CN109679901B

CN109679901B - Adipogenic induced differentiation culture solution and adipogenic induced differentiation method

Info

Publication number: CN109679901B
Application number: CN201910127743.8A
Authority: CN
Inventors: 黄飞; 徐晓艳; 李玉秋; 周鹏
Original assignee: Shandong Huasi Biotechnology Co ltd
Current assignee: Shandong Huasi Biotechnology Co ltd
Priority date: 2019-02-21
Filing date: 2019-02-21
Publication date: 2022-06-24
Anticipated expiration: 2039-02-21
Also published as: CN109679901A

Abstract

The invention relates to a adipogenic induction culture solution and an adipogenic differentiation method. The adipogenic induction culture solution comprises: 3-isobutyl-1-methylxanthine, insulin, dexamethasone and triflusal. The combined application of multiple induction factors in the invention can effectively induce the adipogenic differentiation of the adipose-derived stem cells, shorten the adipogenic time and improve the adipogenic quality when being used for human adipose-derived stem cells. The adipogenic differentiation effect is obviously superior to that of the conventional induction method.

Description

Adipogenic induced differentiation culture solution and adipogenic induced differentiation method

Technical Field

The invention relates to a adipogenic differentiation induction culture solution and an adipogenic differentiation induction method.

Background

Stem cells (stem cells) are a type of pluripotent cells with self-replicating capacity (self-rejuvenating). Under certain conditions, it can differentiate into multiple functional cells. The stem cells are classified into embryonic stem cells (ES cells) and adult stem cells (somatic stem cells) according to the development stage of the stem cells. The stem cells are classified into three types according to their developmental potential, Totipotent Stem Cells (TSC), pluripotent stem cells (pluripotent stem cells), and unipotent stem cells (multipotent stem cells). Stem cells (Stem cells) are insufficiently differentiated and immature cells, have the potential function of regenerating various tissues, organs and human bodies, and are called "universal cells" in the medical field.

Human adipose-derived stem cells (HADSCs), a type of stem cell having a multi-differentiation potential, which has been recently isolated from adipose tissue. Mainly recovers the repairing function of tissue cells, promotes the regeneration of cells, recovers the young face, simultaneously fully improves the body function, effectively improves diseases such as sub-health, premature senility and the like, and really and effectively resists aging from inside to outside. HADSCs are adult stem cells which are extracted from adipose tissues and have multipotential differentiation potential, have the advantages of sufficient sources, convenient material taking, simple operation, high extraction rate, easy in vitro culture, stable biological properties, low immunogenicity, no ethical limitation and the like, and are widely concerned in recent years.

The existing adipogenesis induction time is generally more than 14 days, the adipogenesis effect of 14 days is generally small liquid, the reaction time is long, and the adipogenesis effect is general.

Disclosure of Invention

Based on the defects of the prior art, the invention provides a adipogenic differentiation induction culture solution. So that the fat forming time is short and the fat forming amount is high.

A adipogenic differentiation induction culture solution comprises the following raw material components: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, and triflusal.

Preferably, the raw material components and the dosage of the culture solution comprise: 0.25-1umol/L of 3-isobutyl-1-methylxanthine, 1-10mg/L of insulin, 0.5-1.5umol/L of dexamethasone and 50-250umol/L of triflusal.

Preferably, the raw material components and the dosage of the culture solution comprise: 3-isobutyl-1-methylxanthine 0.5umol/L, insulin 10mg/L, dexamethasone 1umol/L and triflusal 200 umol/L.

The invention also provides a adipogenic induction differentiation method, which comprises the following steps:

(1) adopting low-sugar DMEM culture solution containing 10% fetal calf serum to amplify the adipose-derived stem cells;

(2) inoculating the third generation cells of the adipose-derived stem cells of step (1) into a 6-well plate, adding a low-sugar DMEM culture solution containing 10% fetal bovine serum, starting to add the adipogenic differentiation culture solution of any one of claims 1 to 3 when the cells grow to 100%, and replacing the adipogenic differentiation culture solution every three days.

