CN117327660A - Immortalized mesenchymal stem cell line for simultaneously expressing IL1-RN and COL17A protein and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to an immortalized mesenchymal stem cell line for simultaneously expressing IL1-RN and COL17A proteins, and a preparation method and application thereof. The method specifically comprises the following steps: (1) isolating and culturing mesenchymal stem cells; (2) constructing pMSCV-BMI-P2A-TERT virus; (3) Infecting the mesenchymal stem cells in the step (1) and carrying out passage to obtain immortalized mesenchymal stem cells; (4) constructing pMSCV-IL1-RN-P2A-COL17A1 virus; (5) Infecting the immortalized mesenchymal stem cells of step (3) with the pMSCV-IL1-RN-P2A-COL17A1 virus, and passaging. The cell supernatant of the cell line provided by the invention has the function of promoting the repair of skin injury, and can be applied to functional cosmetics and skin repair medicines.

Description

An immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins and Its preparation method and application

Technical field

The present invention relates to the field of genetic engineering technology, and in particular to an immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins and its preparation method and application.

Background technique

Mesenchymal stem cells are adult stem cells with multi-lineage differentiation potential and are widely distributed in many parts of the body, such as fat, bone marrow, umbilical cord, placenta and other tissues. It not only participates in the self-renewal of various tissues and organs, repair of tissue damage, etc., but also has extensive anti-inflammatory functions. In particular, umbilical cord-derived mesenchymal stem cells have been widely used in clinical treatment trials for a variety of diseases, and can be used to treat osteoarthritis, diabetes, and host rejection of organ transplants. Mesenchymal stem cells can secrete a variety of immunomodulatory and anti-inflammatory factors, such as IDO, PGE2, IL-10, IFN-, etc., which inhibit the activity of T cells, inflammatory macrophages, and NK cells.

In 2019, Professor Emi Nishimura of the University of Tokyo in Japan published blockbuster research results in the top scientific journal "Nature" and found that human skin collagen COL17A1 plays an extremely critical supporting role in skin anti-aging and anti-hair loss. The COL17A1 collagen in the basal layer of the skin decreases with age. By supplementing collagen, it can promote the self-renewal and regeneration of skin stem cells, effectively delay skin aging, and even promote the regeneration of hair follicles and hair. COL17A1 collagen is relatively large, and it is difficult for prokaryotic cell expression to reproduce its appropriate three-dimensional structure and modification, which affects its anti-aging function. Using mesenchymal stem cell lines to express this collagen through genetic engineering has become a feasible strategy.

The interleukin receptor 1 antagonist IL1-RN is a broad-spectrum anti-inflammatory factor. It can bind to interleukin receptor 1 (IL-1R) and inhibit the inflammatory response in tissues. Using its overexpression to prepare genetically engineered products can be used to treat a series of inflammatory diseases, such as osteoarthritis and rheumatoid arthritis.

Skin aging is not only related to the decline in the regeneration capacity of skin stem cells, but also to the increase in the level of local chronic inflammation in the skin stimulated by external factors. Studies have shown that exosomes from mesenchymal stem cells can mediate and secrete a large number of anti-inflammatory and regenerative factors. These exosomes can inhibit skin aging and promote skin regeneration. Therefore, genetic engineering of mesenchymal stem cells to optimize their anti-aging and anti-inflammatory factor secretion capabilities has become one of the important directions for the development of advanced skin regeneration products and advanced cosmetic raw materials.

Contents of the invention

The purpose of the present invention is to provide an immortalized genetically engineered bovine umbilical cord mesenchymal stem cell line that can continuously and simultaneously highly express COL17A1 anti-aging collagen and IL1-RN anti-inflammatory factors. Through the mouse skin damage test, the present invention proves that the cell supernatant has the function of promoting skin damage repair and can be used as a raw material for functional cosmetics and skin repair drugs.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The invention provides a method for preparing an immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins, including the following steps:

(1) Isolate and culture mesenchymal stem cells;

(2) Construct plasmid pMSCV-BMI-P2A-TERT, transfect plasmids pMSCV-BMI-P2A-TER, pMD2.G and pMDLg-pRRE into cells to obtain pMSCV-BMI-P2A-TERT virus;

(3) Use the pMSCV-BMI-P2A-TERT (as shown in SEQ NO: 10) virus to infect the mesenchymal stem cells described in step (1) and passage them to obtain immortalized mesenchymal stem cells;

(4) Construct plasmid pMSCV-IL1-RN-P2A-COL17A1 (as shown in SEQ NO: 11), transfect plasmid pMSCV-IL1-RN-P2A-COL17A1, pMD2.G and pMDLg-pRRE into cells to obtain pMSCV-IL1-RN-P2A-COL17A1 virus;

(5) Use the pMSCV-IL1-RN-P2A-COL17A1 virus to infect the immortalized mesenchymal stem cells described in step (3) and passage them to obtain an immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins.

