CN113717929B

CN113717929B - Pharmaceutical composition containing gene-edited adipose-derived stem cells

Info

Publication number: CN113717929B
Application number: CN202111037481.XA
Authority: CN
Inventors: 杨骏; 朱成光
Original assignee: Shaanxi Beitian Biotechnology Co ltd
Current assignee: Shaanxi Beitian Biotechnology Co ltd
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2022-07-12
Anticipated expiration: 2041-09-06
Also published as: CN113717929A

Abstract

The present invention relates to pharmaceutical compositions comprising gene-edited adipose-derived stem cells. According to the invention, after gamma secretase in adipose-derived mesenchymal stem cells is knocked out by using a CRISPR technology, a specific monoclonal antibody aiming at the gamma secretase is used for specific differentiation induction to obtain the pancreatic islet-like cells, and mouse experiments prove that the pancreatic islet-like cells and chitooligosaccharides have better blood sugar reducing effect.

Description

Pharmaceutical composition containing gene-edited adipose-derived stem cells

Technical Field

The present application relates to the field of biology. More particularly, it relates to a pharmaceutical composition containing a gene-edited adipose-derived stem cell.

Background

Diabetes mainly is a chronic systemic metabolic disease which is caused by relative or absolute lack of insulin due to interaction of factors such as heredity, environment, spirit and the like, and takes continuous hyperglycemia as a main biochemical characteristic, and injection and drug therapy of insulin become a current main method for clinically controlling diabetes and reducing the occurrence of complications of diabetes, but the diabetes cannot be fundamentally cured. The research proves that the transplantation of the islet cells in the in vitro experiment can effectively reduce the blood sugar and reduce the occurrence of complications thereof. Stem cell transplantation is a more ideal cell replacement therapy for the treatment of type 1 diabetes due to its self-renewal, proliferation and multipotentiality.

MSCs share the commonality of stem cells: self-renewal, multi-directional differentiation and homing ability. In the experiment, under a specific induction condition, the MSCs can be differentiated to various tissues or cells, and the plasticity is strong. In vitro MSCs are differentiated into pancreatic progenitor cells or stem cells firstly in the induction process and then into islet-like cells with a secretion function, and the coexistence of the stem cells or progenitor cells and mature islet cells detected in cells successfully induced currently proves that insulin-secreting cells can be induced and differentiated from the stem cells. The conditions for induction were different, and the cells differentiated from bone marrow mesenchymal stem cells (BM-MSCs) were also different. Recent research shows that BM-MSCs have great potential for inducing differentiation into islet-like cells to treat diabetes; BM-MSCs can differentiate to various tissue cells under specific induction conditions. In the test, the BM-MSCs are induced and differentiated by a specific inducer and then implanted into the body to control blood sugar and improve the immune state of the body, can delay the occurrence of complications of the BM-MSCs and is closely related to diabetic angiopathy and retinopathy.

There are many methods for inducing differentiation of MSCs into islet-like cells, and the chemical induction method, which is the most commonly used method at present, promotes differentiation of BM-MSCs into islet-like secretory cells by using a chemical agent. It is found that MSCs can be directionally induced to differentiate into islet-like cells in vitro through drug induction or by simulating the microenvironment of islet cells in vivo, and can express a plurality of functional characteristics of the islet cells. The most commonly used inducers are cytoregulin, activin A, nicotinamide, basic fibroblast growth factor (bFGF), Endothelial Growth Factor (EGF), Hepatocyte Growth Factor (HGF), B77, mercaptoethanol, etc. Chen et al separates rat MSCs, after passage for several times in vitro, mercaptoethanol and nikkamide are added into serum-containing DMEM for pre-induction for 24H, mercaptoethanol and nikkamide are added into serum-free H-DMEM culture solution for 10H, cell morphology changes, nestin protein and insulin amount change, the induced cells are transplanted into a diabetic rat, transplanted cells aggregated in pancreas of the diabetic rat can be observed, the shape is similar to that of islet-like cells, and trace insulin secretion is found through detection, so that blood sugar of the diabetic rat can be controlled. It was thus demonstrated that MSCs can differentiate into cells capable of secreting insulin by induction and can lower blood glucose in diabetic mice.

