阅读说明：本技术 脂肪来源干细胞在局限性硬皮病中的用途 (Use of adipose derived stem cells in localized scleroderma ) 是由 龙笑 王晓军 陈洁 李竹君 崇煜明 张文超 王晨羽 俞楠泽 夏泽楠 黄久佐 于 2020-05-09 设计创作，主要内容包括：本发明公开了脂肪来源干细胞在制备辅助脂肪移植治疗局限性硬皮病的产品中的用途。发明人通过构建博来霉素硬皮病裸鼠模型,研究脂肪干细胞+脂肪移植可降低硬皮病组小鼠的TGF-β1含量及Ⅲ型胶原含量,减轻硬皮病的症状。(The invention discloses application of adipose-derived stem cells in preparation of a product for assisting adipose transplantation in treating limited scleroderma. The inventor researches that the content of TGF-beta 1 and the content of type III collagen of mice in a scleroderma group can be reduced by constructing a bleomycin scleroderma nude mouse model and performing adipose stem cell and adipose transplantation so as to relieve the symptoms of scleroderma.)

1. Use of adipose-derived stem cells in the preparation of a product for assisting fat transplantation in treating localized scleroderma.

2. The use according to claim 1, wherein the product is a stem cell preparation.

3. The use of claim 2, wherein the adipose-derived stem cell immunophenotype is negative for CD34, CD45, and positive for CD29, CD 44.

4. The use of claim 3, wherein the adipose-derived stem cells are used to assist fat transplantation in improving adipose survival while reducing dermal collagen deposition in scleroderma patients.

5. The use of claim 4, wherein the adipose-derived stem cells reduce the levels of TGF- β 1 and type III collagen in scleroderma patients.

6. A stem cell preparation for assisting fat transplantation in treating local scleroderma, which is an adipose-derived stem cell preparation.

7. The stem cell preparation of claim 6, comprising 1 x 10 per unit of said stem cell preparation⁵To 1X 10⁶And (3) adipose-derived stem cells.

8. The stem cell preparation of claim 7, wherein said stem cell preparation is an intravenous injection.

9. Use of a stem cell preparation according to any one of claims 6 to 8 in the manufacture of a medicament for the treatment of localized scleroderma.

10. The use of claim 9, wherein the medicament further comprises another medicament for treating localized scleroderma.

Technical Field

The invention relates to the technical field, in particular to application of adipose-derived stem cells in limited scleroderma.

Background

Scleroderma is a collagenous disease characterized by fibrosis and sclerosis of skin and tissue organs, and is called Localized Scleroderma (LS), also called hard spot disease (Morphea), and diffuse skin accumulation with multiple visceral organ systems called Systemic scleroderma (SSc). Early treatment of scleroderma is primarily directed to immune, vascular and collagen abnormalities, based on anti-inflammation, immunosuppression, immunomodulation, improved blood circulation and reduced fibrosis. Deformities such as skin sclerosis, subcutaneous lipoatrophy, local depressions and the like are left in the later stage, and orthopedic treatment is often needed. Orthopedic treatment of locally depressed deformities currently includes autologous tissue transplantation with and without the vascular pedicle. The tissue transplantation operation wound with the vascular pedicle is large, the microssuturing risk is high, and the autologous fat transplantation becomes a treatment scheme most popular for patients and plastic doctors because of small operation wound.

The autologous fat transplantation operation is simple to operate, the operation wound is small, operation scars are not basically left after the operation, extra supply area loss is not increased, the source is wide, rejection reaction does not exist, the injection can be repeated, autologous adipose tissue defects are filled, the shape and the texture of the autologous adipose tissue defects are consistent with those of surrounding tissues, and therefore the autologous fat transplantation operation method becomes the most common operation method for treating facial depression deformity caused by limited scleroderma at present, and is particularly suitable for patients with light and moderate limited scleroderma. In the case of fat transplantation of patients with limited scleroderma, many scholars have given experience in treatment themselves, detailed description of the surgical mode and surgical effect, and our science has accumulated some experiences in long-term application of fat transplantation to treat limited scleroderma: for example, in the active areas such as a chin area, a cleft-shaped sunken area, a red lip and the like with tightly adhered tissues, the fat survival rate is low, and a satisfactory effect can be obtained only by 5-6 times of injection and transplantation; for the region with tight tissue adhesion, a small needle knife is adopted to be close to the deep surface of dermis to fully release adhesion so as to increase the survival space of fat particles and facilitate the shaping of fat; in addition, the fat particles are injected into the deep tissues in multiple points and layers as much as possible, and then a proper amount of fat particles are injected into the separated cavities to reduce the re-adhesion. These surgical procedures have improved the effectiveness of the surgery, but the low fat survival rate and the need for multiple surgeries remain the most common problems in clinical practice.

