Ultraviolet damage sensitive cell model as well as preparation method and application thereof

Abstract

The invention relates to an ultraviolet damage sensitive cell model as well as a preparation method and application thereof. The cell model is a cell with UVSSA gene function deletion. According to the invention, UVSSA genes in human epidermal Hacat cells are knocked out by utilizing a CRISPR-Cas9 gene editing technology, and a cell model sensitive to medium-wave-band ultraviolet (UVB) is constructed and is successfully used for screening active raw materials with a light protection effect.

Description

Technical Field

[0001] The invention relates to the technical field of cell models, and in particular to an ultraviolet damage sensitive cell model and a preparation method and application thereof. Background Art

[0002] Sunlight generally consists of ultraviolet rays (UVA, UVB and UVC), visible light and infrared rays (IR-A, IR-B and IR-C). Long-term and excessive exposure to sunlight can damage the skin, increase skin sensitivity, accelerate skin aging and increase the risk of tumors.

[0003] During DNA replication, exposure to excessive UVB will cause DNA damage reactions, mainly causing adjacent pyrimidines on the same DNA chain to form dimers with covalent bonds, producing a large number of pyrimidine dimers, interfering with the normal physiological process of DNA polymerase. UVB irradiation can also cause damage such as DNA chain breaks. If the damage is not repaired or repaired in time, the normal expression of genes will be disturbed, resulting in incorrect protein coding. If it involves genes related to controlling cell growth, it can lead to cancer.

[0004] Once photodamage occurs, cells repair it through photoreactivation and nucleotide excision repair (NER). In photoreactivation, a single photolyase uses the energy generated by photons to reverse the damage. Photolyases specific for CPD and (6-4)PP have been identified in bacteria, yeast, insects, plants, and marsupial mammals; however, in other mammals (including humans), no photolyases or analogs have been identified, so nucleotide excision repair has become the only way to remove photodimers.

[0005] In mammals, nucleotide excision repair is performed in multiple steps by at least 11 enzymes (composed of 30 polypeptides). Repair is mainly divided into three steps: (1) DNA damage recognition, which can be performed directly (global repair) or in a transcription-coupled manner (transcription-coupled repair); (2) excision of the 3' and 5' ends of the damage, removing the nucleotide fragments and leaving a single-stranded gap; (3) DNA synthesis, filling this gap, connecting, and finally restoring the complete DNA double strand. Loss-of-function mutations in excision repair-related genes can lead to serious genetic diseases, including xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). The protein encoded by the UVSSA gene participates in the transcription-coupled nucleotide excision repair (TC-NER) pathway. It can interact with the NER complex and participate in the ubiquitination and dephosphorylation of RNA polymerase II subunits, thereby repairing and removing DNA damage that blocks transcription.

[0006] However, in the prior art, the common method for constructing a UV-sensitive cell model is to directly irradiate with UVB, but different irradiation time, irradiation intensity and cell state will have a great impact on the final cell model. Therefore, how to achieve a stable UV-sensitive cell model has become a problem that needs to be solved urgently. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a UV damage sensitive cell model and a preparation method and application thereof. The UVSSA gene in human epidermal Hacat cells is knocked out by CRISPR-Cas9 gene editing technology, and a cell model sensitive to medium-band ultraviolet UVB is constructed, which is successfully used to screen active raw materials with photoprotective effects.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a UV damage sensitive cell model, wherein the cell model is a cell with UVSSA gene function deficiency.

[0010] In the present invention, it was found through experimental verification that the proliferation activity of cells with UVSSA gene function loss did not change significantly under conventional culture conditions, but its proliferation activity was significantly reduced after exposure to ultraviolet irradiation, and the expression level of aging marker genes was significantly increased, which proved that cells with UVSSA gene function loss can be used as an efficient and stable light-damaged cell model.

[0011] Preferably, the source of the cells includes any one of skin, blood, tissue fluid or liver cells, or a combination of at least two of them.

[0012] Preferably, the cells include any one of Hacat cells, hsf cells, HDF cells, BJ cells or HFF cells, or a combination of at least two of them.

[0013] Preferably, the nucleic acid sequence of the UVSSA gene includes the sequence shown in SEQ ID No.1.

