阅读说明：本技术 一种重金属铀诱导仓鼠卵巢细胞损伤的研究方法 (Research method for inducing damage of hamster ovary cells by heavy metal uranium ) 是由 尹晶晶 李建国 潘泽轩 高洁 黄立群 刘欢 秦秀军 于 2021-07-19 设计创作，主要内容包括：本发明属于辐射防护技术领域,涉及一种重金属铀诱导仓鼠卵巢细胞损伤的研究方法。所述的研究方法依次包括如下步骤：(1)建立重金属铀诱导仓鼠卵巢细胞损伤模型；(2)分别提取正常仓鼠卵巢细胞RNA和步骤(1)所得损伤模型的仓鼠卵巢细胞RNA；(3)对正常仓鼠卵巢细胞RNA和损伤模型的仓鼠卵巢细胞RNA分别进行转录组测序；(4)对正常仓鼠卵巢细胞和损伤模型的仓鼠卵巢细胞进行转录水平差异比较分析,筛选出差异表达基因；(5)进行差异表达基因的功能富集分析。利用本发明的研究方法,能够筛选出重金属铀致仓鼠卵巢细胞损伤相关基因,并对差异表达基因进行功能富集分析,为重金属铀致仓鼠卵巢细胞损伤作用机制研究提供分子水平的实验数据。(The invention belongs to the technical field of radiation protection, and relates to a research method for inducing damage of hamster ovary cells by heavy metal uranium. The research method sequentially comprises the following steps: (1) establishing a heavy metal uranium-induced hamster ovary cell damage model; (2) extracting RNA of normal hamster ovary cells and RNA of hamster ovary cells of the damage model obtained in the step (1) respectively; (3) performing transcriptome sequencing on normal hamster ovary cell RNA and hamster ovary cell RNA of an injury model respectively; (4) carrying out comparative analysis on difference of transcription levels of normal hamster ovary cells and hamster ovary cells of an injury model, and screening out a differential expression gene; (5) and performing functional enrichment analysis on the differentially expressed genes. By utilizing the research method, the genes related to the damage of the heavy metal uranium caused hamster ovary cells can be screened out, the function enrichment analysis is carried out on the differentially expressed genes, and the experimental data of the molecular level is provided for the research on the mechanism of the damage of the heavy metal uranium caused hamster ovary cells.)

1. A research method for inducing hamster ovary cell damage by heavy metal uranium is characterized by sequentially comprising the following steps:

(1) establishing a heavy metal uranium-induced hamster ovary cell damage model;

(2) extracting RNA of normal hamster ovary cells and RNA of hamster ovary cells of the damage model obtained in the step (1) respectively;

(3) performing transcriptome sequencing on normal hamster ovary cell RNA and hamster ovary cell RNA of an injury model respectively;

(4) carrying out comparative analysis on difference of transcription levels of normal hamster ovary cells and hamster ovary cells of an injury model, and screening out a differential expression gene;

(5) and performing functional enrichment analysis on the differentially expressed genes.

2. The method of investigation of claim 1, wherein: in the step (1), a hamster ovary cell damage model is established by using 50-1000 mu mol/L heavy metal uranium for 4-48 h.

3. The method of investigation of claim 1, wherein: in the step (2), an RNA extraction kit is used for extracting RNA, and the RNA is extracted according to the using method of the RNA extraction kit; and performing quality detection on the extracted RNA.

4. The method of investigation of claim 1, wherein: in the step (3), ribosome RNA is removed from the extracted hamster ovary cell RNA, the RNA is fragmented, then reverse transcription is carried out to synthesize cDNA, then terminal repair is carried out, A tail is added, a joint is added, and finally sequencing is carried out after PCR amplification.

5. The method of investigation of claim 1, wherein: in the step (4), screening the differential expression genes by adopting DESeq software for standardization, calculating the difference multiple, and carrying out the difference significance test by adopting a negative binomial distribution test mode, wherein the difference screening condition is that P is less than 0.05 and the difference multiple is more than 2.

6. The method of investigation of claim 1, wherein: and (5) performing functional enrichment analysis on the differentially expressed genes by adopting GO and KEGG.

7. The method of investigation of claim 6, wherein: the GO functional enrichment analysis is to use all genes as backgrounds and differentially expressed genes as candidate lists screened from the background lists, calculate the enrichment significance by using super-geometric distribution test, and use the fisher algorithm to perform enrichment analysis on the differentially expressed genes in biological processes, cell components and molecular functions.

8. The method of investigation of claim 6, wherein: the KEGG functional enrichment analysis is to perform channel analysis on differentially expressed genes by using a KEGG database, and calculate the significance of differential gene enrichment in each channel by adopting a hyper-geometric distribution test method.