Has the beneficial effects that: the invention improves the adipogenesis effect of the adipose-derived stem cells on the premise of shortening the adipogenesis time. The technology can obviously shorten the fat-forming time and improve the fat-forming quality. The invention can achieve the effect of traditional fat induction for more than 14 days when the induction is carried out for 9 days, thereby saving the reaction time and improving the fat forming effect.

Drawings

FIG. 1 is a CD44 and CD105 specific marker profile.

FIG. 2 is a map of CD34 and CD45 specific markers.

FIG. 3 is a photograph of the cells of example 1 stained under an oil red O staining microscope after 9 days of induction.

FIG. 4 is a photograph showing the staining of the cells of comparative example 1 observed under an oil red O staining microscope after 9 days of induction.

FIG. 5 is a photograph of the cells of comparative example 2 stained under an oil red O staining microscope after 9 days of induction.

FIG. 6 is a photograph showing the staining of the cells of comparative example 3 observed under an oil red O staining microscope after 9 days of induction.

FIG. 7 is a photograph showing the staining of the cells of comparative example 4 observed under an oil red O staining microscope after 9 days of induction.

Detailed Description

The invention provides a adipogenesis induction differentiation culture solution, which comprises the following raw material components: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, and triflusal.

As an embodiment of the invention, the raw material components and the dosage of the culture solution specifically comprise: 3-isobutyl-1-methylxanthine 0.25-1umol/l, insulin 1-10mg/l, dexamethasone 0.5-1.5umol/l and triflusal 50-250 um.

Example 1

A adipogenic induced differentiation method comprising the steps of:

1.1 adipose-derived Stem cells extraction and culture

5g of subcutaneous adipose tissues of lying-in women after caesarean delivery in obstetrical department of hospitals affiliated to Binzhou medical colleges, the lying-in women have no other basic diseases and are not treated by special medicines, and informed consent of the lying-in women is obtained before material drawing. Fully rinsing with PBS containing 500U/ml double antibody for 3 times, and removing blood water; removing macroscopic blood vessels and connective tissues from the PBS liquid, and washing; sufficiently cutting the raw materials into pieces by using an ophthalmic scissors; repeatedly washing with PBS to remove erythrocytes as much as possible; sucking the fat fragments into a 50ml centrifuge tube by using a pipette; adding 2 times of 0.2% collagenase type I; after oscillating and mixing uniformly, digesting in a constant-temperature water bath kettle at 37 ℃ for 60min, and shaking once every 10 min; adding isovolumetric low-sugar DMEM containing fetal bovine serum with a volume fraction of 10% to terminate digestion; centrifuging at 1800rpm for 10 min; centrifuging, dividing into 3 layers, wherein the upper layer is grease and undigested complete adipose tissue, the middle layer is supernatant, and the lower layer is adipose stem cells, red blood cells and the like; sucking the upper layer and the middle layer by using a 5ml suction pipe; add 1ml of complete medium to resuspend the cells; filtering with 200 mesh cell filter sieve, and inoculating into 100mm culture dish; then sucking 1ml of complete culture medium, washing the centrifuge tube, and repeating the previous step; culturing at 37 deg.C and 5% CO2 saturation temperature; after 24h, half-amount changing of the culture solution by using a low-sugar DMEM culture solution containing 10% fetal calf serum; after 48 hours, the liquid is changed in full quantity; after that, 10ml of the solution is changed every 3 days. The first generation of adipose-derived stem cells was obtained. And (5) after 80-90% of the cells are grown in the dish for passage.