Preferably, the mesenchymal stem cells in step (1) are derived from bovine umbilical cord.

Preferably, the mass ratio of pMSCV-BMI-P2A-TER, pMD2.G and pMDLg-pRRE plasmids in step (2) is 3-5:0.5-1.5:0.5-1.5.

Preferably, the mass ratio of pMSCV-IL1-RN-P2A-COL17A1, pMD2.G and pMDLg-pRRE plasmids in step (4) is 3-5:0.5-1.5:0.5-1.5.

Preferably, the number of passages in step (3) is ≥20 generations.

Preferably, the number of passages described in step (5) is 4 to 7 generations.

The invention also provides an immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins and is prepared by the preparation method.

The present invention also provides the application of the immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins in the preparation of cosmetics.

The present invention also provides the use of the immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins in the preparation of drugs that promote skin damage repair.

Preferably, the main component of the drug is the supernatant after culture of the immortalized mesenchymal stem cell line.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention first separates primary bovine umbilical cord mesenchymal stem cells by sterilizing and tissue lysing the newborn fetal bovine umbilical cord. Then, these two proteins were overexpressed through the retroviral vector pMSCV-BMI-TERT to induce cell immortalization. After antibiotic screening and multiple passages, immortalized bovine cell clones were isolated. On this basis, pMSCV-IL1-RN-COL17A1 retrovirus was further used to infect immortalized bovine cell clones, and through antibiotic screening, cell clones overexpressing COL17A1 and IL1-RN were obtained. Through multiple passages and Western blot identification, stable and high-yield genetically engineered stem cell lines were screened. Collect the culture supernatant of this cell line to enrich COL17A1 and IL1-RN proteins.

The immortalized bovine umbilical cord stem cell line obtained by the method of the present invention, as a genetically engineered cell line, can separate the cell culture supernatant and produce anti-aging collagen and anti-inflammatory factors. The supernatant can be widely used as a raw material for high-end cosmetics to perform anti-aging and local inflammation-inhibiting effects on skin tissue.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only These are embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on the provided drawings without exerting creative efforts.

Figure 1 is a diagram of the WB detection results of Example 1;

Legend: Marker, protein molecular weight gradient marker; 1, 10 μg/mL drug screening bovine cell sample of the 4th generation; 2, 15 μg/mL drug screening 4th generation bovine cell sample; 3, 10 μg/mL drug screening 7th generation 293T cells Samples; 4, bovine cell samples from 7 generations of drug screening at 10 μg/mL; 5, bovine cell samples from 7 generations of drug screening at 15 μg/mL;

Figure 2 is a diagram of mouse skin damage and repair test in Example 1.

Figure 3 is a structural diagram of plasmid pMSCV-BMI-P2A-TERT.

Figure 4 is a structural diagram of plasmid pMSCV-IL1-RN-P2A-COL17A1.

Detailed ways

The technical solutions provided by the present invention will be described in detail below with reference to the examples, but they should not be understood as limiting the protection scope of the present invention.

Example 1

1. Isolation of primary bovine umbilical cord mesenchymal stem cells

(1) Preparation: 75% disinfectant alcohol; umbilical cord cleaning solution: 1% four antibodies (penic/streptomycin, amphotericin B, fluconazole) added to normal saline; sterilized scissors and ophthalmic tweezers; umbilical cord preservation bottle (500ml jar, PBS buffer added with 1% tetra-antibodies (penic/streptomycin, amphotericin B, fluconazole)); product numbers are as follows: PBS (Servicebio, item number G4202); 10% fetal bovine serum FBS (BioAgrio, Cat. No. S1350-500); L-Glutamine: (Gibico, 25030-081); Penicillin/Streptomycin (Yisheng, Cat. No. 60162ES76); Amphotericin B (Yuanpei, Cat. No. S120JV); Fluconazole ( Guizhou Tiandi Pharmaceutical, 0.2g/100ml dissolved in physiological saline); complete culture medium DMEM (Hakata, product number A19008); 10cm culture dish (Nest, product number 704001); T75 culture bottle (Corning, product number 430641)

(2)Collect cow umbilical cord

After the cow gives birth naturally, use ophthalmic tweezers to clamp the umbilical cord, lift it up, and quickly cut the calf's umbilical cord (30 to 40cm in length). #Note: Do not take the umbilical cord directly with your hands, and when cutting the umbilical cord, the umbilical cord should not come into contact with the cow's external skin, the ground and other unclean environments.