9. The method of claim 1, wherein the method further comprises the following steps after step (5):

(6) and selecting genes with obvious differential expression, and verifying the reliability of the transcriptome sequencing data.

10. The method of investigation of claim 9, wherein: and (6) selecting genes with obvious differential expression, detecting by adopting a real-time fluorescence quantitative PCR technology, and comparing with the expression trend of the genes in the transcriptome sequencing data to verify the reliability of the transcriptome sequencing data.

Technical Field

The invention belongs to the technical field of radiation protection, and relates to a research method for inducing damage of hamster ovary cells by heavy metal uranium.

Background

Uranium, as a heavy metal element, has strong heavy metal toxicity and radiotoxicity. Uranium mainly exists in the form of compounds in nature, and the soluble uranium is easily absorbed by organisms and enters human bodies along food chains, so that internal radiation is caused to the human bodies, and the human health is seriously harmed.

There have been many studies reporting the chemical toxicity of uranium, but uranium has received less attention as a reproductive toxicity for mammals. Reproductive developmental toxicity of the heavy metal uranium is mainly based on its chemical toxicity rather than radiotoxicity.

Hamster ovary cells are cell strains commonly used in experiments, the culture conditions are simple, the adherent strength is moderate, and therefore a reliable cell model can be established by using the hamster ovary cells.

The transcriptome sequencing technology is a newly developed technology for analyzing transcriptome by using a high-throughput sequencing platform, and becomes one of important means for researching transcriptomics. The technology can comprehensively and quickly obtain the sequence information and the expression information of almost all transcripts of a specific cell or tissue under a certain state, including mRNA (messenger ribonucleic acid) of coded protein and various non-coded RNAs, the expression abundance of different transcripts generated by gene alternative splicing and the like. The advantages of transcriptome sequencing are that transcriptome of any species can be analyzed without knowing the gene information of the target species, the detection flux is high, and the detection range is wide.

Disclosure of Invention

The invention aims to provide a method for researching damage of hamster ovary cells induced by heavy metal uranium, which can screen out related genes of damage of hamster ovary cells induced by heavy metal uranium, perform function enrichment analysis on differentially expressed genes and provide molecular level experimental data for researching the mechanism of action of damage of hamster ovary cells induced by heavy metal uranium.

To achieve the purpose, in a basic embodiment, the invention provides a research method for inducing damage of hamster ovary cells by heavy metal uranium, which sequentially comprises the following steps:

(1) establishing a heavy metal uranium-induced hamster ovary cell damage model;

(2) extracting RNA of normal hamster ovary cells and RNA of hamster ovary cells of the damage model obtained in the step (1) respectively;

(3) performing transcriptome sequencing on normal hamster ovary cell RNA and hamster ovary cell RNA of an injury model respectively;

(4) carrying out comparative analysis on difference of transcription levels of normal hamster ovary cells and hamster ovary cells of an injury model, and screening out a differential expression gene;

(5) and performing functional enrichment analysis on the differentially expressed genes.

In a preferred embodiment, the invention provides a research method for inducing damage of hamster ovary cells by heavy metal uranium, wherein in the step (1), a hamster ovary cell damage model is established by infecting heavy metal uranium with 50-1000 mu mol/L for 4-48 h.

In a preferred embodiment, the invention provides a research method for inducing damage of hamster ovary cells by heavy metal uranium, wherein in the step (2), RNA is extracted by using an RNA extraction kit, and the extraction is carried out according to the using method of the RNA extraction kit; the extracted RNA is subjected to quality detection (including concentration and quality).

In a preferred embodiment, the invention provides a research method for inducing damage of hamster ovary cells by heavy metal uranium, wherein in the step (3), ribosomal RNA is removed from extracted hamster ovary cell RNA, the RNA is fragmented, then reverse transcription is carried out to synthesize cDNA, then terminal repair is carried out, A tail is added, a linker is added, and finally PCR amplification is carried out and then sequencing is carried out.

In a preferred embodiment, the invention provides a research method for inducing hamster ovary cell damage by heavy metal uranium, wherein in the step (4), screening of the differentially expressed genes is subjected to standardization treatment by using DESeq software, a difference multiple is calculated, and a difference significance test is performed by using a negative binomial distribution test, wherein the difference screening condition is that P is less than 0.05, and the difference multiple is more than 2.

In a preferred embodiment, the invention provides a research method for inducing hamster ovary cell damage by heavy metal uranium, wherein in the step (5), GO and KEGG are used for performing functional enrichment analysis on differentially expressed genes.

In a preferred embodiment, the invention provides a research method for inducing hamster ovary cell damage by heavy metal uranium, wherein GO function enrichment analysis is performed by taking all genes as a background and differentially expressed genes as a candidate list screened from the background list, calculating enrichment significance by using a hyper-geometric distribution test, and performing enrichment analysis on the differentially expressed genes in a biological process, a cell component and a molecular function by using a fisher algorithm.

