CN111269888A

CN111269888A - Adipostem cell modified by adipsin gene, preparation method and application thereof

Info

Publication number: CN111269888A
Application number: CN202010212650.8A
Authority: CN
Inventors: 刘明录; 冯建海; 金海锋; 王立新; 卢永灿; 强邦明; 张传鹏; 王亮; 许淼
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shandong Xinrui Biotechnology Co ltd
Priority date: 2020-03-24
Filing date: 2020-03-24
Publication date: 2020-06-12
Anticipated expiration: 2040-03-24
Also published as: CN111269888B

Abstract

The invention provides an adipsin gene modified fat stem cell, which comprises an adipsin recombinant gene expressing adipsin protein, and also provides a preparation method and application of the fat stem cell.

Description

Adipostem cell modified by adipsin gene, preparation method and application thereof

Technical Field

The invention relates to an adipsin gene modified adipose-derived stem cell, a preparation method and application thereof, and belongs to the technical field of biological medicines.

Background

With the improvement of living standards and the change of dietary structures of people, the incidence of diabetes mellitus in China is rapidly increasing, and recent research data shows that about 1.14 hundred million adults in China have diabetes mellitus, the prevalence rate is as high as 11.6%, and the people are the first to live in the world, the conventional treatment method of diabetes mellitus is oral administration or injection of antidiabetic drugs, hyperglycemia and insulin sensitivity can be temporarily improved, but insulin resistance cannot be reversed (glucose metabolism capability regulated by insulin is reduced), functional failure of insulin β cells cannot be repaired, and long-term use of insulin is easy to form drug resistance.

In the prior art, an in-vitro contact co-culture mode of adipose-derived stem cells (ADSCs) and islet cells is mostly adopted, that is, the isolated and cultured adipose-derived stem cells and the purified islet cells are in-vitro contact co-cultured, and the influence of the ADSCs on the survival rate of the in-vitro cultured islet β cells and the insulin secretion function is observed.

The cells are co-cultured by the ADSCs and the islet β cells, the cells are easy to agglomerate in the culture process, the cell growth is inhibited, the insulin secretion of the islet β cells is reduced, the blood sugar balance cannot be maintained for a long time, and meanwhile, the adipose-derived stem cells have poor effects of promoting the proliferation of the islet cells and the insulin secretion of the islet cells.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an adipsin gene modified fat stem cell, a preparation method and application thereof. The ADSCs carrying the adipsin gene can secrete HGF, VEGF, TGF-1, b-FGF and other growth factors and can secrete adipsin protein, wherein each nutritional factor can improve the microenvironment of damaged parts and inhibit apoptosis, and the following invention aims are achieved:

promoting the proliferation of islet cells, promoting the secretion of insulin from islet β cells, and improving insulin resistance.

In order to solve the technical problems, the invention adopts the following technical scheme:

an adipsin gene-modified adipose-derived stem cell, which comprises an adipsin recombinant gene expressing adipsin protein.

The method comprises the steps of artificially synthesizing an obtained adipsin gene sequence, constructing the adipsin gene sequence on a pAAV-IRES-ZsGreen1 adenovirus expression vector, transferring the pAAV-IRES-adipsin gene into a fat stem cell to enable the fat stem cell to express adipsin protein, namely the pAAV-adipsin fat stem cell, and verifying the capability of the fat stem cell in promoting insulin secretion of pancreatic islet cells and regulating and controlling glucose metabolism.

The adipsin recombinant gene comprises a signal peptide and an adipsin gene; the adipsin gene is a nucleotide sequence shown as SEQ ID NO.2 in a sequence table.

The amino acid sequence of the adipsin protein is shown as SEQ ID NO.4 in the sequence table.

The patent optimizes a self-inhibiting false substrate region of the Adipsin protein structure, and the region can be combined with a catalytic region sequence to inhibit the kinase activity.

Compared with the FD factor (accession number: NM-001317335) published on NCBI, the invention optimizes the sequence of the Adipsin gene, selects 8 nucleic acid sites for coding a false substrate region, and guides the synthesized protein to be changed by 3 amino acid sites. The optimized pseudo substrate region can be combined with a catalytic region sequence to inhibit kinase activity.

The nucleotide sequence of the Adipsin is specifically optimized to adopt A to replace G at the 15 th site, T to replace C at the 245 th site, A to replace C at the 246 th site, C to replace G at the 447 th site, C to replace T at the 562 th site, A to replace C at the 564 th site, A to replace T at the 650 th site and T to replace C at the 651 th site; the specific comparison results are shown in figure 3.

