Pnpla3 I148M和Tm6sf2 E167K双突变纯合小鼠模型的构建

王孟轲; 刘守胜; 褚雪汝; 王艺奋; 辛永宁

doi:10.3969/j.issn.1001-5256.2022.08.013

Pnpla3 I148M和Tm6sf2 E167K双突变纯合小鼠模型的构建

DOI: 10.3969/j.issn.1001-5256.2022.08.013

王孟轲^1a,
刘守胜^1b,
褚雪汝²,
王艺奋^1a,
辛永宁^1a, , ,

1a.
青岛大学附属青岛市市立医院感染性疾病科，山东青岛 266071
1b.
青岛大学附属青岛市市立医院临床研究中心，山东青岛 266071
2.
中国海洋大学医药学院，山东青岛 266071

基金项目:

国家自然科学基金面上项目 (31770837)

伦理学声明：实验所用动物均按照青岛大学附属医院医学实验动物伦理委员会批准的原则进行。伦理审批编号：AHQU-MAL20180504，伦理通过时间2018年5月4日。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：王孟轲进行课题设计与实施、撰写论文；刘守胜进行课题设计、论文审阅；褚雪汝、王艺奋进行课题实施、数据收集；辛永宁进行课题设计、实验指导和提供经费。

详细信息

通信作者:
辛永宁，xinyongning@163.com

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出版历程
- 收稿日期: 2022-01-21
- 录用日期: 2022-02-25
- 出版日期: 2022-08-20

Construction of Pnpla3 I148M and Tm6sf2 E167K double mutant mouse model

1a.
Department of Infectious Diseases, The Affiliated Qingdao Municipal Hospital of Qingdao University, Qingdao, Shandong 266071, China
1b.
Clinical Research Center, The Affiliated Qingdao Municipal Hospital of Qingdao University, Qingdao, Shandong 266071, China
2.
School of Medicine and Pharmacy, Ocean University of China, Qingdao, Shandong 266071, China

Research funding:

National Natural Science Foundation of China (31770837)

More Information

Corresponding author: XIN Yongning, xinyongning@163.com(ORCID: 0000-0002-3692-7655)