There are many factors that affect the survival rate of fat injection transplantation, including the selection of donor area, blood supply, mobility and adhesion of recipient area, the manner of obtaining fat particles, suction technique, purification of fat particles, and systemic factors of patients. In the technical aspect, physical and chemical factors which may damage fat particles in the processes of fat extraction, cleaning and transplantation are avoided. Meanwhile, in order to promote the survival of fat particles, the contact area between the fat particles and tissues is increased during transplantation. Therefore, multi-level, multi-tunnel, multi-point transplantation has become a common general knowledge in fat transplantation technology. However, most of previous researches are directed to normal fat specimens, studies on scleroderma patients are few, fat transplantation of patients with localized scleroderma is different from fat transplantation of normal people, even if the principles on the fat transplantation technology are followed, the fat survival rate of scleroderma patients is still obviously lower than that of fat transplantation of normal people, the fat transplantation is possibly related to factors such as tissue atrophy and adhesion in a scleroderma-localized lesion area, poor local blood supply, local inflammatory microenvironment and the like, and meanwhile, the fact that scleroderma-localized patients have no biological change in fat in a scleroderma self-supply area needs to be verified.

Research shows that: the Adipose-derived stem cells (ADSCs) can obviously improve the survival rate of fat and improve the blood supply of local tissues, has the function of immunoregulation, can obviously reduce the expression of TGF-beta and the expression of Collagen I, and inhibit the proliferation of fibroblasts, and the characteristics of the stem cells can just make up the defect of treating the facial depression deformity of patients with localized scleroderma by autologous fat transplantation. These related studies on localized scleroderma and ADSCs provide us with a new idea to apply fat transplantation to treat localized scleroderma.

Disclosure of Invention

One aspect of the invention provides the use of adipose derived stem cells for the preparation of a cell preparation for the treatment of localized scleroderma.

In order to achieve the above objects, the present invention provides, in a first aspect, a use of adipose-derived stem cells for preparing a cell preparation for assisting adipose transplantation in treating localized scleroderma.

Preferably, the product is a stem cell preparation.

Preferably, the adipose-derived stem cell immunophenotype is negative for CD34 and CD45, and positive for CD29 and CD 44.

Preferably, the adipose-derived stem cells assisted fat transplantation can not only improve the survival rate of fat, but also reduce the skin collagen deposition of scleroderma patients.

Preferably, the adipose-derived stem cells reduce the TGF- β 1 content and the type iii collagen content in scleroderma patients.

Further, the invention provides a stem cell preparation for assisting fat transplantation in treating local scleroderma, wherein the stem cell preparation is an adipose-derived stem cell preparation.

Preferably, the stem cell preparation comprises 1 × 10 cells per unit⁵To 1X 10⁶And (3) adipose-derived stem cells.

Preferably, the stem cell preparation is an intravenous injection.

Furthermore, the invention provides application of the stem cell preparation in preparing a medicine for treating localized scleroderma.

Preferably, the medicament also comprises other medicaments for treating the limited scleroderma.

Advantageous effects

Besides the improvement of fat survival rate of adipose-derived stem cells, the skin collagen content of scleroderma nude mice after adipose-derived stem cell assisted adipose transplantation is found to be obviously reduced. The inventor researches that the content of TGF-beta 1 and the content of type III collagen of mice in a scleroderma group can be reduced by constructing a bleomycin scleroderma nude mouse model and performing adipose stem cell and adipose transplantation so as to relieve the symptoms of scleroderma.