1.2 plating and Induction of adipose Stem cells

When the first generation of cells grew over 80% -90% of the dish for passage, the cell supernatant was aspirated and rinsed 2 times with PBS, 3ml each time, TE 2ml placed at room temperature for 30min, then resting in a CO2 incubator at 37 ℃ for 4min, and observing cell rounding under a microscope, 3ml of low-sugar DMEM medium containing 10% fetal calf serum was added to the cell dish in a clean bench for termination of digestion. And (4) carrying out passage by two in each dish to obtain the second generation, and when 80-90% of the second generation cells grow over the dish and are passaged by the same method, the obtained third generation cells need to be inoculated on a 6-well plate for fat stem cell induced adipogenesis experiments. The number of cells per well in a 6-well plate was kept at 2X 10 by blood cell count⁴And (4) respectively. Changing the culture solution every 3 days for 3ml each time, and adding the adipogenic differentiation culture solution when the cells grow to 100%. The inducing solution was changed 3ml each time every 3 days, and after continuous culture for 9 days, the cells were stained with oil red O, and the staining state was observed under a microscope and photographed.

The preparation method of the adipogenic differentiation induction culture solution comprises the following steps:

3.9246mg dexamethasone was dissolved in 10ml ethanol to make 1mM stock solution.

20mg of insulin was dissolved in 2ml of 10mM HCl to prepare a 10ug/ml stock solution.

50mg IBMX was dissolved in 270. mu.l of 1M KOH and 4.23ml of double distilled water to prepare a 50mM stock solution.

100mg of triflusal was dissolved in 0.22um of 8.06ml of acetone to prepare a 50mM stock solution (stored under dark conditions). The above inducing components are filtered by 0.22um filter head at-20 deg.C for use.

The dosage of each component in the adipogenic induction culture solution is as follows: 10mg/L of insulin, 1umol/L, IBMX 0.5.5 umol/L of dexamethasone and 200um umol/L of triflusal.

1.3 adipose Stem cell adipogenic staining

Preparing an oil red O dye solution, weighing 0.15g of oil red O powder, fixing the volume to 30ml by using isopropanol, violently shaking up and down to dissolve the oil red O, filtering by using a funnel, obtaining a mother solution of the oil red O dye solution after filtering, and preparing the oil red O mother solution according to the following steps: and preparing oil red O working solution by using double distilled water in a ratio of 9: 6. And (3) absorbing and removing the cell induction liquid of a six-hole plate, washing each hole with PBS for 2 times and 3ml each time, adding 2ml of 4% paraformaldehyde to fix the cells for 1 hour, absorbing and removing the supernatant, washing each hole with PBS for 2 times and 3ml each time, adding 2ml of oil red O working solution to stain for 30min, absorbing and removing the supernatant, and washing each hole with PBS for 2 times and 3ml each time. Decolorizing with 75% ethanol, 1ml per well, 20 s. The supernatant was aspirated and washed with 3 ml/time PBS until the aspirated supernatant was colorless, and finally 1ml PBS was retained in each six-well plate, and the staining was observed under a microscope and photographed as shown in FIG. 3.

1.4 identification of surface markers of adipose-derived Stem cells

And identifying cell surface specific markers CD34, CD44, CD45 and CD105 by adopting third-generation ADSC, wherein the experimental steps are as follows:

1) one tube of PBS was taken for use. The adipose stem cells were trypsinized, centrifuged for 1000r 4min, resuspended in PBS, counted, 3X 105 cells were removed per 2ml EP tube, centrifuged for 1000r 4min, the supernatant was discarded, and a final volume of 100/50ul PBS was added for resuspension.

2) Taking 6 tubes of cells:

a. blank cells 50. mu.l PBS;

b. add 1. mu.l CD44-FITC antibody + 99. mu.l PBS;

c. add 2.5. mu.l CD105-PE antibody + 47.5. mu.l PBS;

d. add 2.5. mu.l CD34-APC antibody + 47.5. mu.l PBS;

e. 2.5. mu.l of CD45-PerCP-cyanine5.5 antibody + 47.5. mu.l PBS was added;

f. 1/5. mu.l of each of the 4 antibodies was added to the cells + 84. mu.l PBS.

