CN113827616A

CN113827616A - Preparation method and application of nano fat extract

Info

Publication number: CN113827616A
Application number: CN202110962840.6A
Authority: CN
Inventors: 单乐天
Original assignee: Hangzhou Sanjiang Shangyu Biotechnology Co ltd
Current assignee: Hangzhou Sanjiang Shangyu Biotechnology Co ltd
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2021-12-24

Abstract

The invention discloses a preparation method of a nano-fat extract and application thereof, wherein the preparation method specifically comprises the steps of 1) extracting adipose tissues; 2) breaking fat tissue; 3) digesting and centrifuging adipose tissues; 4) obtaining nano fat; 5) extracting paracrine substances of the nano fat cells. The invention collects and extracts the paracrine substances of the nano-adipocyte by utilizing the paracrine action mechanism of the nano-adipocyte, and is applied to the treatment research of the knee osteoarthritis.

Description

Preparation method and application of nano fat extract

Technical Field

The invention belongs to the field of medicines, and particularly relates to a preparation method and application of a nano fat extract.

Background

Nano-fat (nanofat) is a mixture of cells obtained from adipose tissue, mainly consisting of mesenchymal stem cells, endothelial progenitor cells, stromal cells and immune cells. The nano fat is widely applied to the field of regenerative medicine, plays a role in promoting tissue regeneration, and plays a role mainly through fat stem cells contained in the nano fat. MSCs are a broad class of adult multipotent stromal cells isolated from adult tissues, with self-renewal capacity, multi-lineage differentiation potential, paracrine effects, and immunomodulatory properties. However, the clinical use of MSCs raises ethical concerns, as the in vitro amplification of MSCs may lead to genetic and epigenetic alterations. In addition, mesenchymal stem cell expansion in vitro is time consuming and presents a potential clinical risk of delayed treatment. Compared with MSCs and other cell therapies, the nano-fat can be directly obtained from adipose tissues, the operation is simple and convenient, and the overnight culture is not needed, so that the nano-fat is safer and less limited compared with MSCs; different types of cells in the nano-fat have synergistic effect in the aspects of immune regulation, anti-inflammation, angiogenesis and the like, and the curative effect of the nano-fat is possibly superior to that of single cell treatment; the homing effect of nano-fat is stronger than MSCs because culture expansion results in the loss of homing properties, which mainly mediates the regeneration mechanism of MSCs. However, in the clinical application process, the adipose-derived stem cells in the nano-adipose lose their activities with the passage of time, and it is difficult to maintain the activities for a long time, so that the therapeutic effects are greatly affected. Nano-fats contain various types of cells, and many soluble factors can be secreted, possibly leading to paracrine results. Therefore, in order to solve the problem, the invention utilizes the action mechanism of the nano-adipocyte paracrine, collects and extracts the paracrine substances of the nano-adipocyte, and is applied to the treatment research of the knee osteoarthritis.

Disclosure of Invention

Aiming at the problems that the activity of adipose-derived stem cells in nano-adipose is lost with the passage of time and the long-term activity maintenance is difficult to realize, so that the treatment effect is poor in the prior art, the invention aims to provide a preparation method of a nano-adipose extract and further aims to provide application of the nano-adipose extract in treatment of knee osteoarthritis.

The invention is realized by the following technical scheme:

the preparation method of the nano fat extract is characterized by comprising the following steps:

1) 3-week-old SD rats are anesthetized by 3% pentobarbital intraperitoneal injection, then killed, placed in 75% alcohol, taken into a cell room, and under the aseptic condition, the adipose tissues at the inguinal position of the SD rats are separated, the taken adipose tissues are placed in a culture dish containing double-antibody PBS, and the culture dish is moved into a clean bench for the next operation;

2) further cleaning fat tissue in a super clean bench, separating, removing muscle and fascia tissue, cleaning for 3 times by PBS containing double antibodies, and finally cutting the fat tissue into minced meat;

3) preparing IV-type collagenase by PBS (phosphate buffer solution) to digest adipose tissues, sucking minced adipose tissues obtained in the step 2) into a centrifugal tube, adding the prepared IV-type collagenase, sealing the centrifugal tube by using a sealing film, and placing the centrifugal tube on a constant-temperature shaking table for digestion;

4) centrifuging the centrifuge tube at room temperature after digestion, removing supernatant after centrifugation, adding PBS for resuspension and precipitation, filtering with a 100 μm cell sieve, and centrifuging the filtered solution to obtain nanometer fat;

5) inoculating the nano fat obtained in the step 4) into a culture dish of 10CM in alpha-MEM (alpha-MEM) culture medium, culturing for 48h, collecting supernatant, centrifuging to remove cell debris, performing sterile filtration by using a 0.22 mu m filter to obtain Nanofat-80 ℃ nano fat cell paracrine substances for later use.

