CN116162585A - Skin cell ultraviolet injury model constructed by HaCaT cells - Google Patents
Skin cell ultraviolet injury model constructed by HaCaT cells Download PDFInfo
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Abstract
The invention provides a skin cell ultraviolet injury model constructed by HaCaT cells, which is applied to the field of skin ultraviolet irradiation injury research. After the skin cell model provided by the invention is stimulated by UVB illumination, the cell activity is obviously reduced, and the secretion amount of inflammatory factors is obviously increased, so that the model is sensitive to the ultraviolet illumination. After the skin model pretreated by the ultraviolet-resistant protective agent retinoic acid is subjected to UVB stimulation, various detection indexes show that the damage degree is reduced, and the model is applicable to the efficacy evaluation and screening of the ultraviolet-resistant protective agent and the research of the skin ultraviolet damage mechanism.
Description
Technical Field
The invention belongs to the technical field of cell models, and particularly relates to an ultraviolet injury skin cell model constructed by HaCaT cells; namely, a skin cell model constructed by taking immortalized keratinocyte HaCaT as a cell source can be applied to the field of skin ultraviolet irradiation damage research.
Technical Field
The skin is used as the largest organ of the human body, is directly contacted with the external environment, and is the first defense line of the human body against external injury. Among the many factors of skin damage, ultraviolet light is one of the most common. Ultraviolet rays can be classified into long wave (UVA), medium wave (UVB) and short wave (UVC) according to their wavelengths. Wherein the UVA wavelength is 320-400 nm, the UVB wavelength is 280-320 nm, and the UVC wavelength is 100-280 nm. UVC has been substantially sequestered by the ozone layer upon entry into the atmosphere; thus, some damage to the skin is caused mainly by UVB and UVA.
Excessive ultraviolet rays damage skin cells, accelerate skin aging, cause skin surface roughness, and skin cancer in severe cases, and cause many skin problems such as horny layer hyperplasia. Wherein, UVB mainly damages the skin surface layer, and can directly cause the surface skin to be sunburned or sunburned, and UVA can directly reach the dermis layer of the skin, damage collagen, elastic fibers and the like in the skin, and accelerate the aging of the skin. Therefore, the cause of skin photodamage and the control method have been research hot spots.
Immortalized human keratinocyte cell line HaCaT is often used as a typical cell line for the in vitro culture of skin keratinocytes due to its highly preserved differentiation capacity. Although cytogenetically abnormal, immortalized keratinocyte cell line HaCaT shows high differentiation potential under in vivo and in vitro conditions, and has been widely used as a substitute for normal human keratinocytes for fundamental research in skin biology. Experiments find that HaCaT cells under conventional culture conditions are sensitive to UVB irradiation, so that HaCaT is also often used as a cell model for research on photodamage of skin epidermis.
However, haCaT cells under conventional culture conditions have significant limitations in simulating real epidermal photodamage. The method is mainly characterized in that:
1) The surface of the real skin is protected by a layer of cuticle naturally formed by dead cells and is of a multi-cell layer structure, and a single-layer HaCaT cell cultured in a plane lacks the layer structure;
2) The traditional cell culture is immersed in a liquid culture medium, and ultraviolet rays need to pass through a liquid layer at first when in out-of-line illumination, so that the direct damage effect of the ultraviolet rays on the skin cannot be truly simulated.
Therefore, it is very significant to build a more simulated HaCaT cell-based skin ultraviolet irradiation damage model.
Disclosure of Invention
The invention provides a skin model taking immortalized keratinocyte HaCaT as a cell source, namely a novel ultraviolet UVB (ultraviolet UVB) versus skin illumination damage model, which is suitable for ultraviolet damage mechanism research and efficacy evaluation and screening of an ultraviolet protection agent.
The invention provides a skin model taking immortalized keratinocyte HaCaT as a cell source, which comprises the following construction method:
1) Mixing human dermal fibroblast HSF and type I rat tail collagen to prepare a gel solution;
the concentration of human dermal fibroblast HSF in the gel solution is 1×10 6 -2×10 6 cell/mL: the concentration of the type I rat tail collagen is 0.5-1.5mg/mL;
2) Adding the gel solution into a culture hole, inoculating the HaCaT cell suspension onto the gel for culture after the gel solution is solidified and hardened, and culturing until the gel is contracted to be stable in size and separated from the culture hole;
the culture conditions are 5% CO at 37 DEG C 2 Culturing in an incubator;
3) Placing a cell screen in a culture hole of a six-hole plate, and adding culture solution until a nylon net of the cell screen is covered; then placing the gel after shrinkage and separation in the step 2) on a screen mesh, enabling the HaCaT cell surface to face upwards, adding a culture solution to enable the gel main body to be immersed in the culture solution, exposing the upper surface of the gel main body to air, and culturing for 2 weeks to prepare a skin cell model;
the aperture of the cell screen is 40 mu m;
in this step, the culture medium is changed every day during the culture;
the culture solution comprises the following specific components: a mixture of DMEM medium and F12 medium (DMEM to F12 medium volume ratio of 3:1), 10% Fetal Bovine Serum (FBS), 1% green streptomycin.