3) Incubate at 4 ℃ for 45min in the dark (shake 1 time every 15 min).

4) After incubation, 1ml of PBS is added for resuspension and centrifugation for 1000r 4min, the supernatant is discarded, 1ml of PBS is added for washing again, 500ul of PBS is added for resuspension, and detection is carried out by an up-flow cytometer.

After the adipose-derived stem cells are matured, specific markers of the adipose-derived stem cells are identified by flow cytometry, CD44 and CD105 are expressed positively, CD34 and CD45 are expressed negatively, and the specific markers indicate that the cells are the adipose-derived stem cells. The results are shown in FIGS. 1 and 2.

Comparative example 1

Comparative example 1 is the same as example 1. The difference is that the dosage of each component in the adipogenic induction culture solution is as follows: 10mg/L of insulin and 1umol/L, IBMX 0.5.5 umol/L of dexamethasone.

Comparative example 2

Comparative example 2 is the same as example 1. The difference is that the dosage of each component in the adipogenic induction culture solution is as follows: 10mg/L of insulin, 1umol/L, IBMX 0.5.5 umol/L of dexamethasone, 200umol/L of triflusal and 200umol/L of indomethacin.

Wherein the configuration of the indometacin is as follows: 100mg of indomethacin was dissolved in 5.59ml of acetone to prepare a 50mM stock solution.

Comparative example 3

Comparative example 3 is the same as example 1. The difference is that the dosage of each component in the adipogenic induction culture solution is as follows: 10mg/L of insulin, 1umol/L, IBMX 0.5.5 umol/L of dexamethasone and 200umol/L of indomethacin.

Comparative example 4

Comparative example 4 is the same as example 1. The difference is that the dosage of each component in the adipogenic induction culture solution is as follows: dexamethasone 1umol/L, IBMX 0.5.5 umol/L, triflusal 200umol/L, and indometacin 200 umol/L.

The adipogenic induction culture fluids and the amounts thereof of the examples and comparative examples of the present invention are shown in Table 1.

TABLE 1 adipogenic induction culture medium and its amount for each example of the present invention and comparative example

As shown in figures 3-7, after the cells are induced for 9 days, oil red O staining is carried out to observe cell adipogenesis, and the effect of the triflusal is more obvious than that of indomethacin, so that the time for inducing adipogenesis is shorter, the fat drop is larger, and the adipogenesis effect is better. As shown in fig. 5, when triflusal and indomethacin were used together, a significant decrease in the adipogenic effect was observed. Triflusal is not only an inhibitor of cyclooxygenase, but also an inhibitor of phosphodiesterase, a key pathway in adipogenesis; its promoting effect is better. Although indomethacin can promote adipogenesis of adipose stem cells, the excessive concentration of indomethacin can not promote adipogenesis of adipose stem cells, but can inhibit the adipogenesis effect.

The effect of the triflusal is more obvious than that of indometacin, and the triflusal can be induced to form fat in shorter time, and has larger fat drops and better fat forming effect.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments of the invention are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A adipogenic induction differentiation culture solution is characterized by comprising the following raw material components in parts by weight: 0.25-1umol/L of 3-isobutyl-1-methylxanthine, 1-10mg/L of insulin, 0.5-1.5umol/L of dexamethasone and 50-250umol/L of triflusal.

2. The culture solution according to claim 1, wherein the culture solution comprises the following raw material components in parts by weight: 3-isobutyl-1-methylxanthine 0.5umol/L, insulin 10mg/L, dexamethasone 1umol/L and triflusal 200 umol/L.

3. A adipogenic induced differentiation method comprising the steps of:

(2) inoculating the third generation cells of the adipose-derived stem cells of step (1) into a 6-well plate, adding a low-sugar DMEM culture solution containing 10% fetal bovine serum, starting to add the adipogenic differentiation culture solution of any one of claims 1-2 when the cells grow to 100%, and replacing the adipogenic differentiation culture solution every three days.