CN113151182B

CN113151182B - Preparation method and application of gene recombinant cell based on MSC

Info

Publication number: CN113151182B
Application number: CN202110416022.6A
Authority: CN
Inventors: 张明杰; 马云坤
Original assignee: Shenzhen Mingjie Bioengineering Co ltd
Current assignee: Shenzhen Mingjie Bioengineering Co ltd
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2022-12-06
Anticipated expiration: 2041-04-19
Also published as: CN113151182A

Abstract

The invention discloses a preparation method of a gene recombinant cell based on MSC, which comprises the following steps: s101: cloning the coding genes of IL-35, IL-22 and FGF1 into a eukaryotic expression vector pDisplay with a transmembrane region structure respectively to form IL-35, IL-22 and FGF1 cell membrane surface display plasmids (pDIL 35, pDIL22 and pDFGF 1); s102: preparing hUC-MSC cells; s103: equal amounts of pDIL35, pDIL22, pDFGF1 plasmids were mixed and transfected into MSC cells, yielding MSCd12235 cells. The invention expresses a plurality of cytokines with known biological activity on the surface of a functional cell to form a gene recombinant cell with novel biological function. The functional cell provides a solid-phase action surface for the active factor, and the active factor endows the original cell with new functions and is applied to the treatment of diabetes.

Description

Preparation method and application of gene recombinant cell based on MSC

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of a gene recombinant cell based on MSC.

Background

Diabetes is a group of metabolic diseases characterized by hyperglycemia. It is also a common disease of endocrine system, which is a syndrome of disorder of metabolism of sugar, fat and protein mainly caused by sugar metabolism due to deficiency of insulin in vivo or failure of insulin to exert normal physiological action due to its own quality and other reasons. Hyperglycemia, which is present in long-term diabetes, leads to chronic damage to, dysfunction of, various tissues, particularly, the eyes, kidneys, heart, blood vessels, nerves.

Diabetes is mainly classified into four types: type I diabetes (T1 DM), type II diabetes (T2 DM), gestational diabetes, and specific type diabetes.

In the treatment of stem cells of diabetes, in addition to the direct action of inducing differentiation to prepare islet beta cells, mesenchymal Stem Cells (MSCs) from tissues such as umbilicus, bone marrow, fat and the like have certain curative effects on T1D and T2D and diabetic complications. The mechanism of MSC for treating diabetes mainly acts through the paracrine action of various cytokines (MCP-1, IL-6 and the like) with immunosuppressive property secreted by MSC, and comprises the functions of promoting the regeneration of islet beta cells, inhibiting inflammation, improving insulin resistance, improving the autophagy capability of the islet beta cells, improving the autophagy function of the liver, improving the glucose uptake and glycogen synthesis capability of the liver and the like. At present, MSC has the effect of improving complications to a certain extent, can reduce the dosage of other hypoglycemic drugs, but cannot reach the degree of drug interruption.

Disclosure of Invention

The invention mainly aims to provide a preparation method and application of a gene recombinant cell based on MSC; the gene recombinant cell is applied to the treatment of diabetes, is effective to both T1DM and T2DM, and has better curative effect than the MSC cell without gene recombination.

In order to achieve the above objects, the present invention provides a method for preparing a recombinant cell based on MSC, comprising the steps of:

s101: cloning coding genes of IL-35, IL-22 and FGF1 into a eukaryotic expression vector pDislay with a transmembrane region structure respectively to form cell membrane surface display plasmids (pDIL 35, pDIL22 and pDIGF 1) of IL-35, IL-22 and FGF1;

s102: preparing hUC-MSC cells;

s103: equal amounts of pDIL35, pDIL22, pDFGF1 plasmids were mixed and transfected into MSC cells, yielding MSCd12235 cells.

Preferably, in S101, the method includes:

s1011: constructing an IL-35 membrane surface display plasmid (pDIL 35);

s1012: constructing an IL-22 membrane surface display plasmid (pDIL 22);

s1013: an FGF1 membrane surface display plasmid (pDFGF 1) was constructed.

