Method for promoting proliferation and differentiation of cervical squamous epithelial cells

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a method for promoting proliferation and differentiation of cervical squamous epithelial cells. The method comprises the following steps: S1, extracting and culturing primary cervix uteri squamous epithelial cells from cervix uteri tissues to obtain primary cervix uteri squamous epithelial cells; s2, adding the primary cervical squamous epithelial cells into a cell proliferation induction culture solution to induce cell proliferation, so as to obtain proliferated cervical squamous epithelial cells; and S3, uniformly mixing the proliferated cervical squamous epithelial cells with differentiation matrigel, and inducing cell differentiation by using a cell differentiation induction culture solution to obtain differentiated cervical squamous epithelial cells. Proliferation and differentiation of the cervical squamous epithelial cells are promoted by preparing a proper culture medium, and influence factors of proliferation and differentiation of the cervical squamous epithelial cells are researched by culturing the cervical squamous epithelial cells in vitro, so that the pathogenesis of cervical erosion and cervical cancer can be clarified; therefore, a direction is provided for research and development of medicines for treating cervical erosion and cervical cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for promoting proliferation and differentiation of cervical squamous epithelial cells. Background Art

[0002] Squamous epithelium is mainly found in the peritoneum, pleura, skin, cervix, vagina and other parts. The squamous epithelium located in the cervix and vagina is also called stratified squamous epithelium, which has a strong regenerative ability and can also prevent bacterial invasion to a certain extent, thus playing a role in protecting the cervix. Cervical squamous epithelium is the epithelial tissue covering the surface of the cervix, which is generally stratified squamous epithelium. Cervical squamous epithelial cells are cells of the cervix itself. Under normal circumstances, cervical squamous epithelial cells are pink and generally appear polygonal or irregular in shape under a microscope, and the edges of the cells are usually serrated.

[0003] The transition area between stratified squamous epithelium and cervical columnar epithelium is a common site for cervical cancer, and the proliferation and differentiation of stratified squamous epithelial cells in this area are closely related to cervical erosion and cervical cancer. Therefore, by culturing cervical squamous epithelial cells in vitro and studying the factors affecting the proliferation and differentiation of cervical squamous epithelial cells, it is helpful to clarify the pathogenesis of cervical erosion and cervical cancer, thereby providing direction for the development of drugs to treat cervical erosion and cervical cancer. Summary of the invention

[0004] The purpose of the present invention is to provide a method for promoting the proliferation and differentiation of cervical squamous epithelial cells, so as to promote the proliferation and differentiation of cervical squamous epithelial cells in vitro by preparing a suitable culture medium and combining a suitable culture method.

[0005] To this end, the present invention provides the following technical solutions.

[0006] One aspect of the present invention provides a method for promoting proliferation and differentiation of cervical squamous epithelial cells, the method comprising the following steps:

[0007] S1: Extract and culture primary cervical squamous epithelial cells from cervical tissue to obtain primary cervical squamous epithelial cells;

[0008] S2: adding primary cervical squamous epithelial cells to a cell proliferation induction culture medium to induce proliferation of the primary cervical squamous epithelial cells, thereby obtaining proliferating cervical squamous epithelial cells;

[0009] S3: The proliferating cervical squamous epithelial cells are mixed with the differentiation matrix gel and plated on a well plate, and a cell differentiation induction culture medium is used to induce differentiation of the proliferating cervical squamous epithelial cells to obtain differentiated cervical squamous epithelial cells.

[0010] In some preferred embodiments, step S1 comprises:

[0011] S11: Cervical tissue was cut into pieces under sterile conditions, and then digested in a digestive enzyme solution. After digestion, the cells were centrifuged and washed and centrifuged. This was repeated three times to obtain a cell precipitate.

[0012] S12: Resuspend the cells in primary culture medium to a cell concentration of 2-4×10 5 / mL, and cultured at 5% CO2 and 37°C for 5-10 days, wherein the culture medium was changed every 2-3 days to obtain primary cervical squamous epithelial cells.

[0013] In some preferred embodiments, in step S11, the digestive enzyme solution is composed of: 10-20 mg collagenase type I, 10-20 mg trypsin and 30-50 mg sodium chloride per 100 mL PBS buffer.

[0014] In some preferred embodiments, in step S11, the digestive enzyme solution is added in an amount of 100-200 μL of the digestive enzyme solution per 1 mg of cervical tissue.

[0015] In some preferred embodiments, in step S11, the digestion conditions are: 37° C., 60 min.

[0016] In some preferred embodiments, in step S12, the primary culture medium is composed of: DMEM culture medium, to which are added: transforming growth factor-β 10-20 μg / mL, transferrin 50-100 μg / mL, epidermal growth factor 10-20 μg / mL, sericin phosphopeptide 40-80 ng / mL, laurocapram 10-20 ng / mL, and ribose 10-30 ng / mL.

[0017] In some preferred embodiments, step S2 comprises:

[0018] S21: dispersing, centrifuging and digesting the obtained primary cervical squamous epithelial cells to obtain single cells;

[0019] S22: Resuspend the cells in cell proliferation induction medium to obtain a cell concentration of 1 to 4 × 10 6 The cells were suspended in a suspension of 100 cells / mL and cultured in the conditions of 5% CO2 and 37°C. The culture medium was replaced every 2-4 days. The cells were cultured until the confluence reached 80%, which was recorded as the P1 generation. The same steps were followed to subculture to the P10 generation to obtain proliferating cervical squamous epithelial cells.

