中华鳖胚胎肝成纤维细胞的原代培养及其聚肌苷酸胞苷酸(Poly I:C)刺激模型构建

doi:10.3969/j.issn.1004-1524.2022.01.08

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (1): 60-69.DOI: 10.3969/j.issn.1004-1524.2022.01.08

中华鳖胚胎肝成纤维细胞的原代培养及其聚肌苷酸胞苷酸(Poly I:C)刺激模型构建

王水涛¹(), 何盛盛¹, 江玲丽²^,^*(), 高有领¹^,^*()

1.浙江万里学院生物与环境学院,浙江宁波 315100
2.宁波卫生职业技术学院,浙江宁波 315100

收稿日期:2021-03-27 出版日期:2022-01-25 发布日期:2022-02-05
通讯作者: 江玲丽,高有领
作者简介:高有领,E-mail: gaoyl@zwu.edu.cn
* 江玲丽,E-mail: jllgrace@163.com;
王水涛(1993—),男,湖北黄冈人,硕士研究生,研究方向为水产动物营养与免疫。E-mail: 2335361898@qq.com
基金资助:
宁波市科技局项目(202002N3048);浙江省自然科学基金(LY19C180001);浙江省一流学科自设课题(ZS2020003)

Primary culture of fibroblast from embryonic liver of Chinese soft-shelled turtle (Pelodiscus sinensis) and establishment of polyinosinic acid cytidylic acid (Poly I:C) stimulation cell model

WANG Shuitao¹(), HE Shengsheng¹, JIANG Lingli²^,^*(), GAO Youling¹^,^*()

1. College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
2. Ningbo College of Health Sciences, Ningbo 315100, Zhejiang, China

Received:2021-03-27 Online:2022-01-25 Published:2022-02-05
Contact: JIANG Lingli,GAO Youling

摘要/Abstract

摘要：

为建立中华鳖胚胎肝成纤维细胞的体外分离培养体系,研究聚肌苷酸胞苷酸(Poly I:C)刺激对细胞干扰素生成通路相关因子的影响,从而最终构建基于该成纤维细胞的干扰素生成通路激活模型,以发育至23期的中华鳖胚胎为实验材料,采用胰蛋白酶消化法得到肝成纤维细胞,结合细胞形态学、生长曲线和PCR法进行鉴定,并分析其生物学特性。之后,用Poly I:C转染细胞24 h,检测细胞形态和干扰素生成信号通路相关因子的基因和蛋白表达水平。结果表明:应用构建的体外分离培养体系得到的中华鳖胚胎肝成纤维细胞生长状况良好,成功传至20代,该细胞的生长曲线为“S”形,符合细胞增殖规律,可检测到成纤维细胞标记基因VIM和ACTA2,确定为成纤维细胞。冻存前后,细胞存活率分别为90.51%和82.95%。Poly I:C转染后,RIG-Ⅰ和MAVS蛋白的表达量上调,RIG-Ⅰ基因的表达量上调,m⁶A和YTHDF2基因的表达量下调,并引起细胞形态的变化。综上,本研究成功建立了中华鳖胚胎肝成纤维细胞的体外分离培养体系,并构建了基于该细胞的干扰素生成通路激活模型。

关键词: 中华鳖, 原代培养, 成纤维细胞, 干扰素

Abstract:

The aims of the present study were to establish an in vitro isolation and culture system for the embryonic liver fibroblast of Chinese soft-shelled turtle(Pelodiscus sinensis), and to study the effect of polyinosinic acid cytidylic acid (Poly I:C) stimulation on the key factors of the cellular interferon production pathway, finally, to establish the activation model of interferon production pathway based on the fibroblast. The embryo developed to stage 23 was used to obtain the liver fibroblast by trypsin digesting. After that, the cell was identified by PCR method. Its biological characteristics including cell morphology and growth pattern were analyzed. Besides, the gene and protein expression levels of the factors related to the interferon production signal pathway were detected at 24 h after cell transfection with Poly I:C. The results showed that the obtained embryonic liver fibroblasts were in good growth condition and had been successfully passed to the 20^th generation. The growth curve of the cell was S-shaped, which conformed to the cell proliferation law. Fibroblast marker genes VIM and ACTA2 were confirmed. Before and after cryopreservation, the cell survival rates were 90.51% and 82.95%, respectively. After Poly I:C stimulation, the protein expressions of RIG-Ⅰ and MAVS were up-regulated, the gene expression of RIG-Ⅰ was up-regulated. Whereas, the gene expressions of m⁶A and YTHDF2 were down-regulated. In addition, the change of cell morphology was recorded. In conclusion, the study successfully established an in vitro isolation and culture system for embryonic liver fibroblasts and constructed an activation model of interferon production pathway based on embryonic liver fibroblasts of Chinese soft-shelled turtle.