Although the immunomodulatory role of MSCs is currently unknown, its broad source, low immunogenicity and the effect of the immune response make it the most promising tool for the treatment of severe refractory autoimmune diseases. Although numerous studies have shown that MSCs derived from various tissues can induce differentiation into IPCs, many key problems remain to be further studied, such as how to control the growth and differentiation of MSCs, how to improve the efficiency of induced differentiation of MSCs, how to change cell surface antigens after induction of differentiation thereof, and the tendency of cells to have no degeneration after long-term culture, long-term effect after transplantation, and the like. In addition, the mechanism of action of the differentiated cells in vivo also needs to be further studied and solved.

The gene editing technology refers to the mechanism of recognizing specific sites of a genome by using programmable nucleases, mediating double-strand breaks of DNA, and then inducing non-homologous end-joining (NHEJ) or homologous recombination repair (HDR) of the DNA, so as to realize site-directed modification of DNA sequences, including targeted knockout or knock-in of genes. A Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas system established in recent years has the advantages of high efficiency, simple operation, easy modification and optimization and the like, is the most important gene editing tool at present, and is widely applied to the fields of gene expression regulation, cell imaging, nucleic acid labeling, epigenetic modification, disease treatment, functional gene screening and the like. However, at present, only a few studies have been conducted on improvement of differentiation into islet-like cells by editing adipose mesenchymal stem cells using CRISPR.

Disclosure of Invention

The invention relates to a method for improving induction of adipose-derived mesenchymal stem cells into islet-like cells.

More particularly, the application relates to a preparation method of a gamma secretase knockout adipose-derived mesenchymal stem cell. The method is to perform the knockout by using CRISPR technology.

Specifically, CRISPR targets are: acctgggcattcttagctgcggg

The sgRNA sequence is:

accugggcauucuuagcugcGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU(SEQ ID NO：1)

still further, the present invention provides monoclonal antibodies specific for gamma secretase. The monoclonal antibody is prepared by using an epitope peptide VELGQVALRTSLELWMHTDPVSQKNESVRNQVEDLLATLEK of gamma-secretase as an immunogen through a conventional monoclonal antibody preparation method.

The monoclonal antibody cell line secreted IgG1 as antibody and Kappa chain as light chain. The light chain variable region (SEQ ID NO: 2) and the heavy chain variable region (SEQ ID NO: 3).

Further, the present invention provides a method for inducing differentiation of transgenic adipose-derived stem cells into islet-like cells, comprising (1) preparing a knockout adipose-derived mesenchymal stem cell; the knockout adopts a CRISPR method, and a specific sgRNA sequence is shown as SEQ ID NO:1 is shown in the specification; (2) when the gene-knocked-out adipose-derived mesenchymal stem cells prepared in the step (1) are cultured and fused to 80% -90%, the beta-nerve growth factor with the composition of 100 mu g/L, 4nmol/L of activin A, 10mmol/L of nicotinamide, 25 mu g/L of epidermal growth factor and 50 mu g/L of monoclonal antibody are subjected to induced culture for 10d by using 10% FBS DMEM culture solution, and the solution is changed once every 2d half; then the culture medium is changed into DMEM with 10% FBS and the components of 10mmol/L nicotinamide, 10 mug/L basic fibroblast growth factor, 50 mug/L monoclonal antibody and 1% insulin-transferrin-selenium, the induction is continued for 10 days, and the total induction is 20 days; wherein the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 2, the heavy chain variable region sequence is shown as SEQ ID NO: 3, respectively.

An islet-like cell, characterized in that it is prepared by the method for inducing and differentiating transgenic adipose-derived stem cells into islet-like cells.

A pharmaceutical composition characterized by comprising the islet-like cell represented above.

The application of the islet-like cells in preparing a pharmaceutical composition for treating diabetes.

Specifically, the number of therapeutic cells of islet-like cells is 1X 10⁹1 x10 of¹¹One per kg.

