CN111549000B - Recombinant adipose-derived stem cell for over-expression of Hpgds, preparation method and application thereof - Google Patents

Abstract

The invention provides a recombinant adipose stem cell overexpressing Hpgds gene, a preparation method and application thereof, and belongs to the technical field of genetic engineering medicine. The preparation method includes the following steps: constructing a lentiviral vector expressing the Hpgds gene; extracting adipose stem cells; infecting the adipose stem cell with the lentiviral vector expressing the Hpgds gene to obtain recombinant adipose stem cells; Proliferation on the immune regulation gene Hpgds can be safely and persistently expressed to accelerate the healing of diabetic-damaged skin and shorten the healing time.

Description

A kind of recombinant adipose stem cell overexpressing Hpgds, preparation method and application thereof

technical field

The invention relates to the technical field of genetic engineering drugs, in particular to a recombinant adipose stem cell expressing Hpgds, a preparation method and its application in the repair of diabetic wounds.

Background technique

In the course of the onset of diabetes, 30% of patients will have skin infections, which often recur and persist for a long time. For people with diabetes, once a skin infection occurs, the symptoms of skin ulceration will be aggravated, and it is not easy to control. If the treatment is not timely, it will develop in depth, and even lead to amputation, which will seriously affect the patient's quality of life. According to statistics, amputations caused by diabetic ulcers account for more than 50% of non-traumatic amputation patients. Therefore, the occurrence of skin ulcers in diabetic patients has attracted more and more attention from medical workers and researchers.

In the state of diabetes, the number and function of neutrophils are abnormal, the function and phenotypic switch of macrophages are disordered, and the inflammatory complex is activated to form a continuously expanding inflammatory response, thus resulting in obstacles to wound healing.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a recombinant adipose stem cell overexpressing Hpgds, a preparation method and application thereof. Play a role.

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

A recombinant adipose stem cell overexpressing Hpgds, the recombinant adipose stem cell is obtained by transfecting the gene expressing Hpgds into the adipose stem cell.

In a preferred embodiment, the adipose stem cells are derived from human adipose tissue.

In one embodiment, the vector expressing the Hpgds gene is an adenovirus vector, preferably, the adenovirus vector is pCDH-CMV-MCS-EF1-copGFP.

In one embodiment, a method for preparing recombinant adipose stem cells is provided, comprising the following steps:

Construct a lentiviral vector expressing the Hpgds gene;

Infecting adipose stem cells with the lentiviral vector expressing the Hpgds gene;

Recombinant adipose stem cells expressing Hpgds gene were obtained.

In one embodiment, the method further includes the step of extracting adipose stem cells, and the adipose stem cells are extracted by collagenase digestion; preferably, the temperature of collagenase digestion is 36-33° C.; and the digestion time is 1-1.5 hours.

In one embodiment, the step of extracting adipose stem cells further includes identifying the adipose stem cells obtained by the extraction, preferably including identification of osteogenesis and adipogenicity.

In one embodiment, the multiplicity of infection for infection by the lentiviral vector expressing the Hpgds gene is 300-400.

The invention also provides the application of the recombinant adipose stem cells in the preparation of medicines for repairing biological tissues.

In one embodiment, the biological tissue is diabetic wound tissue.

The present invention also provides a preparation method of a medicine for repairing diabetic wound tissue, comprising the following steps:

a. Construction of recombinant adipose stem cells overexpressing Hpgds gene;

b. Mixing the recombinant adipose stem cells with a hydrogel scaffold, preferably an animal-derived RGD peptide-modified polysaccharide-free three-dimensional scaffold, to obtain a drug for repairing diabetic wound tissue.