The operation process is as follows:

a) Use ophthalmic tweezers to transfer the bovine umbilical cord to a 10cm sterile dish, and add 50ml of umbilical cord cleaning solution to clean the umbilical cord.

b) Repeat step a) four more times

c) Use ophthalmic tweezers to transfer the cow umbilical cord to 75% sterile alcohol and soak it for 1 minute.

d) Clamp the umbilical cord into a 10cm sterile dish and add 50ml of umbilical cord cleaning solution to clean the umbilical cord.

e) Repeat step d) four more times

f) Clamp the umbilical cord into an umbilical cord storage bottle, cover it, and pack it in 2 layers with a sterile bag. Ship back to the laboratory in a cryogenic shipping box.

(3) Umbilical cord tissue isolation, inoculation and culture

Prepare the following consumables and reagents: tissue cleaning solution (1% four-antibody saline), complete culture medium (DMEM, 10% FBS, 1% L-Glutamine, 1% four-antibody), 75% disinfecting alcohol, sterilized instruments ( Straight cutter*1, curved cutter*1, hooked tweezers*2), T75 culture bottle, centrifuge tube, pipette, 10cm petri dish, stainless steel bucket.

The operation process is as follows:

a) Add physiological saline to 5 stainless steel buckets (1L/each)

b) Use tweezers to pick up the umbilical cord and wash it step by step 5 times in 5 stainless steel buckets

c) Use tweezers to pick up the umbilical cord and put it into a petri dish, soak it in 75% disinfecting alcohol for 1 minute and disinfect it.

d) Use tissue scissors, tweezers and tissue cleaning solution to clean the blood stains inside and outside the umbilical cord 10 times

e) Cut off both ends of the umbilical cord, cut the umbilical cord into small sections of about 2cm, and use tissue scissors to cut along the blood vessels

f) Peel off Wharton's glue (peeling off blood vessels), and wash the obtained tissue 10 times with tissue cleaning solution.

Drain

g) Transfer the tissue into a centrifuge tube and cut it into 1 mm ³ -sized tissue pieces with curved scissors.

The chopped tissue pieces were inoculated into culture bottles (T75 culture bottles) according to the tissue volume of 1ml/bottle, and the complete culture medium was added to 10ml.

(4) Collection of umbilical cord mesenchymal stem cells

Prepare the following consumables and reagents: complete culture medium (DMEM, 10% FBS, 1% L-Glutamine, 1% tetra-antibody (penic/streptomycin, amphotericin B, fluconazole)), T75 culture bottle, centrifuge tube ,Pipette.

Here's how to do it:

a) For the first time, change the fluid by half the amount

b) The second medium change should be carried out according to the method of full volume change (if there are signs of bacterial contamination in the culture system, full volume change, and during the medium change process, the tissue block should be washed with physiological saline and re-inoculated)

c) Observe the cell growth every day, and perform passage/cryopreservation operation when the cell confluence reaches 50% (usually on the 11th to 12th day).

2. Construction of pMSCV-BMI-P2A-TERT plasmid

(1) Synthetic fragment SV40-P2A (Qingke Biotechnology)

SV40-P2A fragment:

CCCAAGAAGAAGCGCAAGGTGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT (as shown in SEQ NO: 1)

(2) Using the pOTB7-BMI1 (human) (Miaoling Company plasmid, P16771) plasmid as a template, use a high-fidelity PCR kit (Norwegian, Cat. No. P505-d1) to amplify the fragment, and use a kit (Norwegian, Cat. No. P505-d1) to amplify the fragment. Catalog number: DC301-01) purified PCR product.

(3) The reaction procedures and systems of high-fidelity PCR are as shown in Table 1 and Table 2

5’ primers to amplify BMI1:

TTCTCTAGGCGCCGGAATTAGATCTATGCATCGAACAACGAGAATCAAGA (as shown in SEQ NO: 2)

3’ primers to amplify BMI1:

TTCCCACCTTGCGCTTCTTCTTGGGACCAGAAGAAGTTGCTGATGACCCA (as shown in SEQ NO: 3)

(4) Use pLV-hTERT-IRES-hygro (Miaoling Company plasmid, #P18830) plasmid as template, high-fidelity PCR kit (Norwegian, Cat. No. P505-d1) to amplify the fragment, and use the kit (Norwegian, Cat. No. P505-d1) to amplify the fragment. , Catalog No.: DC301-01) purified PCR product.

