烧伤大鼠肝组织关键基因的筛选

doi:10.3877/cma.j.issn.1674-6880.2023.06.002

目的

通过基因表达综合（GEO）数据库分析，筛选烧伤大鼠肝组织新的标志物，为其治疗提供理论基础。

方法

采用GEO数据库GSE1860和GSE802数据集，分析24 h烧伤后肝组织的差异基因及共同表达的差异基因。选择3只健康成年雄性Sprague Dawley大鼠，脱毛麻醉后，应用台式超级控温烫伤仪以98 ℃在大鼠背部致伤10 s，制备大鼠烧伤模型。对照组3只大鼠模拟致假伤，脱毛麻醉后以37 ℃在大鼠背部停留10 s。伤后24 h，取大鼠肝脏行逆转录-聚合酶链反应（RT-PCR）检测共同的差异基因的表达量。选择大鼠肝脏组织，使用Tabula Muris数据库定位差异基因在其肝脏组织中单个细胞中的表达情况。

结果

GSE1860数据集烧伤后24 h筛选出81个差异基因；GSE802数据集烧伤后24 h筛选出186个差异基因。共同的差异基因2个，分别为Cast、Col11a1。伤后24 h，烧伤组大鼠Cast mRNA [（1.20 ± 0.14）vs.（1.00 ± 0.04），t = 5.490，P = 0.005]和Col11a1 mRNA [（3.74 ± 0.15）vs.（1.00 ± 0.27），t = 41.224，P < 0.001]相对表达量均较对照组显著升高。基于Tabula Muris数据库获得枢纽基因在单细胞中的表达情况，结果显示Cast几乎可以表达于全部肝脏细胞中，然而Col11a1仅有少量表达于肝脏细胞。

结论

通过GEO数据库分析，发现烧伤24 h肝组织中Cast、Col11a1基因表达增加，有可能成为诊断和治疗烧伤后肝脏损伤的靶点。

关键词: 烧伤, 肝组织, 基因表达综合数据库, 差异基因表达

Objective

By analyzing the Gene Expression Omnibus (GEO) database, new markers in liver tissue of burned rats were screened to provide a theoretical basis for treatment.

Methods

The burn-related microarray sets of GSE1860 and GSE802 in the GEO database were analyzed for differential genes in liver tissue at 24 h after the injury. Three healthy adult male Sprague Dawley rats were selected for each group. After dehairing and anesthesia, a burn model was prepared by applying a benchtop super temperature-controlled scalding instrument to the back of the rats at 98 ℃ for 10 s. In the control group, a sham injury was simulated, and the rats were anesthetized by dehairing and then incubated at 37 ℃ for 10 s on the back. The expression of common differential genes was verified by reverse transcription-polymerase chain reaction (RT-PCR) at 24 h after the injury. The liver tissue was selected and the expression of differential genes in a single cell of liver tissue was located using the Tabula Muris database.

Results

The GSE1860 dataset was screened for 81 differential genes, while the GSE802 dataset was screened for 186 differential genes at 24 h after the injury. There were two common differential genes, namely Cast and Col11a1. At 24 h after the injury, the relative expression of Cast mRNA [(1.20 ± 0.14) vs. (1.00 ± 0.04), t = 5.490, P = 0.005] and Col11a1 mRNA [(3.74 ± 0.15) vs. (1.00 ± 0.27), t = 41.224, P < 0.001] was significantly higher in the burn group compared to the control group. About the expression of hub genes in a single cell based on the Tabula Muris database, Cast could be expressed in almost all liver cells; however, Col11a1 was only expressed in a small amount in liver cells.

Conclusion

The GEO database reveals increased expression of Cast and Col11a1 genes in liver tissue at 24 h of injury, which could potentially be a target for the diagnosis and treatment of post-burn liver injury.

Key words: Burn, Liver tissue, Gene Expression Omnibus dataset, Differential gene expression

表1 引物序列

目标基因		引物序列（5'→3'）
Cast	正向反向	AGTCAGGCGTGAATGCTGCTTTG
Cast	正向反向	CAAGGTAGGTAGAATCCATTGGGTCC
Col11a1	正向反向	ATCTCACCATCTCAACCCTCGTC
Col11a1	正向反向	CTGGGCTTCCTCGGTAACTCTAT
GAPDH-rat	正向反向	TTCAGCTCTGGGATGACCTT
GAPDH-rat	正向反向	TGCCACTCAGAAGACTGTGG

表1 引物序列

目标基因		引物序列（5'→3'）
Cast	正向反向	AGTCAGGCGTGAATGCTGCTTTG
Cast	正向反向	CAAGGTAGGTAGAATCCATTGGGTCC
Col11a1	正向反向	ATCTCACCATCTCAACCCTCGTC
Col11a1	正向反向	CTGGGCTTCCTCGGTAACTCTAT
GAPDH-rat	正向反向	TTCAGCTCTGGGATGACCTT
GAPDH-rat	正向反向	TGCCACTCAGAAGACTGTGG

表2 GSE1860和GSE802数据集差异基因Cast和Col11a1表达情况

数据集	Cast		Col11a1
数据集	P值	\|LogFC\|	P值	\|LogFC\|
GSE1860	0.031	2.074	0.049	2.054
GSE802	0.001	3.138	0.015	2.121

表2 GSE1860和GSE802数据集差异基因Cast和Col11a1表达情况

数据集	Cast		Col11a1
数据集	P值	\|LogFC\|	P值	\|LogFC\|
GSE1860	0.031	2.074	0.049	2.054
GSE802	0.001	3.138	0.015	2.121

图1 单细胞测序聚类分布图注：tSNE. t-分布随机邻域嵌入，是一种降维方法；CPM.每百万映射读取的reads数，为基因表达量的标准化；NK.自然杀伤；颜色越深代表基因在相应的细胞群表达水平越高

图2 Cast（a）和Col11a1（b）基因在肝脏不同细胞中的表达分布注：tSNE. t-分布随机邻域嵌入，是一种降维方法；CPM.每百万映射读取的reads数，为基因表达量的标准化；NK.自然杀伤

图3 Cast（a）和Col11a1（b）基因在肝脏不同细胞中的表达情况注：NK.自然杀伤；CPM.每百万映射读取的reads数，为基因表达量的标准化

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Screening of key genes in liver tissue of burned rats

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Screening of key genes in liver tissue of burned rats