阅读说明：本技术 丹参酚酸b在制备提高自体脂肪移植存活率的药物中的应用 (Application of salvianolic acid B in preparation of medicine for improving autologous fat transplantation survival rate ) 是由 余力 郑丹宁 张艺凡 孙佳铭 侯家康 高雅 钟钊豪 于 2021-07-08 设计创作，主要内容包括：本发明提出了一种丹参酚酸B在制备提高自体脂肪移植存活率的药物中的应用。本发明通过实验验证丹参酚酸B可有效增强3T3-L1(小鼠前脂肪细胞细胞系)和h-ADSC(人脂肪干细胞原代细胞)增殖能力及分化水平；并可改善裸鼠皮下脂肪移植的存活率。本发明药物辅助下的脂肪移植,给药方式简单,且效果明显,且无明显副作用,有着显著的社会效益与经济效益,对于自体脂肪移植的应用具有推进作用。(The invention provides application of salvianolic acid B in preparing a medicament for improving the survival rate of autologous fat transplantation. Experiments prove that the salvianolic acid B can effectively enhance the proliferation capacity and differentiation level of 3T3-L1 (mouse preadipocyte cell line) and h-ADSC (human adipose-derived stem cell primary cells); and can improve the survival rate of the nude mice subcutaneous fat transplantation. The fat transplantation under the assistance of the medicine has the advantages of simple administration mode, obvious effect, no obvious side effect, obvious social benefit and economic benefit and propulsion effect on the application of autologous fat transplantation.)

1. Application of salvianolic acid B in preparing medicine for improving autologous fat transplantation survival rate is provided.

2. The use of claim 1, wherein the salvianolic acid B is 10-100 μmol/L.

3. The use of claim 1, wherein the salvianolic acid B is at a concentration of 50 μmol/L.

4. The use of claim 1, wherein the medicament further comprises a pharmaceutical excipient or other compatible medicament.

5. The use of claim 1, wherein the medicament is an injectable solution.

6. Application of salvianolic acid B in preparing medicine for promoting proliferation of adipose-derived stem cells is provided.

7. Application of salvianolic acid B in preparing medicine for promoting adipogenic differentiation by up-regulating PPAR gamma, CEBP alpha and FABP 4.

Technical Field

The invention relates to the technical field of medicines, in particular to application of salvianolic acid B in preparing a medicine for improving the survival rate of autologous fat transplantation.

Background

Autologous fat is used in clinic as an ideal filling material for soft tissue defects. Autologous fat transplantation has a problem of high absorption rate after transplantation. In experimental studies, the absorption rate of fat transplantation reaches as high as 80%, and the retention rate in clinical trials is only about 34%. In the current research, the method for improving the survival rate of autologous fat transplantation is mainly realized by optimizing the processing flow of granular fat, stem cell assisted transplantation, growth factors assisted transplantation and the like. The technologies used in the current research for improving the survival rate of fat transplantation are all complex, and have certain biological safety problems and risk of tumor change, so that the wide clinical application of the technology is limited. On the other hand, even if these aids are applied, the survival rate of large-volume fat transplantation is still unsatisfactory. At present, no effective small molecule compound which is definitely used for improving the fat survival rate after the fat transplantation exists in the market.

Disclosure of Invention

The invention provides application of salvianolic acid B in preparing a medicament for improving the survival rate of autologous fat transplantation, and solves the problem of high fat transplantation absorption rate in the prior art.

The technical scheme of the invention is realized as follows:

application of salvianolic acid B in preparing medicine for improving autologous fat transplantation survival rate is provided.

In some embodiments, the concentration of the salvianolic acid B is 10-100 μmol/L.

In some embodiments, the concentration of salvianolic acid B is 50 μmol/L.

In some embodiments, the medicament further comprises a pharmaceutic adjuvant or other compatible medicaments.

In some embodiments, the medicament is an injection.

Application of salvianolic acid B in preparing medicine for promoting proliferation of adipose-derived stem cells is provided.

Application of salvianolic acid B in preparing medicine for promoting adipogenic differentiation by up-regulating PPAR gamma, CEBP alpha and FABP 4.

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

(1) experiments prove that the salvianolic acid B can effectively enhance the proliferation capacity and differentiation level of 3T3-L1 (mouse preadipocyte cell line) and h-ADSC (human adipose-derived stem cell primary cells); and can improve the survival rate of the nude mice subcutaneous fat transplantation.

(2) The fat transplantation under the assistance of the medicine has the advantages of simple administration mode, obvious effect, no obvious side effect, obvious social benefit and economic benefit and propulsion effect on the application of autologous fat transplantation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive exercise.

FIG. 1: the cytotoxicity and proliferation effect of salvianolic acid B in example 1 on 3T3-L1 and ADSC are shown. Wherein, fig. 1A: the chemical structural formula of the salvianolic acid B; FIG. 1B: detecting cytotoxicity; FIG. 1C: edu the proliferation level was measured.

FIG. 2: the flow-type apoptosis test in example 1 was performed to examine the effect of salvianolic acid B at different concentrations on apoptosis of 3T3-L1 and h-ADSC.