Preferably, in S1011, the method includes:

s10111: primer design

1) Primer 1 IL35EBI3upSacII GACGTCCCGCGGatgacccgcagcttcctg;

2) Primer 2 IL35EBI3dnSalI GACGTCGTCGACCttgccccaggctcttgtggca;

3) Primer 3 IL35IL12AupBglII GACGTCAGATCTatggtcgtccaacacagcc;

4) Primer 4 IL35IL12AdnSalI GACGTCGTCGACggcgagctcagatagcccat;

s10112: preparation of RNA: collecting 50ml of peripheral blood of a volunteer, separating lymphocyte (PBMC) by using lymphocyte separating medium, and preparing total RNA by using a TRIzol reagent;

S10113： RT- PCR：

1) Using total RNA as a template, using a primer 1 and a primer 2 as primers, preparing an IL35EBI3 gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit, and purifying by using an EZ Spin column type PCR product purification kit;

2) Taking total RNA as a template, taking a primer 3 and a primer 4 as primers, preparing an IL35IL12A gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit, and purifying by using an EZ spin column type PCR product purification kit;

s10114: pDisplay vector digested by restriction enzyme and PCR product

1) Digesting pDisplay vectors by SacI/SalI and BglII/SalI restriction enzymes respectively, and purifying by EZ-10 Spin Column DNA PAGE Gel Extraction Kit respectively to obtain pDisplay SacII/SalI and pDisplay BglII/SalI 2 restriction enzyme digestion vector products;

2) Digesting the IL35EBI3 gene segment by SacII/SalI restriction enzyme respectively, digesting the IL35IL12A gene segment by BglII/SalI restriction enzyme, and purifying by EZ-10 Spin Column DNA PAGE Gel Extraction Kit respectively to obtain 2 enzyme digestion gene products of the SacII/SalI segment of the IL35EBI3 gene and the BglII/SalI segment of the IL35IL12A gene;

s10115: connection of

1) Connecting the pDisplay SacII/SalI and the SacII/CalI fragment of the IL35EBI3 gene by using DNA ligase to obtain a pD-IL35EBI3 product;

2) Connecting pDisplay BglII/SalI and IL35IL12A gene BglII/SalI fragment by using DNA ligase to obtain a pD-IL35IL12A product;

s10116: transfecting an allelopathic bacterium:

transfecting DH5 alpha competent bacteria with a pD-IL35EBI3 product and a pD-IL35IL12A product respectively;

s10117: selecting bacterial colonies and verifying:

selecting positive colonies, carrying out small-scale culture, extracting plasmid DNA, carrying out primary selection by using corresponding restriction enzyme digestion, and finally carrying out sequencing confirmation. Storing the recombinant pD-IL35EBI3 and pD-IL35IL12A bacteria;

s10118: culturing pD-IL35EBI3 and pD-IL35IL12A recombinant bacteria to prepare pD-IL35EBI3 and pD-IL35IL12A recombinant plasmids.

Preferably, in S1012, the method includes:

s10121: primer design

1) Primer 5 IL22upBglII GACGTCAGATCTatggccgctgcagaatct;

2) Primer 6 IL22dnSalI GACGTCGTCGACaatgcaggcattctcagga;

s10122: preparation of RNA:

collecting 50ml of peripheral blood of a volunteer, separating lymphocyte (PBMC) by using lymphocyte separating medium, and preparing total RNA by using a TRIzol reagent;

S10123： RT- PCR

preparing an Il-22 gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit by using total RNA as a template and a primer 5 and a primer 6 as primers, and purifying by using an ES Spin column type PCR product purification kit;

s10124: pDisplay vector digested by restriction endonuclease and PCR product

1) Digesting the pDisplay vector by a BglII/SalI restriction enzyme, and purifying by using an EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain a pDisplay BglII/SalI restriction enzyme digestion vector product;

2) Digesting the IL-22 gene fragment by BglII/SalI restriction enzyme, and respectively purifying by using an EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain an IL-22 gene BglII/SalI fragment enzyme digestion gene product;

s10125: connecting, namely connecting the pDisplay BglII/SalI and the IL-22 gene BglII/SalI fragment by using DNA ligase to obtain a pD-IL22 product;

s10126: transfecting an infected bacterium: transfecting DH5 alpha competent bacteria with the pD-IL22 product;

s10127: selecting bacterial colonies and verifying: selecting positive colonies, carrying out small-scale culture, extracting plasmid DNA, carrying out primary selection by using corresponding restriction enzyme digestion, finally carrying out sequencing confirmation, and storing the pD-IL22 recombinant bacteria;

s10128: and (3) culturing the pD-IL22 recombinant bacteria to prepare the pD-IL22 recombinant plasmid.