[0014] SEQ ID No.1:

[0015]

[0016] The present invention finds that cells with UVSSA gene function loss can be used as an efficient and stable light-damaged cell model. It can be understood that the UVSSA gene function loss means that the protein encoded by it loses part or all of its normal function. The common gene function loss methods in the art are applicable to the present invention, such as gene mutation or knockout, and commonly used tools include CRISPR-Cas editing system, etc.

[0017] Preferably, the UVSSA gene functional loss is the loss of the 400th adenine deoxyribonucleotide in the UVSSA gene.

[0018] Preferably, the wavelength of the ultraviolet rays is a medium wavelength band.

[0019] Preferably, the wavelength of the ultraviolet light is in the range of 280-320 nm, for example, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm or 320 nm.

[0020] Ultraviolet rays can be divided into near-ultraviolet rays (low frequency, UVA), far-ultraviolet rays (medium-wavelength, UVB), ultra-short ultraviolet rays (high-wavelength, UVC) and extreme ultraviolet rays (ultra-high-wavelength, EUV) according to their wavelengths, but UVB is more harmful to cells, especially during DNA replication. Therefore, the cell model proposed by Hou Jian in the present invention is mainly aimed at cell damage caused by UVB.

[0021] In a second aspect, the present invention provides a method for preparing a UV damage-sensitive cell model according to the first aspect, characterized in that the preparation method comprises the steps of: transfecting sgRNA-F and sgRNA-R into cells after a plasmid vector, and performing PCR identification after screening with antibiotics.

[0022] Preferably, the nucleic acid sequence of the sgRNA-F includes the sequence shown in SEQ ID No.2.

[0023] SEQ ID No. 2: CACCTTGGCACGCCACGTATGGAGAGG.

[0024] Preferably, the nucleic acid sequence of the sgRNA-R includes the sequence shown in SEQ ID No.3.

[0025] SEQ ID No. 3: AAACCCTCTCCATACGTGGGCGTGCCAA.

[0026] Preferably, the plasmid vector comprises any one of PX459, PX330, phU6-gRNA or lentiCRISPR v2, or a combination of at least two of them.

[0027] Preferably, the plasmid is connected to the nucleic acid sequence of Cas9.

[0028] Preferably, the antibiotic comprises any one of puromycin, kanamycin or ampicillin, or a combination of at least two of them.

[0029] Preferably, the nucleic acid sequence of the upstream primer identified by PCR includes the sequence shown in SEQ ID No.4.

[0030] SEQ ID No. 4: GACCCAGCTGAACCAAGAGC.

[0031] Preferably, the nucleic acid sequence of the downstream primer identified by PCR includes the sequence shown in SEQ ID No.5.

[0032] SEQ ID No. 5: GATATGTTTCTCGGCTCCTGCA.

[0033] In the present invention, a CRISPR-Cas9 gene editing method is designed for the UVSSA gene, which can efficiently perform gene editing.

[0034] Preferably, the cells with UVSSA gene functional deficiency identified by PCR are also irradiated with UVB to cause light damage.

[0035] In a third aspect, the present invention provides an application of the UV damage sensitive cell model according to the first aspect in screening active raw materials with photoprotective effects.

[0036] Preferably, screening active raw materials with photoprotective effects refers to treating the UV damage-sensitive cell model prepared by the present invention with the active raw materials to be screened, and evaluating the photoprotective effect of the active raw materials to be screened by examining the influence of the active raw materials to be screened on the proliferation activity and / or the expression level of aging marker genes.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] The present invention knocks out the UVSSA gene in Hacat cells to construct a cell model sensitive to mid-band ultraviolet UVB. Experimental verification shows that the proliferation activity of the cell model is not affected, but it is highly sensitive to light damage and is successfully used to screen active raw materials with light protection effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a comparison chart of sequencing results of monoclonal cell lines after knockout.

[0040] Figure 2This is a comparison chart of the cell proliferation activity of Hacat-uvssa cells and Hacat cells before and after UVB irradiation.

[0041] Figure 3 This is a comparison chart of the transcriptional expression levels of gene p16 in Hacat-uvssa cells and Hacat cells before and after UVB irradiation.

