Effect Of Cell Proliferation And Apoptosis In The CHG-5 Cell Treated By Ultrasonic Microbubble Intensifier And Survivin Antisense Oligonucleotides Transfection

PART ONE Effect of expression of survivin protein in the CHG-5cell treated by ultrasonic microbubble intensifier and survivin antisense oligonucleotides transfectionOBJECTIVE: To study the role of inhibition of survivin mRNA and protein in the human glioma cell line CHG-5 cell treated by ultrasonic microbubble intensifier and survivin antisense oligonucleotides transfectionMETHODS: Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. There were five regimen groups treated by different agents. Group A, survivin antisense oligonucleotides combined with ultrasonic microbubble intensifier transfection and ultrasound irradiation﹙ ASODN + UM + U﹚ . Group B, survivin antisense oligonucleotides combined with lipofectamine transfection and ultrasound irradiation﹙ ASODN + lipo + U﹚ . Group C, survivin antisense oligonucleotides combined with lipofectamine transfection﹙ASODN+lipo﹚ . Group D,survivin antisense oligonucleotides combined with ultrasonic microbubble intensifier transfection﹙ASODN+UM﹚. Group E, blank control﹙Control﹚. The mRNA expression of survivin in different groups was measured by RT-PCR. The protein expression of survivin in different groups was measured by immunocytochemistry staining, confocal laser scanning microscopy and Western blotting.RESULTS: The inhibition ratio of survivin mRNA was 67.76﹪in group A, however the inhibition ratio of survivin mRNA in groups of B and C was 48.70﹪and 44.03﹪respectively, which was lower than that in the group A(P﹤0.05), the three disposal groups had significant difference compared with the blank control respectively(P﹤0.05). The inhibition ratio of survivin mRNA was 3.84﹪in the group D, which had not significant difference compared with the blank control(P﹥0.05). The expression of survivin protein was down regulation in group A ,B and C, but the expression of survivin protein in group A dropped most obviously(P﹤0.05), the three disposal groups had significant difference compared with the blank control respectively(P﹤0.05). The expression of survivin protein in group D had not significant difference compared with the blank control(P﹥0.05). This changes of survivin protein expression was same as the changes of survivin mRNA.CONCLUSIONS: The sequence of survivin antisense oligonucleotides was specific. The level of survivin mRNA and protein was dropped obviously after transfection of survivin antisense oligonucleotides, but survivin ASODN combined with ultrasonic microbubble intensifier transfection and ultrasound irradiation dropped most obviously. Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively. PART TWOEffect of cell proliferation and apoptosis in the CHG-5 cell treated by ultrasonic microbubble intensifier and survivin antisense oligonucleotides transfectionOBJECTIVE: To study the effect of cell proliferation and apoptosis in the human glioma cell line CHG-5 cell by ultrasonic microbubble intensifier and survivin antisense oligonucleotides transfection.METHODS: Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. There were five regimen groups treated by different agents. Group A, survivin antisense oligonucleotides combined with ultrasonic microbubble intensifier transfection and ultrasound irradiation﹙ ASODN + UM + U﹚ . Group B, survivin antisense oligonucleotides combined with lipofectamine transfection and ultrasound irradiation﹙ ASODN + lipo + U﹚ . Group C, survivin antisense oligonucleotides combined with lipofectamine transfection﹙ASODN+lipo﹚ . Group D,survivin antisense oligonucleotides combined with ultrasonic microbubble intensifier transfection﹙ASODN+UM﹚. Group E, blank control﹙Control﹚. MTT assay was used to measure the changes of proliferation, and the changes of apoptosis ratio was detected by flow cytometry.RESULTS:﹙1﹚As time increasing the cell growth inhibition ratio, displayed a increasing trend at 24h, 48h, 72h after transfection in the same experimental group, and the inhibition ratio at 72h was the most largest. At the same time point, the difference of cell growth inhibition ratio between the group B and group C was not significant (P﹥0.05), the group D had not significant difference compared with the blank control(P﹥0.05),but group A had significant difference compared with the rest groups(P﹤0.05). ﹙2﹚The apoptosis ratio of group A was (54.97±1.89)﹪measured by flow cytometry , which had significant difference compared with the group B(35.98±3.51)﹪and group C(33.74±1.77)﹪(P﹤0.05), and the three disposal groups had significant difference compared with the blank control respectively(P﹤0.05), the group D﹙7.76±1.08﹚﹪had not significant difference compared with the blank control(P﹥0.05).CONCLUSIONS: Survivin antisense oligonucleotides combined with ultrasonic microbubble intensifier transfection and ultrasound irradiation can significantly inhibit cell proliferation, and promote cell apoptosis.