(5) The reaction procedures and systems of high-fidelity PCR are as shown in Table 1 and Table 2

5’ primer to amplify TERT:

AGACGTGGAGGAGAACCCTGGACCTATGCCGCGCGCTCCCCGCTGCCGAG (as shown in SEQ NO: 4)

3’ primer to amplify TERT:

GGTAGAATTCGGATCCGTCGACTCAGTCCAGGATGGTCTTGAAGTCTGAG (as shown in SEQ NO: 5)

Table 1 High-fidelity PCR amplification reaction system

Table 2 High-fidelity PCR amplification reaction procedure

(6) Enzyme digest pMSCV-Blasticidin plasmid (Addgene plasmid #75085), use 1ug Bgl II (NEB, R0144V) and 1ug Sal I (NEB, R0144V) for enzyme digestion, react at 37°C for 15 minutes, and then use the kit (Novi Like, product number: DC301-01) purified enzyme digestion product.

(7) Use homologous recombinase (YEASEN, 10912ES10) to perform homologous recombination of the three inserted genes and the backbone (referring to SV40-P2A; pOTB7-BMI1; pLV-hTERT-IRES-hygro; pMSCV-Blasticidin) to construct pMSCV -BMI-P2A-TERT vector.

(8) After homologous recombination is completed, plasmid transformation is performed and plated. Transform the homologous recombination product into Trelief ^TM 5α E. coli competent strain (Qingke, Cat. No. TSC-C01), select clones on LB plates containing 100 μg/mL ampicillin, and use the 5' primer of TERT and the 5' primer of BMI. Primer sequencing identified the inserted clone (the P2A sequence is only 60 bp, and it can be extended and verified using BMI's primer sequencing).

2. pMSCV-BMI-P2A-TERT virus packaging and collection

(1). Culture 293T (ATCC, Cat. No. CRL-3216) cells. Before transfection, inoculate 2*10 ⁶ cells per bottle into T75 bottles for culture. To allow the cell density to reach about 70-80%, inoculate 4 bottles with 15 ml culture medium (DMEM+1% L-GLUTAMINElutamine+10% FBS) in each bottle.

(2). Before performing the transfection step, replace each bottle containing cells with fresh culture medium (complete culture medium containing serum).

(3). Refer to Table 3, take a clean sterile centrifuge tube, add 750 μl of serum-free DMEM culture medium to each bottle of cells to be transfected, and mix pMSCV-hBMI-P2A-hTERT, pMD2.G (Miao The three plasmids (Plasmid from Miaoling Company, #P0262) and pMDLg-pRRE (Plasmid from Miaoling Company, #P0685) were transfected into 293T cells at a mass ratio of 4:1:1.

Table 3: Mixing ratio of viral DNA and transfection reagent

T75 culture bottle 4 bottles Serum-free culture medium DMEM 750μl 3ml DNA 14μg+3.5μg+3.5μg 56μg+14μg+14μg Lipo8000 ^TM transfection reagent 34μl 136μl

Lipo8000 ^TM transfection reagent (Beyotime, Cat. No. C0533)

(4). Regardless of adherent cells or suspended cells, according to the dosage of 750 μl Lipo8000 ^TM transfection reagent-DNA mixture in each T75 bottle, drop it evenly into the entire well, and then mix gently. Continue to culture and change the medium once every 6 hours and 24 hours.

(5). Virus collection: 60 hours after plasmid transfection, add 1/3 volume of virus supernatant of 40% PEG8000 to each bottle, that is, 5 ml per bottle, mix well and place in a 4°C refrigerator overnight to wait for the precipitation of virus particles. After standing, the virus supernatant mixture was centrifuged to collect the settled virus particles. Centrifuge with a pre-cooled 4°C centrifuge (Thermo Scientific, ST40R), adjust the speed to 3000 rpm, and centrifuge for 30 minutes. After centrifugation, discard the supernatant and resuspend the virus in DMEM or PBS. One T75 bottle was resuspended in 0.5 ml DMEM (without serum).

Note: 40% PEG8000 (40g/100mL, Sangon, Cat. No. A100159-0500) is fully dissolved in ultrapure water, and then sterilized at high temperature.

3. Screening of immortalized bovine umbilical cord mesenchymal stem cell lines

(1) Viral infection: Culture primary bovine umbilical cord mesenchymal stem cells in a six-well cell culture plate (Nest, Cat. No. 703001). When the cell density reaches 80%, the pMSCV-BMI-P2A-TERT virus can be infected. During virus infection, you need to add polybrene with a final concentration of 8 μg/mL (Yisheng, product number 40804ES76, stock solution 10 mg/mL), 2.5 ml DMEM medium + 2 μl polybrene + 250 μl virus. During the virus infection process, no antibiotics were added to the cell culture medium. After 24 hours of infection, the culture medium was replaced with normal complete culture medium.

DMEM, culture for 3 days.

