ESTABLISHMENT OF A PREADIPOCYTE CELL LINE OF SPOTTED SEA BASS (LATEOLABRAX MACULATUS) AND OLEIC ACID-INDUCED DIFFERENTIATION
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摘要:
花鲈(Lateolabrax maculatus)前脂肪细胞系为探索鱼类脂肪细胞分化和脂肪沉积的调控作用及相关分子机制提供了一种新的体外模型。研究将花鲈腹腔脂肪组织用胶原酶消化后原代培养, 再以胰酶消化后传代, 总共经过150次传代培养后, 得到花鲈前脂肪细胞系。进而, 染色体核型分析表明该细胞系长期培养仍能保持正常表型, 进一步使用线粒体cox1和cytb基因鉴定该细胞系来源于花鲈。用pEGFP-C1质粒转染细胞系, 观察到明显的绿色荧光, 表明可用于研究外源基因的表达。用不同浓度的油酸培养细胞系, 3d后细胞有明显脂滴聚集, 7d后脂滴达到最大; 油红O染色表明, 400 μmol/L油酸诱导7d后细胞即可正常分化成熟。综上所述, 实验建立了花鲈前脂肪细胞系, 该细胞系形态典型、可正常分化, 为后续研究鱼类脂肪细胞分化及脂肪沉积提供了良好的实验材料。
Abstract:The proadipocyte line of spotted sea bass (Lateolabrax maculatus) provides a new in vitro model for exploring the regulation of adipocyte differentiation and adipocyte deposition in fish and the related molecular mechanisms. This experiment aimed to establish a proadipose cell line for the spotted sea bass. Peritoneal adipose tissue was digested using collagenase and pancreatic enzyme, and after multiple passages, a preadipose cell line was successfully obtained following 150 times of culture cycles. karyotype analysis showed confirmed the maintenance of a normal phenotype in the cell line during long-term culture. Additionally, the mitochondrial cox1 and cytb genes were further used to identify the cell line as originating from the spotted seabass. The established cell lines were subjected to transfection with the pEGFP-C1 plasmid, resulting in observable green fluorescence, indicating their suitability for studying the expression of foreign genes. Furthermore, when cultured with different concentrations of oleic acid, the cell lines exhibited prominent lipid droplet aggregation after 3d, reaching its maximum accumulation after 7d. Oil red O staining demonstrated that the cells could differentiate and mature normally after 7d of induction with 400 μmol/L oleic acid. In summary, this experiment successfully established a preadipose cell line for the spotted sea bass, characterized by typical morphology and normal differentiation. This cell line serves as valuable experimental material for future studies on adipose cell differentiation and adipose deposition in fish.
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Keywords:
- Preadipocyte /
- Cell line /
- Cell culture /
- Induction differentiation /
- Lateolabrax maculatus
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图 1 花鲈前脂肪细胞系的表观形态
A. 第0代培养24h的原代细胞; B—E. 是培养24h后的第1、10、50和100代的细胞; F. 冻存前第54代细胞; G—I. 冻存1、3和6个月后复苏培养24h的细胞
Figure 1. The apparent morphology of proadipose cell line of the spotted sea bass
A. Primary cells cultured for 24h in the 0 generation; B—E. is the 1st, 10th, 50th, and 100th generation of cells cultured for 24h; F. Cryopreservation of pre-54th generation cells; G—I. Cells frozen 1, 3, and 6 months later are resuscitated and cultured for 24h
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图 2 不同培养条件下原代细胞的生长曲线
A. 不同培养基条件下细胞的生长曲线; B. 不同血清浓度的细胞生长曲线; *表示显著性差异
Figure 2. Growth curves of primary cells under different culture conditions
A. Cell growth curves under different medium conditions; B. Cell growth curves at different serum concentrations; *indicates significant difference
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图 3 花鲈前脂肪细胞分裂中期染色体及染色体数目分布