Beneficial effects of the present invention:

The present invention constructs a recombinant lentiviral vector containing Hpgds gene, and after transfecting human adipose stem cells, the immunoregulatory protein Hpgds is highly expressed, and works together with the stem cells to create a microenvironment conducive to skin regeneration. The recombinant adipose stem cells can safely, lastingly and effectively express the immune regulation gene Hpgds, and have a good application prospect for promoting the healing of diabetic wounds. The adipose stem cells constructed by expressing Hpgds in the present invention have good healing effect on back wounds of mice, significantly reduce the number of white blood cells and CD8 T cells in the new tissue, and significantly improve the M2 repair macrophage in the new tissue. At the same time, the content of new blood vessels and collagen tissue in the new tissue is also increased, which helps to promote the transition of the wound from the inflammatory phase to the proliferative phase. The wound heals faster and the healing effect is better. The recombinant adipose stem cells expressing Hpgds and the preparation method of the invention provide reference for basic research and clinical application of wound repair.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the description of the specific embodiments or the prior art will be briefly introduced below. The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Figure 1 shows the morphology of ADSCs under an inverted microscope, where A: primary culture for 48h; B: 3rd generation ADSCs for 48h;

Figure 2 shows the induction identification of ADSCs under an inverted microscope, where A: Alizarin red staining after 14 days of osteogenic induction; B: Oil red O staining after 7 days of adipogenic induction;

Figure 3 shows the results of flow cytometry detection of cell surface markers in the third passage;

Figure 4 shows the morphology of ADSCs cells after infection by inverted fluorescence microscope, where A: normal ADSCs for 48h; B/C: pCDH-Hpgds-GFP infection for 48h; D: pCDH-Hpgds-GFP infection for 96h;

Figure 5 is the effect of lentivirus infection on the proliferation of ADSCs in Example 1;

Figure 6 is the expression level of Hpgds gene and the content of PGD2 in the culture supernatant after 48 hours of recombinant adipose stem cells in Example 1;

Fig. 7 is animal model among the embodiment 1;

Figure 8 is the immunohistochemical staining of neutrophils and CD8 T cells in the wound on the seventh day after the wound healing of the diabetic mouse in Example 1;

Figure 9 is the immunofluorescence staining of M2 macrophages in the wound on the 7th day of wound healing in Example 1;

Figure 10 is the immunofluorescence staining of proliferating cell nuclear antigen (PCNA) in the wound on the 10th day of wound healing in Example 1;

Figure 11 is the immunohistochemical staining of collagen type I (Collagen I) and vascular endothelial cell marker (CD31) in the wound on the 10th day of wound healing in Example 1;

Figure 12 is the healing process of the diabetic wound in the mouse in Example 1;

13 is the histological observation and thickness statistics of the wound on the 17th day of the healing of the diabetic wound of the mouse in Example 1. FIG.

Detailed ways

The present invention provides a recombinant adipose stem cell overexpressing Hpgds, the recombinant adipose stem cell is obtained by infecting the adipose stem cell with a lentiviral vector expressing the Hpgds gene.

In the present invention, the adipose stem cells are preferably derived from adipose tissue in clinical liposuction. In the present invention, the gene vector is pCDH-CMV-MCS-EF1-CoGFP, which is then transferred together with psPAX2 and pMD2G into 293T cells to package lentivirus; the pCDH vector preferably expresses green fluorescent protein. The source of the lentiviral vector is not particularly limited in the present invention, and the conventional method in the art can be used to prepare it or a commercially available conventional lentiviral vector can be used.

The present invention provides a method for preparing recombinant adipose stem cells, comprising the following steps: constructing a lentiviral vector expressing Hpgds gene; extracting adipose stem cells; infecting the adipose stem cell with the lentiviral vector expressing Hpgds gene to obtain recombinant adipose stem cells.

In the present invention, for the construction of a lentiviral vector expressing Hpgds gene, it is preferable to package the Hpgds gene with a lentiviral vector to obtain a lentiviral vector expressing Hpgds gene. In the present invention, for the construction method of the lentiviral vector expressing the Hpgds gene, refer to the French Polyplus transfection kit.

In the present invention, the extraction of adipose stem cells preferably adopts collagenase digestion method. In the specific implementation process of the present invention, the following steps are included: S1) collecting adipose tissue in liposuction; S2) cleaning and shredding the adipose tissue, and then digesting the adipose tissue with collagenase to obtain digested cells; S3) removing the The digested cells are filtered through a mesh screen and centrifuged to collect solid-phase components, which are adipose stem cells.

