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Ultrasound combined with LHRHa targeted microbubble mediated lipid wild-type p53 gene transfection of ovarian cancer cells in experimental study A2780/DDP

Author: GuoJuan
Tutor: ChangShuFang
School: Chongqing Medical University
Course: Obstetrics and Gynaecology
Keywords: Lipid microbubbles targeted LHRHa Ultrasound Gene transfection Ovarian Cancer
CLC: R737.31
Type: Master's thesis
Year: 2011
Downloads: 60
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Abstract

The first part LHRHa lipid microbubbles targeted Preparation and in vitro preparation of targeting the purpose of identification of human ovarian carcinoma targeted ultrasound contrast agent, observed in vitro homing capability. Methods biotin - streptavidin-biotin prepared luteinizing hormone releasing hormone analogue (LHRHa) targeted lipid microbubbles, by immunofluorescence staining verify LHRHa lipid microbubbles combined with the situation, and in general lipid microbubbles as a control, was observed under an inverted microscope LHRHa lipid microbubbles targeted on human ovarian cancer A2780/DDP vitro homing capability. Results LHRHa lipid microbubbles targeted immunofluorescence positive; homing in vitro experiments showed that LHRHa lipid microbubbles targeted to work with LHRH receptors present on the surface of A2780/DDP cell-specific binding, while the ordinary lipid microbubbles can not be combined. Conclusion biotin - streptavidin-biotin method can successfully prepared LHRHa targeting lipid microbubbles, the targeted contrast agent with in vitro homing capability. The second part of different microbubble ultrasound intensity and concentration on the ovarian cancer cell activity A2780/DDP Objective To investigate the influence of different microbubble ultrasound intensity and concentration on cell growth activity A2780/DDP screened microbubbles optimal parameter combination, for subsequent gene transfection experiments basis. Method 1 would A2780/DDP cells were divided into nine experimental groups were given 0.5W/cm ~ 2 (30s, 50s, 70s), 1.0W/cm ~ 2 (30s, 50s, 70s), 1.5W/cm ~ 2 (30s, 50s, 70s), with untreated cells as the control group, MTT assay different ultrasonic intensity and time on the A2780/DDP cell growth activity. Screening had no significant effect on cell viability ultrasound intensity and duration. 2 A2780/DDP cell into the three experimental groups, the concentration of microbubbles into a 10:1,15:1,20:1 cell concentration ratio, combined screening effect ultrasonic intensity and duration of the experimental group, to the extent not treatment, control group, MTT assay cell growth activity of each group screened on A2780/DDP cell activity was less affected microbubble ultrasound parameters and concentration combinations. Results 1. 0.5W/cm ~ 2,30 s sound intensity ultrasound irradiation, cell viability gt; 90%. 2 and cell concentration ratio of 10:1 microbubble concentration joint 0.5W/cm ~ 2,30 s ultrasonic irradiation, cell viability gt; 90%. Conclusion Ultrasound microbubble irradiation A2780/DDP cells inhibited the proliferation, and with the ultrasonic intensity, time, and the increasing concentration of microbubbles on cell proliferation increases. In order to affect cell growth activity was not obvious under the premise of gene transfection, we chose to 0.5 W / cm ~ 2,30 s ultrasound parameters combined with cell concentration ratio of 10:1 microbubble concentration as the subsequent gene transfection experiment conditions . The third part of the joint LHRHa targeted microbubble ultrasound-mediated transfection of wild-type p53 gene transfection A2780/DDP cells and apoptosis shadow on the purpose of the second part of the screening effect of irradiation parameters on A2780/DDP cells to explore Ultrasound combined with LHRHa targeted microbubble mediated lipid wild-type p53 gene transfection efficacy, and after transfection on cell apoptosis. Method 1 would A2780/DDP cells were divided into six groups: A: plasmid group; B: non-targeted microbubbles plasmid group; C: Targeted microbubbles plasmid group; D: Ultrasound plasmid group; E. ultrasonic non-target micro- bubble plasmid group; F. ultrasound targeted microbubble plasmid group, respectively, in each experimental group to join the appropriate treatment factors were observed under a fluorescence microscope fluorescence expression cells in each group, each group were compared using flow cytometry transfection efficiency, RT- PCR expression on wtp53mRNA semi-quantitative analysis. Two would A2780/DDP cells were divided into seven groups: A. blank group; B. ultrasound group; C: Ultrasonic non-targeted microbubble group D: ultrasound targeted microbubble group E: Ultrasound Ultrasound non-target plasmid group F. microbubble plasmid group G: Ultrasound targeted microbubble plasmid group, respectively, in each group to join the appropriate treatment factors, the use of flow cytometry to detect cell apoptosis. 3 The cells were divided into two groups A2780/DDP: A: ultrasound microbubbles targeted plasmid group B: blank control group, the cells were treated accordingly, the use of flow cytometry cell cycle, apoptosis TEM . Results 1. Inverted fluorescence microscope ultrasound targeted microbubble ultrasound plasmid group and non-targeted microbubble plasmid group were seen more bright green fluorescent cells, ultrasonic plasmid group only see a very small amount of fluorescent cells, almost did not see the rest of the group fluorescent cells. 2 transfection efficiency by flow cytometry: ultrasound targeted microbubble ultrasound plasmid group and non-targeted microbubble its wtp53 plasmid gene transfection rate was (43.90 ± 6.19)% and (25.33 ± 4.44 ring)%, In comparison, the difference was statistically significant (P lt; 0.05), ultrasound combined with plasmid transfection rate (7.24 ± 5.15)%, the rest of the group transfection rate lt; 1%; 3.RT-PCR results Show: targeted microbubble ultrasound LHRHa plasmid group and non-targeted microbubble ultrasound plasmid expression could be detected in higher wtp53mRNA, both compared to the former than the latter, the difference was statistically significant (P lt; 0.05) ultrasound detected only very low plasmid group wtp53mRNA expression, the rest of the group did not detect wtp53mRNA expression. 4 cell apoptosis was detected by flow cytometry: ultrasound targeted microbubble LHRHa plasmid group and non-targeted microbubble ultrasound plasmid apoptosis rate was (39.67 ± 5.95)% and (24.54 ± 3.68)%, two were compared, the difference was statistically significant (P lt; 0.05). 6 cell cycle analysis by flow cytometry: Ultrasound combined with LHRHa targeted microbubble mediated wild-type p53 gene was transfected into cells, (62.79 ± 4.65)% of the cells remain in the G1 phase, compared with the control group, difference was statistically significant (P lt; 0.05). 7 TEM cell apoptosis: wild-type p53 gene transfection cells were apoptotic after, compared with normal cells, chromatin margination, nuclear budding, chromatin condensation forming wrap prolapse apoptotic bodies, cell cytoplasm vacuoles increased. Conclusion Ultrasound combined with LHRHa targeted microbubbles can effectively transfected with wild-type P53 gene into human ovarian cancer A2780/DDP cells and transfected with wild-type p53 gene can induce apoptosis A2780/DDP, is expected to become an effective ovarian cancer Targeted gene therapy.

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