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Apoptosis and Proliferation as Well as Regulation Mechanism of Hela and Bovine Granulosa Cells Transfected with Eukaryotic Expression Plasmid Encoding Two Copies of Inhibin a (1-32) Fragments

Author: ZhenYanHong
Tutor: YangLiGuo
School: Huazhong Agricultural University
Course: Animal Genetic Breeding and Reproduction
Keywords: Inhibin α Bovine granulosa cells / Hela cells Apoptosis / cycle
CLC: S823
Type: Master's thesis
Year: 2010
Downloads: 73
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Abstract


In this study, gene cloning, plasmid extraction, cell culture, liposomal transfection, reverse transcriptase RT-PCR, real-time fluorescence quantitative PCR, confocal microscopy positioning, flow cytometry, tetramethylazodicarboxamide wow blue (MTT ), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) technology, aimed at: ① The detection built inhibit the quality of grain quality analysis of its mechanism of action, to lay the foundation for the preparation of inhibin DNA vaccine; ② investigate the inhibition factors overexpression granulosa cell proliferation and apoptosis and its mechanism, to lay the foundation for further elucidate the regulation mechanism of follicular development, breeding new technology developed to promote follicular development; ③ analysis inhibin overexpression of cervical cancer cells (Hela cells) effect and mechanism of apoptosis and proliferation, and to explore the role of inhibin uterus, lay the theoretical foundation for development to improve the rate of embryonic development, and new technologies for anti-cervical cancer disease. The content and the results are as follows: 1. Resistance eukaryotic expression vector pVAX-asd and the nuclear expression plasmid pEGISI of inhibin really successfully constructed the no resistance inhibin eukaryotic expression plasmid pVAX-asd-ISI-EGFP was (hereinafter referred to as pXAISI-EGFP). The subunit fragment the plasmid containing the dual copy inhibin a (1-32), hepatitis B surface antigen gene HBsAg-S and the enhanced green fluorescent protein gene EGFP2. Taken primary bovine granulosa cells and passaged Hela cells, the application of liposomal transfection stained pXAISI-EGFP plasmid reverse transcriptase RT-PCR method detected inhibin 3 at the transcriptional level. bovine granulosa cells and Hela cells transfected pXAISI-EGFP, the application of confocal microscope subcellular localization, was found to inhibit the hormone fusion protein in the Hela cell nucleus expression, and the expression in bovine granulosa cells of cytoplasmic 4. applications V-FITC apoptosis detection kit and flow cytometry after transfection apoptosis in Hela cells transfected 48h blank control group, pXAISI-EGFP experimental group pVAX-asd-EGFP negative control group, the apoptosis rate were 8.20 ± 3.92%, 24.86 ± 5.38%, 31.83 ± 8.37%, the difference was significant (p lt; 0.05) shows that transfected Hela cell apoptosis can promote inhibin can inhibit Hela cell apoptosis compared with the negative control. 48h transfected bovine granulosa cells, microscopic observation showed inhibin can promote apoptosis of bovine granulosa cells 5. 48h after transfection, MTT assay to detect cell proliferation after transfection, bovine granulosa cells of the experimental group and blank control group and the negative control group compared to the inhibition rates were 41.86 ± 1.24% and 26.31 ± 0.78%; inhibition rate of Hela cells in the experimental group compared with the blank control group and negative control group was 74.35 ± 1.51% and 38.64 ± 3.60%, respectively; The results showed that cattle The proliferation of granulosa cells and Hela cells by suppression 6. 48h propidium iodide-treated cells to detect cell cycle changes after transfection using flow cytometry analysis. The results showed that: of GCs cell blank control group G0/G1 phase, S phase and G2 / M phase ratio were 64.92% ± 1.75,15.53% ± 1.98 and 19.55% ± 1.32; experimental group pXAISI-EGFP were 55.07% ± 1.55, 25.50% ± 1.48 and 19.44 ± 1.71; pVAX-asd-EGFP group were 62.71% 4 ± 1.21,4.37% ± 1.37 and 32.91% ± 1.28. Hela cells in G0/G1 phase of the blank control group, the proportion of S phase and G2 / M phases were 56.27 ± 2.57%, 30.24 ± 1.36% and 13.49 ± 3.94%; experimental group pXAISI-EGFP were 55.75 ± 2.70%, 39.41 ± 2.71 % and 4.84 ± 0.04%; pVAX-asd-EGFP group were 56.11 ± 2.69%, 36.20 ± 1.48% and 7.67 ± 1.25%. Found in bovine granulosa cells or Hela cells, inhibin can make the S-phase cells increased 7 by real-time quantitative PCR assay of apoptosis-related genes (Bcl-2, Bax, NFKB, p53) and inhibin by expression of the gene (TGFβR3), inhibiting apoptotic gene Bcl-2 and NFκB expression level was significantly reduced in Hela cells, the relative expression of the amount of 0.62 ± 0.02 and 0.38 ± 0.01, respectively, pro-apoptotic gene Bax and p53 expression levels increase, respectively, 3.24 ± 0.12 and 2.58 ± 0.06; bovine granulosa cells both inhibit apoptosis (Bcl-2, Bcl-xl) gene or the amount of the relative expression of pro-apoptotic genes (Bax, p53) are raised were 1.514 ± 0.11,1.92 ± 0.15,1.59 ± 0.09,1.23 ± 0.12. Inhibition of the receptor gene TGFβR3 the expression level was significantly upregulated relative expression levels of bovine granulosa cells and Hela cells were 16.15 ± 0.14 (p lt; 0.01) and 1.23 ± 0.04 (p lt; 0.05), the results suggest that inhibin a (1-32) overexpression in granulosa cells and Hela cells receptor gene (TGFβR3), can significantly increase the expression of 8 24 h after transfection, 48h, 72h, 96h by RIA and enzyme-linked immunosorbent assay detection of bovine particles cell culture fluid related hormones (estradiol, progesterone, estradiol / progesterone, inhibin) levels change. The results showed that estradiol and estradiol / progesterone 24h after transfection increased after is decreasing; progesterone secretion levels were decreased after transfection, and 48 h after transfection secretion; inhibin hormone levels compared to the blank control group and negative control group is always in a higher state transfected 72h lowest results of these studies indicate that inhibit the prime of bovine granulosa cells and Hela cells, cell proliferation, cycle, apoptosis, and apoptosis-related genes, inhibiting receptor a significant role in the regulation and related hormone levels.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Cow
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