Further, 1% penicillin-streptomycin double-antibody mixed solution is added into the PBS in the step 1).

Further, the collagenase type IV in the step 3) has a concentration of 0.2%, and is preferably added in an amount of 2-3ml more than that of the adipose tissue under the digestion conditions of 37 ℃ and 400rpm for 30 min.

Further, the centrifugation time in the step 4) is 5 minutes, and the centrifugation rotating speed is 1000 rpm/min.

Further, the seeding density of the nano-fat in the step 5) is 1x10⁶And each dish, wherein the alpha-MEM culture medium contains 10% FBS, the centrifugation time is 10min, and the centrifugation rotating speed is 1500 rpm/min.

The application of the nano fat extract in preparing the medicine for treating knee osteoarthritis is disclosed.

The invention collects and extracts the paracrine substances of the nano-adipocyte by utilizing the paracrine action mechanism of the nano-adipocyte, and is applied to the treatment research of the knee osteoarthritis.

Drawings

FIG. 1 is a flow detection analysis chart of nano-fat and ADSCs;

FIG. 2 is a graph of SO staining, H & E staining and Col2 immunohistochemical staining of knee joints;

FIG. 3 is a quantification chart of OARSI scores, MMT and TWL in the NC, Model, Nanofat and ADSCs panel;

FIG. 4 shows that Nanofat-CM promotes chondrocyte proliferation and induces IL-1 β -treated chondrocyte migration; wherein, FIG. 4A shows the proliferation of chondrocytes after treatment with Nanofat-CM; FIG. 4B is a cell scratch; FIG. 4C is a quantification of a cell migration assay;

FIG. 5 shows the effect of Nanofat and Nanofat-CM on IL-1 β -induced chondrocytes; wherein, figure 5A is qPCR analysis of chondrocyte mRNA expression co-cultured with Nanofat; FIG. 5B is a qPCR analysis of mRNA expression from chondrocytes treated with Nanofat-CM; fig. 5C is the expression of the target protein in chondrocytes.

Detailed Description

The invention is further described in the following with reference to the drawings and specific test examples to better explain the technical scheme of the invention.

The preparation process of the nano fat extract comprises the following steps:

1) SD rats of 3 weeks old were first anesthetized by intraperitoneal injection with 3% pentobarbital, then sacrificed, placed in 75% alcohol, and taken into the cell house. Under aseptic conditions, the adipose tissues at the inguinal region were carefully separated with an ophthalmic scissors, the fascia tissue was carefully isolated, and the removed adipose tissues were placed in a petri dish containing PBS to which 1% diabesin (Penicillin-Streptomycin Solution) mixture was added. The culture dish is moved to an ultra clean bench for further operation.

2) The adipose tissue was further cleaned in a clean bench with ophthalmologic forceps and ophthalmologic scissors, separated, muscle and fascia tissue removed, and washed 3 times with PBS containing double antibody. Finally, the adipose tissues are cut into meat paste by an ophthalmic scissors.

3) Adipose tissue was digested with collagenase type iv formulated with PBS at 0.2%. Sucking the cut adipose tissues into a 15ml centrifuge tube by using a 1ml tip with the tip cut off, and adding the prepared IV type collagenase, wherein the addition amount is 2-3ml more than that of the adipose tissues. The tube was then sealed with a sealing film, placed on a constant temperature shaker, and digested at 37 ℃ and 400rpm for 30 min.

4) After digestion, the tube was centrifuged at 1000rpm/min for 5min at room temperature. After the centrifugation is finished, the supernatant is removed, PBS is added to resuspend the precipitate, then the filtration is carried out by using a cell sieve with the particle size of 100 mu m, and finally the filtered solution is centrifuged (1000 rpm/min, 5 min) to obtain the Nanofat.

5) The Nanofat obtained in the step 4 is mixed with 1x10⁶The cells were plated at a density of 10cm in a culture dish, and the medium was α -MEM (containing 10% FBS). After 48h incubation, the supernatant was collected, centrifuged at 1500rpm/min for 10min to remove cell debris, and sterile filtered through a 0.22 μm filter to obtain Nanofat-CM, which was stored at-80 ℃ for further use.

