Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (3): 312-321.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0784
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SHAN Xin-yu(), LI Tai-chun, YANG Ruo-chen, DUAN Xiang-ru, KANG Jia, ZHANG Ying-jie, LIU Yue-qin()
Received:
2023-08-14
Online:
2024-03-26
Published:
2024-04-08
Contact:
LIU Yue-qin
E-mail:1126954588@qq.com;Liuyueqin66@126.com
SHAN Xin-yu, LI Tai-chun, YANG Ruo-chen, DUAN Xiang-ru, KANG Jia, ZHANG Ying-jie, LIU Yue-qin. Effect of γ-aminobutyric Acid on Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells[J]. Biotechnology Bulletin, 2024, 40(3): 312-321.
基因Gene | 引物序列Primer sequence(5'-3') | 退火温度Annealing temperature/℃ | 产物长度Size/bp |
---|---|---|---|
GAPDH | F: GGTCGGAGTGAACGGATTTG | 60 | 222 |
R: CTTGACTGTGCCGTGGAACTT | |||
CCND1 | F:GGCGTTCGTAATCGTGTTTTG | 60 | 185 |
R:CAGGTAGAAGGAGTGAGGAATGTGT | |||
PCNA | F:GCCGAGGAGAACAAGCAGA | 60 | 123 |
R:GAGGGTGGGTTGGAAATGAA | |||
CCNB1 | F:GGCAGGAGGACTATGACATT | 60 | 222 |
R:TCAGGCACCCACTTCTCTTTC | |||
Bax | F:CGAGACCTGAAGCCAGAGAC | 60 | 183 |
R:GCAAAGATACAGCCAACACC | |||
Caspase-3 | F:CCCATCCTCACCATCATCAC | 60 | 275 |
R:GCACAAACACGCACCTCAA | |||
Bcl-2 | F:ACTTGGACCTGTCGCTGTCC | 60 | 120 |
R:CCTGCGTTTGGAGTGGTAGA | |||
CYP11A1 | F:AACATGGAGCTGCAGAGGAT | 60 | 120 |
R:CCAATGTCCAGCCCATGATG | |||
StAR | F:TCTTTGAGTTCGGAGGGGTC | 60 | 270 |
R:GGAGAAATCAAACAGGGGCC | |||
3β-HSD | F:TGGACCCGTCGATCTGAAAA | 60 | 283 |
R:GCGTACAAGAAGTCTGCCTC | |||
CYP19A1 | F:AATGCTGCCCCAGGTGTT | 60 | 379 |
R:CGGCGTCTCTGCGATTTT |
Table 1 Primer sequence
基因Gene | 引物序列Primer sequence(5'-3') | 退火温度Annealing temperature/℃ | 产物长度Size/bp |
---|---|---|---|
GAPDH | F: GGTCGGAGTGAACGGATTTG | 60 | 222 |
R: CTTGACTGTGCCGTGGAACTT | |||
CCND1 | F:GGCGTTCGTAATCGTGTTTTG | 60 | 185 |
R:CAGGTAGAAGGAGTGAGGAATGTGT | |||
PCNA | F:GCCGAGGAGAACAAGCAGA | 60 | 123 |
R:GAGGGTGGGTTGGAAATGAA | |||
CCNB1 | F:GGCAGGAGGACTATGACATT | 60 | 222 |
R:TCAGGCACCCACTTCTCTTTC | |||
Bax | F:CGAGACCTGAAGCCAGAGAC | 60 | 183 |
R:GCAAAGATACAGCCAACACC | |||
Caspase-3 | F:CCCATCCTCACCATCATCAC | 60 | 275 |
R:GCACAAACACGCACCTCAA | |||
Bcl-2 | F:ACTTGGACCTGTCGCTGTCC | 60 | 120 |
R:CCTGCGTTTGGAGTGGTAGA | |||
CYP11A1 | F:AACATGGAGCTGCAGAGGAT | 60 | 120 |
R:CCAATGTCCAGCCCATGATG | |||
StAR | F:TCTTTGAGTTCGGAGGGGTC | 60 | 270 |
R:GGAGAAATCAAACAGGGGCC | |||
3β-HSD | F:TGGACCCGTCGATCTGAAAA | 60 | 283 |
R:GCGTACAAGAAGTCTGCCTC | |||
CYP19A1 | F:AATGCTGCCCCAGGTGTT | 60 | 379 |
R:CGGCGTCTCTGCGATTTT |
一抗名称Name of primary antibody | 品牌及货号Brand and coale | 稀释度Proportion of dilution | 分子量Molecular weight/kD |
---|---|---|---|
Caspase-3 | Abcam ab13847 | 1∶1 000 | 34 |
StAR | Abcam ab133657 | 1∶1 000 | 32 |
3β-HSD | Abcam ab55268 | 1∶1 000 | 42 |
Bax | Abcam ab32503 | 1∶1 000 | 21 |
Bcl-2 | Abcam ab182858 | 1∶1 500 | 26 |
CCNB1 | Abcam ab181593 | 1∶1 500 | 55 |
CCND1 | Abcam ab16663 | 1∶200 | 33 |
CYP11A1 | Abcam ab272494 | 1∶1 000 | 60 |
CYP19A1 | Abcam ab18995 | 1∶800 | 55 |
PCNA | Abcam ab29 | 1∶800 | 30 |
β-actin(C4)(内参) | Santa Cruz SC-47778 | 1∶1 500 | 43 |
Table 2 Primary antibody information
一抗名称Name of primary antibody | 品牌及货号Brand and coale | 稀释度Proportion of dilution | 分子量Molecular weight/kD |
---|---|---|---|
Caspase-3 | Abcam ab13847 | 1∶1 000 | 34 |
StAR | Abcam ab133657 | 1∶1 000 | 32 |
3β-HSD | Abcam ab55268 | 1∶1 000 | 42 |
Bax | Abcam ab32503 | 1∶1 000 | 21 |
Bcl-2 | Abcam ab182858 | 1∶1 500 | 26 |
CCNB1 | Abcam ab181593 | 1∶1 500 | 55 |