Preferably, in S1013, the method includes:

s10131: primer design

1) Primer 7 FGF1upBglII GACGTCAGATCTatggctgaagggaaaatcaccc;

2) A primer 8 FGF1dnSalI GACGTCGTCGACatcagagagaactggcaggg;

s10132: preparation of RNA: collecting 50ml of peripheral blood of a volunteer, separating lymphocyte (PBMC) by using lymphocyte separating medium, and preparing total RNA by using a TRIzol reagent;

s10133: RT-PCR: taking total RNA as a template, taking a primer 5 and a primer 6 as primers, preparing an FGF1 gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit, and purifying by using an ES Spin column type PCR product purification kit;

s10134: pDisplay vector digested by restriction enzyme and PCR product

1) Digesting the pDisplay vector by BglII/SalI restriction enzyme, and purifying by using an EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain a pDisplay BglII/SalI restriction enzyme digestion vector product;

2) Digesting FGF1 gene segments by BglII/SalI restriction enzymes, and respectively purifying by using an EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain FGF1 gene BglII/SalI segment enzyme digestion gene products;

s10135: connecting, namely connecting pDisplay BglII/SalI and FGF1 gene BglII/SalI fragments by using DNA ligase to obtain a pD-FGF1 product;

s10136: transfecting an infected bacterium: transfecting DH5 alpha competent bacteria with the pD-FGF1 product;

s10137: selecting bacterial colonies and verifying: selecting positive colonies, carrying out small-scale culture, extracting plasmid DNA, carrying out primary selection by using corresponding restriction enzyme digestion, and finally carrying out sequencing confirmation. Storing the pD-FGF1 recombinant bacteria;

s10138: culturing the pD-FGF1 recombinant bacteria to prepare pD-FGF1 recombinant plasmids.

Preferably, in S102, the method includes the following steps:

s1021: the umbilical cord is taken from a healthy pregnant woman born by caesarean section in term of term, the pregnant woman has negative detection of HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-treponema pallidum antibody, mycoplasma, anti-cytomegalovirus antibody and the like, and all examinations of the pregnant woman in pregnancy period, including TORCH, down's screening and the like, are normal;

s1022: collecting umbilical cord under aseptic condition, cutting the umbilical cord into small segments, cutting into 1cm each segment, removing arteriovenous (two arteries and 1 vein), cutting into pieces in a glass bottle, placing in a 15ml centrifuge tube (2 small segments and 2cm each tube), adding collagenase to digest (IV type) by 8ml, digesting for 3h, filtering with a 100-mesh filter screen (or centrifuging at 500rpm for 3-5 min), discarding undigested tissue blocks, transferring the supernatant to a 15ml centrifuge tube, adding PBS to 10ml each tube, centrifuging at high speed (3000-4000 rpm), discarding the supernatant, taking the precipitate, transferring the precipitate to a culture dish for culture, placing at 37 ℃, incubating in a 5 CO2 saturation humidity incubator, changing liquid for half 48h, changing liquid for full amount after 4d, and discarding a large amount of suspended cells;

s1023: after every 3-4d, the medium color is changed, and after the primary cells reach 60-80% of the covered culture dish, 1:2, passage;

s1024: identification and use criteria of umbilical cord mesenchymal stem cells: adopting 3 rd-10 th generation cells, when the number of the cells reaches 108, flushing the cells for 2 times by using PBS, dripping 0.05 percent Ttypsin-EDTA solution for digestion, adding a culture medium for terminating digestion to prepare 1-3 multiplied by 106 cell suspension, and detecting by using a flow cytometer to show that: CD14 or CD11b, CD79a or CD19, CD34, CD45, CD109, HLA-DR negative <2%; the positive rates of CD29, CD44, CD73, CD90 and CD105 are all more than 95 percent, and the positive rates of EB virus, cytomegalovirus, HIV virus, hepatitis B virus, mycoplasma, bacterial culture and fungal culture are all negative;

s1025: directly using, or storing in liquid nitrogen for later use.