Further, the pharmaceutical composition further comprises a second therapeutic agent.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

Advantageous effects

According to the invention, after gamma secretase in adipose-derived mesenchymal stem cells is knocked out by using a CRISPR technology, a specific monoclonal antibody aiming at the gamma secretase is used for specific differentiation induction, so that islet-like cells are obtained, and mouse experiments prove that the islet-like cells have a good blood sugar reducing effect.

Drawings

FIG. 1 map of plasmid pTrcHis2B

FIG. 2 is a graph showing the results of the monoclonal antibody subtype

FIG. 3 is a graph showing the results of detecting protein expression of islet cell-associated markers by Western blotting

FIG. 4 result graph of fasting blood glucose level after treatment

FIG. 5 result graph of fasting blood glucose level after treatment

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of adipose-derived mesenchymal stem cells

15ml of human fat particles are centrifuged at 1000r/min for 5 min. Taking the upper adipose tissue and the lower cells. Trypsin and collagenase were added to the adipose tissue at a final concentration of 0.1%. Digesting in a water bath shaker at 37 deg.C for 30 min. Centrifuging at 700g/min for 5 min. And (4) reserving the lower layer cells, mixing the two cells uniformly, adding a PBS buffer solution to resuspend the cells, and centrifuging again for 700g/min and 5 min. After the cells were resuspended in the α -MEM culture medium, the cells were inoculated into a flask and cultured in a 5% carbon dioxide incubator at 37 ℃. Is marked as P0 generation. The growth of the cells was observed. Half of the liquid is changed on the first 3 days, and then every 3 days, the cells are subcultured when the cells reach 90% fusion, digested by 0.05% trypsin and subcultured according to the proportion of 1: 5. And transferring to the 3 rd generation for identification. Expression of hADSCs surface markers CD13, CD29, CD31, CD34, CD45, CD44, CD73, CD90, CD105 and HLA-DR was determined by flow cytometry. The result of the hADSCs immunophenotyping test shows that the CD90 and CD105 expressions are positive, and the positive rate is more than 95.9%; CD34, CD45 and HLA-DR expression are negative, and the positive rate is less than 1.5 percent. And storing the separated adipose-derived mesenchymal stem cells for later use.

Example 2 preparation of gamma secretase knockout adipose-derived mesenchymal stem cells

The sequences in the present invention are all 5 '-3'.

Dozens of targets were obtained by optimizing the target sequence based on the sequence of γ secretase APH1B, and the identification revealed that target 1 had the best effect. Specifically targets 1 and 2 are exemplified.

Target 1: acctgggcattcttagctgcggg

sgRNA1 sequence:

target 2: tagtcagtgtctctgggttttgg

sgRNA2 sequence:

uagucagugucucuggguuuGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

preparation of ascipf 1 protein:

vector construction plasmid pTrcHis2B (purchased from Invitrogen) was constructed, and the plasmid map is shown in FIG. 1, and the 3' end of the AsCpf1 gene sequence plus his tag sequence was inserted between the NcoI and XhoI cleavage sites of the plasmid, to obtain recombinant expression vector pTrcHis2B-AsCpf 1. The prokaryotic expression vector is transferred into an expression host bacterium, and a single clone is selected and inoculated into a fresh LB culture medium (containing ampicillin). AsCpf1 expression was induced by addition of 0.8mM IPTG (isopropyl-B-D-thiogalactose) after 8 hours or so of bacterial amplification. After induction for 6 hours, the cells were collected, and then added with a disruption buffer (pH7.520mM Tris-HCl, 50mM NaCl, 1% Triton-100, 20% glycerol) to carry out ultrasonication to take the supernatant, the supernatant was passed through a nickel column, after completion of the loading, the nickel column was eluted with an eluent (20mM Tris-HCl pH7.5, 50mM NaCl, 0.1% TritonX-100, 500mM imidazole, 20% glycerol) to collect the eluent, and the collected protein was dialyzed into 20mM Tris,50mM NaCl pH7.5, 20% glycerol, and after completion of the dialysis, the protein AsCpf1 was obtained by ultrafiltration concentration.