Drawings

FIG. 1 shows general observation and histological sectioning of normal nude mice and bleomycin scleroderma nude mice, thickening and hardening of skin of model-making group, thickening, increasing and disorganizing of collagen fiber, and successful model making;

FIG. 2 shows that the survival rate of CAL group is higher than that of single fat transplantation in both normal nude mice and scleroderma nude mice;

FIG. 3 HE staining of transplanted fat;

FIG. 4Masson staining shows skin changes after fat transplantation or CAL in normal and scleroderma nude mice;

FIG. 5 TGF-. beta.1 immune index: the normal nude mice have no obvious change after fat transplantation or fat stem cell + fat transplantation. However, for scleroderma mice, fat transplantation or CAL can reduce the TGF-beta 1 content of the scleroderma mice, and particularly the CAL effect is more obvious;

fig. 6 collagen type III immune index: the normal nude mice have no obvious change after fat transplantation or fat stem cell + fat transplantation. However, in scleroderma mice, fat transplantation or CAL can reduce the type III collagen content of scleroderma mice, and particularly the CAL effect is more obvious.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 culture and expansion of adipose-derived Stem cells

Adipose tissue was obtained from waste adipose tissue after Peking cooperative hospital plastic surgery and liposuction, and informed consent was obtained.

1. Adipose-derived stem cell extraction

(1) Adding 20ml of fat obtained by liposuction into a 50ml centrifuge tube, adding D.hang ks to 40ml, and centrifuging for 3min at 800 r;

(2) sucking off liquid at the bottom, transferring fat into a new centrifugal tube, washing for 2 times by D.hangks, and centrifuging for 3min at 800 r;

(3) adding collagenase into the tube, wherein the volume of the collagenase is 1/3-1/4 of the volume of fat, shaking the tube at 37 ℃, and rotating the tube at 200rpm for 30 min;

(4) after the shaking table is taken out, filtering by a 100nm filter screen (Dhangks wets the filter screen, which is beneficial to filtering);

(5) centrifuging the filtrate at 1500r for 8 min;

(6) after centrifugation, sucking off the upper layer of grease, and pouring off (slightly pouring) the remaining D.hang ks;

(7) blowing up the cells by 10ml of D.hangks, and pouring the cells into a new centrifuge tube;

(8) adding D.hangks to the mixture until the volume is 40ml, and centrifuging at 1500r for 8 min;

(9) decanting the supernatant of the centrifugate to obtain precipitated cells, which are SVF enriched in adipose-derived stem cells;

(10) adding culture medium (containing 58% DMEM/F12+ 40% MCDB-201, 2% Fetal Calf Serum (FCS), 10ng/ml EGF, 10ng/ml PDGF, 1 × Insulin-Transferrin-selenious acid (Insulin-Transferrin-Selenium, ITS), 1 × linoleic acid-bovine serum albumin (linoleic acid-bovineser albumin, LA-BSA), 50 μ M β -mercaptoethanol, 2mM L-glutamine, 100 μ g/ml penicillin and 100U/ml streptomycin sulfate) into the cells, mixing, culturing in a T75 flask at 37 deg.C (one flask per 10ml fat seed), and culturing in a culture box with cells growing at about 5 × 10⁶And (4) cells.

2. Cell resuspension

Blowing the cells cultured in the previous day by using a pipette, and sucking the culture medium into another new centrifugal tube; centrifuging at 1000r for 5 min; after centrifugation, the supernatant was poured off and 1 flask of cells was resuspended in 2 flasks; washing the original bottle with D.hang ks for 1-2 times, and adding a fresh culture medium into the original bottle; 2T 75 heavy suspension 1, add the original 2, total to get 3T 75. (adipose-derived stem cells grow adherently, blood cells, endothelial cells and the like grow in a suspension manner, and the blood cells and the endothelial cells can be washed away in the process of replacing and suspending the liquid, so that the adherently-growing adipose-derived stem cells are remained).

3. Cell passage

Pour out old media from T75, wash 1-2 times with PBS (note not to blow liquid onto cells); preparing 0.25% pancreatin from a new clean centrifugal tube; adding 5ml prepared pancreatin into each T75, and digesting for 1-2 min; observing that most cells are changed from an adherent strip shape into a suspended circular shape under a microscope, and adding serum to stop digestion; blowing and beating by using a liquid transfer gun, and adding into a centrifugal tube; centrifuging at 1000r for 5 min; dispersing the precipitated cells, adding a culture medium, and inoculating the cells into T75; one second, 3 original T75 gave 6T 75 (here, 1 generation cells).