Preferably, in S103, the method includes the following steps:

s1031 preparation of cells: taking each well of a 6-well plate as an example, a proper amount (5X 105) of MSC cells are suspended in 2ml of culture solution for transfection when the cell density is 50-80% full one day before transfection;

s1032, 1ug of each 4 plasmids of pDIL22, pDIGF 1 and pDIL35EBI3 + pDIL35IL12A are mixed evenly;

s1033 preparation of transfection reagent: add 4. Mu.g plasmid DNA into 600. Mu.l Opti-MEM low serum culture medium, mix well; mix Lipofectamine LTX reagent gently, add 5 μ l into DNA tube; mixing, and incubating at room temperature for 30min;

s1034, adding about 100 mu l of DNA-Lipofectamine LTX compound into each hole of cell, and slightly shaking the culture plate back and forth and left and right to mix evenly;

s1035:37oC,5% CO2 incubator, 24 hours later, the transfected MSCd12235 cells were taken, respectively added with mouse anti-human FGF1 (Abcam), IL22 (Abcam) and IL35 (Abcam) monoclonal antibodies, incubated at 37 ℃ for 1 hour, washed 2 times, then added with FITC fluorescent labeled rabbit anti-mouse secondary antibody (Abcam), incubated at 30 ℃ for 30 minutes, washed 2 times, and then suspended in 50. Mu.l of physiological saline;

s1036 fluorescence microscope (Orlingbas) observation result shows that the positive rate of FGF1, IL22 and IL35 is about 55%.

The invention also discloses application of the MSC-based gene recombinant cell in treating diabetes.

The invention displays (expresses) IL-35 which has known therapeutic activity on T1DM, IL-22 which is effective on T2DM and FGF1 which is effective on both T1DM and T2DM on the surface of MSC cells which are effective on both T1DM and T2DM, thereby generating gene recombinant FGF1IL22IL35DMSC (MSCd 12235, MSC displaced FGF1IL22 IL-35) cells which simultaneously display IL-35, IL-22 and FGF1. The MSCd12235 cells which simultaneously display IL-35, IL-22 and FGF1 on the surface not only retain the function of MSC for treating diabetes, but also provide support equivalent to solid phase for the factors by anchoring IL-35, IL-22 and FGF1 on the surface of the MSC, improve the stability of the factors and prolong the half-life period, and are more effective than free factors. MSCd12235 was effective against both T1DM and T2DM and was more efficacious than MSC cells.

Drawings

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

FIG. 1 is a flowchart of a method for preparing a MSC-based recombinant cell according to an embodiment of the present invention;

FIG. 2 is a pDisplay plasmid map provided by an embodiment of the present invention;

FIG. 3 is a graph of the effect of different MSC cell preparations on lowering blood glucose in diabetic rats provided by an embodiment of the present invention;

the implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

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

It should be noted that all directional indicators (such as up, down, left, right, front, back \8230;) in the embodiments of the present invention are only used to explain the relative positional relationship between the components, the motion situation, etc. in a specific posture (as shown in the attached drawings), and if the specific posture is changed, the directional indicator is changed accordingly.

In addition, the descriptions relating to "first", "second", etc. in the present invention are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.

In the embodiment of the present invention, referring to fig. 1, the method for preparing the MSC-based recombinant gene cell includes the steps of:

s102: preparing hUC-MSC cells;

Further, in S101 above, the method includes:

s1011: constructing an IL-35 membrane surface display plasmid (pDIL 35);

IL-35 is composed of IL-12 alpha and IL-27 beta chain dimers encoded by two genes, IL12A and EBI 3. In tissues, IL-35 is not stably expressed, but is expressed after induction by pro-inflammatory stimuli. IL-35 has a selective regulatory effect on different T cell subsets, inducing the proliferation of Tregs populations but reducing the activity of Th17 cell populations.

The plasmid map of pDisplay is shown in FIG. 2, and the present example uses pDisplay as a vector, but is not limited to pDisplay vector; but includes eukaryotic expression vectors consisting of any promoter, transmembrane region, and intracellular signaling structure.