Gene knockout is carried out by electrotransfer:

adipose-derived mesenchymal stem cells prepared in example 1 were collected, centrifuged to discard the medium in a 20. mu.l system/cuvette, washed with 1640 incomplete medium, and resuspended in electrotransport solution (PostElectroporationMediumPEM-2) to adjust the cell density to 2.0X10⁶Add AsCpf1 protein and sgRNA (ratio 1. mu.g: 1. mu.g, sequence of sgRNAs shown in SEQ ID NO:1 or 2, respectively) to 20. mu.l, add to the cuvette, and take care that no air bubbles are present. The electric transfer condition is 520V,25ms, and parameters are adjusted before electric transfer to preheat. After the electrotransformation is completed, the cells after the electrotransformation are taken and put into electrotransformation buffer (electroporation buffer103) for enrichment and standby.

And collecting the transgenic enriched adipose-derived stem cells, and extracting genomic DNA. Conventional PCR amplification was performed using the primer pair APH1BF1: agcatttataatcctggtgc and R1: agaacaccttgggtattcct. After the PCR reaction is finished, collecting PCR products, performing agarose gel electrophoresis, and then, preparing gel to recover and purify DNA fragments. And entrusts Shanghai's company to carry out gene sequencing, discovers that a section of sequence is deleted from a target gene according to a sequencing result, and successfully knocks out the gene. Wherein the knockout results in a frameshift essentially at the gRNA position of the gene and protein translation is terminated. No knockdown was detected in the negative control. This illustrates that SEQ ID NO: the sgRNA of 1 has a good knockout effect. PCR-identified SEQ ID NO:1, and subculturing the adipose-derived stem cells successfully knocked out by the sgRNA of the gene 1 for later use.

EXAMPLE 3 preparation of Gamma-secretase monoclonal antibody

(1) Protein immunization and antibody preparation

Screening to obtain high immunity epitope peptide sequence, i.e. epitope peptide of gamma-secretase

VELGQVALRTSLELWMHTDPVSQKNESVRNQVEDLLATLEK, the polypeptide is emulsified and mixed with Freund's complete adjuvant, BALB/c mice are immunized subcutaneously, and after 3 weeks, booster immunization is carried out 2 times, and the dose and method of immunization are the same as the first time. Before 3d fusion, additional immunization was performed by intraperitoneal injection with antigen without adjuvant. The method of fusion of cells is prepared by methods conventional in the art. And (3) performing detection and screening by using indirect ELISA to obtain positive cells capable of secreting antibodies, performing subcloning by using a limiting dilution method, performing cloning for 3 times to obtain a stable hybridoma cell line, performing amplification culture on the stable hybridoma cell line, and freezing and storing the stable hybridoma cell line. The hybridoma cell lines were injected intraperitoneally into mice after 1 week. An anti-gamma-secretase antibody 5C6 is successfully prepared.

(2) Monoclonal antibody subtypes

The antibody subtypes in the monoclonal cell culture supernatant and in the ascites were detected using a mouse antibody typing kit, and the results are shown in FIG. 2. As can be seen from FIG. 2, the monoclonal antibody cell line secreted IgG1 as the antibody and Kappa as the light chain.

(3) Cross reaction rate test

Gamma-secretase, BSA, mouse serum and a plurality of samples are serially diluted, indirect competitive ELISA tests are carried out, a curve equation is drawn, half inhibition concentration IC50 of each drug is calculated, cross reaction rates (CR) of each component and the monoclonal antibody are compared, and the specificity of the antibody is judged. The concentration range of the cross reaction product is 0.001-1000 ng/mL. The cross-reactivity (%) ═ IC50(γ -secretase)/IC 50 (competitor) × 100%. the results are shown in table 1.

TABLE 1 Cross-reactivity and inhibitory Effect

As is clear from Table 1, the monoclonal antibody of the present invention showed an inhibition rate of 100% for gamma-secretase, and showed no cross reaction with BSA or mouse serum, indicating that the antibody had good specificity.

(4) Antibody performance characterization

In addition, the binding activity of the gamma-secretase antibody was 0.31nM as measured using antibody binding capacity assays routine in the art.

The variable region protein of the anti- γ -secretase mouse monoclonal antibody was sequenced, and the sequences of the variable regions of 5C6 were as follows.