In a preferred embodiment, the invention provides a research method for inducing hamster ovary cell damage by heavy metal uranium, wherein KEGG functional enrichment analysis is to perform pathway analysis on differentially expressed genes by using a KEGG database, and calculate significance of differential gene enrichment in each pathway by using a hyper-geometric distribution test method.

In a preferred embodiment, the invention provides a research method for inducing damage of hamster ovary cells by heavy metal uranium, wherein the research method further comprises the following steps after the step (5):

(6) and selecting genes with obvious differential expression, and verifying the reliability of the transcriptome sequencing data.

In a preferred embodiment, the invention provides a research method for inducing hamster ovary cell damage by heavy metal uranium, wherein in the step (6), genes with obvious differential expression are selected, a real-time fluorescence quantitative PCR technology is adopted for detection, and the detection is compared with the expression trend of the genes in transcriptome sequencing data to verify the reliability of the transcriptome sequencing data.

The method has the advantages that by using the research method for inducing damage of hamster ovary cells by heavy metal uranium, genes related to damage of hamster ovary cells by heavy metal uranium can be screened out, and differential expression genes are subjected to function enrichment analysis, so that experimental data of molecular level are provided for research of mechanism of action of damage of hamster ovary cells by heavy metal uranium.

Drawings

Fig. 1 is a flow chart of an exemplary method of the invention for studying hamster ovary cell injury induced by the heavy metal uranium.

FIG. 2 is a flow chart of the construction and sequencing of the transcriptome cDNA library of example 1.

Detailed Description

The following description will further describe embodiments of the present invention with reference to the accompanying drawings.

Example 1:

an exemplary method for studying hamster ovary cell injury induced by heavy metal uranium according to the present invention is shown in fig. 1, and comprises the following steps:

1. establishment of heavy metal uranium-induced hamster ovary cell damage model

A hamster ovary cell injury model is established by adopting 500 mu mol/L heavy metal uranium for 24 h.

2. Extraction of Normal hamster ovary cell RNA and hamster ovary cell RNA of injury model

According to the method of the RNA extraction kit, normal hamster ovary cell RNA and hamster ovary cell RNA of the injury model are respectively extracted, the concentration and the quality of the extracted RNA are detected, and the detection result shows that the concentration and the quality of the RNA meet the experimental requirements.

3. Transcriptome sequencing of Normal hamster ovary cell RNA and hamster ovary cell RNA from injury model

For extracted hamster ovary cell RNA, ribosomal RNA is removed, RNA is fragmented, then reverse transcription is carried out to synthesize cDNA, terminal repair is carried out, A tail is added, a linker is added, and finally PCR amplification is carried out and then sequencing is carried out (see figure 2).

4. Performing comparative analysis on difference of transcription levels of normal hamster ovary cells and hamster ovary cells of injury model, and screening out differentially expressed genes

And carrying out comparative analysis on the difference of the transcription levels, and screening out the differentially expressed genes. Screening of the differential expression genes adopts DESeq software to carry out standardization processing, calculate the difference multiple, and adopt a negative two-term distribution test mode to carry out the difference significance test, wherein the differential screening condition is that P is less than 0.05 and the difference multiple is more than 2, 2389 differential expression genes are screened out, wherein 1539 differential expression genes are up-regulated and 850 differential expression genes are down-regulated.

5. Performing functional enrichment analysis of differentially expressed genes

The GO function enrichment analysis of the differential expression genes is to use all the genes as backgrounds and the differential expression genes as candidate lists screened from the background lists, calculate the enrichment significance by utilizing super-geometric distribution inspection, use fisher algorithm to carry out enrichment analysis on the differential expression genes in biological processes, cell components and molecular functions, and find that the differential expression genes are mainly enriched in immune, oxidative damage, apoptosis and other processes.

The KEGG functional enrichment analysis of the differentially expressed genes is to perform channel analysis on the differentially expressed genes by utilizing a KEGG database, calculate the significance of differential gene enrichment in each channel by adopting a hyper-geometric distribution test method and find that signal channels of cellular immunity, oxidative damage and apoptosis have differential expression.

6. Selecting genes with obvious differential expression, and verifying reliability of transcriptome sequencing data

Selecting genes with obvious differential expression, adopting a real-time fluorescence quantitative PCR technology to detect, comparing the expression trend of the genes with the expression trend of the genes in the transcriptome sequencing data, verifying the reliability of the transcriptome sequencing data, and displaying that the PCR amplification result is consistent with the sequencing result by a verification result.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is intended to include such modifications and variations. The foregoing examples or embodiments are merely illustrative of the present invention, which may be embodied in other specific forms or in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the invention should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims should be construed to be included therein.