FIG. 3: the effect of salvianolic acid B in example 1 on adipogenic differentiation of 3T3-L1 and ADSC. Wherein, fig. 3A: detecting the differentiation level by oil red staining; FIG. 3B: and (4) detecting triglyceride level.

FIG. 4: RNA level changes following treatment with salvianolic acid B (50. mu. mol/L) were examined for RNA-Seq in example 1. Wherein, fig. 4A: a gene difference scatter plot; FIG. 4B: gene expression wien map; FIG. 4C: day four and day eight KEGG pathway analysis; FIG. 4D: thermographic analysis of PPAR γ; FIG. 4E: GSEA analysis on day four and day eight.

FIG. 5: the influence of salvianolic acid B on adipogenesis-related genes of adipose-derived stem cells was verified by WB and PCR in example 1. FIG. 5A: PCR verifies the influence of salvianolic acid B on the expression of 3T3-L1 and ADSC adipogenic related genes; FIG. 5B: WB verified the effect of salvianolic acid B on the expression of lipid-associated proteins 3T3-L1 and ADSC.

FIG. 6: the model of subcutaneous fat transplantation in nude mice in example 1 was used. Among them, fig. 6A: the whole animal experiment process; FIG. 6B: subcutaneous fat transplantation part of nude mice.

FIG. 7: the salvianolic acid B in example 1 can improve the survival rate of autologous fat transplantation. Among them, fig. 7A: general picture and volume and weight of transplanted fat; FIG. 7B: HE staining of transplanted fat; FIG. 7C: perlipin (green) stained viable fat.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(I) in vitro experiments

(1) The safe drug concentration range detected by CCK8 experiment is less than or equal to 100 mu mol/L (figure 1B):

the effect of salvianolic acid B on the viability of 3T3-L1 cells and h-ADSCs was tested by Cell Counting Kit-8 (Beyotime, China) according to the manufacturer's instructions. Briefly, 5000 cells/well were seeded in 96-well plates. Culturing the cells in growth medium or lipogenic differentiation medium containing salvianolic acid B (0, 0.1, 0.5, 1, 5, 10, 25, 50, 75, 100, 125 μmol/L) at different concentrations. After 72 h of incubation, 10% CCK-8 reagent was mixed with the medium and added to each well. The 96-well plate was incubated at 37 ℃ for 2 hours. OD values were measured by absorbance at 450nm using a microplate reader (Thermo, USA).

(2) The effect of salvianolic acid B on the proliferation level of the adipose-derived stem cells is detected by Edu at 10,50 and 100. mu. mol/L, and the effect of salvianolic acid B on the proliferation level of the adipose-derived stem cells is found to be capable of promoting the proliferation of the adipose-derived stem cells at the concentration of 10, 50. mu. mol/L (FIG. 1C):

cells were seeded in 24-well plates and incubated with different concentrations of salvianolic acid B under standard conditions. After 24 hours of incubation, cell proliferation was detected using the EdU cell proliferation detection kit (Invitrogen, USA) according to the manufacturer's protocol. Briefly, cells were incubated with 50 μ M EdU for 2 hours before fixation, membrane permeation, and color reaction. Then, the cell nucleus was stained with Hoechst33342(Invitrogen, USA) for 30 minutes. The incorporation of EdU into cells was determined by inverted fluorescence microscopy (Nikon, Japan).

(3) The apoptosis was detected by flow assay, and it was found that 10. mu. mol/L and 50. mu. mol/L did not cause significant apoptotic effects on adipose stem cells (FIG. 2):

cells were seeded in 6-well plates, negative controls were set, groups 0,10, 50,100 μmol/L, and after drug treatment, cells were harvested and washed with cold PBS. 1XAnnexin-binding Buffer and 100. mu.g/ml PI were prepared. The cells were centrifuged, resuspended using Annexin-binding Buffer, and diluted to 1 × 10⁶One per ml. Mu.l of FITC annexin V, 5. mu.l of FITC annexin V and 1. mu.l of PI at 100g/ml were incubated for 15 minutes at room temperature. Adding 400 μ l Annexin-binding Buffer, and detecting on a computer.

(4) The adipogenic differentiation of the adipose-derived stem cells is induced by using three concentrations of 10 mu mol/L, 50 mu mol/L and 100 mu mol/L, and the results of oil red staining and triglyceride measurement show that the three concentrations can promote the adipogenic differentiation of the adipose-derived stem cells, wherein the 50 mu mol/L effect is the best (figure 3):

oil red dyeing: cells were washed with PBS, fixed in 4% paraformaldehyde at room temperature for 30 min, and stained with fresh oil-red O solution (Solarbio, China) for 30 min. The stained cells were then washed twice with PBS. Adipogenic differentiation was observed using an inverted fluorescence microscope (Nikon, Japan).

