MLL2in Diffuse Large B Cell Lymphoma Cell Lines LY-3the Expression And Function

Objective: Through application of mixed lineage leukemia2(MLL2)antibody both in-vitro in diffuse large b-cell lymphoma cell strain LY-3andin-vivo in LY-3-cell-seeded tumor-bearing rats, this research is to explore intothe effect of MLL2antibody on inducing cell apoptosis in diffuse large B celllymphoma.Methods:1Cell Culture and Subculture of DLBL Cell Strain LY-3 LY-3cells are cultured in nutrient solution of10%(volume fraction)fetal bovine serum and1%double-resisted RPMI1640, which is located inincubators of37℃, saturated humidity and5%CO2. The cells culture every48-72hours. The cells grew well, like floating clouds. All experiments in thisstudy adopt LY-3cells in logarithmic phase.2RT-PCR Validation of MLL2Gene Expression’s Existence in LY-3CellsFetch LY-3cell suspension and extract total RNA. Measureconcentration and purity on nucleic acid protein meter. Reverse transcriptRNA into cDNA according to the experimental steps of reverse transcriptionkits. Prepare PCR reaction system with MLL2front primer sequences5’CCAATGCATAAGATCAAG3’and back primer sequences5’CTATGAAAGTCAGCCATC3’. Put it into a Realtime PCR for PCRamplification and do sample point in the electrophoresis tank with5μlMarker，5μl GAPDH，5μl MLL2from left to the right. Then doelectrophoresis under condition of U:120V,A：60A,T：35min and observe theresult through gel imaging system.3MTT Detection of MLL2Antibody’s influence on LY-3CellProliferationInoculate LY-3cell suspension (cell density:1×105/ml) into U96-wellplates(100ul/hole). Randomly divide the suspension into control group andexperimental MLL2antibody group. Set3complex holes in both groups. Add100ul cell culture into each hole of the control group. For the experimentalgroup, inject100ul cell culture with different concentration MLL2antibody(0.32ug/ml、0.16ug/ml、0.08ug/ml、0.04ug/ml、0.02ug/ml) into each hole.Incubate the2groups of suspension in37℃,5%CO2, saturated humidityincubators. Inject20ul MTT solution (5mg/ml) respectively after24and48hours into each hole. After another4hours’ incubation in the incubators,centrifugal culture plate, absorb supernatant, add dimethyl sulfoxide, and put itin an oscillator until the crystallization gets fully dissolved. Measure the absorbance value of each hole by492nm enzyme standard instruments.4Apply MLL2Antibody into LY-3-cell-seeded Tumor-bearing Rats.Observe its anti-tumor effect.Culture LY-3cells in vitor, contain them in250ml plastic culturebottles, and adjust cell density into3-4×105/ml. Collect cells suspension andcentrifugal it after the cells grows into a certain order of magnitude. Abandonthe supernatant. Solely add3.0ml RPMI1640liquid to dilute, then stir andblending. Inoculate the cell suspension into the left armpit skin of15nudemice,0.2ml each (order of magnitudes of tumor cells:107). Six days after theinoculation, tumors grow into the size of soya beans; on the fourteenth day,they grow into1cm long/wide. Rule out three tumor-burdened rats whosetumors are not satisfying enough. Randomly divide the remaining12numbered tumor-bearing rats into blank control group and experimental group,with each group6mice. Subcutaneous inject0.16ug/ml MLL2antibodies oneach tumor-bearing mice in the experimental group. Regularly observe andmeasure the size of tumors. Four weeks after the inoculation of tumor cells,euthanize the mice by breaking necks, strip and weigh the tumors. Extracttotal RNA from the tumor tissue. Obtain RT-PCR amplification products anddo agarose gel electrophoresis, EB dyeing, gel imaging analysis systemanalysis in turn. Extract total proteins of tumor tissue. Test MLL2proteinexpression through the method of Western-blot: after the preparation ofseparation and enrichment adhesive, apply Gel electrophoresis, transfer,blocking of antibody, then stain it. Use infrared imaging system to do analysisand scanning.Results:1There exists MLL2gene expression in LY-3cells.Extract RNA from LY-3cells. Measure its concentration and purity onnucleic acid protein tester with results of432.7ng/ul and260/280=1.92.Then in turn do reverse transcription and PCR amplification and agarose gelelectrophoresis. Gel imaging system shows that there exists MLL2gene expression in LY-3cells.2MLL2antibody could induce cell apoptosis on LY–3cells in vitro.After added into different concentration of MLL2antibody in24and48hours, detect the results by MTT. Each specimen’s absorbance value reducesto some extent; MLL2antibody has anti-proliferative activity, and has theability to induce cell apoptosis. After the same processing time, for this verycase48hours, in certain concentration range, LY-3cell inhibition rateincreased when MLL2antibody concentration increases.3MLL2antibody has anti-tumor effect on in vivo LY-3-cell-seededtumor-bearing mice.Compared with that of the control group, the tumors of the tumor-bearingmice from the MLL2antibody experimental group grew slower and turnedsmaller. With P <0.05, the difference is statistically significant. Strip thetumors, detect MLL2gene expression by RT-PCR, and detect MLL2proteinexpression by Western-blot method. It shows that both MLL2gene expressionand MLL2protein expression in the experimental group reduced.Conclusion:1There exists MLL2gene expression in DLBL Cell Strain LY-3Cells.2In a certain concentration range, with the increase of MLL2antibodyconcentration, LY-3cell inhibition rate increases; MLL2antibody hasanti-proliferative activity, and can induce cell apoptosis.3MLL2antibody can induce cell apoptosis and inhibit tumors’ growth intumor-bearing mice’s body.