Note: The culture medium formula of bovine umbilical cord mesenchymal stem cells is DMEM+1% L-Glutamine+10% FBS.

(2) The virus-infected bovine mesenchymal stem cells were passaged in a 6-well plate every 3 days.

During the passage to the 5th to 10th passage, by observing the cell morphology, the cell clones that become smaller, rounder, and proliferate rapidly are picked out with an inoculation loop, cultured in a 6-well plate, and continued to be passaged.

(3) Based on the standard of being able to grow to nearly 80% saturation every 3 days, transfer the rapidly growing cell clones to a 6-well plate for passage and expansion. Then gradually transfer to 10cm plates and T175 culture bottles to continue passaging and expanding culture.

(4) Cell clones that have been passaged for more than 20 generations on a 10cm culture dish and can maintain rapid proliferation can be used as successfully immortalized bovine umbilical cord mesenchymal stem cell clones. The cells are frozen in liquid nitrogen using a cryopreservation solution. live. The cells can be used for further genetic engineering and collagen overexpression. Note: The cryopreservation solution is 10% DMSO (Sigma Company, Cat. No. D2650) + 90% DMEM culture medium.

4. Construction of pMSCV-IL1-RN-P2A-COL17A1 vector

(1) Use the pOTB7-IL1RN (human) plasmid (Miaoling Company plasmid, P18378) as a template, use a high-fidelity PCR kit (Norwegian, Cat. No. P505-d1) to amplify the fragment, and use a kit (Norwegian, Cat. No. P505-d1) to amplify the fragment. Catalog number: DC301-01) purified PCR product. The reaction procedures and systems of high-fidelity PCR are shown in Table 1 and Table 2.

5’ primer to amplify IL1-RN:

AGGCGCCGGAATTAGATCTCtcgagatggctttagagacgatctgccgac (such as SEQ

NO: shown in 6)

3’ primer to amplify IL1-RN:

TTCCCACCTTGCGCTTCTTCTTGGGctcgtcctcctggaagtagaatttg (as shown in SEQNO: 7)

(2) Use the pEnCMV-COL17A1 (human)-3×FLAG-SV40-Neo (Miaoling Company plasmid, P31251) plasmid as a template, high-fidelity PCR kit (Norwegian, Cat. No. P505-d1) to amplify the fragment, and use The PCR product was purified using a kit (NovoZant, Cat. No.: DC301-01). The reaction procedures and systems of high-fidelity PCR are shown in Tables 1 and 2.

5’ primer to amplify COL17A1:

AGACGTGGAGGAGAACCCTGGACCTatggatgtaaccaagaaaaacaaac (as shown in SEQ NO: 8)

3’ primers to amplify COL17A1:

GCCTCCCCTACCCGGTAGAATTcCTACTTGTCATCGTCATCCTTGTAGTC (as shown in SEQ NO: 9)

(3) Enzyme digestion of pMSCV-puro (Miaoling Company plasmid, P10650) with 1ug Bgl II (NEB, R0144V) and 1ugSal I (NEB, R0144V), react at 37°C for 15 minutes, and then use a kit (Norwegian , Catalog No.: DC301-01) purified enzyme digestion product.

(4) Use homologous recombinase (YEASEN, 10912ES10) to perform homologous recombination of the two inserted genes and the backbone to construct the pMSCV-IL1-RN-P2A-COL17A1 vector.

(5) After homologous recombination is completed, plasmid transformation is performed and plated. The homologous recombination product was transformed into Trelief ^TM 5α E. coli competent strain (Qingke, Cat. No. TSC-C01), and cloned on an LB plate containing 100 μg/mL ampicillin. The 5' primer of IL1-RN and the 5' primer of COL17A1 Primer sequencing identified the inserted clone.

5. pMSCV-IL1-RN-P2A-COL17A1 virus packaging and collection

(1) Cultivate 293T cells. Before transfection, inoculate 2*10 ⁶ cells per bottle into T75 bottles for culture so that the cell density can reach about 70-80%. Inoculate 4 bottles with 15 ml culture medium per bottle ( DMEM+1%L-Glutamine+10%FBS).

(2) Before performing the following transfection steps, replace each bottle containing cells with fresh culture medium (complete culture medium containing serum).

(3) Refer to Table 3, take a clean sterile centrifuge tube, add 750 μl of serum-free DMEM culture medium to each bottle of cells to be transfected, and mix pMSCV-IL1-RN-P2A-COL17A1, pMD2.G( The three plasmids (Miaoling Company plasmid, #P0262) and pMDLg-pRRE (Miaoling Company plasmid, #P0685) were transfected into 293T cells at a mass ratio of 4:1:1. Add the mixture evenly throughout the well and mix gently. Continue to culture and change the medium once every 6 hours and 24 hours.