摘要

摘要: 目的利用Pnpla3^148M/M纯合小鼠与Tm6sf2^167K/K纯合小鼠杂交方法构建Pnpla3^148M/M Tm6sf2^167K/K双突变小鼠模型。方法利用Pnpla3 Ⅰ 148M和Tm6sf2 E 167K单突变纯合小鼠交配出Pnpla3^148M/M Tm6sf2^167K/K双突变杂合小鼠，再通过自交得到Pnpla3^148M/M Tm6sf2^167K/K双突变纯合小鼠。选取同窝的Pnpla3^148M/M Tm6sf2^167K/K(n=6)、Pnpla3^148M/M Tm6sf2^167E/E(n=6)、Pnpla3^148I/I Tm6sf2^167K/K(n=6)和野生(Wt)(n=6)雄性小鼠普通饮食喂养8周，在第8周检测小鼠葡萄糖及脂质代谢等指标。多组间比较采用方差分析，进一步两两比较采用LSD-t检验。结果琼脂糖凝胶电泳和核酸测序结果表明Pnpla3^148M/M Tm6sf2^167K/K双突变小鼠模型构建成功。Pnpla3^148M/M Tm6sf2^167K/K小鼠体质量与其他3种基因型小鼠比较，差异无统计学意义(P值均＞0.05)，Pnpla3^148M/M Tm6sf2^167K/K小鼠肝湿重高于Wt小鼠(P＜0.05)。Pnpla3^148M/M Tm6sf2^167K/K小鼠的空腹血糖低于Tm6sf2^167K/K单突变小鼠和Wt小鼠(P值均＜0.05)，Pnpla3^148M/M Tm6sf2^167K/K小鼠葡萄糖耐受能力较Tm6sf2^167K/K单突变小鼠有所下降(P＜0.05)，四组基因型小鼠的胰岛素水平无明显差异(P值均＞0.05)。Pnpla3^148M/M Tm6sf2^167K/K双突变小鼠的血浆生化指标与其他三种基因型小鼠比较，差异均无统计学意义(P值均＞0.05)。肝脏油红O切片染色结果显示Pnpla3^148M/M Tm6sf2^167K/K双突变小鼠的肝脏较Pnpla3^148M/M单突变小鼠和Wt小鼠更容易发生脂质积累。结论 Pnpla3^148M/M Tm6sf2^167K/K双突变小鼠模型构建成功，Pnpla3 Ⅰ 148M和Tm6sf2 E 167K双突变可引起小鼠葡萄糖代谢异常。
- 非酒精性脂肪性肝病 /
- 点突变 /
- 模型, 动物 /
- 小鼠
Abstract: Objective To construct a Pnpla3^148M/M Tm6sf2^167K/K double mutant mouse model by crossbreeding Pnpla3^148M/M homozygous mice and Tm6sf2^167K/K homozygous mice. Methods Pnpla3^148I/M Tm6sf2^167E/K heterozygous mice were bred by hybridization of Pnpla3^148M/M Tm6sf2^167E/E and Pnpla3^148I/I Tm6sf2^167K/K homozygous mice, and the Pnpla3^148M/M Tm6sf2^167K/K mice were obtained by the self-crossbreeding of Pnpla3^148I/M Tm6sf2^167E/K mice. Male mice of Pnpla3^148M/M Tm6sf2^167K/K (n=6), Pnpla3^148M/M Tm6sf2^167E/E (n=6), and Pnpla3^148I/I Tm6sf2^167K/K (n=6) genotypes and Wt mice (n=6) were fed with normal diet for 8 weeks, and then the glucose and lipid metabolism indices were measured. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison bewteen two groups. Results Agarose gel electrophoresis and nucleic acid sequencing results showed that the Pnpla3^148M/M Tm6sf2^167K/K double mutant mouse model was successfully constructed. There were no significant difference in body weight between the Pnpla3^148M/M Tm6sf2^167K/K mice and the Pnpla3^148M/M Tm6sf2^167E/E, Pnpla3^148I/I Tm6sf2^167K/K, and Wt mice (all P > 0.05). The Pnpla3^148M/M Tm6sf2^167K/K mice had a significantly higher liver wet weight than the Wt mice (P < 0.05). The fasting blood glucose of Pnpla3^148M/M Tm6sf2^167K/K mice was significantly lower than that of Pnpla3^148I/I Tm6sf2^167K/K mice and Wt mice (both P < 0.05). The glucose tolerance of Pnpla3^148M/M Tm6sf2^167K/K mice was significantly reduced compared with the Pnpla3^148I/I Tm6sf2^167K/K mice (P < 0.05). There were no significant differences in insulin level between the four groups of mice (all P > 0.05). Also, there were no significant differences in the serum levels of biochemical indices between the Pnpla3^148M/M Tm6sf2^167K/K mice and the Pnpla3^148M/M Tm6sf2^167E/E, Pnpla3^148I/I Tm6sf2^167K/K, and Wt mice (all P > 0.05). Oil red O staining of the liver showed that more lipid accumulation was observed in the Pnpla3^148M/M Tm6sf2^167K/K mice than in the Pnpla3^148M/M Tm6sf2^167E/E and Wt mice. Conclusion The Pnpla3^148M/M Tm6sf2^167K/K double mutant mouse model was successfully constructed. Pnpla3 Ⅰ 148M and Tm6sf2 E 167K double mutations can cause abnormal glucose metabolism in mice.
- Non-alcoholic Fatty Liver Disease /
- Point Mutation /
- Models, Animal /
- Mice

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图 1 Pnpla3^148M/M Tm6sf2^167K/K双突变纯合小鼠繁育策略