[0020] In some preferred embodiments, in step S22, the cell proliferation inducing culture fluid is composed of: 1 / 2 DMEM medium and 1 / 2 F12 medium, to which are added: 10-50 μg / mL glutamine, 1-10 M dexamethasone, 10-20 μg / mL transforming growth factor-β, 20-50 μg / mL recombinant human epidermal growth factor, 10-20 μg / mL isoproterenol, 300-500 pg / mL interferon-α, 30-80 pg / mL recombinant human interleukin-1β, 10-50 μg / mL bletilla striata polysaccharide, 10-30 μg / mL kaempferol-3-rhamnoside, 1-3 μg / mL paeonol and 1-3 μg / mL titanium oxynitride.

[0021] In some preferred embodiments, step S3 comprises:

[0022] S31: resuspending and centrifuging the proliferating cervical squamous epithelial cells to obtain cell pellets;

[0023] S32: Resuspend the cells in cell differentiation induction medium I to obtain a cell concentration of 1 to 4 × 10 6 Cell suspension of 100 cells / mL;

[0024] S33: The cell suspension and differentiation matrix gel were mixed at a volume ratio of 1:2-3 and plated on a well plate. The mixture was allowed to stand at 37°C for 20-30 min. After the differentiation matrix gel solidified, cell differentiation induction culture medium I was added. The mixture was cultured at 5% CO2 and 37°C for 4-6 days. The culture medium was replaced every 2-3 days. Thereafter, the culture medium was replaced with cell differentiation induction culture medium II. The mixture was cultured at 5% CO2 and 37°C for 8-10 days. The culture medium was replaced every 2-3 days to obtain differentiated cervical squamous epithelial cells.

[0025] In some preferred embodiments, in step S32, the composition of the cell differentiation induction culture medium I is: 1 / 2 DMEM culture medium and 1 / 2 F12 culture medium, to which are added: epidermal growth factor 30-50 ng / mL, fibroblast growth factor-10 10-30 ng / mL, dexamethasone 1-5 M, insulin 10-20 g / mL, hepatocyte growth factor 5-15 ng / mL, carbachol 100-200 nM, isoproterenol 1-5 μM, matrine 1-5 ng / mL, inositol 1-3 g / mL, interleukin 3 10-30 pg / mL, sophora japonica glycoside 10-30 pg / mL and lotus leaf extract 10-30 g / mL.

[0026] In some preferred embodiments, in step S33, the composition of the cell differentiation induction culture medium II is: 1 / 2 DMEM culture medium and 1 / 2 F12 culture medium, to which are added: epidermal growth factor 30-50 ng / mL, fibroblast growth factor-10 10-30 ng / mL, dexamethasone 1-5 M, insulin 10-20 g / mL, heparin sodium 100-200 g / mL, isoproterenol 1-5 μM, 100-300 nM carbachol, lichenic acid 10-30 pg / mL, palmitoyl tripeptide-1 5-10 ng / mL, cedrol 1-5 ng / mL and magnolol 1-3 ng / mL.

[0027] In some preferred embodiments, in step S33, the method for preparing the differentiation matrix gel comprises the following steps:

[0028] S331: dissolving hyaluronic acid in water to obtain a hyaluronic acid solution with a concentration of 1 to 5 mg / mL;

[0029] S332: adding a water-soluble monomer and an initiator to the obtained hyaluronic acid solution, mixing and reacting at 50 to 120° C. for 4 to 16 hours to obtain a modified hyaluronic acid solution;

[0030] S333: Add allantoin and astragalus polysaccharide to the modified hyaluronic acid solution in a mass volume ratio of 1 mg: 5-10 mg: 100-300 mL and stir evenly, then add the cross-linking agent and stir for 4-8 hours. After stirring, let it stand and filter, and dry the obtained product to obtain differentiation matrix gel.

[0031] In some preferred embodiments, in step S332, the water-soluble monomer is selected from any one of acrylamide, methacrylamide, N-isopropylacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-hydroxymethylacrylamide, N-vinylformamide, and N-vinylacetamide.

[0032] In some preferred embodiments, in step S332, the amount of the water-soluble monomer added is 1 to 3 times the mass of the hyaluronic acid.

[0033] In some preferred embodiments, in step S332, the initiator is selected from any one of sodium persulfate, ammonium persulfate, and potassium persulfate.

[0034] In some preferred embodiments, in step S332, the amount of the initiator added is 0.2-0.7% of the mass of the water-soluble monomer.

[0035] In some preferred embodiments, in step S333, the cross-linking agent is selected from any one of catechol, hydroquinone, and resorcinol.

[0036] In some preferred embodiments, in step S333, the amount of the cross-linking agent added is 0.1 to 0.5% of the mass of the hyaluronic acid.

[0037] By means of the above technical solution, the present invention has at least the following advantages:

[0038] 1. The present invention promotes the proliferation and differentiation of cervical squamous epithelial cells by preparing appropriate primary culture medium, cell proliferation induction culture medium and cell differentiation induction culture medium, and studies the influencing factors of cervical squamous epithelial cell proliferation and differentiation by culturing cervical squamous epithelial cells in vitro, which is helpful to clarify the pathogenesis of cervical erosion and cervical cancer, thereby providing direction for the development of drugs for treating cervical erosion and cervical cancer.

[0039] 2. The three culture media of the present invention add combined cell growth factors, hormones, phenols, polypeptides, amino acids, polysaccharides, alcohols, etc. to the original basic culture media. The various components are reasonably matched to stimulate the proliferation and differentiation of cervical squamous epithelial cells and maintain the activity of the cells. Studies have shown that the culture media of the present invention exhibit better proliferation activity and differentiation ability when culturing cervical squamous epithelial cells.