The effect of the prepared paracrine substance of the nano-adipocyte on the aspect of treating knee osteoarthritis is shown in each test example.

Test example 1: flow cytometry

Mixing Nanofat and ADSCs at 10%⁶Individual cells/ml were suspended in PBS at a density and incubated with the following antibodies: FITC-CD29, PE-CD34, APC-CD44, and FITC-CD 90. After incubation for 30 minutes at room temperature, each cell suspension was centrifuged (2000 rpm/min, 5 min). The supernatant was removed and 100. mu.l PBS was added to resuspend the cell pellet for flow cytometry analysis. The results are shown in FIG. 1.

As can be seen from fig. 1, the positive expression rates of CD29, CD34, CD44 and CD90 in Nanofat were 86.43%, 19.13%, 8.53% and 68.77%, respectively, and the positive expression rates of CD29, CD34, CD44 and CD90 in ADSCs were 99.99%, 0.26%, 99.94% and 99.97%, respectively.

Test example 2: immunohistochemistry

All rats were sacrificed 4 weeks after treatment. The joints of each rat were sampled and fixed with 10% formalin for 24 hours and decalcified with 10% EDTA in PBS for 8 weeks. Each sample was embedded in paraffin and cut to 2-3 μm and stained with HE or SO. The grade of OA progression was assessed by double-blind observation according to the OARSI scoring system. Immunohistochemistry was used to detect the expression of collagenase type II (Col 2). Duplicate sample sections were incubated with 0.01 mol/l citrate buffer at 60 ℃ for 4 hours as antigen retrieval, and then incubated overnight at 4 ℃ with 100. mu.l PBS-diluted (1:100) rat Col2 primary antibody. After PBS washing, all sections were incubated with horseradish peroxidase-conjugated secondary antibody for 20 minutes at room temperature, followed by 8 minute colorimetric detection using DAB. Immunoreactivity of Col2 was semi-quantified under light microscopy using Image-Pro Plus 6.0 software. The results are shown in FIGS. 2-3.

As can be seen from fig. 2: the pathological staining and immunohistochemical staining results of the Model group show that cartilage degeneration caused by chondrocyte loss and collagen damage is caused, and the chondrocytes and the collagen are recovered after treatment by the Nanofat or the ADSCs; as can be seen from FIG. 3, the Model cohort significantly reduced TWL and MWT levels compared to the NC cohort (P<0.01). Significantly improved TWL and MWT levels after treatment with Nanofat and ADSCs (both) compared to the Model groupP<0.05). Results of OARSI scores showed a significant increase in OARSI scores for the Model cohort over the NC cohort (P<0.01). The OARSI scores were significantly reduced for the Nanofat and ADSCs groups compared to the Model group (bothP<0.01）。

Test example 3: CCK8

Mixing chondrocytes at 5 × 10³The density of individual cells/well was plated in 96-well plates, cultured in 200. mu.l medium for 24 hours, and then plated with cells from different cell densities (50, 100, 150, 200X 10)⁴Individual cells) for 24 hours and 48 hours. Add 20. mu.l of CCK-8 solution to each well and incubate at 37 ℃ for 2h until the color becomes orange. Optical Density (OD) values were measured at 450nm using a microplate reader. Proliferation rate (%) = (Nanofat-CM treated OD/untreated OD) × 100. Such as 4A in fig. 4.

As shown in FIG. 4A, after the treatment with Nanofat-CM for 24 hours and 48 hours, the proliferation rate of chondrocytes increased with the increase in the concentration thereof

Test example 4: scratch test

In logarithmic growth phaseThe cartilage cells of (2) are at 3X 10⁵The density of individual cells/well was plated in 6-well plates and divided into Control group, IL-1. beta. group and Nanofat-CM group. All groups were scraped using a sterile 200 μ l pipette tip and then gently washed twice with PBS to remove cell debris. Subsequently, the nafat groups were treated with nafat-CM for 0, 12 and 24 hours. Cells were observed under an inverted microscope and imaged. The scratch area was calculated by Image J1.47 software and the results are shown in fig. 4B and 4C.

As is clear from FIG. 4B, the wound surface area of chondrocytes in IL-1. beta. group was larger than that in Control group. Compared with the IL-1 beta group, the wound surface of the chondrocytes treated by the Nanofta-CM is smaller, and the wound surface healing capacity is stronger.

As can be seen from FIG. 4C, after 12h and 24h, the area of the IL-1 β group was larger than that of the Control group, and the area of the Nanofat-CM group was smaller than that of the Control group.