(4) Virus collection: 60 hours after plasmid transfection, add 1/3 volume of virus supernatant of 40% PEG8000 to each bottle, that is, 5 ml per bottle, mix well and place in a 4°C refrigerator overnight to wait for virus particles to precipitate. After standing, the virus supernatant mixture was centrifuged to collect the settled virus particles. Centrifuge in a pre-cooled 4°C centrifuge, adjust the speed to 3000 rpm, and centrifuge for 30 minutes. After centrifugation, discard the supernatant and resuspend the virus in DMEM or PBS. One T75 bottle was resuspended in 0.5 ml DMEM (without serum).

Note: 40% PEG8000 (40g/100mL, Sangon, Cat. No. A100159-0500) is fully dissolved in ultrapure water, and then sterilized at high temperature.

6. Screening of bovine umbilical cord mesenchymal stem cell lines overexpressing IL1-RN and COL17A1 collagen

(1) Virus infection: When the density of cells to be infected (immortalized bovine umbilical cord mesenchymal stem cells) reaches 80%, pMSCV-IL1-RN-P2A-COL17A1 virus infection can be carried out (six wells plate). During virus infection, you need to add polybrene with a final concentration of 8 μg/mL (Yisheng, product number 40804ES76, stock solution 10 mg/mL), 2.5 ml culture medium + 2 μl polybrene + 250 μl virus. During the virus infection process, the best cell culture medium Do not add antibiotics. After 24 hours of infection, change the culture medium to the normal complete medium DMEM and culture for 3 days.

Note: The culture medium formula of bovine umbilical cord mesenchymal stem cells is DMEM+1% L-GLUTAMINE+10% FBS.

(2) The virus-infected bovine mesenchymal stem cells were passaged twice every 3 days. Then start adding different concentrations of Puro antibiotics (10μg/mL, 15μg/mL) and conduct screening in the complete culture medium. After the cells grow to 80% saturation, they are divided into plates and passaged at a ratio of 1:3.

(3) Cells that have been screened with antibiotics and passaged 4 to 7 times (collected at each passage), and a part of the cells are frozen. Collect cells from one well of a 6-well plate and prepare to lyse for Western Blot to determine protein overexpression efficiency.

Note: The cryopreservation solution is 10% DMSO (Sigma Company, Cat. No. D2650) + 90% DMEM culture medium.

(4) Prepare HEK293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP-502), using cells containing 10% fetal calf serum (BioAgrio, catalog number S1350-500), penicillin 100 U/ml, and streptomycin 100 μg/ml (Next St., Cat. No. 60162ES76) DMEM (Hakata, Cat. No. A19008) complete medium, cultured in 37°C, 5% _CO2 environment, in a 10 cm Petri dish (Nest, Cat. No. 704001). When the cell density reaches 80%, the pMSCV-IL1-RN-P2A-COL17A1 virus can be infected.

According to the same virus infection and drug screening processes as (1) to (3) above, 293T cells overexpressing COL17/IL1RN were screened and used for parallel experimental control.

7. Use Western Blot to detect the protein expression efficiency of bovine umbilical cord stem cell lines infected by pMSCV-IL1-RN-P2A-COL17A1 virus

(1) Extraction of cellular proteins:

The protein of pMSCV-ILRN-P2A-COL17A1 virus-infected bovine umbilical cord stem cells and 293T cells was extracted using T-PER protein extraction reagent (Thermo Scientific, Cat. No. 78510). Add 100 μL to each 24 wells and shake at 4°C for 15 minutes. , after sufficient lysis, inhale into the centrifuge tube, centrifuge at 12,000 rpm for 10 minutes at 4°C, absorb the supernatant, and measure the protein concentration using the UV method in a UV spectrophotometer. Take proteins of the same quality and adjust to the same final protein concentration, and add 6xSDS-PAGE protein loading buffer (Beyotime, Cat. No. P0015F). Boil the protein sample in boiling water for 10 minutes and freeze it at -20°C until use.

(2) Configuration of SDS-PAGE gel, electrophoresis solution, transfer solution and TBST buffer:

The formula of SDS-PAGE gel is shown in Table 4:

Table 4 SDS-PAGE gel configuration

First configure the separation gel, add the gel plate and make sure it is flush, then use ddH ₂ O liquid seal, wait for 30 minutes, wait for the separation gel to solidify, then configure the concentration gel, add the gel plate, insert the comb, and wait for 30 minutes for the concentration gel to solidify.