[0040] 3. The present invention also provides a differentiation matrix gel for use in the differentiation process of cervical squamous epithelial cells. The differentiation matrix gel of the present invention uses hyaluronic acid as a raw material, introduces a water-soluble monomer under the action of an initiator, and then mixes with allantoin and astragalus polysaccharide. Allantoin can promote the repair of damaged cells. Astragalus polysaccharide contains rich nutrients such as sugars, and also has certain antibacterial, anti-stress, antioxidant and other effects. In addition, it can also promote the solidification of the gel matrix. The addition of a cross-linking agent can help the polymer molecules to re-form a new three-dimensional spatial structure. The obtained product is used in the process of cell differentiation, can maintain the activity of the cells, provide cells with sufficient energy sources, and at the same time reduce the impact of the external environment on the cells, and improve the survival rate and differentiation ability of the cells.

[0041] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the cell growth situation of Example 1;

[0043] Figure 2 This is the cell growth situation of Example 2;

[0044] Figure 3 This is the cell growth situation of Example 3;

[0045] Figure 4The cell growth condition of comparative example 1 is shown;

[0046] Figure 5 The cell growth condition of comparative example 2;

[0047] Figure 6 The cell growth condition of comparative example 3 is shown;

[0048] Figure 7 This is the cell growth condition of Comparative Example 4. DETAILED DESCRIPTION

[0049] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0051] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0052] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0053] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0054] Unless otherwise specified, the collagenase type I activity used in the following examples is 200 kU·L -1 , trypsin activity is 250 kU·L -1 ; Dispase was dispase I, No. D4818, purchased from Sigma; Astragalus polysaccharide, lotus leaf extract, and Bletilla striata polysaccharide were all purchased from Shanyang Lianfeng Biotechnology Co., Ltd.; Ribose was purchased from Xi'an Musen Bioengineering Co., Ltd.; Lichenic acid was purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd. Other materials and reagents can be obtained from commercial channels.

[0055] Embodiment 1:

[0056] Prepare digestive enzyme solution: Add 15 mg collagenase type I, 15 mg trypsin and 40 mg sodium chloride to every 100 mL PBS buffer to obtain digestive enzyme solution.

[0057] Preparation of primary culture medium: Add the following concentrations of each substance into DMEM culture medium: transforming growth factor-β 15 g / mL, transferrin 75 μg / mL, epidermal growth factor 15 μg / mL, sericin phosphopeptide 60 ng / mL, laurocapram 15 ng / mL, ribose 20 ng / mL, mix well, and sterilize at 121°C for 25 min.

[0058] Prepare cell proliferation inducing culture medium: mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: glutamine 30 μg / mL, dexamethasone 5M, transforming growth factor-β 15 μg / mL, recombinant human epidermal growth factor 35 μg / mL, isoproterenol 15 μg / mL, interferon-α 400 pg / mL, recombinant human interleukin 1β 55 pg / mL, bletilla striata polysaccharide 30 μg / mL, kaempferol-3-rhamnoside 20 μg / mL, paeonol 2 μg / mL and titanium oxynitride 2 μg / mL, mix well, and sterilize at 121°C for 25 min.

[0059] Preparation of cell differentiation induction medium I: Mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: epidermal growth factor 40 ng / mL, fibroblast growth factor-10 20 ng / mL, dexamethasone 3 M, insulin 15 g / mL, hepatocyte growth factor 10 ng / mL, carbachol 150 nM, isoproterenol 3 μM, matrine 3 ng / mL, inositol 2 g / mL, interleukin 3 20 pg / mL, sophora japonica glycoside 20 pg / mL and lotus leaf extract 20 g / mL, mix well, and sterilize at 121°C for 25 min.

[0060] Prepare cell differentiation induction medium II: mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: epidermal growth factor 40 ng / mL, fibroblast growth factor-10 20 ng / mL, dexamethasone 3 M, insulin 15 g / mL, heparin sodium 150 g / mL, isoproterenol 3 μM, 200 nM carbachol, lichenic acid 20 pg / mL, palmitoyl tripeptide-1 7.5 ng / mL, cedrol 3 ng / mL, and magnolol 2 ng / mL.

[0061] Preparation of differentiation matrix gel: Dissolve hyaluronic acid in water to obtain a hyaluronic acid solution with a concentration of 3 mg / mL. Add acrylamide (the amount added is 2 times the mass of hyaluronic acid) and ammonium persulfate (the amount added is 0.5% of the mass of acrylamide) to the obtained hyaluronic acid solution, mix and react at 90°C for 10 hours to obtain a modified hyaluronic acid solution. Add allantoin and astragalus polysaccharide to the modified hyaluronic acid solution in a mass volume ratio of 1 mg: 7.5 mg: 200 mL and stir evenly, then add catechol (the amount added is 0.3% of the mass of hyaluronic acid) and stir for 6 hours. After stirring, let it stand and filter. Wash the obtained solid product with deionized water and dry it to obtain differentiation matrix gel.