4. Cell cryopreservation

Pouring out old culture medium in T75, and cleaning for 1-2 times by using D.hangks; adding prepared 0.25% pancreatin 5mk for digestion for 1-2 minutes; observing that most cells are changed from an adherent strip shape into a suspended circular shape under a microscope, and adding serum to stop digestion; blowing and beating by using a liquid transfer gun, and adding into a centrifugal tube; centrifuging at 1000r for 5 min; the precipitated cells were flicked off, frozen stock solution (DMSO: serum 1: 9) was added, one frozen tube for each 1T 75, 1ml of frozen stock solution was added to each tube; placing the mixture into a freezing storage box, placing the freezing storage box at the temperature of minus 20 ℃ for 2 hours, and then placing the freezing storage box into a refrigerator at the temperature of minus 80 ℃ for freezing storage.

5. Flow cytometry for detecting cell cycle

Fixing 3 rd generation ADSCs close to fusion with 70% cold ethanol at 40 ℃ overnight; washing twice with PBS; RNase (10. mu.g/mL) was added and incubated at 37 ℃ for 30 min; then adding 50 mu g/mL Propidium Iodide (PI) and incubating for 15min at 4 ℃ in the dark; cell cycle was examined by flow cytometry.

Flow cytometry detection of DNA content in ADSCs cells showed that most of the cells were in the resting stage (GO/G1 stage: 85.65%) and only a small fraction were in the dividing stage (G2/M + S stage: 11.22%, where G1/G2 stage: 3.13%).

6. Flow cytometer for detecting cell surface markers

(1) Preparing single cell suspension from 3 rd generation ADSCs, and centrifuging at 800r/min for 5 min; (2) washing with PBS for 2 times; (3) adjusting cell density to 10⁶Each volume is 0.1ml, and the mixture is subpackaged into test tubes; (4) respectively adding 5 μ l of FITC-labeled mouse antihuman CD34, CD45, CD29 and CD 44; (5) incubating at 4 deg.C in dark for 30min, and adding PBS in equal amount to the blank control group; (6) after incubation, washing with PBS 2 times; (7) finally resuspending the cells with lmLPBS; (8) flow cytometry detects cell surface markers.

The results show that the surface markers of the 3 rd generation ADSCs detected by the flow cytometer show that the expression of CD34 (1.51%), CD45 (0.85%) and CD45 is negative; CD44+ (99.1%), CD29+ (97.8%), and positive expression.

Example 3 adipose-derived stem cell-assisted fat transplantation therapy of scleroderma nude mouse model

1. Bleomycin scleroderma nude mouse model

The bleomycin powder is diluted to 200ug/ml by Phosphate Buffer Solution (PBS), and is stored for later use after being filtered and sterilized by a sterile filter membrane of 0.22 um. The model building group injects bleomycin diluent to the back subcutaneous part of a nude mouse, a control group injects PBS liquid to the back subcutaneous part of the nude mouse, 1 time a day, 0.1ml each time, 4 weeks, general changes of back skin elasticity, appearance and the like are observed, 2 experimental model building groups and 2 control groups are killed in 4 weeks, the back skin of an injection area is taken and placed in tissue fixing liquid for fixation, HE staining and Masson staining are respectively carried out, and compared with the PBS control group mouse, whether the model building is successful or not is confirmed.

2. Fat transplantation and Cell-assisted lipotransplantation (CAL) procedure

After 4 weeks, 2% sodium pentobarbital 50mg/kg was used for intraperitoneal injection for anesthesia, and a 1ml syringe was used for subcutaneous injection of fat and stem cells. Normal nude mice and scleroderma molding nude mice are divided into three groups: control group, fat-transplanted group, CAL group. 6 control groups were injected subcutaneously with 0.3ml PBS, 6 fat-transplanted groups were injected subcutaneously into the back with 0.3ml fat, and 6 CAL groups were injected subcutaneously with 0.3ml fat + 1X 10⁶Adipose-derived stem cells were injected subcutaneously into the back. The 1ml screw propelling injector is used for uniformly injecting the transplanted fat into the affected area, and the injected fat is prevented from being inaccurate due to the sudden change of resistance in the injection when the needle is withdrawn, so that the injection is not performed under the condition of rough high pressure. The single-point injection makes the injected fat in an original-ball shape, avoids confusion with the fat tissue of the nude mouse after the injection, and is convenient for postoperative measurement.

Weighing transplanted fat 1 month after operation, cutting skin of injection part of nude mouse, fixing in formaldehyde solution, embedding in paraffin, making tissue section, and observing with hematoxylin-eosin (HE) staining and Masson staining. At the same time, immunohistochemical staining was performed to determine the levels of collagen type III and transforming growth factor TGF-beta 1 in the skin.