In the present invention, after separating and obtaining the adipose tissue, the adipose tissue is washed and cut into pieces, and then digested with collagenase to obtain the digested cells. In the present invention, the washing solution for washing is preferably a PBS solution containing 1% double antibody (double antibody is penicillin and streptomycin) in volume fraction, and the washing times are preferably 3 to 4 times. Preferably, it is carried out in a centrifuge tube; in the specific implementation process of the present invention, the washing should try to wash away the blood vessels and residual substances in the adipose tissue. In the present invention, after the cleaning, the cleaned adipose tissue is cut into pieces. In the present invention, the cleaned adipose tissue is transferred to a new container, and the adipose tissue is cut into pieces; the present invention has no special requirements for the cutting method, and the conventional tissue cutting method in the field is adopted That’s it; in the method in the specific implementation process of the present invention, the shredding time is preferably 10-20 min; the adipose tissue is preferably shredded to a paste state in the present invention. In the present invention, collagenase digestion is performed after the shredding; the collagenase solution is mixed with the shredded adipose tissue for collagenase digestion; the volume ratio of the collagenase solution to the shredded adipose tissue is preferably (1.5-2):1, more preferably 2:1; the concentration of the collagenase solution is preferably 0.5%-1.5% (v/v), more preferably 1% (v/v). In the present invention, the collagenase is preferably type I collagenase. In the present invention, the temperature of the collagenase digestion is preferably 36-38°C, more preferably 37°C; the collagenase digestion time is preferably 1-1.5 hours. In the present invention, during the digestion process, it is preferably shaken in a shaker, and the rotational speed is preferably 80-100 rpm. In the present invention, after the collagenase digestion is completed, the digestion is preferably terminated and fully pipetted. In the present invention, the digestion is preferably terminated by adding an equal volume of DMEM complete medium with 10% FBS to the digested tissue.

In the present invention, the digested cells are filtered through a mesh screen and centrifuged to collect solid phase components, which are adipose stem cells. In the present invention, the pore size of the sieve is preferably 200 meshes; the rotation speed of the centrifugation is preferably 800-1200 rpm, more preferably 1000 rpm; the centrifugation time is preferably 8-12 min, more preferably 10 min. In the present invention, after the centrifugation, the solid phase components are collected to obtain adipose stem cells; the obtained adipose stem cells are preferably resuspended in a complete medium for culture; the culture is preferably inoculated into a culture flask and placed at 37°C, Culture in a 5% CO ₂ incubator. The present invention further includes identifying the fatty liver stem cells obtained by the extraction after the adipose stem cells are extracted. The purpose of the identification described in the present invention is to determine whether the adipose stem cells obtained by extraction have the characteristics of stem cells; the identification preferably includes the identification of osteogenesis and adipogenesis; the present invention adopts the conventional method in the field for the identification method That's it.

In the present invention, after the adipose stem cells are obtained, the adipose stem cells are infected with the lentiviral vector expressing the Hpgds gene to obtain recombinant adipose stem cells. In the present invention, the adipose stem cells are preferably cultured and passaged to the third generation of adipose stem cells; the concentration of the third generation of adipose stem cells is preferably (0.1～5)×10 ⁵ cells/ml, more preferably 0.5～ 2×10 ⁵ pieces/ml, most preferably 1×10 ⁵ pieces/ml. In the specific implementation process of the present invention, it is preferable to inoculate the adipose stem cells of the concentration on a cell culture plate for culture, and perform virus infection when the cells grow to 70-80%. In the present invention, the multiplicity of infection (MOI) value of the virus infection is preferably 300 to 400, more preferably 350. In the present invention, the added amount of the lentiviral vector expressing the Hpgds gene is preferably (1-5)×10 ¹⁰ pfu/mL, more preferably 1.26×10 ¹⁰ pfu/mL; the calculation method of the added virus volume in the present invention is: MOI=virus amount (ml)×virus titer (pfu/mL)÷cell number (piece). In the present invention, after the infected cells, the cell culture plate is preferably shaken, so that the lentiviral vector expressing the Hpgds gene is fully contacted with the adipose stem cells, and the infected cells are replaced with complete medium 4 hours to continue culturing. In the present invention, the fluorescent expression of the cells is preferably observed under a fluorescence microscope 24, 48, and 72 hours after the infected cells, and it is determined that the Hpgds carried by the lentivirus can successfully infect the adipose stem cells.