[0062] A method for promoting proliferation and differentiation of cervical squamous epithelial cells, comprising the following steps:

[0063] (1) Fresh healthy human cervical tissue was minced under sterile conditions, and then 200 μL of digestive enzyme solution was added to each mg of cervical tissue and digested at 37°C for 60 min. After digestion, the cells were centrifuged and washed twice with Hank's balanced salt solution. The digested cells were filtered through a 100 μm filter, and the cell suspension was centrifuged and the supernatant was discarded. This was repeated three times to obtain a cell pellet. The cells were resuspended in primary culture medium to a cell concentration of 3 × 10 5 / mL, and cultured at 5% CO2 and 37°C for 10 days, wherein the culture medium was changed every 2 days, to obtain primary cervical squamous epithelial cells.

[0064] (2) Add 1 mL of dispase to the obtained primary cervical squamous epithelial cells, let stand at 37°C for 30 minutes, gently blow and collect into a centrifuge tube, centrifuge and remove the supernatant, add 1 mL of 0.05% trypsin to digest for 5 minutes to digest into single cells, and terminate the digestion with 1% BSA / PBS to obtain single cells. Resuspend the cells in cell proliferation induction culture medium to obtain a cell concentration of 3×10 6 The cells were suspended in a suspension of 100 cells / mL and cultured in the conditions of 5% CO2 and 37°C. The culture medium was replaced every 2 days. The cells were cultured until the cell confluence reached 80%, which was recorded as the P1 generation. The same steps were followed to subculture to the P10 generation to obtain proliferating cervical squamous epithelial cells.

[0065] (3) The above-mentioned P8 proliferated cervical squamous epithelial cells were resuspended and centrifuged to obtain a cell pellet. The cells were resuspended in cell differentiation induction medium I to obtain a cell concentration of 3×10 6 / mL cell suspension. The cell suspension was mixed with differentiation matrix gel at a volume ratio of 1:2 and plated in a well plate, and allowed to stand at 37°C for 20 to 30 minutes. After the differentiation matrix gel solidified, cell differentiation induction culture medium I was added, and cultured at 5% CO2 and 37°C for 6 days, with the culture medium replaced every 2 days. Thereafter, the culture medium was replaced with cell differentiation induction culture medium II, and cultured at 5% CO2 and 37°C for 10 days, with the culture medium replaced every 2 days, to obtain differentiated cervical squamous epithelial cells.

[0066] During the culture process, the cell growth was counted, see Figure 1 ; Cell viability was detected at P8 of proliferation culture and on day 10 of differentiation culture, see Table 1.

[0067] Embodiment 2:

[0068] Prepare digestive enzyme solution: Add 20 mg collagenase type I, 10 mg trypsin and 50 mg sodium chloride to every 100 mL PBS buffer to obtain digestive enzyme solution.

[0069] Preparation of primary culture medium: Add the following substances at the following concentrations into DMEM culture medium: transforming growth factor-β 10 μg / mL, transferrin 100 μg / mL, epidermal growth factor 10 μg / mL, sericin phosphopeptide 80 ng / mL, laurocapram 10 ng / mL, ribose 30 ng / mL, mix well, and sterilize at 121°C for 25 min.

[0070] Prepare cell proliferation inducing culture medium: mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: 50 μg / mL glutamine, 1 M dexamethasone, 20 μg / mL transforming growth factor-β, 20 μg / mL recombinant human epidermal growth factor, 20 μg / mL isoproterenol, 300 pg / mL interferon-α, 80 pg / mL recombinant human interleukin-1β, 10 μg / mL bletilla striata polysaccharide, 30 μg / mL kaempferol-3-rhamnoside, 1 μg / mL paeonol and 3 μg / mL titanium oxynitride, mix well, and sterilize at 121°C for 25 min.

[0071] Preparation of cell differentiation induction medium I: Mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: epidermal growth factor 30 ng / mL, fibroblast growth factor-10 30 ng / mL, dexamethasone 1 M, insulin 20 g / mL, hepatocyte growth factor 5 ng / mL, carbachol 200 nM, isoproterenol 1 μM, matrine 5 ng / mL, inositol 1 g / mL, interleukin 3 30 pg / mL, sophora japonica glycoside 10 pg / mL and lotus leaf extract 30 g / mL, mix well, and sterilize at 121°C for 25 min.

[0072] Preparation of cell differentiation induction medium II: Mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: epidermal growth factor 50 ng / mL, fibroblast growth factor-10 10 ng / mL, dexamethasone 5 M, insulin 10 g / mL, heparin sodium 200 g / mL, isoproterenol 1 μM, 300 nM carbachol, lichenic acid 10 pg / mL, palmitoyl tripeptide-1 10 ng / mL, cedrol 1 ng / mL, and magnolol 3 ng / mL.

[0073] Preparation of differentiation matrix gel: Dissolve hyaluronic acid in water to obtain a hyaluronic acid solution with a concentration of 1 mg / mL. Add methacrylamide (the amount added is 3 times the mass of hyaluronic acid) and ammonium persulfate (the amount added is 0.7% of the mass of methacrylamide) to the obtained hyaluronic acid solution, mix and react at 90°C for 4 hours to obtain a modified hyaluronic acid solution. Add allantoin and astragalus polysaccharide to the modified hyaluronic acid solution in a mass volume ratio of 1 mg: 5 mg: 100 mL and stir evenly, then add hydroquinone (the amount added is 0.5% of the mass of hyaluronic acid) and stir for 4 hours. After stirring, let it stand and filter. Wash the obtained solid product with deionized water and dry it to obtain differentiation matrix gel.