The formula of Western Blot electrophoresis solution is shown in Table 5:

Table 5 Electrophoresis solution formula

Element Dosage Tris(Biyuntian, item number ST760-2.5kg) 3.03g Glycine (VETEC, Cat. No. V900144-500G) 14.4g SDS (sangon, item number A600485-0500) 1g ddH ₂ O Adjust volume to 1L

The formula of Western Blot transfer solution is shown in Table 6:

Table 6 Transfer film liquid formula

Element Dosage Tris(Biyuntian, item number ST760-2.5kg) 3.03g Glycine (VETEC, Cat. No. V900144-500G) 14.4g Methanol (Sinopharm, CAS No. 67-56-1) 200mL ddH ₂ O Adjust volume to 1L

The 1x TBST buffer recipe is shown in Table 7:

Table 7 1x TBST Buffer Recipe

Element Dosage 1MTris-Hcl (pH=7.5) 50mL Nacl (GeneralReagent, CAS number 7647-14-5) 8.8g Tween-20 (Biyuntian, item number ST825) 400μL ddH ₂ O Adjust volume to 1L

(3) Electrophoresis:

Pour the electrophoresis solution into the electrophoresis tank. Boil the protein for another 5 minutes before loading, and add the same amount to the lane. Electrophoresis is divided into two stages: the first stage uses 80V constant voltage electrophoresis for 35 minutes to concentrate the protein in the stacking gel; the second stage uses 120V constant voltage electrophoresis with an electrophoresis time of 120 minutes.

(4)Transfer film:

The wet transfer method was used, and the membrane was a PVDF membrane (Millipore, Cat. No. IPVH00010), which was activated in methanol (Sinopharm Group, CAS No. 67-56-1) before transfer. Pour the transfer solution into the electrophoresis tank and make a transfer "sandwich" in sequence. The transfer conditions were 300mA and constant current transfer for 100 minutes. Since a large amount of heat will be generated during film transfer, it is necessary to build an ice pack into the film transfer tank and place the film transfer tank in an ice water bath.

(5) Closed:

After the transfer, the PDVF membrane was washed three times with 1x TBST buffer for 10 minutes each time. The membrane was transferred to 5% skimmed milk powder (Sangon, Cat. No. A600669-0250) and shaken at room temperature for 1 hour.

(6) Primary and secondary antibody incubation:

After blocking, the membrane was washed three times with 1x TBST, 10 min each time. Configure rabbit-derived IL1-RN primary antibody (Proteintech, Cat. No. 10844-1-AP), mouse-derived β-actin primary antibody (Trans, Cat. No. HC201-02), and mouse-derived Flag antibody primary antibody (ABclonal, All antibodies (Cat. No. AE005) were diluted with antibody diluent (Sangon, Cat. No. C520011-0250). After adding the antibody, shake at 4°C overnight.

After primary antibody incubation, the membrane was washed three times with 1x TBST buffer for 10 min each time. Anti-mouse secondary antibody (Proteintech, Cat. No. SA00001-1) was prepared at a ratio of 1:10000, and anti-rabbit secondary antibody (Proteintech, Cat. No. SA00001-1-2) was prepared at a ratio of 1:10000. The antibodies were all prepared using antibody diluent (Biotech, No. SA00001-1). (Product No. C520011-0250) diluted. After adding the antibody, incubate on a shaker at room temperature for 1 hour.

(7) ECL luminescent substrate development:

After incubation with the secondary antibody, wash the membrane three times with 1x TBST for 10 minutes each time. Using the ECL luminescence kit (Shenger Biotech, Cat. No. SB-WB012), place the membrane in the prepared developer for 1 min, and then image using a Tanon 5200 chemiluminescence imager. The IL1-RN band is predicted to be about 20KD, the COL17A1 band is about 150KD, and the β-actin band is about 42KD.

Results: Bovine umbilical cord stem cells and 293T cells infected with pMSCV-IL1-RN-P2A-COL17A1 virus were able to efficiently express IL1- RN anti-inflammatory protein and COL17A collagen.

The WB test results are shown in Figure 1 (From the WB results, the expression effect of 293T cells is indeed better than that of bovine umbilical cord stem cells. However, bovine umbilical cord stem cells themselves secrete other anti-inflammatory factors. Although the concentrations of each component are not high, they can comprehensively repair The anti-inflammatory effect is better than that of 293T cells. This is the reason for preparing this cell line).

8. Experiment on using bovine umbilical cord stem cell culture supernatant to promote skin damage repair

(1) Take 6 black-haired mice 42 to 60 days after birth (purchased from Zhejiang Branch of Vitong Lihua Experimental Animal Technology Co., Ltd.). This stage is in the resting period of hair growth. At this stage, the hair stops growing. For skin damage experiments, both male and female rats can be used as experimental materials.