The skin model is used for drug screening and drug effect evaluation.
Compared with the HaCaT single cell layer, the skin model provided by the invention has the advantages of enhanced ultraviolet irradiation tolerance and higher skin simulation. After being excited by UVB illumination, the cell activity is obviously reduced, and the secretion amount of inflammatory factors is obviously increased, which proves that the model has good relevance to the ultraviolet illumination. After the skin model pretreated by the retinoic acid as an ultraviolet protective agent is subjected to UVB stimulation, various detection indexes show the reduction of the damage degree, which indicates that the model is suitable for drug screening and drug effect evaluation.
Drawings
Fig. 1: schematic of the culture of skin cell model, wherein 1, cell culture plate; 2. cell screening; 3. collagen gel; 4. a liquid level position line of the culture solution;
fig. 2: culturing a mature skin cell model physical map;
fig. 3: MTT cell viability detection result diagram;
fig. 4: and (3) detecting the secretion amount of inflammatory factors IL-1 alpha and IL-1 beta in the cell culture solution.
Detailed Description
The invention takes the cell line HaCaT as a cell source to construct the multi-cell layer skin tissue, and after injury stimulation is carried out by using UVB illumination, the multi-cell physiological index is obviously changed similar to the real skin photoinjury reaction, so that the invention can be used for researching the mechanism of skin ultraviolet injury and evaluating the protection and repair effects of a tested object on the skin photoinjury.
The skin cell model of the invention is a collagen gel prepared by mixing human dermal fibroblast HSF and I-type rat tail collagen. After the gel is solidified and hardened, the suspension of HaCaT cells is inoculated onto the gel for culture, and the gel is contracted to be stable in size and separated from the pore plate. The cell screen was taken, placed in a new six-well plate, and medium was added to the wells until the nylon mesh of the cell screen was covered. The gel tray after shrinkage separation was then placed on a screen with HaCaT cells facing up and the culture level was adjusted to just below the upper edge of the gel, allowing the gel to be fully immersed in the culture while exposing its surface to air. Fresh broth was changed daily throughout the experiment. After 2 weeks of culture, a skin cell model was obtained.
Further ultraviolet irradiation stimulation experiments can be performed, and endpoint index detection can be performed, wherein the endpoint detection index can be selected:
and (3) detecting the activity of the epidermal cells: the treated cells were incubated in MTT solution for 3h by MTT method, skin tissue was removed using a punch, and absorbance was measured at 490nm with sufficient dissolution in DMSO.
Detecting inflammatory factors: the concentration of inflammatory factors in the culture solution is detected by ELISA method, and the inflammatory factors can be IL-1 alpha and IL-1 beta.
The culture medium is composed of DMEM/F12 (DMEM and F12 are mixed in a volume ratio of 3:1), 10% Fetal Bovine Serum (FBS) and 1% green streptomycin. However, other media used to culture HaCaT cells may also be used.
One preparation method of the collagen gel comprises the following steps: taking 1mL of 1mg/mL of gel as an example, human dermal fibroblast HSF is prepared to be suspended in the culture solution at a cell concentration of 1X 10 6 cells/mL, and placed in an ice bath. 200. Mu.L of rat tail collagen type I (5 mg/mL) was added to 12. Mu.L of 0.1mol/L NaOH and immediately mixed; then 23 mu L of 10 XPBS is added and mixed evenly; adding 760 mu L of cell suspension, mixing, and immediately adding into a culture vessel; after the culture vessel is left at room temperature for 20 minutes to be gelled and fixed, the subsequent experimental operation is carried out.
The seeding density of the HaCaT cells is 1×10 6 Six well plates per well.
The ultraviolet light irradiation stimulation experiment method comprises the steps of culturing a mature skin model, opening a cover, adjusting the irradiation dose, and irradiating under a UVB lamp. After the illumination is finished, culturing in a 5% carbon dioxide incubator at 37 ℃ for 24 hours, and carrying out subsequent detection.