Wherein, the first and the second end of the pipe are connected with each other,

the IL-35EBI3 amino acid sequence is: MTPQLLLALVLWASCPPCSGKGPPALLPRVQCRASRYPIAVDCSWTLPAPPNSTS

PVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSS

SFVPFITEHIIKPDPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGA

ARFHRVGPIEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK

The IL-35EBI3 gene sequence is as follows:

atgaccccgcagcttctcctggcccttgtcctctgggccagctgcccgccctgcagtgga

aggaaagggcccccagcagctctgacactgccccgggtgcaatgccgagcctctcggtac

ccgatcgccgtggattgctcctggaccctgccgcctgctccaaactccaccagccccgtg

tccttcattgccacgtacaggctcggcatggctgcccggggccacagctggccctgcctg

cagcagacgccaacgtccaccagctgcaccatcacggatgtccagctgttctccatggct

ccctacgtgctcaatgtcaccgccgtccacccctggggctccagcagcagcttcgtgcct

ttcataacagagcacatcatcaagcccgaccctccagaaggcgtgcgcctaagccccctc

gctgagcgccagctacaggtgcagtgggagcctcccgggtcctggcccttcccagagatc

ttctcactgaagtactggatccgttacaagcgtcagggagctgcgcgcttccaccgggtg

gggcccattgaagccacgtccttcatcctcagggctgtgcggccccgagccaggtactac

gtccaagtggcggctcaggacctcacagactacggggaactgagtgactggagtctcccc

gccactgccacaatgagcctgggcaag

the amino acid sequence of IL-35 IL-12A is:

MVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSR

NLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRE

TSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGML

VAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA

the IL-35 IL-12A gene sequence is:

atggtcagcgttccaacagcctcaccctcggcatccagcagctcctctcagtgccggtcc

agcatgtgtcaatcacgctacctcctctttttggccacccttgccctcctaaaccacctc

agtttggccagggtcattccagtctctggacctgccaggtgtcttagccagtcccgaaac

ctgctgaagaccacagatgacatggtgaagacggccagagaaaaactgaaacattattcc

tgcactgctgaagacatcgatcatgaagacatcacacgggaccaaaccagcacattgaag

acctgtttaccactggaactacacaagaacgagagttgcctggctactagagagacttct

tccacaacaagagggagctgcctgcccccacagaagacgtctttgatgatgaccctgtgc

cttggtagcatctatgaggacttgaagatgtaccagacagagttccaggccatcaacgca

gcacttcagaatcacaaccatcagcagatcattctagacaagggcatgctggtggccatc

gatgagctgatgcagtctctgaatcataatggcgagactctgcgccagaaacctcctgtg

ggagaagcagacccttacagagtgaaaatgaagctctgcatcctgcttcacgccttcagc

acccgcgtcgtgaccatcaacagggtgatgggctatctgagctccgcc

further, in S1011 described above, the method includes:

s10111: primer design

1) Primer 1 IL35EBI3upSacII GACGTCCCGCGGatgacccgcagcttcctg;

2) Primer 2 IL35EBI3dnSalI GACGTCGTCGACCttgccccaggctcttgtggca;

3) Primer 3 IL35IL12AupBglII GACGTCAGATCTatggtcgtccaacacagcc;

4) Primer 4 IL35IL12AdnSalI GACGTCGTCGACggcgagctcagatagcccat;

s10112: preparation of RNA: collecting 50ml of peripheral blood of a volunteer, separating lymphocyte (PBMC) by using lymphocyte separation liquid, and preparing total RNA by using a TRIzol reagent;

S10113： RT- PCR：

1) Preparing an IL35EBI3 gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit and purifying by using an EZ Spin column PCR product purification kit by using total RNA as a template and a primer 1 and a primer 2 as primers;

2) Using total RNA as a template, using a primer 3 and a primer 4 as primers, preparing an IL35IL12A gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit, and purifying by using an EZ spin column type PCR product purification kit;

s10114: pDisplay vector digested by restriction endonuclease and PCR product

s10115: connection of

1) Connecting pDdisply SacII/SalI and IL35EBI3 gene SacII/CalI fragments by using DNA ligase to obtain a pD-IL35EBI3 product;

s10116: transfecting an allelopathic bacterium:

s10117: selecting bacterial colonies and verifying:

selecting positive colonies, carrying out small-scale culture, extracting plasmid DNA, carrying out primary selection by using corresponding restriction enzyme digestion, and finally carrying out sequencing confirmation. Storing pD-IL35EBI3 and pD-IL35IL12A recombinant bacteria;