3. Taking specimen section

3.1 specimen fixation

In the selection of the fixative, neutral 10% formalin is generally used as a conventional fixative.

3.2 running Water rinsing

After formalin fixation, the cells should be fully washed with running tap water for about 6 to 8 hours.

3.3 dehydration

The time is 10 hours from 70 percent alcohol to 80 percent alcohol to 95 percent alcohol to 100 percent alcohol.

3.4 transparency

Placing the material in a mixture of xylene and absolute ethyl alcohol according to the proportion of 1: 1, and then placing in xylene. The total time is about 2 hours.

3.5 wax dipping

The transparent material is put into normal paraffin in a dissolved state, so that the paraffin is immersed into tissues.

3.6 embedding

Firstly, injecting molten paraffin into an embedding box, then taking out a tissue block from the paraffin of 60 ℃ by using a forceps, immediately putting the tissue block into an embedding mould, enabling a section to be downward for vertical embedding, fixing the tissue in the center of the paraffin by using the forceps for a while, enabling the tissue to stand when the paraffin is solidified, achieving the requirement of vertical embedding, and slowly cooling and solidifying the tissue.

3.7 slicing

In order to maintain the hardness of the wax block during slicing, the wax block can be placed in a freezer. When cutting, the tissue wax block should be trimmed to size before slicing. When slicing, the left hand holds the brush pen and the right hand rotates the rotating wheel of the slicer. The thickness of the slice is preferably 5um to 10um, and the tilt angle is preferably 20 DEG to 30 DEG in general.

3.8 sticking and baking sheet

And lightly flatly spreading the wax sheet with the right side facing upwards on the water surface of a 40-45 ℃ film spreading box, and naturally spreading the slightly-wrinkled wax belt by means of the tension and the temperature of water. After the to-be-cut sheet is fully flattened on the constant temperature water surface, the wax sheet is fished to the middle section of the glass slide, and the residual water on the glass slide is poured off. After being taken out, the slices can be baked after being almost completely dried. Generally, the slices are baked for one hour on a slice baking instrument or placed in a constant temperature box at 60-65 ℃, and part of paraffin wax in the slices is removed.

4. HE staining

Hematoxylin-eosin staining method (HE staining method for short), one of the staining methods commonly used in paraffin section technology. The hematoxylin staining solution is alkaline, and mainly makes chromatin in cell nucleus and ribosome in cytoplasm bluish; eosin is an acid dye that primarily reddens components in the cytoplasm and extracellular matrix.

Reagent configuration

A: 0.5-1% eosin alcohol solution. Weighing 0.5-1 g of eosin Y, adding a small amount of distilled water for dissolving, and then dropwise adding glacial acetic acid until the paste is formed. Filter with filter paper, bake the filter residue in the oven, dissolve with 100ml of 95% alcohol.

B: the formula of hematoxylin staining solution comprises: (3000 ml can be prepared, and can be reduced according to the proportion) 6g of hematoxylin; 100ml of absolute ethyl alcohol; 150g of aluminum potassium sulfate; 2000ml of distilled water; 1.2g of sodium iodate; 120ml of glacial acetic acid; and 900ml of glycerol.

The preparation method comprises the following steps: dissolving hematoxylin in anhydrous ethanol, dissolving aluminum potassium sulfate in distilled water, mixing glycerol, and adding glacial acetic acid and sodium iodate.

C: 1% hydrochloric acid alcohol differentiation solution: adding 1ml of concentrated hydrochloric acid into 99ml of 70% alcohol.

And (3) dyeing:

(1) xylene (I) for 15 min; (2) xylene (II) for 15 min; (3) 100% ethanol (I) for 5 min; (4) 100% ethanol (II) for 5 min; (5) 80% ethanol for 5 min; (6) distilled water for 5 min; (7) staining sappan wood semen for 5 min; (8) slightly washing the sappan wood semen for 1-3s with running water; (9) 1% hydrochloric acid ethanol for 1-3 s; (10) slightly washing with water for 10-30 s; (11) enabling the blue promoting liquid to return blue for 10-30 s; (12) flushing with running water for 10-15 min; (13) washing with distilled water for 1-2 s; (14) dyeing with 0.5% eosin solution for 1-3 min; (15) slightly washing with distilled water for 1-2 s; (16) slightly washing with 80% ethanol for 1-2 s; (17) 95% ethanol (I) 2-3 s; (18) 95% ethanol (II) for 3-5 s; (19) absolute ethyl alcohol for 5-10 min; (20) xylene carbolate for 5-10 min; (21) xylene (I) for 2 min; (22) xylene (II) for 2 min; (23) xylene (III) for 2 min; (24) and (5) sealing by using neutral gum.

