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

doi:10.3969/j.issn.1004-1524.2022.01.08

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (1): 60-69.DOI: 10.3969/j.issn.1004-1524.2022.01.08

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S917.4

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.

Figures/Tables 10

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

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

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.

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

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

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

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

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.

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

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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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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