In the present invention, after the adipose stem cells are obtained, they are resuspended in a serum-free stem cell medium to a density of 5×10 ⁵ cells/mL, and uniformly mixed with the hydrogel scaffold at 1:4 (v/v). The recombinant adipose stem cells proliferate on the RGD peptide-modified polysaccharide three-dimensional biomaterial scaffold, can safely and persistently express the immune regulation gene Hpgds, and cooperate with the adipose stem cells to work together.

The present invention also provides the application of the recombinant adipose stem cells in the preparation of a medicine for repairing biological tissue, preferably, the biological tissue is diabetic wound tissue. In the present invention, the recombinant adipose stem cells can express the gene of human Hpgds.

The present invention also provides a preparation method of a medicine for repairing diabetic wound tissue, comprising the following steps:

a. Construction of recombinant adipose stem cells overexpressing Hpgds gene;

b. Mixing the recombinant adipose stem cells with a hydrogel scaffold, preferably an animal-derived RGD peptide-modified polysaccharide-free three-dimensional scaffold, to obtain a drug for repairing diabetic wound tissue.

Example

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

1. Extraction of adipose-derived stem cells (ADSCs)

Take the adipose tissue after liposuction (both male and female), put it in a petri dish containing 1% double antibody in PBS and wash it 3-4 times. In the beaker, cut it into pieces (until it becomes a paste) for about 15 minutes. Transfer the chopped adipose tissue into a 50ml centrifuge tube, add 2 times the volume of 1% (v/v) type I collagenase to it, seal the tube with parafilm and place it in a 37°C water bath to digest for 1 hour. Shake in a shaker, and the rotation speed is preferably in the range of 80-100 rpm. After the adipose tissue was completely digested (no lumpy tissue was observed), an equal amount of DMEM complete medium with 10% FBS was added to terminate the digestion. Filter through a 200-mesh sieve, centrifuge at 1000 rpm for 10 min, add 5 ml of complete medium to resuspend, inoculate in a 25 cm ² culture flask, and culture in a 37°C 5% CO ₂ incubator. Figure 1 shows the induction of differentiation (osteogenesis, adipogenicity) of ADSCs under an inverted microscope.

The extracted primary adipose stem cells were identified as adipose stem cells by adipogenic and osteogenic differentiation experiments, immunohistochemical identification and flow cytometry phenotype. The third-generation adipose-derived stem cells with strong growth and differentiation potential were used. Figure 2 shows the induction identification of ADSCs under an inverted microscope, where A: Alizarin red staining after 14 days of osteogenic induction; B: Oil red O staining after 7 days of adipogenic induction; Figure 3 is the result of flow cytometry detection of cell surface markers at the third passage.

2. Recombinant Lentiviral Vectors

The Hpgds gene was constructed by Qingke Biotechnology (Shanghai) Co., Ltd. and inserted into the pCDH-CMV-MCS-EF1-CoGFP vector, and then transferred into 293T cells together with psPAX2 and pMD2G to package lentivirus.

