粒细胞集落刺激因子在羊成纤维细胞中的表达及对细胞增殖和凋亡的影响

[目的]研究粒细胞集落刺激因子(granule cell stimulating factor,GCSF)在羊成纤维细胞体外培养中对其增殖、周期和凋亡的影响,为今后基于羊GCSF为靶标诱导全能干细胞进行分子遗传育种研究提供理论依据.[方法]将羊GCSF真核表达质粒pRTL1-GCSF和对照载体质粒pRTL1分别转染到1...

Transient Expression and the Effect on Proliferation and Apoptosis of Granule Cell Stimulating Factor in Ovarian Fibroblasts

【Objective】 The purpose of this paper is to study the transient expression of granule cell stimulating factor (GCSF) in ovarian fibroblast cells, and the influence of GCSF on proliferation, cell cycle, and apoptosis, to provide theoretical basis for molecular genetic breeding of sheep pluripotent stem cells induced by GCSF in the future. 【Method】 The sheep GCSF eukaryotic expression plasmid pRTL1-GCSF and the control vector plasmid pRTL1 were transfected into 1×10⁵ cells·mL^-1 sheep fibroblasts respectively. After 48 h of culture, the total RNA was extracted by Trizol method and reverse transcribed into cDNA. The transient expression level of sheep GCSF in fibroblasts was detected by real-time quantitative PCR. GCSF dependent cell line NFS-60 was used for the biological activity of GCSF secreted and expressed in the supernatant of sheep fibroblasts 48 hours after transfection, which was determined by cell viability detection reagent alamarBlue. The HEK 293F suspension culture was used to express the secreted GCSF protein. The GCSF protein expressed in the cell culture medium was purified by Ni-NTA resin and detected by SDS-PAGE. After adding the 30 ng·mL^-1 purified GCSF protein, the proliferation of sheep fibroblasts was detected by alamarBlue at 24 h and 48 h, and the cell cycle and apoptosis of sheep fibroblasts were detected by flow cytometry. 【Result】 The expression level of GCSF in sheep fibroblasts was significantly increased after transfection for 48 h. In sheep fibroblasts, the expression level of GCSF transfected with pRTL1-GCSF plasmid was 50 615.92 ± 4 738.83 of that of pRTL1 empty control group. The fluorescence intensity of NFS-60 in the experimental group and positive control group was significantly higher than that in the negative control group and blank control group (P<0.01), but there was no significant difference between the experimental group and the positive control group (P>0.05). The results showed that sheep GCSF could significantly stimulate the proliferation of NFS-60 cells, indicating that the GCSF expressed in sheep fibroblasts had biological activity. After eukaryotic expression of secretory GCSF protein in HEK 293F cell line, the sheep GCSF protein was purified. After 30 ng·mL^-1 sheep GCSF was added to sheep fibroblasts, the cell viability of GCSF test group was not significantly different from that of culture medium dilution control group for 24 h and 48 h, but the distribution of cell cycle was significantly changed. At 24 h, compared with the control group, the proportion of G1 phase cells increased from (55.29±1.68)% to (69.37±0.24)%, the difference was very significant (P<0.01); the proportion of S phase cells changed from (15.99±0.38)% to (15.39±0.60)%, the difference was not significant (P>0.05); G2/M phase cells increased significantly (P<0.05), and the proportion increased from (22.88±1.00)% to (26.76±0.82)%. The results showed that 24 hours after the addition of sheep GCSF, the number of cells in division and interphase increased significantly. At 48 h, compared with the control group, the proportion of G1 phase cells decreased from (65.96±0.37)% to (45.69±0.26)%, the difference was very significant (P<0.01); the proportion of S phase cells increased from (13.45±1.33)% to (37.87±2.43)%, the difference was very significant (P<0.01); the proportion of G2/M phase cells changed from (16.42±1.29)% to (21.80±1.86)%, the difference was not significant (P>0.05). The results showed that the number of cells in interphase was significantly decreased and the number of cells in DNA replication state increased significantly at 48 h after adding GCSF. Compared with the control group, the apoptosis rates of the control group (Ctr) and the experimental group (GCSF) were (7.51±0.38)% and (9.16±0.46)% respectively at 24 h culture. At 48 h, the apoptosis rates of the control group and the experimental group were (5.73±0.29)% and (5.39±0.27)%, respectively. At 72 h, the apoptosis rates of control group (Ctr) and experimental group (GCSF) were (8.88±0.45)% and (5.41±0.27)%, respectively. There was a significant difference between 24 h and 72 h (P<0.01), but there was no significant difference at 48 h (P>0.05). The results showed that GCSF promoted the apoptosis within 24 hours, and the apoptosis was inhibited with the prolongation of time. 【Conclusion】 In conclusion, sheep fibroblasts can express GCSF instantaneously and have biological activity. GCSF did not affect the proliferation of sheep fibroblasts, but could regulate its cell cycle and affect cell apoptosis. The results laid a foundation for breeding sheep with high immunity and disease resistance by GCSF mediated by sheep fibroblasts.