[0042] Figure 4 This is a comparison chart of the proliferation activity of tea polyphenols on photodamaged Hacat-uvssa cells.

[0043] Figure 5 This is a comparison chart of the transcriptional expression levels of gene p16 in photodamaged Hacat-uvssa cells induced by tea polyphenols. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0045] The actual sources used in the following examples are:

[0046] Lipofectamine TM 3000 Kit: ThermoFisher, L3000008.

[0047] Hacat cells: Beijing Sai Ao Company.

[0048] OMEM medium: Gibco.

[0049] Example 1

[0050] This example designs the gene sequence

[0051] First, the nucleotide sequence of the target gene UVSSA was searched on the online website NCBI, and the specific sequence is shown in SEQ ID No. 1. Then it was input into the online website CHOPCHOP to design the sgRNA sequence for knocking out the adenine deoxyribonucleotide at position 400 of the UVSSA gene. The nucleic acid sequence of sgRNA-F includes the sequence shown in SEQ ID No. 2. The nucleic acid sequence of sgRNA-R includes the sequence shown in SEQ ID No. 3.

[0052] Suzhou Hongxun Biotechnology was commissioned to synthesize the recombinant PX459 vector. The specific operation was: the pSpCas9(BB)-2A-Puro(PX459) plasmid vector was cut with the endonuclease BbsI, and then sgRNA-F and sgRNA-R were connected to PX459 to obtain the recombinant plasmid PX459-UVSSA-sgRAN.

[0053] Example 2

[0054] In this example, the recombinant plasmid was transfected

[0055] In a 6-well plate, 2 × 10 5 Hacat cells were inoculated at a density of 100 cells / well and incubated overnight in a CO2 incubator at 37°C. Transfection was started when the cells grew to 50%. Lipofectamine was diluted with OMEM medium. TM 3000, add 125 μL of OMEM medium and 3.75 μL of Lipo3000 to the centrifuge tube, mix well, and obtain mixed solution 1. In 125 μL OMEM medium, add 2.5 μg of the corresponding volume of recombinant plasmid PX459-UVSSA-sgRAN and 5 μL P3000 TM Reagents, mix well to obtain mixed solution 2. Mix mixed solution 1 and mixed solution 2 in a ratio of 1:1 to form a liposome mixed solution, and let stand at room temperature for 15 min.

[0056] After washing the Hacat cells with OMEM medium, add 250 μL of liposome mixture to each well, shake gently, incubate at 37°C for 3 hours, replace with complete medium, and continue culturing for 3 days. When the cells grow to 80%, screen for monoclonal cell lines.

[0057] Screening of monoclonal cell lines: 48 hours after transfection, add DMEM medium containing 0.3 μg / mL puromycin for 7 days, trypsinize the cells and adjust the concentration to 100 cells / mL, take a 96-well plate, inoculate 100 μL of cell suspension in each well according to the proportional gradient dilution, observe the wells containing 1 cell under a microscope, and mark them with a marker. Keep the cells in good condition, and transfer them to 24-well and 6-well plates in turn until the density grows to 70% to continue culturing. The monoclonal cells Hacat-UVSSA with good growth are frozen and passaged.

[0058] Example 3

[0059] This example is used to identify monoclonal cells

[0060] The Hacat-UVSSA strain obtained in Example 2 was subjected to PCR detection, with 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 63°C annealing for 30 s, 72°C extension for 1 min, 35 cycles; 72°C extension for 5 min. The primers for PCR detection were the sequences shown in SEQ ID No. 4 and SEQ ID No. 5. The sequence for PCR amplification was the 153rd to 695th positions of the UVSSA gene, totaling 543 bp.

[0061] The PCR product was sequenced by Beijing Qingke Biotechnology Co., Ltd., and the sequencing results were compared with the original genome sequence to screen for the correct knockout mutant. Figure 1 As shown, Qurey is the original genome sequence and Sbjct is the sequencing result sequence. It can be seen that the monoclonal Hacat-UVSSA strain has a deletion mutation at the 248th adenine deoxyribonucleotide of the Hacat-UVSSA detection sequence and a deletion mutation at the 400th adenine deoxyribonucleotide of the UVSSA full-length gene, which is very likely to cause irreversible effects on the normal transcription and expression of the gene and is named Hacat-uvssa.

