cells were implanted stereotactically into the right caudate nucleus of SD rats. Rats bearing well-established cerebral gliomas defined by MRI at 6 days postimplantation were divided into 4 groups, including control group, p53 treated group, gamma knife radiosurgery treated group and cpmbination of P53 and gamma knife treated group. Treated with ad-p53 at 6 and 8 days after implantation, the 4-mm collimator of 201-source cobalt 60 gamma unit (Leksell Gamma Knife, model C) was used for radiosurgery. The 50% isodose line (15Gy) matched the approximate tumor size at 10 days after implantation. The general behavior and survival of the rats were observed, the dynamic MRI and histopathological changes of the tumors and the expression of p53, PCNA, CyclinDl, p21 and apoptosis in glioma were examined.Part II Firstly the expression of Ku70 was demonstrated in C6 cell line with semiquantitative -reverse transcription-polymerase chain reaction(SQ-RT-PCR), thentransfects siRNA sequence into C6 glioma cell line according to the supplier's.Results:Part I With Western Blot analysis, it was found that ad-p53 were successfully transfected into C6 gliomas in vivo. In C6 control group, the mean survival was 18.7±0.45 days. The survival time of p53 group and Gamma Knife group was prolonged, 2 and 4 rats kept alive in 56 days after implantation respectively, while the survival of rats of combined group was significantly prolonged, 6 rats kept alive during observation period. MRI indicated that the tumor volumes of the rats treated with ad-p53 and gamma knife radiosurgery were less than the control group, while the combined group was the least. In HE, the necrosis area of glioma in the gamma knife treated group was smaller than that of combined group, but it was obviously larger than that of control group, there is no difference between p53 treated group and control group. Histopathological analysis showed that PCNA precipitating cells in tumor of control group was 84.33±4.93. The PCNA precipitating cells in p53 group and gamma knife group were much less than that of control group, and that of the combined group was the least. The CyclinDl expression was downregulated, while the expression of P21 was upregulated, the change of P21 and CyclinDl was most prominent in the rats with combined therapy. A lot of apoptotic cell could be found in transfected group and therapeutic group, and little in control group.Part II C6 cell expressed high level of Ku70 mRNA determined by SQ-RT-PCR, Thereafter, siRNA sequence targeting Ku70 were transfed into C6 cell. The results indicated that siRNA sequence 1 obviously reduced the expression of Ku70. As analyzed with Bandscan and SPSS softwares, a significant difference were found between interfering sequence 1 and control. Conclusion:Part I These findings in the present study suggests that the p53 gene couldimprove the radiosensitity, and the combined p53 and gamma knife maybe a novel therapeutic approach for malignant gliomas.Part II There is expression of Ku70 in C6 glioma cell line, and the expression of Ku70 can be reduced by siRNA technology.
|
PDF Full Text Request