Light chain variable region (SEQ ID NO: 2)

DIVITQSPALMAASPGEKVTITCWDQYLACLIIKNWYQQKSGISPKPWIYADNWYCSGVPARFSGSGSGTSYSLTITSMEAEDAATYYCWVYPRFKNLFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESGTELARPGASVKLSCKASGYIFSCFQTTWIKQRPGQGLEWIGFPVNMEWWLELKIPTQGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGNGMYSMEWGLGTTLAVSS。

Example 4 Induction experiment of adipose-derived mesenchymal Stem cells

Transgenic adipose-derived stem cell monoclonal antibody induction group: taking the fat mesenchymal stem cells with the knocked-out genes prepared in the example 2, respectively fusing the cells to 80% -90%, and performing induction culture for 10 days by using DMEM culture solution of 10% FBS with the composition of 100 mu g/L beta-nerve growth factor, 4nmol/L activin A, 10mmol/L nicotinamide and 25 mu g/L epidermal growth factor, and 50 mu g/L monoclonal antibody of the example 3, and half replacing the solution every 2 days; then the medium was changed to DMEM with 10% FBS containing 10mmol/L nicotinamide, 10. mu.g/L basic fibroblast growth factor, 50. mu.g/L monoclonal antibody of example 3, 1% insulin-transferrin-selenium, and induction continued for 10 days for a total of 20 days.

Human umbilical cord mesenchymal stem cell monoclonal antibody induction group: the normal adipose-derived mesenchymal stem cells of example 1, when fused to 80% -90% respectively, were induced to culture for 10d with DMEM culture solution of 10% FBS having a composition of 100. mu.g/L of beta-nerve growth factor, 4nmol/L of activin A, 10mmol/L of nicotinamide, 25. mu.g/L of epidermal growth factor, 50. mu.g/L of monoclonal antibody of example 3, and half of the solution was changed every 2 d; then the medium was changed to DMEM with 10% FBS containing 10mmol/L nicotinamide, 10. mu.g/L basic fibroblast growth factor, 50. mu.g/L monoclonal antibody of example 3, 1% insulin-transferrin-selenium, and induction continued for 10 days for a total of 20 days.

Transgenic adipose-derived stem cell induction group: taking the fat mesenchymal stem cells with the knocked-out genes prepared in the embodiment 2, respectively fusing the cells to 80% -90%, and carrying out induction culture for 10d by using DMEM culture solution of 10% FBS (bovine serum albumin) with the components of 100 mug/L beta-nerve growth factor, 4nmol/L activin A, 10mmol/L nicotinamide and 25 mug/L epidermal growth factor, wherein half of the solution is changed every 2 d; then the culture medium is changed into 10mmol/L nicotinamide and 10 mug/L basic fibroblast growth factor, and the induction is continued for 10 days, and the total induction is continued for 20 days.

Human umbilical cord mesenchymal stem cell induction group: the normal adipose-derived mesenchymal stem cells of example 1, when fused to 80% -90% respectively, were induced to culture for 10 days with DMEM culture solution of 10% FBS having the composition of 100 μ g/L β -nerve growth factor, 4nmol/L activin a, 10mmol/L nicotinamide, 25 μ g/L epidermal growth factor, with half-way replacement every 2 days; then the culture medium is changed into DMEM with 10% FBS and 10mmol/L nicotinamide, 10 mug/L basic fibroblast growth factor and 1% insulin-transferrin-selenium, and the induction is continued for 10 days, and the total induction is continued for 20 days.

Dithizone (DTZ) staining: cells were induced followed by DTZ staining if the cell pellet was stained red-brown

(positive for DTZ), indicating that the induced cytoplasm is rich in zinc ions, indicating the presence of islet-like cells. Specific induction rates are shown in table 2.

TABLE 2 islet-like cell induction results

Grouping	Inductivity (%)
		Transgenic adipose-derived stem cell monoclonal antibody induction group	99.13±0.11
Non-transgenic mesenchymal stem cell monoclonal antibody induction group	87.51±0.28
		Transgenic adipose-derived stem cellsInduction group	90.33±0.39
Non-transgenic mesenchymal stem cell induction group	76.28±0.30

The results in table 2 show that after the gamma-secretase gene of the adipose-derived stem cell is knocked out and is induced by a specific gamma-secretase monoclonal antibody, the induction effect of the islet-like cell close to 99.13 +/-0.11 can be realized within a short induction time of 20d, and the pancreatic islet-like cell induction method has an excellent application value.