3. Infection of adipose tissue stem cells with recombinant adenovirus vector

The third-generation cells of adipose tissue stem cells were taken to prepare a single cell suspension, and the density was adjusted to 1×10 ⁵ /ml and seeded on a 6-well plate. When the cells grow to 70-80%, take the multiplicity of infection (MOI) value of 350. The recombinant lentiviral vector (1.26 x ¹⁰¹⁰ pfu/mL) was added to the cells. The calculation method of the added virus volume is: MOI=virus amount (ml)×virus titer (pfu/mL)÷cell number (unit). The 6-well plate was gently shaken to make the virus liquid fully contact with the cells, and the complete medium was replaced for 24 hours to continue the culture. 24h, 48h and 72h after virus infection of cells, the fluorescence expression of cells was observed under a fluorescence microscope (ADSCs capable of expressing fluorescence are ADSCs successfully transfected by adenovirus). Figure 4 shows the morphology of ADSCs cells after infection by inverted fluorescence microscope, where A: normal ADSCs for 48h; B/C: pCDH-Hpgds-GFP infection for 48h; D: pCDH-Hpgds-GFP infection for 96h.

4. Detection of cell proliferation after transfection by CCK-8 method

For the ADSC-Hpgds group (ADSCs transfected with recombinant lentiviral vector of Hpgds gene) and the ADSC group (normally cultured ADSCs without any treatment), the cell proliferation after transfection was detected by CCK-8 method, as shown in Figure 5 shown, the result shows:

The cells in both groups entered the logarithmic growth phase after 3 days of culture, and entered the plateau phase after 5 days. The cells in both groups showed an inverted "S" curve growth; there was no statistical difference in the OD value between the two groups within 6 days. Significance (P>0.05).

5. RT-PCR detection of Hpgds gene expression in ADSC-Hpgds group cells after infection.

RT-PCR step

Total RNA was extracted from two groups of cells by TRIZOL method

The specific operation of the RNA extraction process is as follows

①Rinse the culture flask 2-3 times with PBS solution, suck up the liquid inside, add 1 mL of TRIZOL to the flask, put it on ice for 20 minutes, shake the culture flask at the same time to make the liquid fully contact with the cells, pipette the cells, and make the cells fall off with TRIZOL The liquid was mixed evenly and transferred to a 1.5ml enzyme-free EP tube.

② Add 200 μL of chloroform to the EP tube, invert it up and down manually for 15 s to mix it, and let it stand for 15 min at room temperature.

③ Centrifuge at 12000g at 4°C for 15min.

④After centrifugation, the liquid in the EP tube is divided into 3 layers. The colorless water sample in the uppermost layer is RNA. Take 400-500 μL of it and transfer it to a new non-enzyme EP tube (be careful not to inhale the middle layer to avoid RNA degradation. or pollution).

⑤ Add an equal volume of pre-cooled isopropanol (the amount should be greater than or equal to the inhaled amount), invert it up and down for 3 times, and let it stand for 10 minutes at room temperature.

⑥ Centrifuge at 12000g at 4°C for 15min, and prepare 1mL of 75% ice ethanol and place it in a 4°C refrigerator to pre-cool (750μl of anhydrous ethanol + 250μl of DEPC).

⑦ After centrifugation, place at room temperature for 5 min to completely precipitate the RNA, discard the supernatant, add 1 mL of 75% ice ethanol and invert several times to wash the RNA precipitate.

⑧ Discard the supernatant, dry at room temperature for about 10 minutes (to ensure that there is no residual ethanol in the tube, and avoid excessive drying of the RNA), and dissolve the RNA in 10 μL of enzyme-free DEPC-treated water.

⑨ Pipet 2 μL of all RNA samples with a micropipette, and measure the RNA concentration and OD value at 260/280nm with a NanoDrop micro UV spectrophotometer. A260/280 between 1.8 and 2.0 is regarded as reaching the standard, and the total cell RNA is detected on the machine. concentration. RNA was reverse transcribed into cDNA after determination of RNA concentration. Using GAPDH as an internal reference, a pair of specific primers (Table 1) were designed, and the primer synthesis was completed by Shanghai Qingke Bioengineering Company, and the specific operations for PCR amplification of specific sequences were as follows.

Table 1 Primer sequences

①Primer dilution: Centrifuge the EP tube containing the upstream primer at 4000rpm for 4-5min, slowly open the tube cap, add DEPC water to dilute to 10μM, cover the cap, shake well and mix well for later use.

② PCR was performed with cDNA as the template, 3 replicate wells in each group, and a total reaction system of 20 μL was constructed.