The amino acid sequence of Adipsin is specifically optimized to replace P by L at position 82, replace S by P at position 188 and replace I by N at position 217, and the specific comparison result of the amino acid sequence is shown in figure 4.

The signal peptide is a nucleotide sequence shown as SEQ ID NO.1 in a sequence table.

A preparation method of an adipsin gene modified adipose-derived stem cell comprises the steps of preparing a plasmid containing an adipsin gene expression vector, preparing the adipose-derived stem cell, packaging and detecting the titer of adenovirus, and infecting the adipose-derived stem cell with adenovirus.

The plasmid containing the adipsin gene expression vector is prepared by inserting a fusion gene comprising a signal peptide and a nucleic acid artificial sequence of adipsin into an adenovirus expression vector pAAV-IRES-ZsGreen1, and extracting and purifying the plasmid after correct conversion and sequencing to obtain the plasmid containing the adipsin gene expression vector.

Preparing the adipose-derived stem cells, wherein when the primary cell fusion degree is 78-82%, digestion passage is carried out, and the cells are transferred to P3 or P4 generations; performing flow cytometry instrument surface marker detection on P3 or P4 generation human adipose-derived stem cells, and targeting the cells (about 1X 10)⁶Individual cells) were added with anti-mouse anti-human monoclonal antibodies: CD29, CD34, CD44, CD105, CD90 and HLA ⁃ DR, when the expression rate of CD29, CD44, CD105 and CD90 is more than 98 percent and the expression rate of CD34 and HLA ⁃ DR is less than 1 percent by using flow cytometry detection analysis, the fat stem cell is identified;

the adenovirus packaging and titer detection, namely packaging the plasmid containing the adipsin gene expression vector by adopting adenovirus to obtain recombinant adenovirus; the titer of the recombinant adenovirus is 3.36 multiplied by 10⁷pfu/mL；

The adenovirus infects adipose-derived stem cells, and the method comprises the following steps:

(1) digesting and counting adipose-derived stem cells, and diluting to density of 4.5-5.5 × 10⁵Culturing the cells per mL for 20-28 h;

(2) taking recombinant adenovirus liquid (the virus titer is 3.3-3.4 × 10)⁷pfu/mL) is added with polybrene with the final concentration of 7.5-8.5 mug/mL to obtain virus liquid to be infected;

(3) and removing the culture medium from the adipose-derived stem cells, adding a virus solution to be infected, continuously culturing for 20-28h, removing the virus solution, adding a complete culture medium for culturing, simultaneously adding 2.8-3.2ug/mL puromycin for screening, continuously culturing for 4-6 days, and obtaining the adipose-derived stem cells infected by the adenovirus, which are named as pAAV-adipsin-adipose-derived stem cells. The flow cytometer detects the GFP expression rate of the pAAV-adipsin-adipose-derived stem cells, and the result shows that 23.5% of cells are GFP positive (the adenovirus vector expresses GFP protein), which indicates that the adipsin constructed by the invention can be expressed on the surface of the adipose-derived stem cells, and the transfection efficiency is 23.5%.

The application of the adipsin gene modified adipose-derived stem cell is characterized in that the adipsin gene modified adipose-derived stem cell and islet cells are subjected to non-contact co-culture; in the non-contact co-culture, adipsin gene modified fat stem cells are inoculated in the lower chamber of a culture plate, islet cells are inoculated in the upper chamber of the culture plate, the fat stem cells and the islet cells are adjusted to the same cell density by 9.5-10.5% FBS RPMI-1640 culture medium, and then culture is carried out, and the liquid is changed periodically.

The culture plate is a Transwell6 pore plate with an insertion layer, and the cell density of the adipsin gene modified adipose-derived stem cells and the islet cells is 0.8-1.2 multiplied by 10⁴Individual cells/well; the islet cells are INS-1 cells.

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

(1) the invention adopts adipostem cells modified by adipsin gene, which can improve the activity of islet cells, and compared with common adipose stem cells and islet cells, the activity of the islet cells is improved by 1 time after the adipostem cells modified by adipsin gene and the islet cells are co-cultured.

(2) The invention adopts adiposin gene modified fat stem cells, utilizes the paracrine action and the regeneration and repair action of the fat stem cells to secrete a plurality of growth factors, so that the proliferation capacity of the islet β cells is doubled, and the secreted adiposin protein can increase the insulin secretion of the islet cells, wherein the insulin secretion amount is 1.78-2.1 times that of the fat stem cells which are not modified by the genes.