注：Wt为野生小鼠；×代表杂交；⊗代表自交。

Figure 1. Breeding strategy of Pnpla3^148M/M Tm6sf2^167K/K double mutant homozygous mice

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图 2 Pnpla3 I148M和Tm6sf2 E167K突变小鼠基因型鉴定和测序结果

注：a，Tm6sf2 E167K突变小鼠基因型鉴定电泳结果；b，Pnpla3 I148M突变小鼠基因型鉴定电泳结果；c、d和e为Pnpla3 I148M突变小鼠基因型鉴定测序结果(c，Pnpla3^148M/M；d，Pnpla3^148I/M；e，Pnpla3^148I/I)。

Figure 2. Genotype identification and sequencing results of Pnpla3 I148M and Tm6sf2 E167K mutant mice

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图 3 四种基因型小鼠在8周龄时的体质量、肝湿重和肝指数比较

Figure 3. Comparison of body weight, liver weight, and liver index of mice with four genotypes at 8 weeks of age

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图 4 四种基因型小鼠各个器官的形态比较

注：a, Pnpla3^148M/M Tm6sf2^167K/K基因型小鼠; b, Pnpla3^148M/M Tm6sf2^167E/E基因型小鼠；c, Pnpla3^148I/I Tm6sf2^167K/K基因型小鼠; d, Wt小鼠。

Figure 4. Morphological comparison of organs in mice with four genotypes

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图 5 四种基因型小鼠葡萄糖耐量比较

注：*P＜0.05;* *P＜0.01。

Figure 5. Comparison of glucose tolerance of mice with four genotypes

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图 6 四种基因型小鼠血浆生化水平

Figure 6. Plasma biochemical levels of mice with four genotypes

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图 7 四组基因型小鼠肝脏HE染色(×200)

注：a，Wt；b，Pnpla3^148M/M Tm6sf2^167E/E; c，Pnpla3^148I/I Tm6sf2^167K/K; d，Pnpla3^148M/M Tm6sf2^167K/K。

Figure 7. HE staining of liver of mice with four genotypes (×200)

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图 8 四组基因型小鼠肝脏油红O染色(×200)

注：a，Wt；b，Pnpla3^148M/M Tm6sf2^167E/E; c，Pnpla3^148I/I Tm6sf2^167K/K; d，Pnpla3^148M/M Tm6sf2^167K/K。

Figure 8. Oil red O staining of liver of mice with four genotypes (×200)

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表 1 小鼠基因型鉴定引物序列

Table 1. Primer sequences for mouse genotypic identification

PCR引物名称	引物序列(5′-3′)
Pnpla3-wt-tF1	ATCTCTGTGAGTTCGATTGCCAG
Pnpla3-wt-tR1	AGTGTATCCAACAGACAGCAGGC
Tm6sf2-wt-tF1	GGCCTTTCCTAGACTCCTCA
Tm6sf2-wt-tR1	CCTTCTCAGATGTTCCTCCCT

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表 2 Pnpla3小鼠基因型测序引物序列

Table 2. Primer sequences for Pnpla3 mouse genotype sequencing

PCR引物名称	引物序列(5′-3′)
Pnpla3-wt-tR1	AGTGTATCCAACAGACAGCAGGC

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表 3 Pnpla3^148I/M Tm6sf2^167E/K小鼠自交后子代各基因型数量

Table 3. Number of progenies with each genotype after Pnpla3^{148I/ M}TM6SF2^167E/K mice self-breeding

小鼠基因型	数量	占总体的百分比(%)
Pnpla3^148M/M Tm6sf2^167K/K	13	6.2
杂合小鼠	151	71.9
Pnpla3^148M/M Tm6sf2^167E/E	11	5.2
Pnpla3^148I/I Tm6sf2^167K/K	18	8.6
Wt小鼠	17	8.1

下载: 导出CSV

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