The Research For Ruthenium (â…¡) Pyridine Complexes Joint X-Rays To Osteosarcoma Cell Apoptosis Entrainment Of Experiments

Background: Osteosarcoma is a originated in the bone mesenchymal primary malignant bonetumors, about three-quarters of patients in the age10to30years old.It is the youth of the mostcommon malignant tumor bone, onset acute and pulmonary metastasis early and thecharacteristics of the high mortality rate[1]. Osteosarcoma clinical treatment effect is poor,especially the transfer or recurrence of patients, nearly50years did not have a lot of treatmentprogress, and long-term surial less than25%[2]. Therefore, to seek new ways of treatingosteosarcoma has become urgent clinical needs.Chemotherapy is common treatment for osteosarcoma. In the1970s, cisplatin as antitumordrugs widely used in clinic, the metal complexes anti-tumor activity be biological medicine ofresearch in the field of one of the hotspots. Platinum also become one of the four classicchemotherapy drugs for osteosarcoma[3].However the platinum drugs exists serious adversereactions （neurotoxicity, kidney toxicity, etc） and resistance （innate character or leading）[4]. Inorder to make up for these disadvantages, people have other metal compounds in search forplatinum kind of alternatives. Ruthenium complexes with its cancer cell toxicity, similar platinumcompounds ligand exchange ability, and cisplatin no cross resistance and through the irontransport mechanism low toxic to characteristics of attention, it is generally think rutheniumcomplexes will become one of the most promising cancer drugs[5-7]. The European Union since1997has set up the ruthenium cancer drug research and development group, further strengthenruthenium cancer drug research, some ruthenium metal complexes has entered a phase of clinical [8,9]. But ruthenium complexes of tumor cells are also different inhibition effect is differ, and lackof target of wait for blemish. In recent years, there are reports that the new synthesis[Ru（dmb）₂（DBHIP）]（ClO₄）₂can induce a wide variety of tumor cell apoptosis, BEL-7402, C-6,HepG-2and MCF-7[10].Radiation apoptosis is the tumor radiation biology research hot spot[11,12]. Apoptosis is irradiatedcells after some important ways of cell death, and apoptotic body is the judgment of theimportant morphological index cell apoptosis[11]. Radiation cause cancer cell apoptosis biologymechanism is not clear。Research reports that many cancer gene and tumor-suppressor genesinvolved in the regulation of cell apoptosis, including Bcl-2and Bax. Bcl-2is the most valuedapoptosis study of one of cancer genes. At present， more combination treatments to the patientswho is in the clinical late for osteosarcoma and after the operation in treating osteosarcoma. Thecombination treatments often include chemotherapy, radiation therapy, and biological treatmentmeans.This research in different concentrations [Ru（dmb）₂（DBHIP）]（ClO₄）₂joint different doses ofgamma rays of osteosarcoma MG-63cells in different time after processing, observe the cellgrowth inhibition of osteosarcoma, inducing apoptosis, and observe ruthenium （Ⅱ） of pyridinecomplexes osteosarcoma MG-63cells have radiotherapy sensitization effect, treatingosteosarcoma in order to have a new discovery.Objectives: Research ruthenium （Ⅱ） of pyridine complexes osteosarcoma MG-63cell apoptosisinduction function, as well as for osteosarcoma MG-63cells have sensitization effect of radiation,and the mechanism of the preliminary research.Methods: First：The research in the Ruthenium （Ⅱ） pyridine complexes induce the MG-63cellapoptosis.1、Join different concentration of ruthenium （Ⅱ） pyridine complexes[Ru（dmb）₂（DBHIP）]（ClO₄）₂in the system of medium to treat osteosarcoma MG-63cells, use CCK-eight method tocounts the cell growth curve, and the concentration gradient method of the drug LD50.2、Cell colony forming method observation cell growth inhibition, flow cytometric testing the cellcycle and cell apoptosis, Annexin V-FITC/PI double dyeing measured cell apoptosis,Hoechst-33258dyeing observation of the nucleus shape variation.Second：Research for different doses of gammarays irradiation in treating osteosarcoma cell apoptosis entrainment.1After different radiation dose radiation treatment to MG-63cells and continue to cultivatedifferent period,use CCK-8method to counts the cell growth curve.2, Cell colony forming method observation cell growth inhibition, flow cytometric testing the cellcycle, Hoechst-33258dyeing observation of the nucleus shape variation.Three： Research for ruthenium （Ⅱ） pyridine complexes joint radiation treatment forosteosarcoma MG-63cell apoptosis entrainment1、After different concentrations of ruthenium （Ⅱ） pyridine complexesdifferent radiation doseradiation treatment to MG-63cells and continue to cultivate different period,use CCK-8methodto counts the cell growth curve.2、Cell colony forming method observation cell growth inhibition, flow cytometric testing the cellcycle and cell apoptosis, Annexin V-FITC/PI double dyeing measured cell apoptosis, Wester-blotapproach to measure the cell apoptosis.Hoechst-33258dyeing observation of the nucleus shapevariation.Four：statistics analysis: show the data in mean±standard deviation （`x±s）. All data via softwareSPSS17.0. The design of repetitive measure analysis of variance