The adipostem cell modified by the adipsin gene and the islet cell (INS-1 cell) are cultured together, and under the stimulation of glucose, the secretion of insulin is 23.3mIU/L at the 3 rd time, 29.5mIU/L at the 7 th time and 28.2mIU/L at the 14 th time.

Drawings

FIG. 1 is a schematic diagram of the structure of pAAV-IRES-adipsin expression vector;

FIG. 2 is a cell morphology diagram of a P3 generation adipose-derived stem cell under a light microscope;

FIG. 3 is a comparison of the nucleotide sequence of adipsin of the present invention and the existing FD factor with accession number NM-001317335;

FIG. 4 is a comparison of the adipsin amino acid sequence of the present invention and the existing FD factor with accession number NM-001317335;

FIG. 5 CellTiter-Glo method for detecting the activity of islet cells.

FIG. 6 is a graph comparing insulin secretion content in low sugar and high sugar cultures.

FIG. 7 is a flow chart of the expression level of the molecular marker CD29 on the surface of adipose-derived stem cells;

FIG. 8 is a flow chart showing the expression level of the molecular marker CD44 on the surface of adipose-derived stem cells;

FIG. 9 is a flow chart of the expression level of the adipose-derived stem cell surface molecular marker CD 105;

FIG. 10 is a flow chart showing the expression level of the molecular marker CD90 on the surface of adipose-derived stem cells;

FIG. 11 is a flow chart showing the expression level of the molecular marker CD34 on the surface of adipose-derived stem cells;

FIG. 12 is a flow chart showing the expression level of an adipose-derived stem cell surface molecular marker HLA-DR;

FIG. 13 is a flow chart showing the detection of the expression amount of GFP in pAAV-adipsin-adipose stem cells by a flow cytometer;

FIG. 14 is an adipsin nucleic acid artificial sequence (SEQ ID NO. 2);

FIG. 15 is the amino acid sequence of adipsin protein (SEQ ID NO. 4).

Detailed Description

The invention is further illustrated by the following specific examples. However, the use and purpose of these exemplary embodiments are merely to exemplify the present invention, and do not set forth any limitation in any way on the actual scope of the present invention, and the scope of the present invention is not limited thereto.

Example 1: construction of gene expression vector containing adipsin

The fusion gene of the modified adipose-derived stem cell comprises a signal nucleic acid artificial sequence (SEQ ID NO. 1) and an adipsin nucleic acid artificial sequence (SEQ ID NO. 2), the whole expression frame is synthesized by the nucleic acid coding sequences of SEQ ID NO.1 and SEQ ID NO.2 of the molecular biology engineering (Shanghai) Limited company, inserted into a BamHI-EcoRI site (shown in the attached figure 1) of an adenovirus expression vector pAAV-IRES-ZsGreen1 (SEQ ID NO. 3) and transformed into E.coli (DH5 α), and after the sequencing is correct, plasmids are extracted and purified by using a plasmid purification kit of the Invitrogen company, so that high-quality plasmids of recombinant expression vectors are obtained.

In more detail, the preparation method provided in this example includes the following steps:

the nucleic acid artificial sequence of the fusion gene adipsin was synthesized by Compton Bioengineering (Shanghai) Co., Ltd for its entire expression cassette and inserted into a standard vector pUC, thus named pUC-adipsin, while the pUC-adipsin and pAAV-IRES-ZsGreen1 vectors were subjected to double digestion with Fast Digest BamHI (available from ThermoFisher) and Fast Digest EcoRI (available from ThermoFisher) at 37 ℃ for 1 hour. The 100 mul enzyme digestion system is as follows: 10 × buffer: 10 mu l; 6 mug of DNA; BamHI enzyme: 3 mu l; EcoRI enzyme: 3 mu l; deionized water to make up the volume. The agar parts containing the DNA fragment of adipsin and the linearized pAAV-IRES-ZsGreen1 DNA fragment were cut out by agar electrophoresis and placed in two centrifuge tubes.

The DNA was dissolved from the agar using a DNA extraction kit (available from Thermo Fisher Co.) and concentrated by first adding 500ml DF buffer to the centrifuge tube and allowing to act at 55 ℃ for 10 minutes, shaking every 2-3 minutes until the agar was completely dissolved. The agar solution was then aspirated into the DF Column and covered with the Collection Tube (Collection of filtrate). Centrifuge at 8000rpm for 1 minute and pour off the filtrate. 500ml of Wash Buffer was added thereto, and the mixture was centrifuged at 8000rpm for 1 minute, and the filtrate was decanted. Centrifugation at 12000rpm for 2 minutes ensured that ethanol was removed. And finally transferring the DF Column to another clean micro-centrifuge tube, adding 25 mu l of precipitation Buffer, standing at room temperature for 2 minutes, and centrifuging at 12000rpm for 2 minutes, wherein the liquid in the micro-centrifuge tube is the purified adipsin fragment and the linearized pAAV-IRES-ZsGreen1 DNA fragment.