Key words: Chinese soft-shelled turtle, primary culture, fibroblast, interferon

中图分类号:

S917.4

王水涛, 何盛盛, 江玲丽, 高有领. 中华鳖胚胎肝成纤维细胞的原代培养及其聚肌苷酸胞苷酸(Poly I:C)刺激模型构建[J]. 浙江农业学报, 2022, 34(1): 60-69.

WANG Shuitao, HE Shengsheng, JIANG Lingli, GAO Youling. Primary culture of fibroblast from embryonic liver of Chinese soft-shelled turtle (Pelodiscus sinensis) and establishment of polyinosinic acid cytidylic acid (Poly I:C) stimulation cell model[J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 60-69.

图/表 10

表1 引物序列

Table 1 Sequences of primers

引物名称 Primer name	上游引物序列 Forward primer sequnces(5'→3')	下游引物序列 Reverse primer sequnces(5'→3')	产物长度 Product length/bp
VIM	TACCGCAGACAAATACA	TAAACCTTCAGGGAGAG	633
ACTA2	CAACCGAGAGAAAATGA	ACAGGGAGGCAAGAATA	707
GAPDH	TGGCATAGTGGAAGGTCT	GATGGATGAGTGGCTGTC	379
RIG-Ⅰ	CCTGGCAAGAAAGGTTTACCCGAT	GTTGTTTAGCTTCAAAGTTCGCTGT	304
m⁶A	CAGCCTAACTCTGTTCCAAGTGGT	ATAGAACGGAAAGCACTGTCCAAGC	196
YTHDF2	CCACCCATGCCCTATTTAACATCGT	TTCAATGCTGCCATCCCTTGGTC	315
β-actin	TCCTGACAGAAAGAGGCTACA	GGATGGCTGGAAGAGGG	320

图1 原代培养和传代培养的中华鳖胚胎肝成纤维细胞形态

Fig.1 Morphology of primary culture and subculture of fibroblast from embryonic liver of Chinese soft-shelled turtle

图2 中华鳖胚胎肝成纤维细胞冻存前(a)、后(b)台盼蓝染色结果正常细胞染色后呈白色,死亡细胞染色后呈蓝色。

Fig.2 Trypan blue staining of embryonic liver fibroblast of Chinese soft-shelled turtle before (a) and after (b) cryopreservation Living cells display white color, while the dead ones display blue color.

图3 中华鳖胚胎肝成纤维细胞的生长曲线

Fig.3 Growth curve of embryonic liver fibroblast of Chinese soft-shelled turtle

图4 中华鳖胚胎肝成纤维细胞染色体

Fig.4 Chromosome of embryonic liver fibroblast of Chinese soft-shelled turtle

图5 中华鳖胚胎肝成纤维细胞的分子鉴定 M,DNA分子量标准;1,ACTA2;2,VIM;3,GAPDH;4,H2O。

Fig.5 Molecular identification of embryonic liver fibroblast of Chinese soft-shelled turtle M, DNA marker; 1, ACTA2; 2, VIM; 3, GAPDH; 4, H2O.

图6 Poly I:C转染中华鳖胚胎肝成纤维细胞24 h后的形态

Fig.6 Morphology of embryonic liver fibroblast of Chinese soft-shelled turtle transfected with Poly I:C for 24 h

图7 Poly I:C转染对中华鳖胚胎肝成纤维细胞RIG-Ⅰ、m6A和YTHDF2基因相对表达量的影响标*或**的分别表示对照组与转染组差异显著(P<0.05)或极显著(P<0.01)。

Fig.7 Relative gene expression of RIG-Ⅰ, m6A and YTHDF2 in embryonic liver fibroblast of Chinese soft-shelled turtle transfected with Poly I:C * and ** indicate significant difference between the control group and the transfection group at P<0.05 and P<0.01, respectively.

图8 Poly I:C转染对中华鳖胚胎肝成纤维细胞RIG-Ⅰ、MAVS和IRF1蛋白表达的影响

Fig.8 Protein expression of RIG-Ⅰ, MAVS and IRF1 in embryonic liver fibroblast of Chinese soft-shelled turtle transfected with Poly I:C

表2 Western blot条带量化灰度值

Table 2 Quantified gray value of Western blot

处理Treatment	RIG-Ⅰ	MAVS	IRF1
对照组	0.42±0.02 b	0.37±0.05 b	0.83±0.10
Control group
转染组	1.47±0.11 a	1.03±0.02 a	1.01±0.03
Transfection group

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中华鳖胚胎肝成纤维细胞的原代培养及其聚肌苷酸胞苷酸(Poly I:C)刺激模型构建

Primary culture of fibroblast from embryonic liver of Chinese soft-shelled turtle (Pelodiscus sinensis) and establishment of polyinosinic acid cytidylic acid (Poly I:C) stimulation cell model

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