Triglyceride determination: different concentrations of salvianolic acid B were added to the medium during differentiation. On days 4 and 8 of differentiation, triglyceride levels of the cells were measured by a triglyceride assay kit (Nanjing, China, institute for bioengineering) according to the manufacturer's instructions. Briefly, the assay begins with enzymatic hydrolysis of triglycerides by lipases to produce glycerol and free fatty acids. The glycerol released was then measured by a microplate reader at a wavelength of 510 nm. Protein concentration was measured using BCA kit (Boster, China). Triglyceride content (mmol/gprot) ═ triglyceride concentration (mmol/L)/protein concentration (gprot/L).

(5) PPAR pathways were found to be significantly upregulated in the drug group by collecting day 4 and day 8 adipose stem cell samples from the control and drug treated groups (at a concentration of 50 μmol/L) and sending RNA-Seq (fig. 4):

adding salvianolic acid B or solvent into h-ADSCs, and continuously differentiating for 4 days and 8 days to obtain RNA-Seq samples. RNA expression and quality were measured using a NanoDrop ND-1000 instrument (Thermo Fisher Scientific, Waltham, Mass.). A cDNA library was constructed using the KAPA Stranded RNA-Seq library preparation kit (Illumina, San Diego, Calif.).

(6) The sequencing results were verified by WB and PCR, and found that the expression levels of PPAR γ, CEBP α and FABP4 were increased, and the effect was best at 50. mu. mol/L (FIG. 5):

western-blot: cultured cells were lysed with RIPA buffer (Beyotime, china) supplemented with protease inhibitors (PMSF, Biosharp, china). Briefly, 20. mu.g of protein were separated by 10% or 12% SDS-PAGE and blotted in polyvinylidene fluoride membranes (Millipore Sigma, USA). The membrane was blocked with 5% skim milk for 1 hour at room temperature. The isolated proteins were then immunoblotted and tested with anti-GAPDH antibodies (Proteintetech, China; #10494-1-AP, 1:5,000), anti-CEBP α antibodies (Abcam, UK; ab40764, 1:1,000), anti-PPAR γ antibodies (Abcam, UK; ab59256, 1:1,000) and anti-FABP 4 antibodies (Abcam, UK; ab92501, 1:1,000) overnight at 4 ℃. The following day, a secondary antibody (Abcam, UK; ab205718, 1:10,000) was used for incubation at room temperature for 1 hour, followed by 3 washes with TBST for 10 minutes. ImageJ software was used to quantify the immunoreactive bands.

(II) in vivo experiments

(1) Constructing a nude mouse subcutaneous human granular fat transplantation model (fig. 6):

all animal experiments were approved by the ninth national hospital affiliated with Shanghai university of transportation medical school. Female nude mice (6 to 8 weeks old) were housed in individual cages with a 12 hour light/dark cycle and provided with standard food and water. Mice were randomized into three groups (6 per group): saline, 10. mu. mol/L and 50. mu. mol/L groups. 0.2ml of Coleman fat was injected subcutaneously on both the left and right sides of the back of each mouse using a 1ml syringe with a blunt infiltration cannula. The graft is injected in a spherical shape. Mice were injected topically every two days with 0.2ml of saline or salvianolic acid B (10. mu. mol/L, 50. mu. mol/L). On day 14, the left dorsal grafts were harvested and carefully separated from the surrounding tissue and their volume and weight were measured. The wound is sutured with 6-0 nylon suture, and the affected part is coated with antibiotic ointment for 1 week to prevent topical infection. Histological and immunohistochemical evaluation was performed on each harvested sample. On day 28, the fat grafts were scanned by CT, harvested and carefully separated from the surrounding tissue, and their volume and weight were measured. Histological and immunohistochemical evaluation was performed on each harvested sample.

(2) The fat retention in the drug-treated group at week 4 (50 μmol/L) was found to be higher than that in the control group by collecting samples 2 weeks, 4 weeks after transplantation (fig. 7A, 7B):

graft volume was measured using the drainage method and weight was weighed using an electronic balance.

(3) HE and Perlipin histological staining suggested more complete viable adipose structure in drug-treated groups (fig. 7C, 7D):

tissues were fixed with paraformaldehyde overnight, paraffin embedded, cut to 5 μm thick, and stained with hematoxylin and eosin. For immunofluorescent staining, tissue sections were incubated overnight at 4 ℃ with primary antibodies against Perilipin (Proteitech, #15294-1-AP, China, 1:200) diluted in blocking solution. After incubation with Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (Invitrogen, # A-21206, USA, 1:500), the nuclei were stained with DAPI (Southern Biotech, USA). Image-Pro Plus 6.0 software was used for quantitative analysis.

The mechanism of the invention is as follows: the Salvianolic acid B is observed to promote the proliferation of adipose-derived stem cells by treating the adipose-derived stem cells with the Salvianolic acid B (Sal B), and promotes adipogenic differentiation by up-regulating PPAR gamma, CEBP alpha and FABP 4. The promotion of the proliferation and differentiation of the adipose-derived stem cells is particularly important for improving the survival rate of fat transplantation, and the survival rate of the transplanted fat can be effectively improved by treating the transplanted fat with salvianolic acid B, so that the method has wide application prospects in the aspects of improving the treatment of soft tissue defects and the like of clinical autologous fat transplantation.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.