The two DNA fragments were ligated overnight at 16 ℃ to form pAAV-IRES-adipsin plasmid. The connecting system is as follows: 10 × buffer: 1 mu l; t4 ligase: 1 mu l; adipsin: 4 mu l; linearized pAAV-IRES-ZsGreen1 DNA: 4 mul.

The pAAV-IRES-adipsin is transformed into E.coli (DH5 α), positive clones are screened and identified, the positive clones are selected to shake bacteria at the temperature of 37 ℃ and the speed of 250rpm (12 h-16 h), pAAV-IRES-adipsin plasmids are extracted according to a plasmid extraction and purification kit (purchased from Invitrogen company), the specific steps are shown in an instruction book, the pAAV-IRES-adipsin plasmids are sequenced by committee biotechnology (Shanghai) and are used for later use after the sequencing is correct.

Example 2: preparation of adipose-derived stem cells

Extracting adipose tissue from abdomen of volunteer, washing erythrocyte with PBS under aseptic condition, cutting into fine particles with scalpel, adding trypsin and collagenase (v/v 1: 1), collecting liquid after shaking digestion, 1500rpm/10min, resuspending the culture medium, precipitating, filtering with 200 mesh sieve, 1500rpm/10min, resuspending the obtained cell extract with 10% FBS + DMEM culture solution, placing at 37 deg.C, and 5% CO₂Culturing in an incubator, and changing the culture solution for the first time after 5 days. After that, the solution was changed once in 3 days, when the primary cell confluence was 80%, the digestion passage was passed to P3 for substitution in subsequent experiments, and the cell growth and morphology were observed under an optical microscope (see FIG. 2). Flow cytometry surface marker detection was performed on P3-generation human adipose-derived stem cells to cells (about 1 × 10)⁶Individual cells) were added with anti-mouse anti-human monoclonal antibodies: CD29, CD34, CD44, CD105, CD90 and HLA ⁃ DR, when the expression rate of CD29, CD44, CD105 and CD90 is more than 98% and the expression rate of CD34 and HLA ⁃ DR is less than 1% by using flow cytometry detection analysis, the cell is identified as an adipose-derived stem cell (see attached figures 7-12).

Example 3: adenovirus packaging and titer detection

(I) recovery of 293T cells

1) The frozen 293T cells were removed from the liquid nitrogen tank, quickly dropped into a 37 ℃ water bath and quickly shaken until the cell solution was completely dissolved.

2) The cell solution was transferred to a 50mL centrifuge tube and 10 mL of fresh complete medium was added to the tube, mixed well and centrifuged at 1500rpm for 5 min.

3) The supernatant was removed and 3mL of fresh DMEM medium was added to resuspend the cells, and the cells were transferred to six well plates on average, each well being replenished to 3mL of medium.

4) Placing the six-hole plate at 37 deg.C and 5% CO₂And 95% relative humidity.

5) Cell viability was observed the next day and fresh medium was changed. The growth of the cells is observed every day, and the cells are subcultured when the cells are fully paved on the bottom of the hole, and are used for experiments when the cells are fully paved on 80% of the bottom of the bottle.

(II) adenovirus packaging

Taking two 1.5ml sterile EP tubes, adding 200 mul serum-free DMEM into one EP tube, and adding 2 mug auxiliary plasmid and 1 mug gpAAV-IRES-adipsin plasmid; 200 μ l serum free DMEM was added to another EP tube, 6 μ l lipoLP2000 was added, and the tube was allowed to stand for 5 min. Then fully mixing the two pipes uniformly, and standing for 15-20 min; dropwise adding the 400 mul of mixed solution into a cell culture dish, slightly shaking the culture dish, uniformly mixing, and then placing the culture dish in a thermostat containing 37 ℃ for incubation; after 6h, the medium was aspirated, a suitable amount of DMEM complete medium preheated to 37 ℃ was added, and the cells were incubated in a 37 ℃ incubator. And collecting the supernatant containing the adenovirus 24h, 48 h and 72h after transfection, filtering by using a 0.45um small filter, collecting the filtrate, concentrating and purifying, and storing in a low-temperature refrigerator at-80 ℃ for later use.