5. Masson staining

The Masson dyeing can better display the distribution of muscles, collagen and elastic fibers in tissues and vividly show the characteristics of tissue structures compared with an HE dyeing method.

Preparing a reagent:

(1) hematoxylin staining solution (see HE staining method);

(2) masson complex staining solution: ponceau 2R 0.7g, acid fuchsin 0.3g, distilled water 99ml, glacial acetic acid 1 ml;

(3) bright green staining solution: 1g of light green, 1ml of glacial acetic acid and 99ml of distilled water;

(4) 1% aqueous phosphomolybdic acid solution: phosphomolybdic acid (phosphomolybdydic acid)1g, distilled water was added to 100 ml.

And (3) dyeing:

(1) xylene (I) for 15 min; (2) xylene (II) for 15 min; (3) 100% ethanol (I) for 5 min; (4) 100% ethanol (II) for 5 min; (5) 80% ethanol for 5 min; (6) distilled water for 5 min; (7) dyeing the hematoxylin for 5-10 min; (8) washing with running water, and differentiating with 1% hydrochloric acid; (9) flushing for several minutes with running water; (10) masson composite staining solution for 5-10 min; (11) slightly washing with distilled water; (12) treating with 1% phosphotungstic acid solution for about 5 min; (13) directly re-dyeing with brilliant green dyeing solution (or aniline blue solution) for 5min without washing; (14) treating with 1% glacial acetic acid water for 1 min; (15) slightly washing with 80% ethanol for 1-2 s; (16) 95% ethanol (I) 2-3 s; (17) 95% ethanol (II) for 3-5 s; (18) absolute ethyl alcohol for 5-10 min; (19) xylene carbolate for 5-10 min; (20) xylene (I) for 2 min; (21) xylene (II) for 2 min; (22) xylene (III) for 2 min; (23) and (5) sealing by using neutral gum.

6. TGF-beta 1 and III collagen immunohistochemical staining

(1) Baking slices:

selecting a section with complete tissue, and heating for 30min by a baking machine at 70 ℃;

(2) dewaxing and hydrating:

xylene 15min → absolute ethanol 3min → 95% ethanol 3min → 85% ethanol 3min → 75% ethanol 3min → pure water 3min → 3% H2O2, 10min (for blocking endogenous peroxidase) → pure water 2 min;

(3) antigen retrieval:

adding sufficient antigen repairing solution (1 × CB), and heating at 95 deg.C for 15 min;

cooling to room temperature (about 20 ℃);

(4) and (3) sealing:

dropping goat serum covering tissues → incubating in an incubator at 37 ℃ for 30 min;

(5) add I antibody (dilute antibody according to I antibody specification)

4 ℃ overnight (12-16h) → return to room temperature;

(5) cleaning:

washing with 1 × PBS for three times, each for 3 min;

(6) adding II antibody:

adding a secondary antibody reagent 1 → incubating at 37 ℃ for 20min → washing with 1 XPBS for three times, 3min each time;

adding a secondary antibody reagent 2 → incubating at 37 ℃ for 20min → washing with 1 XPBS for three times, 3min each time;

(7) DAB (3-3 diaminobenzidine) color development:

diluting to 1 × DAB liquid, dripping the DAB liquid into the tissue for 10s, and observing the color development degree under a microscope;

(8) washing with distilled water:

washing for three times, each time for 1 min;

(9) counterdyeing:

hematoxylin counterstain for 5min → tap water rinse;

(10) 1% hydrochloric acid alcohol solution differentiation:

differentiating tissues by using 1% hydrochloric acid alcohol solution for 10 s;

(11) soaking in tap water for 15min

(12) Dehydration and transparent treatment:

distilled water 3min → 75% ethanol 3min → 85% ethanol 3min → 95% ethanol 3min → absolute ethanol 3min → xylene, 8 min;

(13) sealing neutral gum into a sheet;

(14) and (6) microscopic examination.

7. Results

7.1 bleomycin scleroderma Bo nude mouse model

The Taisoma observation shows that the daily subcutaneous injection of bleomycin can induce skin sclerosis, acute skin reaction and scabbing appear after 3 days to 1 week of injection, the skin of an injection part after 1 month has thickened, hardened and poor elasticity, and the back skin of a PBS control group mouse has no obvious change.