[0074] A method for promoting proliferation and differentiation of cervical squamous epithelial cells, comprising the following steps:

[0075] (1) Fresh healthy human cervical tissue was minced under sterile conditions, and then 200 μL of digestive enzyme solution was added to each mg of cervical tissue and digested at 37°C for 60 min. After digestion, the cells were centrifuged and washed twice with Hank's balanced salt solution. The digested cells were filtered through a 100 μm filter, and the cell suspension was centrifuged and the supernatant was discarded. This was repeated three times to obtain a cell pellet. The cells were resuspended in primary culture medium to a cell concentration of 3 × 10 5 / mL, and cultured at 5% CO2 and 37°C for 10 days, wherein the culture medium was changed every 2 days, to obtain primary cervical squamous epithelial cells.

[0076] (2) Add 1 mL of dispase to the obtained primary cervical squamous epithelial cells, let stand at 37°C for 30 minutes, gently blow and collect into a centrifuge tube, centrifuge and remove the supernatant, add 1 mL of 0.05% trypsin to digest for 5 minutes to digest into single cells, and terminate the digestion with 1% BSA / PBS to obtain single cells. Resuspend the cells in cell proliferation induction culture medium to obtain a cell concentration of 1×10 6 The cells were suspended in a suspension of 100 cells / mL and cultured in the conditions of 5% CO2 and 37°C. The culture medium was replaced every 2 days. The cells were cultured until the cell confluence reached 80%, which was recorded as the P1 generation. The same steps were followed to subculture to the 10th generation to obtain proliferating cervical squamous epithelial cells.

[0077] (3) The above-mentioned P8 proliferated cervical squamous epithelial cells were resuspended and centrifuged to obtain a cell pellet. The cells were resuspended in cell differentiation induction medium I to obtain a cell concentration of 3×10 6 The cell suspension was mixed with the differentiation matrix gel at a volume ratio of 1:2 and then plated in a well plate, and allowed to stand at 37°C for 30 minutes. After the differentiation matrix gel solidified, the cell differentiation induction culture medium I was added, and the culture was cultured at 5% CO2 and 37°C for 6 days, and the culture medium was replaced every 2 days. After that, the culture medium was replaced with the cell differentiation induction culture medium II, and the culture was cultured at 5% CO2 and 37°C for 10 days, and the culture medium was replaced every 2 days to obtain differentiated cervical squamous epithelial cells.

[0078] During the culture process, the cell growth was counted, see Figure 2 ; Cell viability was detected at P8 of proliferation culture and on day 10 of differentiation culture, see Table 1.

[0079] Embodiment 3:

[0080] Prepare digestive enzyme solution: add 10 mg collagenase type I, 20 mg trypsin and 30 mg sodium chloride per 100 mL PBS buffer to obtain digestive enzyme solution.

[0081] Preparation of primary culture medium: Add the following concentrations of each substance into DMEM culture medium: transforming growth factor-β 20 μg / mL, transferrin 50 μg / mL, epidermal growth factor 20 μg / mL, sericin phosphopeptide 40 ng / mL, laurocapram 20 ng / mL, ribose 10 ng / mL, mix well, and sterilize at 121°C for 25 min.

[0082] Preparation of cell proliferation inducing culture medium: Mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: glutamine 10 μg / mL, dexamethasone 10 M, transforming growth factor-β 10 μg / mL, recombinant human epidermal growth factor 50 μg / mL, isoproterenol 10 μg / mL, interferon-α 500 pg / mL, recombinant human interleukin 1β 30 pg / mL, bletilla striata polysaccharide 50 μg / mL, kaempferol-3-rhamnoside 10 μg / mL, paeonol 3 μg / mL and titanium oxynitride 1 μg / mL, mix well, and sterilize at 121°C for 25 min.

[0083] Preparation of cell differentiation induction medium I: Mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: epidermal growth factor 50 ng / mL, fibroblast growth factor-10 10 ng / mL, dexamethasone 5 M, insulin 10 g / mL, hepatocyte growth factor 15 ng / mL, carbachol 100 nM, isoproterenol 5 μM, matrine 1 ng / mL, inositol 3 g / mL, interleukin 3 10 pg / mL, sophora japonica glycoside 30 pg / mL and lotus leaf extract 10 g / mL, mix well, and sterilize at 121°C for 25 min.

[0084] Preparation of cell differentiation induction medium II: Mix 1 / 2 DMEM medium and 1 / 2 F12 medium, and add the following concentrations of each substance: epidermal growth factor 30 ng / mL, fibroblast growth factor-10 30 ng / mL, dexamethasone 1 M, insulin 20 g / mL, heparin sodium 100 g / mL, isoproterenol 5 μM, 100 nM carbachol, lichenic acid 30 pg / mL, palmitoyl tripeptide-15 ng / mL, cedrol 5 ng / mL, and magnolol 1 ng / mL.

[0085] Preparation of differentiation matrix gel: Dissolve hyaluronic acid in water to obtain a hyaluronic acid solution with a concentration of 5 mg / mL. Add N-isopropylacrylamide (the amount added is 1 times the mass of hyaluronic acid) and ammonium persulfate (the amount added is 0.2% of the mass of N-isopropylacrylamide) to the obtained hyaluronic acid solution, mix and react at 90°C for 16 hours to obtain a modified hyaluronic acid solution. Add allantoin and astragalus polysaccharide to the modified hyaluronic acid solution in a mass volume ratio of 1 mg: 10 mg: 300 mL and stir evenly, then add hydroquinone (the amount added is 0.1% of the mass of hyaluronic acid) and stir for 8 hours. After stirring, let it stand and filter. Wash the obtained solid product with deionized water and dry it to obtain differentiation matrix gel.