(III) Virus Titer assay

1) The 293T cells with good growth state are digested and counted, and then diluted to 1 × 10⁵PermL, add 96-well plates, 100. mu.L/well, prepare 6 wells for each virus dilution. Culturing at 37 deg.C in 5% CO2 incubator.

2) The following day, 6 1.5mL EP tubes were prepared, and 10. mu.L of virus solution was added to the first EP tube, followed by 10-fold gradient dilution with 6 serial dilutions. The original culture medium in the 96-well plate is sucked, and diluted virus solution is added and marked.

3) On the third day, 100. mu.L of complete medium was added to each well to facilitate cell growth.

4) On the fifth day, virus titers were calculated at 10-30% fluorescence% wells and percent GFP fluorescence was measured using flow cytometry. According to the formula: titer (pfu/mL) = number of cells × fluorescence percentage × MOI (1) × virus dilution × 10³Calculating the virus titer, and the recombinant adenovirus titer obtained in the invention is 3.36 multiplied by 10⁷pfu/mL。

Example 4: adenovirus infected adipose-derived stem cells

Digesting and counting the adipose-derived stem cells in good growth state, and diluting the cells to a density of 5 x10⁵cells/mL were added to a six-well plate and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. 2mL of virus solution (virus titer 3.36X 10) was taken out at-80 deg.C⁷pfu/mL), polybrene (purchased from Sigma) was added to a final concentration of 8. mu.g/mL. Taking out the six-hole plate from the incubator, removing the culture medium in the six-hole plate, adding the prepared virus solution, and putting the virus solution into the incubator to continue culturing for 24 hours. After 24H, the virus solution in the six well plates was removed, complete medium (10% FBS, 1% streptomycin, H-DMEM, all from Gibco) was added for culturing, and 3ug/mL puromycin was added for selection at 37 deg.C and 5% CO₂Culturing in an incubator, changing the culture medium once every 2 days, and culturing for 5 days to obtain viable cells, namely the adipose-derived stem cells infected by the adenovirus, which are named as pAAV-adipsin-adipose-derived stem cells. The flow cytometer detects the GFP expression rate of the pAAV-adipsin-adipose-derived stem cells, and the result shows that 23.5% of cells are GFP positive (the adenovirus vector expresses GFP protein), which indicates that the adipsin constructed by the invention can be expressed on the surface of the adipose-derived stem cells, and the transfection efficiency is 23.5% (see figure 13).

Example 4 pancreatic islet β cell proliferation assay

(I) culture of rat insulinoma cells

Take 1X10⁵Rat insulinoma cells (INS-1, purchased from Shanghai Minghai Bio-technology Co., Ltd.) were subcultured by adding 10 ml of RPMI-1640 medium (containing 10mM HEPES, 2 mM L-glutamine, 1mM sodium pyruvate and 0.05 mM β -mercaptoethanol) containing 10% FBS to 10cm dishes at 37 ℃ in a 5% CO2 cell culture vessel until the cells reached 80% -90% of the cell flasks and adjusting the INS-1 cell concentration to 1X10⁵Individual cells/ml for subsequent experiments.

(II) Co-culture of islet cells and adipose-derived stem cells

A non-contact co-culture system is formed by using a Transwell6 pore plate with an insertion layer and a pore diameter of 0.4um, and the cell ratio is 1:1, adjusting the seeded cell density to 1X10⁴The cells/well are cultured, and the total composition is divided into three groups:

INS-1 cells and pAAV-adipsin-adipose-derived stem cells are used as a co-culture experimental group;

INS-1 cells and adipose stem cells (untransfected) were used as co-culture control;

the same amount of INS-1 cells served as a separate control.

The lower chamber of the Transwell 6-well plate was inoculated with adipose-derived stem cells, INS-1 cells were added and inoculated into the upper chamber of the Transwell 6-well plate, and the adipose-derived stem cells and INS-1 cells were adjusted to the same cell density (1X 10) using 10% FBS RPMI-1640 medium⁴One cell/well), cultured in a 5% CO2 cell incubator at 37 deg.C, and the medium was changed every 3 days for in vitro culture for 14 d. The sampling was repeated 3 times at 3d, 7d, and 14d, respectively.

(II) CellTiter-Glo fluorescent cell activity detection

1) In vitro cultured INS-1 cells of the co-culture experimental group, co-culture control group and individual control group were adjusted to a certain density with 10% FBS-containing RPMI-1640 medium, and inoculated on 96-well culture plates at about 5X 10 per 100. mu.l of cell suspension⁴The cells were pre-cultured for 4 hours at 37 ℃ in a 5% CO2 incubator.