CCND1 | Abcam ab16663 | 1∶200 | 33 |
CYP11A1 | Abcam ab272494 | 1∶1 000 | 60 |
CYP19A1 | Abcam ab18995 | 1∶800 | 55 |
PCNA | Abcam ab29 | 1∶800 | 30 |
β-actin(C4)(内参) | Santa Cruz SC-47778 | 1∶1 500 | 43 |
Fig. 1 Identification of ovine ovarian granulosa cells(400×) A: FSHR staining of ovine ovarian granulosa cells. B: DAPI staining of ovine ovarian granulosa cell nucleus. C: Superimposed dyeing effect
Fig. 2 Effects of different concentrations of GABA on proliferation in ovine granulosa cells The different letters indicate the significant difference(P<0.05), the same below
Fig. 3 Results of apoptosis in ovine granulosa cells treated with GABA A、B、C、D、E is the effects of 0, 10-8, 10-7, 10-6, 10-5 mol/L GABA on the apoptosis of ovine granulosa cells. Q1 was dead cells caused by mechanical injury; Q2 was late apoptosis; Q3 was early apoptosis; Q4 was living cells
项目Item | 正常细胞率Normal cell rate/% | 早期凋亡率Early apoptosis rate/% | 晚期凋亡率Late apoptosis rate/% | 总凋亡率Total apoptosis rate/% |
---|---|---|---|---|
Control | 81.97±0.61a | 1.58±0.22ab | 13.43±0.15a | 15.02±0.26a |
10-8 | 84.97±0.49b | 1.42±0.27a | 11.43±0.38b | 12.85±0.56b |
10-7 | 87.63±0.24c | 1.63±0.21ab | 9.16±0.52c | 10.78±0.31c |
10-6 | 89.27±0.03d | 1.21±0.09a | 7.76±0.24d | 8.97±0.16d |
10-5 | 86.80±0.58c | 2.12±0.16b | 9.70±0.17c | 11.82±0.04e |
Table 3 Effect of different concentrations of GABA on apoptosis rate in ovine granulosa cells
项目Item | 正常细胞率Normal cell rate/% | 早期凋亡率Early apoptosis rate/% | 晚期凋亡率Late apoptosis rate/% | 总凋亡率Total apoptosis rate/% |
---|---|---|---|---|
Control | 81.97±0.61a | 1.58±0.22ab | 13.43±0.15a | 15.02±0.26a |
10-8 | 84.97±0.49b | 1.42±0.27a | 11.43±0.38b | 12.85±0.56b |
10-7 | 87.63±0.24c | 1.63±0.21ab | 9.16±0.52c | 10.78±0.31c |
10-6 | 89.27±0.03d | 1.21±0.09a | 7.76±0.24d | 8.97±0.16d |
10-5 | 86.80±0.58c | 2.12±0.16b | 9.70±0.17c | 11.82±0.04e |
Fig. 4 Effects of GABA on the expressions of proliferation-related genes mRNA and proteins in ovine granulosa cells A: Statistics of mRNA expression results of proliferation-related genes after GABA treatment. B: Western blot results. C: Statistics of protein expression results of proliferation-related genes after GABA treatment
Fig. 5 Effects of GABA on the expressions of apoptosis-related genes mRNA and proteins in ovine granulosa cells A: Statistics of mRNA expression results of apoptosis-related genes after GABA treatment. B: Effect of GABA on ratio of mRNA expression of Bax/Bcl-2. C: Western blot results. D: Statistics of protein expression results of apoptosis-related genes after GABA treatment
项目 Item | 对照组 Control group | 试验组 Test group |
---|---|---|
雌二醇(E2)/(pg·mL-1) | 77.25±1.79a | 61.30±0.74b |
孕酮(P4)/(pmol·L-1) | 344.45±7.03a | 400.66±14.78b |
Table 4 Effects of 10-6 mol/L GABA on steroid hormone secretion of GCs in ovine granulosa cells
项目 Item | 对照组 Control group | 试验组 Test group |
---|---|---|
雌二醇(E2)/(pg·mL-1) | 77.25±1.79a | 61.30±0.74b |
孕酮(P4)/(pmol·L-1) | 344.45±7.03a | 400.66±14.78b |
Fig. 6 Effects of GABA on the expressions of steroid hormone synthesis-related genes and proteins in ovine granulosa cells A: Statistics of mRNA expression results of steroid hormone synthesis-related genes after GABA treatment. B: Western blot results. C: Statistics of protein expression results of steroid hormone synthesis-related genes after GABA treatment
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