[0062] Example 4

[0063] This example conducts a light damage experiment under UVB irradiation

[0064] Hacat-uvssa cells and Hacat cells were seeded into 96-well plates at 1000 cells / well. After culturing for 24 hours, the cells were washed twice with PBS, and PBS was added to each well after washing. 2 The cells were irradiated with UVB, while the control group was not irradiated with UVB (UVB(-)) (one well plate) and cultured for 24 hours under standard conditions. 10uL CCK8 reagent was added to each well and incubated for 2 hours at 37°C. The optical density [OD(450)] value of each well at 450nm was measured using an enzyme-linked immunosorbent assay to calculate the relative cell proliferation activity of each group.

[0065] Cell proliferation activity = [OD (450) value of each experimental group] / [OD (450) value of Hacat control group].

[0066] Specific results such as Figure 2 As shown in the figure, under normal culture conditions, the loss of UVSSA gene had no significant effect on the proliferation activity of Hacat cells. However, after light damage caused by UVB irradiation, the proliferation activity of Hacat-uvssa was significantly reduced compared with wild-type cells.

[0067] Example 5

[0068] In this example, the transcriptional expression level of the aging-related protein p16 was detected.

[0069] After direct UVB irradiation, the transcriptional expression level of the aging-related protein p16 was detected by qRT-PCR. p16 is a cyclin-dependent kinase inhibitor. A large number of studies have shown that the expression of p16 is positively correlated with aging and is a more reliable aging marker in cells, mice and human tissues.

[0070] 1×10 per well 7 The cells were evenly plated in 6 plates at a density of 100 cells / well. After 24 hours of culture, the cells were washed twice with PBS. After washing, PBS was added to each well. 2 The cells were irradiated with UVB ultraviolet light, and the control group was not irradiated (a separate well plate). After irradiation, PBS was aspirated and discarded, and serum-free DMEM medium was added to culture the cells for 24 hours. After 24 hours, the medium was discarded, and total mRNA was extracted using the RNAiso Plus Total RNA extraction kit, and reverse transcribed into complementary DNA using the PrimeScriptTM RT kit, and qRT-PCR amplification was performed using TB GreenTM Premix Ex TaqTMⅡ polymerase. The procedures were all carried out according to the reagent instructions. The mRNA level was calculated by qRT-PCR, and 2 -△△Ct Methods The relative expression of genes was calculated.

[0071] The sequences of the qRT-PCR primers for amplifying the p16 gene are shown in SEQ ID No.6 and SEQ ID No.7, the internal reference is the GAPDH gene, and the sequences of the internal reference qRT-PCR primers are shown in SEQ ID No.8 and SEQ ID No.9.

[0072] SEQ ID No. 6:TCGGGTAGAGGAGGTGCG.

[0073] SEQ ID No. 7: ATCGGGGATGTCTGAGGG.

[0074] SEQ ID No. 8: CAGGAGGGCATTGCTGATGAT.

[0075] SEQ ID No. 9: GAAGGCTGGGGCTCATTT.

[0076] Specific results such as Figure 3As shown in the figure, under normal culture conditions, the loss of the UVSSA gene led to a slight increase in the transcriptional expression level of the p16 gene in Hacat cells. After light damage caused by UVB irradiation, the transcriptional expression level of the p16 gene in Hacat-uvssa increased by about 2 times compared with wild-type cells, indicating that the aging of the cell samples has intensified.

[0077] In summary, when Hacat cells were subjected to photodamage caused by UVB irradiation, the deletion of the UVSSA gene led to a significant decrease in the proliferation activity of Hacat cells and a significant increase in the transcriptional expression level of the p16 gene, indicating that compared with the Hacat wild-type cell line, the mutant Hacat-uvssa was more sensitive to photodamage caused by UVB irradiation and could be used as a more efficient photodamaged cell model.