2) The buffer solution of CellTiter-Glo is dissolved at room temperature and then poured into a brown bottle filled with substrate freeze-dried powder to be mixed evenly, and the pre-cultured cell culture plate is taken out and balanced for 30min at room temperature.

3) To each well was added 100. mu.l CellTiter-Glo reagent (from Promega).

4) Shaking and mixing uniformly for 2min, incubating for 10min, taking a cell sample, and measuring the fluorescence luminescence value (RLU) of each hole in the ELISA plate by using an ELISA reader.

Cells were divided into 4 groups according to the experiment, each group having 3 replicates:

a: the zero-adjusted group was supplemented with RPMI-1640 medium (containing 10% FBS)

B: control group alone, INS-1 cells alone

D: co-culture control group, INS-1 cells and adipose Stem cells (untransfected)

C: co-culture experimental group, INS-1 cell and pAAV-adipsin-adipose-derived stem cell co-culture

The results show that the islet cell viability is gradually enhanced along with the increase of time, the INS-1 cell activity in the co-culture experimental group is higher than that of the INS-1 cell in the culture control group, is obviously higher than that of the INS-1 cell in the single culture group, and the cell viability of the INS-1 cell is 3 times that of the single culture group and is 2 times that of the INS-1 cell and adipose-derived stem cell (untransfected) co-culture group. Thus, adipostem cells expressing adipsin had a significant effect on INS-1 cell proliferation (see fig. 5).

Example 4: insulin secretion test

Islet cells of co-culture experimental group, control group and islet cells of culture only group, cultured for 3 days, 7 days and 14 days, respectively, were seeded into 24-well plates at about 2 × 10 per well according to the culture method of example 3⁵Incubating the cells for 24 h; centrifuging, discarding the supernatant, and pre-incubating in a sugar-free RPMI-1640 medium for 2 hours; 1500rpm, 5min, plate centrifugation, and supernatant discarded; adding 1ml of low-sugar KRBH solution (2.7mmol/L glucose) into each hole once, and incubating for 1 hour; centrifuging to remove low-sugar KRBH, and adding 1ml of high-sugar KRBH (16.7mmol/L glucose) solution culture box into each hole for further incubation for 2 hours; collecting cell supernatant to carry out ELISA detection, detecting insulin secretion (miU/L) according to a kit operation method, and analyzing the influence of adipostem cells modified by adipsin genes on insulin secretion.

Cells were divided into the following 3 groups, each of 3 replicates, according to the experiment:

a: control group alone, INS-1 cells alone

B: co-culture control group, INS-1 cells were co-cultured with adipose Stem cells (untransfected)

Comparison of insulin secretion amount: the results are shown in figure 6, the insulin secretion of the INS-1 cell and the pAAV-adipsin-adipose-derived stem cell group of the co-culture experimental group cultured by low-sugar KRBH and high-sugar KRBH is reduced to 29.5mIU/L when the insulin secretion is 7d from 23.3mIU/L when the insulin secretion is 3d, and is reduced to 28.2mIU/L when the insulin secretion is 14d, the insulin secretion of the co-culture control group is respectively 13.1mIU/L, 15.8mIU/L and 13.6mIU/L, the insulin secretion of the independent control group is respectively 9.2mIU/L, 11.6mIU/L and 10.6mIU/L, the insulin secretion of the INS-1 cell of the co-culture experimental group is obviously higher than that of the INS-1 cell and the INS-1 cell of the independent culture group, the difference is more than 2 times, the statistical significance is achieved, and the 3d, the insulin secretion of the group of the invention is 1.78 times that of the insulin of the co-culture control group, the insulin secretion amount of the group of the invention is 1.87 times of that of the co-culture control group, and at 14d, the insulin secretion amount of the group of the invention is 2.1 times of that of the co-culture control group.

Therefore, the adipostem cell modified by the adipsin gene can promote insulin secretion of islet cells and maintain blood glucose balance.

In conclusion, the adipostem cell modified by the adipsin gene prepared by the invention obviously increases the proliferation capacity and insulin secretion capacity of islet β cells, preliminarily proves that the adipostem cell modified by the adipsin gene can promote the proliferation of islet cells and the insulin secretion function of the islet cells, and provides a theoretical basis for clinical application of stem cells to treatment of diabetes.

Unless otherwise specified, the proportions used in the present invention are mass proportions, and the percentages used are mass percentages.

