Depletion Of Cell Cycle Checkpoint Kinase1/2on Glioblastoma Stem-like Cells Influences The Apoptotic Sensitivity To Irradiation

Objective To obtain glioma stem-like cells (GSCs) primary and identify the stem cellcharacteristics in vivo and in vitro, establish the foundation for glioma targeted therapy.Methods Specimens of high grade glioma were obtained from operations, the grade wasconfirmed by pathological examination, WHOⅢ or above. The specimens were digested bytrypsin for extraction cells, suspension neurosphere culture to cultivate glioma stem-likecells. Immunofluorescence assay and RT-PCR assay for detection of GSCs surface markers.GSCs were planted into brain of nude mice to detect the tumor formation of GSCs. Thedata were analyzed by SPSS17.0.Results The GSCs were successfully cultured in the suspension neural stem cell cultureconditions, CD133(11.02%~33.55%) were expressed highly in the primary GSCsaccording to the RT-PCR, western blotting and immune fluorescent. Nestin was alsoexpressed highly in protein level. The GSCs were planted into brain of nude mice, the brainbearing tumor after8weeks, the model of brain tumor was built successfully.Conclusions The GSCs could be obtained primary from high grade glioma specimens withthe way of suspension neurosphere culture and the GSCs exhibited the properties of thestem-like cell. The GSCs cell line was tumorigenic. Objective Construct the lentiviral expression vectors of shRNA interference specific toChk1or Chk2gene, then to screen the stably transfected cells of GSCs. And study on theinterference Chk1or Chk2gene to GSCs.Methods The DNA sequences of Chk1or Chk2were obtained from the Genbank and theshRNA were designed and synthesized, and one scrambled shRNA sequences served asnegative control. The shRNA were inserted into plasmids of pLKO.1. The recombinantlentiviral vectors were transfected into GSCs, and the stably transfected GSCs wereobtained after being screened by puromycin. Reverse transcriptase-polymerase chainreaction (RT-PCR) and Western blotting were adopted to detect the inhibitory effect onChk1or Chk2at the mRNA level and the protein level respectively.Results The expressions of Chk1or Chk2were significantly down-regulated by shRNA oflentiviral vectors in the stably transfected Chk1-shRNA and Chk2-shRNA GSCs. Theresults were tested by Western blotting and RT-PCR.Conclusions The expression of Chk1or Chk2in the GSCs can be significantlydown-regulated by RNA interference (RNAi) which mediated by the shRNA of lentiviralexpressing vectors. The stably transfected GSCs were obtained for further study. Objective Study on lentivirus transfected the interfered Chk1/Chk2gene to primarygloblastoma stem-like cells (GSCs) for therapy of radiosensitization. it would be a new methodcan be chosed to treatment of glioma.Methods The DNA sequences of Chk1or Chk2gene were obtained from the Genbank andshRNA were designed and synthesized. The shRNA lentiviral vectors were constructed andtransfected into GSCs. Reverse transcriptase-polymerase chain reactions (RT-PCR) andWestern blotting were used to detect the inhibitory effect on Chk1or Chk2at the mRNAlevel and the protein level in the GSCs. X-ray treated these GSCs, then cell cycle andapoptosis were detected.Results After radiation, most of group radiation and group radiation＆interfere Chk2wereblocked in G2/M; The rate of apoptosis after radiation: Group interfere Chk1higher thangroup control and group interfere Chk2.Conclusions Lentiviral transfection interfere with Chk1and Chk2could down-regulateChk1and Chk2genes expression, which could improve the sensitivity of GSCs inradiotherapy. It may be a good method in the treatment of glioblastoma.