③20μL reaction system:

cDNA 1 μg,

Forward primer 0.6 μL,

Reverse primer 0.6 μL,

2 x SYBER Green Master Mix 10 μL,

Make up to 20 μL of ddH ₂ O.

⑤ Reaction conditions: 95°C for 5 min; (94°C for 15s, 60°C for 30s, 72°C for 45s) (40 cycles).

The RT-PCR results are shown in Figure 6 (left).

6. ELISA method to detect the expression level of PGD2 antigen in cell supernatant

The ELISA kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. The specific method is shown in the kit instructions. The test results are shown in Figure 7. The results of the PGD2 level in the supernatant of infected cells in the ADSC group and the ADSC-Hpgds group showed that: 72h after infection with ADSCs in the two groups The expression of PGD2, the catalytic product of Hpgds, could be detected in the inner cell supernatant, and there was a statistically significant difference in the expression of PGD2 between the _two groups (P<0.05). The results are shown in Figure 6 (right).

Animal experiment

1. All laboratory animal handling follows the American guidelines for the management and use of laboratory animals. All efforts are aimed at minimizing pain and use of mice. 10 mice were normally raised for a period of time. After anesthesia, after shaving the mice, a 1cm*1cm full-thickness skin wound was incised on the back of the mice (Fig. 7 shows the mouse animal model). Mice were randomly divided into (1) control group (PBS injection group, Control); (2) adipose stem cell group transfected with empty virus without target gene; (3) recombination infected with lentiviral vector carrying Hpgds gene Adipose stem cell group, 5 mice in each group.

2. Recombinant adipose stem cell wound transplantation: The target cells containing the target gene vector (ie, the recombinant adipose stem cells infected with the lentiviral vector carrying the Hpgds gene, ADSC-Hpgds), the control group (PBS injection group, Control) and those without the target Gene empty virus-transfected adipose stem cell group (ADSC-PCDH)) (at a density of 5×10 ⁵ cells/ML) and hydrogel scaffolds (at 1:4 (v/v)) (purchased from TheWell bioscience in the United States) After compounding, the transplant is covered on the wound surface of the mouse;

All wounds were covered with clear material and secured with bandages. Animals were reared under the same conditions, and the wound healing and animal status were observed every day, including changes in wound area, local inflammation, granulation tissue hyperplasia, epithelial healing, and post-healing skin conditions. On days 0, 3, 7, 10, 14, and 17, the mice were sacrificed by CO ₂ inhalation, and the specimens were taken for histological and real-time quantitative PCR analysis.

3. HE staining

①Baking slices: bake the paraffin slices in a 65 degree oven for 180 minutes;

②Dewaxing: successively put into xylene, xylene, xylene: absolute ethanol (1:1), each step 5-10 minutes;

③Immersion in water: put in 100% alcohol, 95% alcohol, 85% alcohol, 70% alcohol in sequence, each step for 2-5 minutes; finally put it in distilled water and transfer it into dye solution for dyeing;

④ Transfer to hematoxylin for staining for 5-15 minutes;

⑤ Wash the excess dye solution on the slide with water and separate the color with 0.5-1% hydrochloric acid alcohol for a while. Observed under the microscope, the nucleus and the chromatin in the nucleus are clear, and it is terminated, about 10 seconds;

⑥ Rinse with running water for 15 to 30 minutes, or alkalize or blue in lithium carbonate saturated solution for a short time, that is, the nucleus is blue;

⑦Distilled water cleaning;

8. Dye with 0.1-0.5% eosin dye solution for 1-5 minutes. If it is difficult to dye, add 1-2 drops of glacial acetic acid to every 100 ml of dye solution to make it easy to color and not easy to decolorize;

⑨ Go through 70% alcohol, 85% alcohol, 95% alcohol, 100% alcohol in sequence, 2-3 minutes per step;

⑩Xylene is transparent twice for about 10 minutes in total;

Mounting: Wipe off the excess xylene around the specimen, quickly add an appropriate amount of neutral gum, and seal it with a cover glass to avoid air bubbles;

Microscopy: Observe the staining results under a microscope.

