Transfection Of Chk1/2 Antisense Oligonucleotide To Glioma Influences The Apoptotic Sensitivity To Irradiation

PartⅠStudy on Expression of Chk1 and Chk2 in Glioma and Brain Tissue and Their Clinical SignificaneObject The distribution of Chk1 and Chk2 protein in Glioma and Brain Tissue were explored to evaluate the feasibility of Chk1 and Chk2 genes as the target genes of gene therapy.Methods The expressions of Chk1 and Chk2 genes were detected by immune-histochemistry in the paraffin-embeded sections of 80 gliomas and 18 cases normal brain tissue.Results Chk1 and Chk2 protein were widely expressed in different glioma and normal brain tissues. The positive expression rate of Chk1 protein was higher in glioma than normal brain tissues significantly (P=0.027). There was no significant difference in expression of Chk2 protein between glioma and normal brain tissues (P=0.173). The positive expression rate of Chk1 and Chk2 protein was no significant difference in high-grade glioma(WHOⅢ～Ⅳ) and low-grade glioma(WHOⅠ～Ⅱ) (P=0.301,0.135).There was positive correlation between Chk1 and Chk2 protein(r=0.795,P<0.001). Conclusion Chk1 protein was overexpressed and could be considered as the target genes of gene therapy in glioma. There was positive correlation between Chk1 and Chk2 protein, and so it was important to block the Chk1 and Chk2 genes together to increase the sensitivity to irradiation of glioma.PartⅡEffect on Cell Cycle and Expression of Chk1 and Chk2 after Irradiation in Glioma cellsObject The changes of cell cycle and the apoptosis of unsynchronized U251 cell line and effect on Chk1 and Chk2 protein were studied after the irradiation.Methods The changes of cell cycle and apoptosis in U251 cell line which were treated with irradiation after different intervals by flow cytometry were observed. The expression of Chk1 and Chk2 protein was measured by flow cytometry and Western blot.Results G2/M cell arrest was induced in unsynchronized tumor cells after treated with irradiation. Cell cycle arrest was increased with the increased dosage of irradiation, and apoptosis happened after cell cycle arrest and had inverse relationship with the activating cell cycle. Irradiation had no effect on expression of Chk1 and Chk2 protein.Conclusion G2/M cell arrest models were successfully induced in unsynchronized U251 cell line after treated with irradiation.PartⅢTransfection of Chk1/2 Antisense Oligonucleotide to Glioma Increases the Apoptotic Sensitivity to Irradiation Object Effect on expression of Chk1 and Chk2 and changes of cell cycle after radiation in U251 cell line with antisense oligodeoxynucleotide (ASON) were observed.Methods The U251 cell line was transfected with Chk1/2 sense and antisense chain-lipofectamine PLUS complex alone or recombinatively, and the transfection rate was checked. Then we irradiated it and measured the changes of the cell cycle and apoptosis with flow cytometry in order to compare the diference between the Chk1/2 sense and antisense chain, the single and recombinative tansfection of Chk1/2. The expression of Chk1 and Chk2 was measured by Western blot, and Chk1 and Chk2 mRNA were measured by real time PCR.Results Transfecting U251 cell line with Chk1, Chk2 antisense oligonucleotide alone or Chk1/Chk2 together could significantly increase apoptosis induced by irradiation and markedly decrease cell cycle arrest. The expression of Chk1 and Chk2 protein and mRNA markedly decrease after ransfecting U251 cell line with Chk1, Chk2 antisense oligonucleotide alone or Chk1/Chk2 together.Conclusion No matter inactivated Chk1, Chk2 alone or Chk1/Chk2 together, the cell cycle was broken and the sensitivity to apoptosis was excessively increased after treated with irradiation.