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

doi:10.3864/j.issn.0578-1752.2021.11.015

中国农业科学 ›› 2021, Vol. 54 ›› Issue (11): 2434-2444.doi: 10.3864/j.issn.0578-1752.2021.11.015

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

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

李闰婷^1,²(),陈龙欣²,张丽萌^1,²,何海迎¹,王泳¹,杨若晨¹,段春辉¹,刘月琴¹,王玉琴³,张英杰¹()

¹河北农业大学动物科技学院,河北保定 071001
²郑州师范学院分子生物学实验室,郑州 450044
³河南科技大学动物科技学院,河南洛阳 471023

收稿日期:2020-05-11 接受日期:2020-10-29 出版日期:2021-06-01 发布日期:2021-06-09
通讯作者: 张英杰
作者简介:李闰婷,E-mail：rtli1672@126.com。
基金资助:
国家现代农业（肉羊）产业技术体系建设专项(CARS-38);国家现代农业（肉羊）产业技术体系建设专项(CARS-39);国家重点研发计划项目(2018YFD0502100)

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

LI RunTing^1,²(),CHEN LongXin²,ZHANG LiMeng^1,²,HE HaiYing¹,WANG Yong¹,YANG RuoChen¹,DUAN ChunHui¹,LIU YueQin¹,WANG YuQin³,ZHANG YingJie¹()

¹College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, Hebei
²Molecular Biology Laboratory, Zhengzhou Normal University, Zhengzhou 450044
³College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, Henan

Received:2020-05-11 Accepted:2020-10-29 Online:2021-06-01 Published:2021-06-09
Contact: YingJie ZHANG

摘要/Abstract

摘要：

【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1×10⁵个细胞/mL的羊成纤维细胞中,培养48 h后,利用Trizol法分别提取总RNA并反转录为cDNA,通过实时荧光定量PCR检测羊GCSF在羊成纤维细胞中的瞬时表达水平。通过GCSF依赖型细胞系NFS-60,利用细胞活力检测试剂alamarBlue测定转染48 h后羊成纤维细胞培养上清中分泌表达的GCSF的生物学活性。通过HEK 293F悬浮培养细胞系真核表达分泌型羊GCSF蛋白后,用Ni-NTA凝胶对细胞表达至细胞培养基中的GCSF蛋白进行纯化,并SDS-PAGE检测。加入纯化的30 ng·mL^-1 GCSF蛋白后在24和48 h时,通过alamarBlue测定羊成纤维细胞的增殖状态,利用流式细胞术检测羊成纤维细胞的细胞周期和凋亡变化。【结果】羊GCSF真核表达质粒转染羊成纤维细胞48 h,检测发现在羊成纤维细胞中GCSF表达量得到显著提高。在羊成纤维细胞中,转染了pRTL1-GCSF质粒的羊GCSF表达量是转染了pRTL1空载对照组的（50 615.92±4 738.83）倍（P<0.01）;羊成纤维细胞瞬时分泌表达的含有GCSF蛋白的培养基上清加入到GCSF依赖型细胞系NFS-60后,试验组和阳性对照组中的NFS-60的荧光强度与阴性对照组和空白对照组相比显著升高（P<0.01）,试验组中的NFS-60的荧光强度与阳性对照组相比均差异不显著（P>0.05）,结果显示羊GCSF能显著刺激NFS-60细胞的增殖,表明在羊成纤维细胞中表达的羊GCSF具有生物学活性。用HEK 293F悬浮培养细胞系真核表达分泌型羊GCSF蛋白后,纯化得到羊GCSF蛋白。在羊成纤维细胞中添加30 ng·mL^-1的羊GCSF后,体外培养24和48 h,GCSF试验组与培养基稀释液对照组相比,细胞活力变化差异不显著,而细胞周期的分布出现显著改变。24 h时,与对照组相比试验组的G1期细胞比例由(55.29±1.68)%增加到(69.37±0.24)%,差异极显著（P<0.01）;S期细胞比例由(15.99±0.38)%变为(15.39±0.60)%,差异不显著（P>0.05）;G2/M期细胞显著增多（P<0.05）,比例由(22.88±1.00)%增大到(26.76±0.82)%。表明在羊成纤维细胞中加入羊GCSF的24 h后,处于分裂状态和间期的细胞显著增多。48 h时,与对照组相比试验组G1期细胞比例由(65.96±0.37)%减少为(45.69±0.26)%,差异极显著（P<0.01）;S期细胞比例由(13.45±1.33)%增加为(37.87±2.43)%,差异极显著（P<0.01）;G2/M期细胞比例由(16.42±1.29)%变为(21.80±1.86)%,差异不显著（P>0.05）。表明加入GCSF的羊成纤维细胞在48 h时,处于间期的细胞显著减少,同时DNA复制状态的细胞显著增多。试验组凋亡率和对照组相比,培养24 h时对照组（Ctr）和试验组（GCSF）的凋亡率分别是(7.51±0.38)%和(9.16±0.46)%。48 h时对照组和试验组的凋亡率分别是(5.73±0.29)%和(5.39±0.27)%。72 h时对照组（Ctr）和试验组（GCSF）的凋亡率分别是(8.88±0.45)%和(5.41±0.27)%,24 h和72 h凋亡率差异极显著（P<0.01）,48 h检测时凋亡率差异不显著（P>0.05）,表明在GCSF添加的24 h内促进了细胞凋亡,随着时间的延长,细胞的凋亡受到抑制。【结论】羊成纤维细胞中可以瞬时过量表达羊GCSF,并具有生物学活性。GCSF不影响羊成纤维细胞的增殖,但可调控其周期,影响细胞凋亡。该结果为今后通过羊成纤维细胞介导GCSF培育具有高免疫力、高抗病性的羊进行分子遗传育种奠定了基础。

关键词: GCSF, 羊, 转染, 生物学活性, 细胞周期, 细胞凋亡

Abstract:

【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.

Key words: GCSF, sheep, transformation, bio-activity, cell cycles, apoptosis

李闰婷,陈龙欣,张丽萌,何海迎,王泳,杨若晨,段春辉,刘月琴,王玉琴,张英杰. 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[J]. 中国农业科学, 2021, 54(11): 2434-2444.

LI RunTing,CHEN LongXin,ZHANG LiMeng,HE HaiYing,WANG Yong,YANG RuoChen,DUAN ChunHui,LIU YueQin,WANG YuQin,ZHANG YingJie. Transient Expression and the Effect on Proliferation and Apoptosis of Granule Cell Stimulating Factor in Ovarian Fibroblasts[J]. Scientia Agricultura Sinica, 2021, 54(11): 2434-2444.

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引物名称 Primer names	序列 Sequences
GCSF-pR	GAA GGC CGA CGT GAA GGT
GCSF-pF	TCA CGA ATT CGA CCC CCC TTG GCC CTG C
GAPDH-pF	CAA GTT CCA CGG CAC AGT CA
GAPDH-pR	CTC AGC ACC AGC ATC ACC C

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

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

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