The Effects Of HTR Antisense Oligodeoxynucleotide On Detection Of Cell Apoptosis,expression Of Apoptosis Induciong Factor And Mitochondria Transmembrane Potential In Colorectal Carcinoma SW480 Cells

Objective:To explore the effects of sealing the human telomerase RNA antisense oligodeoxynucleotide (hTR ASODN) on telomerase activation, cell apoptosis, expression of apoptosis inducing factor (AIF) and mitochondria transmembrane potential in human colorectal cancer SW480 ce11.Methods:1.Observing the transfection circumstances of colorectal cancer SW480 cell ,which transfected by total thio-modified hTR ASODN with the best concentration of transfection(2.5μmol/L) marked by 5'-FAM after transfected different days by inverted fluorescence microscopy;2.Observing the diversity of telomerase activation after colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection(2.5μmol/L) after different days by the detected method of TRAP-PCR-ELISA;3.Detecting the apoptosis ratio of telomerase activation after colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection (2.5μmol/L)after different days by flow cytometry; 4.Assessing if the transfection can cause the release of AIF after colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection(2.5μmol/L) after different days by RT-PCR detecting expression of AIF;5.Detecting diversity of the mitochondria transmembrane potential after colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection (2.5μmol/L)after different days by flow cytometry.Results:1.After colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection(2.5μmol/L), it showed good transfection when green fluorescent distribute equally in cells by inverted fluorescence microscope.2.After colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection(2.5μmol/L), observing the telomerase activation of every groups by TRAP-PCR-ELISA after transfected 24, 48, 72h respectively: the blank group 0.806±0.019, 0.813±0.024 and 0.810±0.011, the sense group 0.810±0.012, 0.797±0.014 and 0.794±0.009, the anti-sense group 0.657±0.014, 0.411±0.020 and 0.396±0.018. The telomerase activation of the anti-sense group descended obviously compared with the blank group or the sense group, the difference had statistical significance (P< 0.05), while the difference of the blank group and the sense group had no statistical significance (P> 0.05);3.After colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection (2.5μmol/L), observing the apoptosis ratio of every groups by flow cytometry after transfected 24, 48, 72h respectively: the blank group (3.07±0.11)%, (3.71±0.63)% and (4.23 ±0.41)%, the sense group (4.28±0.41)%, (4.49±0.56)% and (5.13±0.56)%, the anti-sense group (13.11±0.32)%, (22.04±0.21)% and (16.71±0.82)%. The apoptosis ratio of the anti-sense group compared with the blank group or the sense group, the difference had statistical significance (P< 0.05), while the difference of the blank group and the sense group had no statistical significance (P> 0.05);4.After colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection(2.5μmol/L), observing the PCR outputs of every groups by flow cytometry after transfected 48h respectively: the blank group (0.1527±0.0021), the sense group (0.158 0±0.0078), the anti-sense group (0.3683±0.0109). The PCR outputs of the anti-sense group compared with the blank group or the sense group, the difference had statistical significance (P< 0.05), while the difference of the blank group and the sense group had no statistical significance (P> 0.05);5.After colorectal cancer SW480 cell transfected by hTR ASODN with the best concentration of transfection(2.5μmol/L), observing the mitochondria transmembrane potential of every groups by flow cytometry after transfected 24, 48, 72h respectively: the blank group 51.68±0.55, 51.63±0.74 and 50.72±0.60, the sense group 51.35±0.79, 50.73±0.73 and 50.02±0.53, the anti-sense group 41.24±0.87, 33.63±0.67 and 34.68±0.67. The telomerase activation of the anti-sense group descended obviously compared with the blank group or the sense group, the difference had statistical significance (P< 0.05), while the difference of the blank group and the sense group had no statistical significance (P> 0.05).Conclusion:Sealing hTR ASODN,which can decrease elomerase activation, reinforce expression of AIF , decrease mitochondria transmembrane potential in colorectal cancer SW480 ce11,accordingly restrain the growth and increase of neoplasms cell,inducing apoptosis,and which afford evidence to cancer of gene therapy.