Western blot to detect protein expression of islet cell-associated markers: harvesting the induced cells according to a digestion passage method, extracting total protein from RIPA cell lysate, uniformly mixing by blowing, centrifuging for 15min at 4 ℃ and 1000g, collecting supernatant, determining the concentration of the total protein by a BCA method, boiling for 3-5 min by boiling water at 100 ℃, adding a sample buffer solution after full denaturation, carrying out electrophoresis on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) denaturing gel, electrically transferring onto a nitrocellulose membrane, and sealing 5% skimmed milk at room temperature for 1.5 h. The cells were incubated with mouse anti-rat insulin (1: 1000), mouse anti-rat glucagon (1: 100), rabbit anti-rat Ngn3 (1: 100), respectively, overnight at 4 ℃ and HRP-goat anti-rabbit or mouse IgG secondary antibodies for 2 h. And (3) tabletting, developing and exposing by using ECL luminous liquid in a dark room. Based on the relative expression amount of GAPDH, the results are shown in FIG. 3.

Expression of cellular islet-associated protein after induction: the Western blot results show that: the expression of insulin, glucagon and Ngn3 protein which are markers of islet cell maturation is the highest in the expression level of the transgenic adipose-derived stem cell and monoclonal antibody induction group.

(5) After the cells were cultured with 33.0mmol/L glucose after induction of glucose-stimulated insulin secretion, the insulin content of the cell culture supernatant was measured by ELISA, according to the instructions of the kit.

The results of the effect of glucose stimulation on insulin secretion by cells after induction are shown in the table below.

TABLE 3 results of the Effect of glucose stimulation on insulin secretion from cells after Induction

Grouping	Insulin concentration ng/mg
		Transgenic adipose-derived stem cell monoclonal antibody induction group	28.88±0.15
Non-transgenic mesenchymal stem cell monoclonal antibody induction group	23.01±0.17
		Transgenic adipose-derived stem cell induction group	22.28±0.15
Non-transgenic mesenchymal stem cell induction group	15.28±0.16

As can be seen from the results in Table 3, the concentration of insulin in cell supernatants was measured by ELISA after the cell coculture with 33.0mmol/L glucose and induction, and the highest value of the transgenic adipose-derived stem cell monoclonal antibody induction group was 28.88 + -0.15 ng/mg protein. The results show that the transgenic stem cells and the antibody have better differentiation effect under the combined action, and the cells have obvious insulin secretion reaction to glucose.

Example 5 mouse model test

Establishing a diabetes animal model: after the rats are subjected to adaptive feeding for 3 days, 10 rats are randomly selected as normal control groups, and the rest rats are used for establishing a diabetes model. Firstly, the mixture is buffered by 0.1mol/L citric acid-sodium citrateThe solution (pH4.2-4.5) is prepared by preparing 0.1% injection with streptozotocin, and injecting 70mg/kg intraperitoneally after rat fasting for 18h in a dark ice bath. Blood sugar is detected 3d after injection, and the blood sugar concentration is higher than 16.7mol/L for 3 times continuously to indicate that the molding is successful. Cell transplantation: after 10 days of modeling, the cells of the four groups of induction groups were transplanted with 2 mL/cell suspension (cell number 2X 10) via tail vein⁶One). The normal control group and the model control group were injected with 2 mL/mouse of culture solution without any cells via tail vein. The transplantation was performed 1 time every 2 weeks and 2 times in total. After the 2 nd transplantation, observation is continued for 4 weeks, and then the tail end of the rat is disinfected by ethanol with the volume fraction of 75% during blood glucose detection, and then the fasting blood glucose level of the rat is detected by tail vein blood collection by using a Roche glucometer. The results are shown in FIG. 4.