Day 0

1) Prepare avertin anesthetic (Beijing Yoshida, product number JT0781, concentration 10 μl/mg). Weigh the body weight of the experimental mice, and calculate the injection volume according to the anesthesia dose of 30 μl/g. Use a 1ml syringe to absorb the anesthetic, and inject the mouse intraperitoneally to anesthetize it. The mouse will enter anesthesia in about 5 minutes. The back of the mouse was shaved and disinfected with alcohol cotton.

Use a skin punch to punch the mouse's back skin in the middle of the line connecting the two forelimbs (punch size 4 mm). Correct operation should avoid bleeding and scabbing that may affect observation. The expected wound healing time is 7 days. Take photos and record (date, number, aperture, ruler reference)

2) Apply medicine: Dip a cotton swab into the reagent (about 250-300 μl), carefully apply it to the wound, and try to complete it while the mouse is anesthetized.

Control group (3 mice): The reagent is physiological saline (Yaojiujiu, product number AYR004854CCQ1);

Test group (3 mice): The reagent is the cultured supernatant (bovine cell supernatant) of an immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins. The culture method is: the cells grow to more than 80% saturation. degree, when passage, directly suck it out with a pipette. Collect the supernatant and centrifuge it at 5000 rpm to remove the cell residue at the bottom. The remaining supernatant can be frozen directly at -20°C. The target proteins IL1-RN and COL17A in the supernatant , measured by ELISA).

3) Resuscitate and wait for the anesthetized mice to wake up. Keep them in separate cages as much as possible.

From now on, the mice will be anesthetized, photographed, and medicated according to the method on day 0. Apply medicine to the wound once a day.

Figure 2 shows the results of the mouse skin damage and repair test.

Among them, the wound diameter of the mice treated with physiological saline on the second day was about 6 mm, and the wound diameter of the mice in the test group was about 3 mm.

On the third day, the wound diameter of the mice treated with physiological saline was approximately 5 mm, while the wound diameter of the mice in the experimental group was approximately 2 to 3 mm.

As shown in Figure 2, starting from the 2nd and 3rd days, it can be clearly seen that the cell supernatant of the cell line prepared by the present invention has a promoting effect on wound healing.

The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (10)

1. A method for preparing an immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins, which is characterized by comprising the following steps: (1) Isolate and culture mesenchymal stem cells; (2) Construct plasmid pMSCV-BMI-P2A-TERT, transfect plasmids pMSCV-BMI-P2A-TER, pMD2.G and pMDLg-pRRE into cells to obtain pMSCV-BMI-P2A-TERT virus; (3) Use the pMSCV-BMI-P2A-TERT virus to infect the mesenchymal stem cells described in step (1) and passage them to obtain immortalized mesenchymal stem cells; (4) Construct plasmid pMSCV-IL1-RN-P2A-COL17A1, transfect plasmid pMSCV-IL1-RN-P2A-COL17A1, pMD2.G and pMDLg-pRRE into cells to obtain pMSCV-IL1-RN-P2A-COL17A1 Virus; (5) Use the pMSCV-IL1-RN-P2A-COL17A1 virus to infect the immortalized mesenchymal stem cells described in step (3) and passage them to obtain an immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins. 2. The method according to claim 1, characterized in that the mesenchymal stem cells in step (1) are derived from bovine umbilical cord. 3. The method according to claim 1, characterized in that the mass ratio of pMSCV-BMI-P2A-TER, pMD2.G and pMDLg-pRRE plasmids in step (2) is 3~5:0.5~1.5:0.5 ~1.5. 4. The method according to claim 1, characterized in that the mass ratio of pMSCV-IL1-RN-P2A-COL17A1, pMD2.G and pMDLg-pRRE plasmids in step (4) is 3~5:0.5~1.5 :0.5～1.5. 5. The method according to claim 1, characterized in that the number of passages in step (3) is ≥ 20 generations. 6. The method according to claim 1, characterized in that the number of passages in step (5) is 4 to 7 generations. 7. An immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A proteins and is prepared by the preparation method according to any one of claims 1 to 6. 8. Application of the immortalized mesenchymal stem cell line expressing IL1-RN and COL17A proteins simultaneously in the preparation of cosmetics according to claim 7. 9. Application of the immortalized mesenchymal stem cell line expressing IL1-RN and COL17A proteins simultaneously as claimed in claim 7 in the preparation of drugs for promoting skin damage repair. 10. Application of the immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A protein according to claim 8 in the preparation of cosmetics or the immortalized mesenchymal stem cell line that simultaneously expresses IL1-RN and COL17A protein according to claim 9. The application of mesenchymal stem cell lines in the preparation of medicines that promote skin damage repair is characterized in that the main component of the medicine is the supernatant after culture of the immortalized mesenchymal stem cell lines.