Figure 3. Chromosomes distribution and chromosomes number in the metaphase of preadipocyte division of spotted sea bass
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图 4 前脂肪细胞cox1和cytb扩增产物琼脂糖凝胶电泳
Figure 4. Agarose gel electrophoresis of cox1 and cytb amplification products from preadipocytes
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图 5 pEGFP-C1质粒转染后48h的荧光蛋白表达
A. 荧光下观察, 绿色代表荧光蛋白表达; B. 明场观察, 黑色箭头表示发出绿色荧光的细胞位置
Figure 5. Expression of fluorescent protein 48h after transfection with pEGFP-C1 plasmid
A. Fluorescent observation, green represents the expression of fluorescent protein; B. Bright field observation, red arrow indicates the location of the cell emitting green fluorescence
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图 6 不同浓度油酸诱导前脂肪细胞油红O染色图
Figure 6. Oil red O staining of preadipocytes induced by different concentrations of oleic acid
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图 7 花鲈前脂肪细胞油酸诱导分化过程中相关基因的表达水平
不同字母表示显著性差异(P<0.05); n=4
Figure 7. Expression levels of related genes during oleic acid-induced differentiation in preadipocytes of spotted sea bass
Different letters indicate significant difference (P<0.05); n=4
表 1 实时荧光定量PCR引物序列
Table 1 Primers sequence of Real-time fluorescent quantitative PCR
基因
Gene引物Primer (5′—3′) 退火温度
Tm (℃)ppar-γ ACGCCGTGGACCTGTCAGAG
CGTGATGGAGGAGGAGGAGATGG60 c/ebp α AAGATGCGCAACATGGAGAC
TCTGAAGATGCCCCGTAACG60 pgc-1α GTTCCTCCGAACTCCCAGTG
GCAACACCCCTCCAACTACA60 pgc-1β GTTCCTCCGAACTCCCAGTG
GCAACACCCCTCCAACTACA60 tfam GGTCAGACATTTCACTGGGTTG
GCGGCTCTAATGCGTGAAT60 cpt-1 CCTCAATGATACATCGGAACCC
CTGCGGCTCATCATCTAACG60 atgl CTTCCTCTCCGCAACAAGTC
TGGTGCTGTCTGGAGTGTTC60 hsl CGAAACACAGAGACGGTCCA
TCATGACATCTACCAGCCGC60 fas AAACTGAAGCCCTGTGTGCC
CACCCTGCCTATTACATTGCTC60 β-actin CAACTGGGATGACATGGAGAAG
TTGGCTTTGGGGTTCAGG60 
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表 2 细胞冻存后复苏存活率
Table 2 Cell resuscitation rate after cryopreservation
冻存时间(月)
Freezing time (month)细胞存活率
Cell viability (%)1 96.33±0.88a 3 95.00±2.08a 6 92.00±1.73a 注: 同列数据肩标不同小写字母表示差异显著(P<0.05), 下同Note: In the same column, values with different small letter superscripts mean significant difference (P<0.05), the same applies below
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表 3 不同培养基条件下原代细胞的倍增时间
Table 3 Multiplication time of primary cells under different medium conditions
培养基
Medium细胞密度Cell density (×105 cell/well) 培养时间Δt
Cultivation time (h)倍增时间T2
Doubling time (h)起始数 N0
Starting number最终数
Final number增长数ΔN
Growth numberDMEM/F12 1.45 5.30±0.04c 3.85±0.04c 144 77.01±0.40a DMEM高糖 1.45 5.11±0.07c 3.66±0.07c 79.23±0.88a L15 1.45 4.45±0.05b 3.00±0.05b 89.03±0.79b M199 1.45 3.40±0.09a 1.95±0.09a 117.57±3.63c
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表 4 不同血清浓度条件下原代细胞的倍增时间
Table 4 Multiplication time of primary cells under different serum concentrations
血清浓度
Serum concentration细胞密度Cell density (×105 cell/well) 培养时间Δt
Cultivation time (h)倍增时间T2
Doubling time (h)起始数 N0
Starting number最终数
Final number增长数ΔN
Growth number5 1.45 3.35±0.10a 1.90±0.10a 144 119.63±4.31c 10 1.45 4.16±0.24b 2.71±0.24b 95.49±5.08b 15 1.45 4.89±0.12c 3.44±0.12c 82.26±1.64a 20 1.45 5.50±0.10d 4.05±0.10c 74.95±1.02a
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