Histologically (Masson staining) modeling showed thick dermis, thickening, disorganizing collagen fibers, thickening or even vascular occlusion of the hypodermis vessels, thinning and disappearance of the fat layer, while the normal dermis was thin and aligned collagen fibers. The type III collagen immunohistochemistry index, bleomycin-induced scleroderma model nude mice (2519.17 ± 774.19) was significantly increased compared to normal nude mice (623.85 ± 113.84) (fig. 1, table 1).

TABLE 1 type III collagen immunization index of model and control groups

The laser speckle contrast imaging technology (laser speckle contrast imaging system, perimeter) is applied to measure the blood flow of normal nude mice and scleroderma nude mice, and the results show that: the blood flow of the injection site of the normal nude mice is 251.72 +/-43.82, the blood flow of the injection site of the scleroderma nude mice is 241.89 +/-22.76, and the results are not different significantly.

7.2 fat survival comparison of fat transplantation with CAL

The fat weight of the fat transplantation group of the normal nude mice is 102.97 +/-17.87 mg, and the fat survival rate is 36.8%; the weight of CAL group fat is 135.05 + -27.28 mg, and the fat survival rate is 48.2%; the weight of transplanted fat of the scleroderma nude mice is 77.54 +/-12.40 mg, and the fat survival rate is 27.7%; the weight of CAL group fat is 102.48 + -23.16 mg, and the fat survival rate is 36.6%. The survival rate of CAL group was higher than that of single fat transplantation group in both normal nude mice and scleroderma nude mice (Table 2, FIG. 2).

TABLE 2 fat transplantation and CAL fat survival comparison

HE staining results showed that adipose tissue was composed of a adipocytes, vascular endothelial cells, fibroblasts and connective tissue, as well as macrophages and lymphocytes, etc. Adipose tissue is filled with voluminous fat cells, with scarce connective tissue and a rich network of capillaries. Compared with a normal group, the scleroderma group has the highest fibrosis degree, the normal nude mouse CAL group has the largest number of fat cells, clear boundaries and clear outlines, tiny fibrous tissues can be wrapped around the fat cells, and the fibrosis degree is light; the scleroderma fat transplantation group has the least number of fat cells, a plurality of fat cells are mutually fused into a large vacuole, the surrounding fibrous tissues are thick, and the fibrosis degree is the heaviest. Small adipocytes were visible between fibrous tissues, probably transformed with neonatal adipose stem cells (fig. 3).

7.3 skin Change following fat transplantation or CAL

Masson staining showed: the differences among the normal nude mouse control group, the fat transplantation group and the CAL group are not obvious, the fibrosis degree is light, the collagen fibers are blue, and the fibrous tissues are arranged and shaped. Compared with normal nude mice, the nude mice with scleroderma have heavier fibrosis degree, wherein the control group of the nude mice with scleroderma has the thickest dermis, thickened and increased collagen fibers and disordered arrangement, the CAL group has obviously thinner dermis and reduced collagen fiber content compared with the control group, and the fat transplantation group is between the two groups (figure 4).

For normal nude mice, immunohistochemistry shows that the TGF-beta 1 content control group is 939.34 +/-216.92, the fat transplantation group is 974.98 +/-470.18, the CAL group is 967.66 +/-448.74, the type III collagen content control group is 623.85 +/-113.84, the fat transplantation group is 605.73 +/-217.17, and the CAL group is 599.34 +/-89.39; for scleroderma nude mice, the TGF-beta 1 content control group is 1945.95 +/-330.77, the fat transplantation group is 1418.86 +/-376.82, the CAL group is 1132.12 +/-190.69, the III type collagen content control group is 2629.39 +/-746.62, the fat transplantation group is 1531.74 +/-836.41, and the CAL group is 946.92 +/-448.90. Normal nude mice had no significant changes in TGF-. beta.1 and type III collagen content after fat transplantation or fat stem cell + fat transplantation (Table 3, FIG. 5, FIG. 6). However, in scleroderma mice, fat transplantation or CAL can reduce the TGF-beta 1 content and the III type collagen content of the scleroderma mice, and especially the CAL effect is more obvious.

TABLE 3 TGF-beta 1 and type III collagen content in normal and scleroderma nude mice

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.