[0086] A method for promoting proliferation and differentiation of cervical squamous epithelial cells, comprising the following steps:

[0087] (1) Fresh healthy human cervical tissue was minced under sterile conditions, and then 200 μL of digestive enzyme solution was added to each mg of cervical tissue and digested at 37°C for 60 min. After digestion, the cells were centrifuged and washed twice with Hank's balanced salt solution. The digested cells were filtered through a 100 μm filter, and the cell suspension was centrifuged and the supernatant was discarded. This was repeated three times to obtain a cell pellet. The cells were resuspended in primary culture medium to a cell concentration of 3 × 10 5 / mL, and cultured at 5% CO2 and 37°C for 10 days, wherein the culture medium was changed every 2 days, to obtain primary cervical squamous epithelial cells.

[0088] (2) Add 1 mL of dispase to the obtained primary cervical squamous epithelial cells, let stand at 37°C for 30 minutes, gently blow and collect into a centrifuge tube, centrifuge and remove the supernatant, add 1 mL of 0.05% trypsin to digest for 5 minutes to digest into single cells, and terminate the digestion with 1% BSA / PBS to obtain single cells. Resuspend the cells in cell proliferation induction culture medium to obtain a cell concentration of 3×10 6 The cells were suspended in a suspension of 100 cells / mL and cultured in the conditions of 5% CO2 and 37°C. The culture medium was replaced every 2 days. The cells were cultured until the cell confluence reached 80%, which was recorded as the P1 generation. The same steps were followed to subculture to the P10 generation to obtain proliferating cervical squamous epithelial cells.

[0089] (3) The above-mentioned P8 proliferated cervical squamous epithelial cells were resuspended and centrifuged to obtain a cell pellet. The cells were resuspended in cell differentiation induction medium I to obtain a cell concentration of 3×10 6 / mL cell suspension. The cell suspension was mixed with differentiation matrix gel at a volume ratio of 1:2 and plated in a well plate, and allowed to stand at 37°C for 20 to 30 minutes. After the differentiation matrix gel solidified, cell differentiation induction culture medium I was added, and cultured at 5% CO2 and 37°C for 6 days, with the culture medium replaced every 2 days. Thereafter, the culture medium was replaced with cell differentiation induction culture medium II, and cultured at 5% CO2 and 37°C for 10 days, with the culture medium replaced every 2 days, to obtain differentiated cervical squamous epithelial cells.

[0090] During the culture process, the cell growth was counted, see Figure 3 ; Cell viability was detected at P8 of proliferation culture and on day 10 of differentiation culture, see Table 1.

[0091] Comparative Example 1: Adding fetal bovine serum to the primary culture medium

[0092] The only difference between this comparative example and Example 1 is that:

[0093] Preparation of primary culture medium: Add 2% fetal bovine serum by volume into DMEM culture medium, mix well, and sterilize at 121° C. for 25 min. Other steps are the same as those in Example 1.

[0094] During the proliferation culture, the cell growth was counted, see Figure 4 ; Cell viability was detected at P8 of proliferation culture and on day 10 of differentiation culture, see Table 1.

[0095] Comparative Example 2: Adding fetal bovine serum to the proliferation medium

[0096] The only difference between this comparative example and Example 1 is that:

[0097] Preparation of cell proliferation induction culture medium: 1 / 2 DMEM medium and 1 / 2 F12 medium were mixed, and 2% fetal bovine serum was added and mixed evenly, and then sterilized at 121° C. for 25 min. Other steps were the same as those in Example 1.

[0098] During the culture process, the cell growth was counted, see Figure 5 ; Cell viability was detected at P8 of proliferation culture and on day 10 of differentiation culture, see Table 1.

[0099] Comparative Example 3:

[0100] The only difference between this comparative example and Example 1 is that no Bletilla striata polysaccharide is added to the cell proliferation inducing culture medium, and the rest is consistent with Example 1.

[0101] During the culture process, the cell growth was counted, see Figure 6 ; Cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0102] Comparative Example 4:

[0103] The only difference between this comparative example and Example 1 is that no kaempferol-3-rhamnoside is added to the cell proliferation inducing culture medium, and the rest is consistent with Example 1.

[0104] During the culture process, the cell growth was counted, see Figure 7 ; Cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0105] Comparative Example 5: Adding fetal bovine serum to differentiation medium 1

[0106] The only difference between this comparative example and Example 1 is that:

[0107] Preparation of cell differentiation induction culture medium I: 1 / 2 DMEM culture medium and 1 / 2 F12 culture medium were mixed, and 2% fetal bovine serum was added and mixed evenly, and then sterilized at 121° C. for 25 min. Other steps were the same as those in Example 1.

[0108] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0109] Comparative Example 6:

[0110] The only difference between this comparative example and Example 1 is that no sophoroside is added to the cell differentiation induction culture medium I. The rest is the same as Example 1.

[0111] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0112] Comparative Example 7:

[0113] The only difference between this comparative example and Example 1 is that no lotus leaf extract is added to the cell differentiation induction culture medium I. The other conditions are the same as those in Example 1.

[0114] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0115] Comparative Example 8: Adding fetal bovine serum to differentiation medium 2

[0116] The only difference between this comparative example and Example 1 is that:

[0117] Preparation of cell differentiation induction culture medium II: 1 / 2 DMEM culture medium and 1 / 2 F12 culture medium were mixed, and 2% fetal bovine serum was added and mixed evenly, and then sterilized at 121° C. for 25 min. Other steps were the same as those in Example 1.