[0078] Example 6

[0079] This example uses tea polyphenols to verify the detection effect of the Hacat-uvssa cell model

[0080] Tea polyphenols have unique protective and therapeutic effects on the skin. They can effectively prevent ultraviolet damage to the skin, and also have anti-aging, anti-wrinkle, brown spot removal and anti-allergy effects. Tea polyphenols can be used to verify the Hacat-uvssa light-damaged cell model.

[0081] Hacat-uvssa cell model and Hacat cells were plated at 1×10 7 The cells were evenly plated in 6 plates at a density of 100 μg / well. After culturing for 24 hours in complete medium containing 5 μg / mL tea polyphenols, the cells were washed twice with PBS. After washing, PBS was added to each well. 2 The cells were irradiated with UVB ultraviolet rays, and the control group was not irradiated (a single well plate). After irradiation, PBS was discarded and serum-free medium was added to culture the cells for 24 hours. The cell proliferation activity was detected by the method described in Example 4, and the transcription expression level of the p16 gene was detected by the method described in Example 5.

[0082] Specific results such as Figure 5 As shown in the figure, under normal culture conditions, tea polyphenols had no significant effect on the proliferation activity of Hacat cells and the transcriptional expression level of p16 gene. However, after being exposed to light damage caused by UVB irradiation, the cell proliferation activity of the tea polyphenols-treated group was significantly improved compared with the control group, and the transcriptional expression level of p16 gene also increased significantly, indicating that tea polyphenols can significantly inhibit the light damage response of Hacat-uvssa. This verifies that Hacat-uvssa can be used as an efficient light-damaged cell model

[0083] In summary, the present invention uses CRISPR-Cas9 gene editing technology to knock out the UVSSA gene in human epidermal Hacat cells, constructs a cell model sensitive to mid-band ultraviolet UVB, and successfully uses it to screen active raw materials with photoprotective effects.

[0084] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A UV-sensitive cell model, characterized in that: The cell model is a cell in which the UVSSA gene function is lost.

2. The UV damage sensitive cell model according to claim 1, characterized in that: The source of the cells includes any one of skin, blood, tissue fluid or liver cells or a combination of at least two thereof; Preferably, the cells include any one of Hacat cells, hsf cells, HDF cells, BJ cells or HFF cells, or a combination of at least two of them.

3. The ultraviolet damage sensitive cell model according to claim 1 or 2, characterized in that: The nucleic acid sequence of the UVSSA gene includes the sequence shown in SEQ ID No.

1.

4. The ultraviolet damage sensitive cell model according to any one of claims 1 to 3, characterized in that: The UVSSA gene functional loss is the loss of the 400th adenine deoxyribonucleotide in the UVSSA gene.

5. The ultraviolet damage sensitive cell model according to any one of claims 1 to 4, characterized in that: The wavelength of the ultraviolet rays is a medium wavelength band; Preferably, the wavelength range of the ultraviolet rays is 280-320 nm.

6. A method for preparing a UV damage sensitive cell model according to any one of claims 1 to 5, characterized in that: The preparation method comprises the steps of: connecting sgRNA-F and sgRNA-R to a plasmid vector and then transfecting them into cells, and performing PCR identification after screening with antibiotics.

7. The method for preparing a UV damage sensitive cell model according to claim 6, characterized in that: The nucleic acid sequence of the sgRNA-F includes the sequence shown in SEQ ID No.2; Preferably, the nucleic acid sequence of the sgRNA-R includes the sequence shown in SEQ ID No.

3.

8. The method for preparing a UV damage sensitive cell model according to claim 6 or 7, characterized in that: The plasmid vector comprises any one or a combination of at least two of PX459, PX330, phU6-gRNA or lentiCRISPR v2; Preferably, the plasmid is connected to a nucleic acid sequence of Cas9; Preferably, the antibiotic comprises any one of puromycin, kanamycin or ampicillin, or a combination of at least two of them.

9. The method for preparing a UV damage sensitive cell model according to claim 6 or 7, characterized in that: The nucleic acid sequence of the upstream primer identified by PCR includes the sequence shown in SEQ ID No.4; Preferably, the nucleic acid sequence of the downstream primer identified by PCR includes the sequence shown in SEQ ID No.

5.

10. Use of the ultraviolet damage sensitive cell model according to any one of claims 1 to 5 in screening active raw materials with photoprotective effects.

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