3. Immunofluorescence detection

The tissue specimens were fixed with 4% paraformaldehyde for 48h, and washed three times with PBS solution. Permeabilized with 0.1% Triton-X100 for 2 min, blocked with 1% BSA for 30 min, incubated with primary antibody at 4°C overnight, rinsed 3 times with PBS containing 1% serum protein serum, and then took the labeled goat anti-mouse IgG secondary antibody. Anti-(1:10001%BSA:PBS) was incubated at room temperature for 45min. After washing with PBS three times, DAPI (1:50001%BSA:PBS) was added, and nuclei were stained for 10 min at room temperature, washed with PBS, and photographed with a fluorescence microscope.

4. Immunohistochemical Staining

Full-thickness skin tissue with a range of 1 cm on both sides at the dorsal incision was taken, fixed with 4% paraformic acid and embedded in paraffin to make paraffin tissue sections for immunohistochemical staining. Specific steps are as follows:

①Put the paraffin sections in the oven for 180 minutes before dewaxing.

② Routinely dehydrated, rinsed with PBS for 5 min.

③ Blocking of endogenous enzymes: add 3% hydrogen peroxide ethanol solution and place at room temperature for 10 minutes to reduce non-specific staining.

④Antigen retrieval after rinsing with PBS for 3 minutes: put the slices into a container containing antigen retrieval solution, add water in a pressure cooker and heat to boiling. The container was then placed in a pressure cooker, heated until a jet of air was generated, continued for 2 minutes, and then cooled naturally.

⑤ Rinse with PBS for 3 minutes and then block: drop goat serum on the tissue piece and incubate at room temperature for 30 minutes. The serum was discarded, the diluted primary antibody was added dropwise, and the cells were incubated overnight at 4°C in a humidified chamber.

⑥ Rinse with PBS for 15 minutes, add HRP-labeled goat anti-rabbit polyclonal antibody dropwise, and incubate at 37°C for 45 minutes.

⑦ Color development after rinsing with PBS for 15 minutes: Add freshly prepared DAB color development droplets to the tissue pieces, and incubate at room temperature for 1-2 minutes.

⑧ Backing dyeing: hematoxylin backing dyeing for 1min, rinsed with tap water for 1min. After hydrochloric acid alcohol differentiation for 2s, rinsed with running water, and returned to blue in the blue-returning solution for 1min.

⑨Dehydration: Dehydrate the glass slides in 75%, 85%, 95%, 100% alcohol at a time, 10s each time.

⑩Xylene I 5min, xylene II 10min for transparent xylene transparent.

Finally, the slides were sealed with neutral gum, and the staining results of neutrophils, CD8T, type I collagen, and new blood vessels were observed under a light microscope.

5. Analysis of inflammatory cells during the healing process of diabetic wounds. On the 7th day after transplantation, the staining results of leukocytes and CD8T cells in the new tissue in the overexpressing Hpgds adipose stem cell group, the empty virus-transfected adipose stem cell group and the control group are shown in the figure. 8.

It can be seen that the number of leukocytes and CD8 T cells in the neonatal tissues of the Hpgds-overexpressing adipose stem cell group was significantly lower than that of the empty virus-transfected adipose stem cell group and the control group.

On the 7th day after transplantation, the staining results of M2 repaired macrophages in the neonatal tissues of the Hpgds-overexpressing adipose stem cell group, the empty virus-transfected adipose stem cell group, and the PBS group are shown in Figure 9.

It can be seen that there are significantly more M2 repair macrophages in the new tissue of the Hpgds adipose stem cell overexpression group than the control group, which helps to promote the transition of the wound from the inflammatory phase to the proliferative phase.