evaluation changes betweengroups, between groups more compares the single factor analysis of variance, P<0.05fordifferences with a statistical significance.Results:1. the first part of this paper, the study shows the concentration gradient method of the drugLD50for175.7μmol/L. Ruthenium （Ⅱ） pyridine complexes can significantly inhibit bonesarcoma cell growth, growth inhibition rate with the drug concentration increases. Drug treatmentof cell cycle when each phase ratio changed, a control group displays obvious G1block. Flowcytometric test results and drugs can obviously induction MG-63cell apoptosis happen, and aconcentration dependence on effect, drug concentration is high, the more obvious early cellapoptosis. Hoechst-33258dyeing results show that the control group in fluorescence microscopecell nucleus intact, fluorescence dispersion into shape, the comparison is bleak, the ruthenium （Ⅱ）pyridine complexes of processing more osteogenic sarcoma cells is condensed chromatin, thenuclear cracking, fluorescence more bright, confirmed ruthenium （Ⅱ） pyridine complexes cancause MG-63cell DNA of degradation, the nucleus is broken, and then induction osteoblasticsarcoma MG-63cells to apoptosis. 2. In the second part of the research results showed that the gamma rays after exposure ofosteosarcoma cells show growth inhibition, and with the time and dose dependent relationship.10GY gamma rays processing four days later, cell growth inhibition rate reached55.44%. Alongwith the increase of the radiation dose, the colony cell number significantly less. The control,2GY,5GY,10GY process cells after14days, cell number respectively mean:（52±3.57）,(56±3.53,(41±2.35,（13±1.24）, cell stick a wall rate （%） were52%,28%,8.2%,1.3%. Gammarays can significantly inhibited shot osteosarcoma cell proliferation, show the G2period block,and present dose dependent on effect, illuminate the higher dose cells in a G2phase of the higherproportion, the control group （3.84±0.15）%increased to2GY group （8.46±0.14）%, add to5GY group （13.19±0.07）%, increase multiples were2.2,3.4times. Different doses of radiationtreatment group compared with controls, the differences are significant （P<0.05）. Hoechst-33258dyeing results show did not deal with group by tumor cells form rules, uniform distributedchromatin, nuclear complete, the gamma rays after irradiation, within a certain range, withincreasing doses, cell apoptosis is more and more, and the apoptotic cells form the more typicalstructure, expression is become pyknotic nuclear chromatin edge collection in nuclear membraneplace, nuclear small body form, etc.3. This paper the third part results show that:（Ⅱ） pyridine ruthenium complexes joint gammarays group processing osteosarcoma cells to grow more individual treatment group obviousinhibition, and with the increase of concentration and the extension of time, the difference biggerdisplays obviously joint effect. colony cells results show that with the increase of theconcentration of the drugs and radiation doses of increase, cell number significantly reduced,percentage is obviously reduced. With150μmol/L joint5GY treatment group, cells formingpercentage is0.8%, and the control group is56%blank, two are nearly70times. Flow cytometrytest results and was cell apoptosis: joint group cell apoptosis rate （61.2±3.98）, is a blank in thecontrol group （0.2±0.02）306times. Hoechst-33258dyeing, according to the results of the jointgroup and individual group, the control group can better cause MG-63cell DNA of degradation,nuclei broken, induction of cells to apoptosis, both union is to promote each other cell had thefunction of apoptosis. Western Blot test of antiapoptotic gene Bcl-2results show that joint groupBcl-2express minimum, blank expression control the strongest, and with the drug concentrationand radiation doses of increasing expression is lower, the tip ruthenium （Ⅱ） pyridinecomplexesjoint gamma rays damage osteosarcoma may is the role of the cells by lowering Bcl-2expression but induce MG-63cell apoptosis happened to realize.Conclusions:1, the concentration gradient method of the drug LD50for175.7μmol/L.Ruthenium （Ⅱ）pyridine complexes make osteosarcoma in cell cycle G1block, and can induceosteosarcoma cell apoptosis happened, along with the drug concentration increases, the strongereffect induced apoptosis.2, gamma rays inhibit bone sarcoma MG-63cell growth, present a dose dependent. Radiationtreatment of osteosarcoma cells show obvious G2period block. In certain scope irradiation range,with increasing doses of radiation, the more obvious cell apoptosis, the microscopic structure ofthe cells become pyknotic performance for the nuclear chromatin edge collection in nuclearmembrane place, nuclear small body form, etc.3, ruthenium （Ⅱ） pyridine complexes [Ru （dmb）₂（DBHIP）]（ClO₄）₂joint and shoot rays canpromote osteosarcoma MG-63happen apoptosis, displays obviously joint effect, inducing cellapoptosis mechanisms may occur with lower Bcl-2expression of the relevant.4, this study for ruthenium （Ⅱ） pyridine complexes can induce osteosarcoma cell had providedlaboratory apoptosis foundation, at the same time also shows that the ruthenium （Ⅱ） of pyridinecomplexes osteogenic sarcoma cells have radiation sensitive function, for the study in relativefield after laid a foundation laboratory.