The present invention will be described in detail with reference to the following examples:
example 1 skin model construction and UVB illumination IC50 value determination experiment
1. Skin cell model construction and detection method
1) Taking out human keratinocyte HaCaT and human dermal fibroblast HSF from a liquid nitrogen tank, resuscitating and subculturing, wherein the culture solution comprises the following components: DMEM/F12 (DMEM to F12 volume ratio 3:1 mixture), 10% fbs, 1% penicillin.
2) When the cell density grows to about 80%, pancreatin is digested into single cell suspension, and the blood cell counting plate is counted for standby.
3) Collagen gel preparation: cell concentration of 1X 10 6 Placing the cells/mL of HSF cell suspension in ice bath for later use, taking 1mL of three-dimensional gel with the concentration of 1mg/mL as an example, adding 200 mu L of rat tail collagen type I (5 mg/mL) into 12 mu L of 0.1mol/L NaOH, and immediately and uniformly mixing; adding 23 mu L of 10 XPBS, and mixing; then 760. Mu.L of HSF cell suspension was added and mixed well.
4) The above collagen solution was rapidly added to a six-well plate with 3mL per well. After the gel is solidified after being placed for 20 minutes at room temperature, the subsequent experimental operation is carried out.
5) After gel setting and hardening, haCaT cells were seeded at 1X 10 6 Density per well inoculated onto gel, 5% co at 37 °c 2 The gel was contracted to volume stability and separated from the well plate after 5 days of culture in an incubator.
6) A 40 μm pore size disposable sterile cell screen (tin-free resistant, cat No.: 258369 After cutting the screen handle with sterile scissors, place in a new six-well plate and add culture medium to the well until the nylon mesh of the cell screen is covered. Placing the gel after shrinkage separation on a screen with HaCaT cell surface facing upwards, and adjusting liquid level to be just below upper surface of the gel to allow the gel body to be fully soaked in culture solution while exposing its surface to air, placing at 37deg.C 5% CO 2 Culturing in an incubator.
7) During the course of the subsequent experiments, fresh culture broth was changed daily. Subsequent testing was performed after 2 weeks of continuous culture.
8) UVB illumination IC 50 Value measurement: the cultured mature skin cell model is opened and irradiated under UVB lamp, and divided into 5 light dose groups of 20, 40, 80, 160, 320mJ/cm respectively 2 And a control group. Each set was set with 3 replicates. After the completion of the irradiation, the cells were cultured in a 5% carbon dioxide incubator at 37℃for 24 hours.
9) More, theChanging culture solution, incubating skin model in culture solution containing MTT with final concentration of 0.3mg/ml for 3 hr, cutting skin tissue from bracket with puncher with diameter of 1cm, placing in 1.5ml EP tube, adding 1ml DMSO, soaking skin tissue under light-shielding condition for 2 hr, vortex oscillating to dissolve purple crystal completely, detecting absorbance value with enzyme-labeled instrument at 490nm, and calculating to obtain UVB illumination IC 50 Values.
2. HaCaT single cell layer preparation and detection method
HaCaT single cell suspension at 1×10 per well 6 Is inoculated in six-hole plates, cultured for 3 days until the cell growth fusion reaches 80%, and is irradiated under UVB lamp after opening the cover, and divided into 5 light dose groups of 20, 40, 80, 160 and 320mJ/cm respectively 2 And a control group. Each set was set with 3 replicates. After the completion of the irradiation, the culture was continued in a 5% carbon dioxide incubator at 37℃for 24 hours. The sample to be tested was replaced with a culture broth containing MTT at a final concentration of 0.3mg/ml and incubated in a 5% carbon dioxide incubator at 37℃for 3h. The supernatant was carefully discarded, 1ml of DMSO was added to each well and incubated at 37℃for 30min to allow the purple crystals to dissolve well and absorbance was measured at 490nm using an microplate reader. Calculation to obtain UVB illumination IC 50 Values.
3. Experimental results:
1) The mature cell model is shown in figure 2, and the surface of the mature skin cell model is dry and compact and has the skin keratinization structure state.
2) The invention constructs a skin model and a UVB illumination IC of a HaCaT single cell layer 50 The result of the value calculation is shown in Table 1
Table 1: UVB illumination IC of skin model and HaCaT single cell layer 50 Value table
IC obtained by calculation 50 The value detection result shows that the skin cell model has good correlation with the UVB photo damage. Compared with a HaCaT single cell layer, the skin model constructed by the invention has improved tolerance to UVBThe physiological performance is more than 2 times and is more similar to that of real skin.