6. On the 10th day, by the detection of PCNA, collagen I and CD31, the immunofluorescence staining of proliferating cell nuclear antigen in the wound on the 10th day of wound healing is shown in Figure 10, and the type I collagen (Collagen I) in the wound on the 10th day of wound healing The immunohistochemical staining of vascular endothelial cell marker (CD31) is shown in Figure 11. The average gray value of the proliferating cell nuclear antigen (Proliferating Cell Nuclear Antigen, PCNA) protein in the overexpression Hpgds adipose stem cell group was higher than that in the blank group (ADSC-PCDH) and the control group (Control) by image analysis. The proliferation state of cells in the overexpression Hpgds adipose stem cell group was significantly higher than that in the control group. At the same time, the content of new blood vessels and collagen tissue in the new tissue was significantly higher than that in the control group.

7. Recovery speed detection

On the 3rd, 5th, 8th, 10th, 14th, and 17th days after the trauma, a sterilized transparent film was used to stick to the wound, and the scope of the wound was delineated along the wound edge, and scanned with a scanner, and the wound area was calculated with ImageJ software. healing rate. Wound healing rate (%)=(initial area-current area)/initial area*100%. Figure 12 shows the healing process of diabetic wounds in mice.

The results showed that the speed of wound healing in the overexpression Hpgds adipose stem cell group was significantly faster than that in the control group.

8. Healing skin thickness statistics

On the 17th day after trauma, the thickness of the new tissue (epidermal + dermis) was measured, 5 measurement points were randomly selected for each field of view, 4 non-consecutive sections were randomly selected in each group, and 5 non-overlapping fields/slices were randomly selected and performed with ImageJ software. Measurement. Figure 13 shows the histological observation and thickness statistics of the wound on the 17th day of healing of the diabetic wound in mice.

The results showed that the thickness of the new tissue in the overexpression Hpgds adipose stem cell group was significantly better than that in the control group.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

Claims (14)

1. A recombinant adipose-derived stem cell over-expressing an Hpgds gene, wherein the recombinant adipose-derived stem cell is obtained by transfecting the Hpgds gene into an adipose-derived stem cell.

2. The recombinant adipose-derived stem cell of claim 1, wherein the adipose-derived stem cell is derived from human adipose tissue.

3. The recombinant adipose-derived stem cell of claim 1, wherein the vector for the Hpgds gene is a lentiviral vector.

4. The recombinant adipose-derived stem cell of claim 3, wherein the lentiviral vector comprises pCDH-CMV-MCS-EF 1-copGFP.

5. The method for producing recombinant adipose-derived stem cells according to any one of claims 1 to 4,

constructing a lentivirus vector for expressing the Hpgds gene;

infecting adipose stem cells with the lentiviral vector expressing the Hpgds gene;

obtaining the recombinant adipose-derived stem cells expressing the Hpgds gene.

6. The method of claim 5, further comprising the step of extracting adipose stem cells using collagenase digestion.

7. The method of claim 6, wherein the temperature of collagenase digestion is 36-38 ℃; the digestion time is 1-1.5 hours.

8. The method according to claim 6, wherein the step of extracting the adipose-derived stem cells is followed by the step of identifying the extracted adipose-derived stem cells, including the identification of osteogenesis and adipogenesis.

9. The method of claim 5, wherein the infection of the lentiviral vector expressing the Hpgds gene has a complex infection value of 300-400.

10. A drug for repairing diabetic wound tissue, comprising the recombinant adipose-derived stem cell according to any one of claims 1 to 4.

11. The method for preparing a medicament for repairing diabetic wound tissue according to claim 10, comprising the steps of:

a. constructing a recombinant adipose-derived stem cell over-expressing the Hpgds gene;

b. and mixing the recombinant adipose-derived stem cells with the hydrogel scaffold to obtain the medicine for repairing the diabetic wound tissues.

12. The method for preparing a drug for repairing diabetic wound tissue according to claim 11, wherein the hydrogel scaffold is an animal-derived RGD peptide-free modified polysaccharide three-dimensional scaffold.

13. Use of the recombinant adipose-derived stem cell according to any one of claims 1 to 4 in the preparation of a medicament for repairing biological tissues.

14. Use of the recombinant adipose-derived stem cells according to any one of claims 1 to 4 in the preparation of a medicament for repairing diabetic wound tissue.

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