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gcattggaac gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata 900

ggcccacaaa aaatgctttc ttcttttaat atactttttt gtttatctta tttctaatac 960

tttccctaat ctctttcttt cagggcaata atgatacaat gtatcatgcc tctttgcacc 1020

attctaaaga ataacagtga taatttctgg gttaaggcaa tagcaatatt tctgcatata 1080

aatatttctg catataaatt gtaactgatg taagaggttt catattgcta atagcagcta 1140

caatccagct accattctgc ttttatttta tggttgggat aaggctggat tattctgagt 1200

ccaagctagg cccttttgct aatcatgttc atacctctta tcttcctccc acagctcctg 1260

ggcaacgtgc tggtctgtgt gctggcccat cactttggca aagaattggg attcgaacat 1320

cgattgaatt ccccggggat cccgcccctc tccctccccc ccccctaacg ttactggccg 1380

aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc 1440

gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag 1500

gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt 1560

tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa 1620

ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc 1680

aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg 1740

gctcacctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat 1800

gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa 1860

cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat 1920

ggcccagtcc aagcacggcc tgaccaagga gatgaccatg aagtaccgca tggagggctg 1980

cgtggacggc cacaagttcg tgatcaccgg cgagggcatc ggctacccct tcaagggcaa 2040

gcaggccatc aacctgtgcg tggtggaggg cggccccttg cccttcgccg aggacatctt 2100

gtccgccgcc ttcatgtacg gcaaccgcgt gttcaccgag tacccccagg acatcgtcga 2160

ctacttcaag aactcctgcc ccgccggcta cacctgggac cgctccttcc tgttcgagga 2220

cggcgccgtg tgcatctgca acgccgacat caccgtgagc gtggaggaga actgcatgta 2280

ccacgagtcc aagttctacg gcgtgaactt ccccgccgac ggccccgtga tgaagaagat 2340

gaccgacaac tgggagccct cctgcgagaa gatcatcccc gtgcccaagc agggcatctt 2400

gaagggcgac gtgagcatgt acctgctgct gaaggacggt ggccgcttgc gctgccagtt 2460

cgacaccgtg tacaaggcca agtccgtgcc ccgcaagatg cccgactggc acttcatcca 2520

gcacaagctg acccgcgagg accgcagcga cgccaagaac cagaagtggc acctgaccga 2580

gcacgccatc gcctccggct ccgccttgcc ctgaaagctt gcctcgagca gcgctgctcg 2640

agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt 2700

tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga 2760

ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt 2820

tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc 2880

acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc 2940

tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg 3000

gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat 3060

ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc 3120

tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg 3180

atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 3240

gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 3300

ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 3360

atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 3420

tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 3480

tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 3540

tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 3600

gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 3660

agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 3720

cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 3780

agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat 3840

ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 3900

caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 3960

ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 4020

cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 4080

tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 4140

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 4200

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 4260

tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 4320

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 4380

agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 4440

tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 4500

tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 4560

atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 4620

ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 4680

tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 4740

acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 4800

ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 4860

aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 4920

ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 4980

cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 5040

gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 5100

actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 5160

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 5220

cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 5280

tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 5340

agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 5400

tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 5460

acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 5520

ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 5580

gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 5640

gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 5700

gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 5760

tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 5820

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 5880

tttgctggcc ttttgctcac atgt 5904

<210>4

<211>233

<212>PRT

<213> ethnic species (Homo sapiens)