S1012: constructing an IL-22 membrane surface display plasmid (pDIL 22);

IL-22 is an alpha-helical protein encoded by the IL-22 gene, a cytokine belonging to the IL-10 family (including IL-19, IL-20, IL-24, IL-26), a potent mediator of cellular inflammatory responses. Its receptor (IL-22R) is composed of two heterogeneous dimeric subunits of IL-10R2 and IL-22R1, and is found only in tissue cells, not in immune cells. IL-22 is produced by activated NK and T cells and initiates an innate immune response against epithelial cells, particularly against pathogens in respiratory and intestinal epithelial cells. IL-22 and IL-17 together are produced by LTi-like cells and have a role in both the innate and adoptive immune systems, as well as autoimmune and tissue regeneration. IL-22 exerts biological functions upon binding to tissue cell surface IL-22R. The complex formed by IL-22 and IL-22R interacts with IL-22BP, and jointly regulates the activity of IL-22. IL-22BP has an amino acid sequence similar to that of the extracellular domain of IL-22R1 (sIL-22R 1). IL-22 acts primarily on hepatocytes, keratinocytes, lung and intestinal epithelial cells outside the hematopoietic system, and it induces pancreatic beta cell proliferation.

It was found that feeding IL-22 to obese and T2DM mice protected their beta cells from stress, fully restored glycemic control, and provided a new approach to the treatment of T2DM (S Hasnain et al, nature Medicine, 02 November 2014; doi: 10.1038/nm.3705). The formation of T2DM is often associated with obesity, and is also associated with a relative lack of insulin production, which increases the stress that beta cells are subjected to. This pressure, on the one hand, is due to the increased levels of fat and sugar in the blood, which increases the demand of the body for cells producing insulin. Another aspect is the stress induced by proteins from the immune system, called cytokines, released into the environment surrounding the beta cells; blocking the action of these cytokines has the advantage of controlling blood glucose, thus providing some new targets for treating T2 DM.

Beta cells accumulate in the pancreas, called islets. In general, the human pancreas contains hundreds of thousands of such islets, each islet containing about 200 beta cells. Each beta cell can produce approximately 100 million molecules of insulin per minute. Beta cells, the most prominent form of stress in diabetes, are oxidative stress, when it occurs, reactive oxygen species are produced intracellularly. Oxidative stress can interfere with cellular metabolism and activate the immune system. Oxidative stress interferes with the proper assembly of proteins (e.g., insulin) into their proper structure within a specialized organelle (the endoplasmic reticulum). In diabetic patients, endoplasmic reticulum stress in the beta cells reduces the amount of insulin produced, thereby inducing immune surveillance systems, eliminating the beta cells, and even triggering beta cell suicide. IL-22 can block these β -cell clearance and β -cell suicide processes by preventing the generation of oxidative stress. It can effectively resist various pressure inducers, close the gene of the coding protein causing pressure, open the gene of the coding antioxidant protein and remove active oxygen free radicals. That is, IL-22 is a powerful natural antioxidant for beta cells.

The application of IL-22 to treat T2DM not only can reduce blood sugar, but also can induce beta cell proliferation and limit disease progression. IL-22 is attractive to solve the fundamental problem of T2D, and to naturally control blood glucose by producing high quality, potent insulin.

Another key characteristic of diabetes is the diminished response of cells in muscle, fat and liver to insulin. Treatment of mice with IL-22 not only restored appropriate insulin release from the pancreas, but also restored insulin sensitivity to normal.

However, IL-22 was found to be ineffective against T1 DM.