[0118] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0119] Comparative Example 9:

[0120] The only difference between this comparative example and Example 1 is that palmitoyl tripeptide-1 is not added to the cell differentiation induction culture medium II. The rest is the same as Example 1.

[0121] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0122] Comparative Example 10:

[0123] The only difference between this comparative example and Example 1 is that cedrol is not added to the cell differentiation induction culture medium II. The other contents are the same as those in Example 1.

[0124] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0125] Comparative Example 11:

[0126] The only difference between this comparative example and Example 1 is that the hyaluronic acid is not modified.

[0127] Preparation of differentiation matrix gel: Dissolve hyaluronic acid in water to obtain a hyaluronic acid solution with a concentration of 3 mg / mL. Add allantoin and astragalus polysaccharide to the hyaluronic acid solution in a mass volume ratio of 1 mg:7.5 mg:200 mL and stir evenly, then add hydroxyquinone (the amount added is 0.3% of the mass of hyaluronic acid) and stir for 6 hours. After stirring, let it stand, filter, and dry the obtained product to obtain differentiation matrix gel.

[0128] The rest is consistent with Example 1.

[0129] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0130] Comparative Example 12:

[0131] The only difference between this comparative example and Example 1 is that no allantoin or astragalus polysaccharide is added to the hyaluronic acid.

[0132] Preparation of differentiation matrix gel: Dissolve hyaluronic acid in water to obtain a hyaluronic acid solution with a concentration of 3 mg / mL. Add acrylamide (the amount added is 2 times the mass of hyaluronic acid) and ammonium persulfate (the amount added is 0.5% of the mass of acrylamide) to the obtained hyaluronic acid solution, mix and react at 90°C for 10 hours to obtain a modified hyaluronic acid solution, then add hydroxyquinone (the amount added is 0.3% of the mass of hyaluronic acid) and stir for 6 hours. After stirring, let it stand and filter, and dry the obtained product to obtain differentiation matrix gel.

[0133] The rest is consistent with Example 1.

[0134] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0135] Comparative Example 13:

[0136] The only difference between this comparative example and Example 1 is that no differentiation matrix gel is added

[0137] The rest is consistent with Example 1.

[0138] The cell viability was detected at the 8th generation of proliferation culture and the 10th day of differentiation culture, see Table 1.

[0139] Table 1 Cell activity at different stages

[0140]

[0141]

[0142] like Figure 1 As shown, compared with Comparative Examples 1 to 2, the cell proliferation and viability during the culture of cervical squamous epithelial cells in Examples 1 to 3 of the present invention are similar, which shows that the culture medium of the present invention can have an equivalent or even higher culture effect than the fetal bovine serum culture medium. Compared with Comparative Examples 2-4, the growth rate during the culture of cervical squamous epithelial cells in Examples 1 to 3 of the present invention is significantly higher, which shows that the bletilla striata polysaccharide and kaempferol-3-rhamnoside in the proliferation culture medium have the effect of promoting cell growth. It should be noted that the conditions of the proliferation process were not changed in Comparative Examples 5-13. Therefore, in theory, the cell growth in Comparative Examples 5-13 is the same as that in Example 1, and the actual statistical situation is consistent with the theory. Therefore, the growth trend is not recorded in the specification.

[0143] As shown in Table 1, compared with Comparative Examples 1-2, 5 and 8, the cell viability of Example 1-3 in both the growth process and the differentiation process is similar, and compared with Comparative Examples 3-4, 6-7 and 9-13, the cell viability of Example 1-3 is higher, which shows that the differences in proliferation medium, differentiation medium and differentiation matrix gel all significantly affect the activity of cells in the proliferation and differentiation process, and the lack of any one of them will affect cell growth and activity. The above results show that the proliferation medium, differentiation medium and differentiation matrix gel involved in the cervical squamous epithelial cell proliferation and differentiation method of the present invention are necessary conditions for promoting the proliferation and differentiation of cervical squamous epithelial cells, and have a synergistic effect.

[0144] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for promoting proliferation and differentiation of cervical squamous epithelial cells, characterized in that: The method comprises the following steps: S1: Extract and culture primary cervical squamous epithelial cells from cervical tissue to obtain primary cervical squamous epithelial cells; S2: adding primary cervical squamous epithelial cells to a cell proliferation induction culture medium to induce proliferation of the primary cervical squamous epithelial cells, thereby obtaining proliferating cervical squamous epithelial cells; S3: The proliferating cervical squamous epithelial cells are mixed with the differentiation matrix gel and plated on a well plate, and a cell differentiation induction culture medium is used to induce differentiation of the proliferating cervical squamous epithelial cells to obtain differentiated cervical squamous epithelial cells.

2. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 1, characterized in that: Step S1 includes: S11: Cervical tissue was cut into pieces under sterile conditions, and then digested in a digestive enzyme solution. After digestion, the cells were centrifuged and washed and centrifuged. This was repeated three times to obtain a cell precipitate. S12: Resuspend the cells in primary culture medium to a cell concentration of (2-4)×10 5 / mL, and cultured at 5% CO2 and 37°C for 5-10 days, wherein the culture medium was changed every 2-3 days to obtain primary cervical squamous epithelial cells.

3. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 2, characterized in that: In step S11, the digestive enzyme solution is composed of: 10-20 mg collagenase type I, 10-20 mg trypsin and 30-50 mg sodium chloride per 100 mL PBS buffer; The digestive enzyme solution is added in an amount of 100 to 200 μL per 1 mg of cervical tissue; The digestion conditions are: 37°C, 60 min.

4. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 2, characterized in that: In step S12, the primary culture medium is composed of: DMEM culture medium, to which are added: transforming growth factor-β 10-20 μg / mL, transferrin 50-100 μg / mL, epidermal growth factor 10-20 μg / mL, sericin phosphopeptide 40-80 ng / mL, laurocapram 10-20 ng / mL, and ribose 10-30 ng / mL.

5. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 1, characterized in that: Step S2 includes: S21: dispersing, centrifuging and digesting the obtained primary cervical squamous epithelial cells to obtain single cells; S22: Resuspend the cells in cell proliferation induction medium to obtain a cell concentration of 1 to 4 × 10 6 The cells were suspended in a suspension of 100 cells / mL and cultured in the conditions of 5% CO2 and 37°C. The culture medium was replaced every 2-4 days. The cells were cultured until the confluence reached 80%, which was recorded as the P1 generation. The same steps were followed to subculture to the P10 generation to obtain proliferating cervical squamous epithelial cells.

6. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 5, characterized in that: In step S22, the cell proliferation inducing culture fluid is composed of: 1 / 2 DMEM culture medium and 1 / 2 F12 culture medium, to which are added: 10-50 μg / mL glutamine, 1-10 M dexamethasone, 10-20 μg / mL transforming growth factor-β, 20-50 μg / mL recombinant human epidermal growth factor, 10-20 μg / mL isoproterenol, 300-500 pg / mL interferon-α, 30-80 pg / mL recombinant human interleukin-1β, 10-50 μg / mL bletilla striata polysaccharide, 10-30 μg / mL kaempferol-3-rhamnoside, 1-3 μg / mL paeonol and 1-3 μg / mL titanium oxynitride.

7. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 1, characterized in that: Step S3 includes: S31: resuspending and centrifuging the proliferating cervical squamous epithelial cells to obtain cell pellets; S32: Resuspend the cells in cell differentiation induction medium I to obtain a cell concentration of 1 to 4 × 10 6 Cell suspension of 100 cells / mL; S33: The cell suspension and differentiation matrix gel were mixed at a volume ratio of 1:2-3 and plated on a well plate. The mixture was allowed to stand at 37°C for 20-30 min. After the differentiation matrix gel solidified, cell differentiation induction culture medium I was added. The mixture was cultured at 5% CO2 and 37°C for 4-6 days. The culture medium was replaced every 2-3 days. Thereafter, the culture medium was replaced with cell differentiation induction culture medium II. The mixture was cultured at 5% CO2 and 37°C for 8-10 days. The culture medium was replaced every 2-3 days to obtain differentiated cervical squamous epithelial cells.

8. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 7, characterized in that: In step S32, the composition of the cell differentiation induction culture medium I is: 1 / 2 DMEM culture medium and 1 / 2 F12 culture medium, to which are added: epidermal growth factor 30-50 ng / mL, fibroblast growth factor-10 10-30 ng / mL, dexamethasone 1-5 M, insulin 10-20 g / mL, hepatocyte growth factor 5-15 ng / mL, carbachol 100-200 nM, isoproterenol 1-5 μM, matrine 1-5 ng / mL, inositol 1-3 g / mL, interleukin 3 10-30 pg / mL, sophora japonica glycoside 10-30 pg / mL and lotus leaf extract 10-30 g / mL.

9. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 7, characterized in that: In step S33, the composition of the cell differentiation induction culture medium II is: 1 / 2 DMEM culture medium and 1 / 2 F12 culture medium, to which are added: epidermal growth factor 30-50 ng / mL, fibroblast growth factor-10 10-30 ng / mL, dexamethasone 1-5 M, insulin 10-20 g / mL, heparin sodium 100-200 g / mL, isoproterenol 1-5 μM, 100-300 nM carbachol, lichenic acid 10-30 pg / mL, palmitoyl tripeptide-1 5-10 ng / mL, cedrol 1-5 ng / mL and magnolol 1-3 ng / mL.

10. The method for promoting proliferation and differentiation of cervical squamous epithelial cells according to claim 7, characterized in that: In step S33, the method for preparing the differentiation matrix gel comprises the following steps: S331: dissolving hyaluronic acid in water to obtain a hyaluronic acid solution with a concentration of 1 to 5 mg / mL; S332: adding a water-soluble monomer and an initiator to the obtained hyaluronic acid solution, mixing and reacting at 50 to 120° C. for 4 to 16 hours to obtain a modified hyaluronic acid solution; S333: adding allantoin and astragalus polysaccharide to the modified hyaluronic acid solution in a mass volume ratio of 1 mg: 5-10 mg: 100-300 mL and stirring evenly, then adding a cross-linking agent and stirring for 4-8 hours, standing and filtering after the stirring, and drying the obtained product to obtain a differentiation matrix gel; In step S332, the water-soluble monomer is selected from any one of acrylamide, methacrylamide, N-isopropylacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-hydroxymethylacrylamide, N-vinylformamide, and N-vinylacetamide; and the amount of the water-soluble monomer added is 1 to 3 times the mass of the hyaluronic acid; In step S332, the initiator is selected from any one of sodium persulfate, ammonium persulfate, and potassium persulfate; the amount of the initiator added is 0.2-0.7% of the mass of the water-soluble monomer; In step S333, the cross-linking agent is selected from any one of catechol, hydroquinone and resorcinol; the amount of the cross-linking agent added is 0.1-0.5% of the mass of the hyaluronic acid.

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