Test example 5: PCR

The medium was first discarded, washed 1 time with PBS and added to RNAioso Plus, blown up and transferred to 1.5ml EP tubes. Standing for 5min, adding 200 μ l of nucleic acid extractive solution, shaking, mixing, and standing for 5 min. Then centrifuged at 12000rpm for 15 minutes at 4 ℃. The upper colorless transparent supernatant was aspirated and transferred to a new EP tube, isopropanol was added, mixed well and left to stand for 10 minutes. The RNA pellet was then precipitated by centrifugation at 12000rpm for 10 minutes at 4 ℃. The supernatant was discarded, and the precipitate was washed with 75% ethanol, centrifuged at 7500rpm at 4 ℃ for 5 minutes, and the supernatant was discarded to obtain RNA. The RNA was dissolved in DEPC water and the purity and integrity of the samples were measured using a NanoDrop2000 spectrophotometer, with a wavelength absorption ratio (260/280 nm) of approximately 2.0 for all samples. Then, reverse transcription was performed to obtain cDNA. As previously described, the final qPCR reaction system was 20. mu.l, including 10. mu.l SYBR, 0.4. mu.l forward primer, 0.4. mu.l reverse primer, 1. mu.l template cDNA and 8.2. mu.lddH₂And O. qPCR reaction conditions included a pre-incubation at 95 ℃ for 5 minutes, 40 cycles, denaturation at 95 ℃ for 10 seconds, annealing at 60 ℃ and extension for 30 seconds. Taking beta-Actin as an internal reference gene and adopting 2^-ΔΔCTThe method measures the relative expression level of mRNA.

Test example 6: western Blot

The chondrocyte culture dish was first placed on ice, the medium was discarded, washed 2 times with PBS, and then fresh PBS was added. The cell suspension was collected in an EP tube using a spatula and centrifuged at 7500rpm for 5 minutes at 4 ℃. The supernatant was discarded, and a lysis buffer (50 mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 1% Triton and 0.1% SDS) containing a mixture of phosphatase and protease inhibitors was added to the chondrocytes, and incubated on ice for 30 minutes. Centrifuging at 4 deg.C for 15 min at 12000rpm, and collecting supernatant to obtain total protein. Protein concentration was assessed by BCA kit.

The target protein was separated by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 6-12%) and transferred to nitrocellulose membrane. Membranes were blocked with 5% skim milk in Tris Buffered Saline Tween (TBST) for 2 hours at 4 ℃, then incubated overnight at 4 ℃ with primary antibodies against β -actin, Col2, Mmp9, Mmp13 and Sox9, below. Subsequently, the membranes were washed 3 times with TBST and incubated with peroxidase-coupled rabbit/mouse antibody for 2 hours at room temperature, detected and scanned using ECL luminophores and X-ray films. The results are shown in FIG. 5.

As can be seen from fig. 5A: nanofat significantly down-regulates COL10, ADAMTS5 and MMP13 in chondrocytes (all of whichP<0.01), Up-Regulation COL2 (P<0.01); as can be seen from fig. 5B: nanofat significantly down-regulates COL10, IL-6, ADAMTS5, MMP3 and MMP13 in chondrocytes (all of whichP<0.01), up-regulation COL2, SOX9 and AGGRECAN (all of whichP<0.01); as can be seen from fig. 5C: nanofat significantly down-regulates MMP9 and MMP13 in chondrocytes (bothP<0.01), up-regulating the expression of COL2 and SOX9 (both)P<0.01）。

Claims

1. The preparation method of the nano fat extract is characterized by comprising the following steps:

2. The method for preparing nano fat extract as claimed in claim 1, wherein 1% penicillin-streptomycin double antibody mixed solution is added to PBS in step 1).

3. The method for preparing nano fat extract according to claim 1, wherein the collagenase type IV is added in an amount of 2-3ml more than the adipose tissue in step 3) at a temperature of 37 ℃, a rotation speed of 400rpm, and a digestion time of 30 min.

4. The method for preparing nano fat extract according to claim 1, wherein the centrifugation time in step 4) is 5 minutes and the centrifugation speed is 1000 rpm/min.

5. The method for preparing nano fat extract as claimed in claim 1, wherein the nano fat seeding density in step 5) is 1x10⁶Person/dish, instituteThe alpha-MEM culture medium contains 10% FBS, the centrifugation time is 10min, and the centrifugation rotating speed is 1500 rpm/min.

6. Use of the nano-fat extract obtained by the method of any one of claims 1 to 5 in the preparation of a medicament for the treatment of knee osteoarthritis.