Example 2 skin cell model Performance evaluation experiment
1. Experimental grouping:
the skin cell model was cultured and matured according to the aforementioned construction method, and divided into 3 groups (3 parallel samples were set for each group), respectively:
control group: groups not receiving UVB light
Illumination group: group receiving UVB light
Treatment group: before UVB light irradiation, tretinoin (retinoic acid) solution was used.
2. The experimental method comprises the following steps:
all the established skin cell models are replaced by fresh culture liquid, 100 mu L of retinoic acid solution with the concentration of 3 mu M is uniformly smeared on the surface of skin tissue in advance in a treatment group model, and the skin tissue is incubated for 2 hours in an incubator. Subsequently, all three groups of samples are taken out from the incubator, and the illumination group and the treatment group model are respectively stimulated by UVB illumination with the illumination dose of 120mJ/cm 2 . After the treatment, all models were again placed in an incubator for stationary culture for 24 hours. After 24 hours, taking out the culture solution at the bottom of each group, and detecting inflammatory factors by ELISA; the upper layer skin cell tissue is incubated for 3 hours in a culture medium containing MTT with the final concentration of 0.3mg/ml, the skin tissue is cut and separated from a bracket by using a puncher with the diameter of 1cm, the bracket is placed in a 1.5ml EP tube, 1ml DMSO is added, the skin tissue is soaked for 2 hours under the light-shielding condition, vortex vibration is carried out, purple crystals are fully dissolved, and the absorbance value is detected under the 490nm of an enzyme-labeled instrument.
Cell viability% = (test sample absorbance-blank absorbance)/(negative control absorbance-blank absorbance)/(x 100)
3. Experimental results:
the cell viability experimental result is shown in fig. 3, the cell viability is obviously reduced after UVB irradiation, and the cell viability is obviously improved after the model is treated by the ultraviolet protective agent retinoic acid before irradiation. The model is sensitive to UVB illumination damage, and the ultraviolet-resistant protective agent has obvious protective effect on the model, so that the model is suitable for effect evaluation and screening of novel protective agents.
The changes in the secretion amounts of inflammatory factors IL-1. Alpha. And IL-1. Beta. In the lower cell culture broth were examined by ELISA, and the results are shown in FIG. 4. The results showed that the secretion of IL-1α increased by more than 2 times and the secretion of IL-1β increased by more than 3 times after UVB irradiation. The secretion of IL-1 alpha and IL-1 beta in the sample treated with retinoic acid before irradiation is significantly reduced.
The two groups of experimental results show that the model is sensitive to UVB illumination damage, the common ultraviolet-resistant protective agent has obvious protective effect on the model, and the model is suitable for effect evaluation and screening of new protective agents.
Claims (10)
1. A skin cell model, wherein the skin cell model is constructed using the following method:
1) Mixing human dermal fibroblast HSF and type I rat tail collagen to prepare a gel solution;
2) Adding the gel solution into a culture hole, inoculating the HaCaT cell suspension onto the gel for culture after the gel solution is solidified and hardened, and culturing until the gel is contracted to be stable in size and separated from the culture hole;
3) Placing a cell screen in a culture hole, and adding culture solution until a nylon net of the cell screen is covered; and then placing the gel after shrinkage and separation in the step 2) on a screen mesh, enabling the HaCaT cells to face upwards, adding a culture solution to enable the gel main body to be immersed in the culture solution, exposing the upper surface to air, and culturing to prepare the skin cell model.
2. The skin cell model of claim 1, wherein the concentration of human dermal fibroblast HSF in the gel solution of 1) is 1 x 10 6 -2×10 6 cells/mL。
3. The skin cell model of claim 1, wherein the concentration of type I rat tail collagen in 1) is 0.5-1.5mg/mL.
4. The skin cell model of claim 1, wherein in 2)Is 5% CO at 37 DEG C 2 Culturing in an incubator.
5. The skin cell model of claim 1 wherein the cell screen of 3) has a pore size of 40 μm.
6. The skin cell model of claim 1, wherein the culture medium is changed daily during the culturing in 3).
7. The skin cell model of claim 1, wherein the culture medium is a mixture of DMEM medium and F12 medium, to which 10% fetal bovine serum and 1% penicillin are added.
8. The skin cell model of claim 7, wherein the volume ratio of DMEM medium to F12 medium is 3:1.
9. use of the skin cell model of claim 1 in screening or efficacy evaluation of a drug for treating skin photo damage.
10. A method of screening for a drug for treating skin photodamage, said method comprising using the skin cell model of claim 1.
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