After cell transplantation treatment, compared with a model control group, the blood sugar of rats in each treatment group is obviously reduced, so that the blood sugar of rats in each treatment group can be reduced to different degrees after cell transplantation, and the blood sugar reducing effect of the transgenic adipose-derived stem cell monoclonal antibody induction group is superior to that of other groups, and the transgenic adipose-derived stem cell monoclonal antibody induction group has a good effect.

Example 6 mouse model synergy test

Establishing a diabetes animal model: after the rats are subjected to adaptive feeding for 3d, 10 rats are randomly selected as normal control groups, and the rest groups are used for establishing a diabetes model. Firstly, 0.1mol/L citric acid-sodium citrate buffer solution (pH4.2-4.5) is used for preparing 0.1% injection with streptozotocin, ice bath is protected from light, and 70mg/kg of streptozotocin is injected into abdominal cavity after rats empty stomach for 18 h. Blood sugar is detected 3d after injection, and the blood sugar concentration is higher than 16.7mol/L for 3 times continuously to indicate that the molding is successful. Cell transplantation: after 10 days of modeling, the cells of the transgenic adipose-derived stem cell monoclonal antibody induction group are transplanted into 2 mL/cell (the number of cells is2 multiplied by 10) of corresponding cell suspension through tail vein respectively⁶One). Transgenic adipose-derived stem cell monoclonal antibody induction group and chitooligosaccharide treatment group, wherein induced differentiated cells are transplanted into corresponding cell suspension 2 mL/cell (cell number 2X 10)⁶Respectively) and simultaneously perfusing 60mg (kg. d) of chitooligosaccharide every day. The normal control group and the model control group were injected with 2 mL/mouse of culture solution without any cells via tail vein. The transplantation was performed 1 time every 2 weeks and 2 times in total. After the 2 nd transplantation, observation was continued for 4 weeks, and then the body was used first in blood glucose measurementThe volume fraction of 75% ethanol was used to disinfect the tail end of the rat, and the fasting blood glucose level of the rat was measured by tail vein blood collection using a Roche glucometer. The results are shown in FIG. 5.

From the results of fig. 5, it can be seen that after the cell transplantation treatment, the blood sugar of rats in each treatment group was significantly reduced compared to the model control group, and after the intragastric lavage chitooligosaccharide and the injected differentiated cells were used together, the blood sugar reducing effect was better than that of the group without intragastric lavage chitooligosaccharide, and the effect was better.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

<110> Luoyang Xuan Zhi Biotech Co., Ltd

<120> pharmaceutical composition containing gene-edited adipose-derived stem cells

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Claims

1. Use of a kit for the preparation of a pharmaceutical composition for the treatment of diabetes in mice, said kit consisting of pancreatic islet-like cells and chito-oligosaccharide; wherein, the islet-like cells are prepared by the following method: the method comprises (1) preparing a knockout adipose-derived mesenchymal stem cell; the gene knockout is carried out by using a CRISPR method and using AsCpf1 protein and gRNA to carry out gene knockout by adopting electric transfer, and a specific sgRNA sequence is shown as SEQ ID NO:1 is shown in the specification; (2) culturing and fusing 80-90% of the gene-knocked-out adipose-derived mesenchymal stem cells prepared in the step (1), and inducing and culturing the cells for 10 days by using DMEM culture solution of 10% FBS (fetal bovine serum) which comprises 100 mu g/L of beta-nerve growth factor, 4nmol/L of activin A, 10mmol/L of nicotinamide, 25 mu g/L of epidermal growth factor and 50 mu g/L of monoclonal antibody, wherein half of the culture solution is replaced every 2 days; then the culture medium is changed into DMEM with 10% FBS and the components of 10mmol/L nicotinamide, 10 mug/L basic fibroblast growth factor, 50 mug/L monoclonal antibody and 1% insulin-transferrin-selenium, the induction is continued for 10 days, and the total induction is 20 days; wherein the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 2, the heavy chain variable region sequence is shown as SEQ ID NO: 3 is shown in the specification; the pharmaceutical composition also comprises a pharmaceutically acceptable carrier; the specific dosage is 1 × 10 therapeutic cell number of islet-like cells⁹1 x10 of¹¹The weight of the chitosan oligosaccharide is 60 mg/kg.