<400>4

Pro Pro Arg Gly Arg Ile Leu Gly Gly Arg Glu Ala Glu Ala His Ala

1 5 10 15

Arg Pro Tyr Met Ala Ser Val Gln Leu Asn Gly Ala His Leu Cys Gly

20 25 30

Gly Val Leu Val Ala Glu Gln Trp Val Leu Ser Ala Ala His Cys Leu

35 40 45

Glu Asp Ala Ala Asp Gly Lys Val Gln Val Leu Leu Gly Ala His Ser

50 55 60

Leu Ser Gln Pro Glu Pro Ser Lys Arg Leu Tyr Asp Val Leu Arg Ala

65 70 75 80

Val Leu His Pro Asp Ser Gln Pro Asp Thr Ile Asp His Asp Leu Leu

85 90 95

Leu Leu Gln Leu Ser Glu Lys Ala Thr Leu Gly Pro Ala Val Arg Pro

100 105 110

Leu Pro Trp Gln Arg Val Asp Arg Asp Val Ala Pro Gly Thr Leu Cys

115 120 125

Asp Val Ala Gly Trp Gly Ile Val Asn His Ala Gly Arg Arg Pro Asp

130 135 140

Ser Leu Gln His Val Leu Leu Pro Val Leu Asp Arg Ala Thr Cys Asn

145 150 155 160

Arg Arg Thr His His Asp Gly Ala Ile Thr Glu Arg Leu Met Cys Ala

165 170 175

Glu Ser Asn Arg Arg Asp Ser Cys Lys Gly Asp Pro Gly Gly Pro Leu

180 185 190

Val Cys Gly Gly Val Leu Glu Gly Val Val Thr Ser Gly Ser Arg Val

195 200 205

Cys Gly Asn Arg Lys Lys Pro Gly Asn Tyr Thr Arg Val Ala Ser Tyr

210 215 220

Ala Ala Trp Ile Asp Ser Val Leu Ala

225 230

Claims

1. An adipsin gene-modified adipose-derived stem cell, which is characterized in that: the adipsin gene modified adipose-derived stem cell comprises an adipsin recombinant gene, wherein the adipsin recombinant gene expresses adipsin protein.

2. The adipsin gene-modified adipose-derived stem cell of claim 1, wherein: the adipsin recombinant gene comprises a signal peptide and an adipsin gene; the adipsin gene is a nucleotide sequence shown as SEQ ID NO.2 in a sequence table.

3. The adipsin gene-modified adipose-derived stem cell of claim 1, wherein: the amino acid sequence of the adipsin protein is shown as SEQ ID NO.4 in the sequence table.

4. The adipsin gene-modified adipose-derived stem cell of claim 2, wherein: the signal peptide is a nucleotide sequence shown as SEQ ID NO.1 in a sequence table.

5. A preparation method of adipostem cells modified by adipsin genes is characterized in that: the preparation method comprises the steps of preparing a plasmid containing an adipsin gene expression vector, preparing adipose-derived stem cells, packaging and detecting the titer of adenovirus, and infecting the adipose-derived stem cells with the adenovirus.

6. The method for preparing the adipsin gene-modified adipose-derived stem cell according to claim 5, wherein the method comprises the following steps: the plasmid containing the adipsin gene expression vector is prepared by inserting a fusion gene comprising a signal peptide and a nucleic acid artificial sequence of adipsin into an adenovirus expression vector pAAV-IRES-ZsGreen1, and extracting and purifying the plasmid after correct conversion and sequencing to obtain the plasmid containing the adipsin gene expression vector.

7. The method for preparing the adipsin gene-modified adipose-derived stem cell according to claim 5, wherein the method comprises the following steps: the above-mentioned

Preparing adipose-derived stem cells, wherein when the primary cell fusion degree is 78-82%, digestion passage is carried out, and the cells are transferred to P3 or P4; and (3) carrying out adenovirus packaging and titer detection, namely packaging the plasmid containing the adipsin gene expression vector by adopting adenovirus to obtain the recombinant adenovirus.

8. The method for preparing the adipsin gene-modified adipose-derived stem cell according to claim 5, wherein the method comprises the following steps: the above-mentioned

Infecting adipose-derived stem cells with adenovirus, comprising the following steps:

(2) adding polybrene with final concentration of 7.5-8.5 μ g/mL into the recombinant adenovirus solution to obtain virus solution to be infected;

(3) removing the culture medium from the adipose-derived stem cells, adding a virus solution to be infected, continuously culturing for 20-28h, removing the virus solution, adding a complete culture medium for culturing, simultaneously adding 2.8-3.2ug/mL puromycin for screening, continuously culturing for 4-6 days, and obtaining the adipose-derived stem cells infected by the adenovirus.

9. The application of adipostem cells modified by adipsin genes is characterized in that: the adipostem cell modified by the adipsin gene and the islet cell are subjected to non-contact co-culture; in the non-contact co-culture, adipsin gene modified fat stem cells are inoculated in the lower chamber of a culture plate, islet cells are inoculated in the upper chamber of the culture plate, the fat stem cells and the islet cells are adjusted to the same cell density by 9.5-10.5% FBS RPMI-1640 culture medium, and then culture is carried out, and the liquid is changed periodically.

10. The use of the adipsin gene-modified adipose stem cells of claim 9, wherein: the culture plate is a Transwell6 pore plate with an insertion layer, and the cell density of the adipsin gene modified adipose-derived stem cells and the islet cells is 0.8-1.2 multiplied by 10⁴Individual cells/well; the islet cells are INS-1 cells.