Wherein the content of the first and second substances,

the IL-22 amino acid sequence is:

MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTF MLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPY MQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLF

MSLRNACI

the IL-22 gene sequence is:

atggccgccctgcagaaatctgtgagctctttccttatggggaccctggccaccagctgc

ctccttctcttggccctcttggtacagggaggagcagctgcgcccatcagctcccactgc

aggcttgacaagtccaacttccagcagccctatatcaccaaccgcaccttcatgctggct

aaggaggctagcttggctgataacaacacagacgttcgtctcattggggagaaactgttc

cacggagtcagtatgagtgagcgctgctatctgatgaagcaggtgctgaacttcaccctt

gaagaagtgctgttccctcaatctgataggttccagccttatatgcaggaggtggtgccc

ttcctggccaggctcagcaacaggctaagcacatgtcatattgaaggtgatgacctgcat

atccagaggaatgtgcaaaagctgaaggacacagtgaaaaagcttggagagagtggagag

Atcaaagcaattggagaactggatttgctgtttatgtctctgagaaatgcctgcatt

further, in S1012 above, the method includes:

s10121: primer design

1) Primer 5 IL22upBglII GACGTCAGATCTatgccgcctgcagaatct;

2) Primer 6 IL22dnSalI GACGTCGTCGACaatgcaggcattctcagga;

s10122: preparation of RNA:

collecting 50ml of peripheral blood of a volunteer, separating lymphocyte (PBMC) by using lymphocyte separation liquid, and preparing total RNA by using a TRIzol reagent;

S10123： RT- PCR

s10124: pDisplay vector digested by restriction endonuclease and PCR product

s10126: transfecting an allelopathic bacterium: transfecting DH5 alpha competent bacteria with the pD-IL22 product;

s10127: selecting bacterial colonies and verifying: selecting positive bacterial colonies, carrying out small-scale culture, extracting plasmid DNA, carrying out primary selection by using corresponding restriction enzyme digestion, finally carrying out sequencing confirmation, and storing the pD-IL22 recombinant bacteria;

s10128: culturing the pD-IL22 recombinant bacteria to prepare pD-IL22 recombinant plasmids.

S1013: an FGF1 membrane surface display plasmid (pDFGF 1) was constructed.

FGF1 is a cytokine with certain therapeutic effect on both T1DM and T2 DM. It was found that mice deficient in FGF1 growth factor were provided with a high fat diet, which soon developed diabetes, suggesting that FGF1 plays a key role in the control of blood glucose levels. FGF1 is injected into obese diabetic mice and given a single dose of FGF1, the blood glucose levels in all diabetic mice are rapidly brought to normal levels. Many previous studies have shown that FGF1 injection has no effect on healthy mice. And when it was injected into diabetic mice, glucose was significantly improved.

FGF1 therapy has many advantages over the hypoglycemic drug pioglitazone (Actos). Pioglitazone can cause weight gain, heart and liver risk problems, and other side effects. Even high doses of FGF1 do not cause these side effects or lead to dangerously low levels of glucose levels. In contrast, FGF1 injection restores the body's own ability to naturally regulate insulin and blood glucose levels, maintains glucose levels within safe ranges, and effectively reverse transcribes some of the core symptoms of diabetes. FGF1 does not cause hypoglycemia or other common side effects.

Wherein the content of the first and second substances,

the FGF1 amino acid sequence is as follows:

MAEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQ

LSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEK

NWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD

the FGF1 gene sequence is as follows:

atggctgaaggggaaatcaccaccttcacagccctgaccgagaagtttaatctgcctcca

gggaattacaagaagcccaaactcctctactgtagcaacgggggccacttcctgaggatc

cttccggatggcacagtggatgggacaagggacaggagcgaccagcacattcagctgcag

ctcagtgcggaaagcgtgggggaggtgtatataaagagtaccgagactggccagtacttg

gccatggacaccgacgggcttttatacggctcacagacaccaaatgaggaatgtttgttc

ctggaaaggctggaggagaaccattacaacacctatatatccaagaagcatgcagagaag

aattggtttgttggcctcaagaagaatgggagctgcaaacgcggtcctcggactcactat

ggccagaaagcaatcttgtttctccccctgccagtctcttctgat

further, in S1013 above, the method includes:

s10131: primer design

1) Primer 7 FGF1upBglII GACGTCAGATCTatggctgaagggaaaatcaccc;

2) Primer 8 FGF1dnSalI GACGTCGTCGACatcagaagacagactggcaggg;

s10132: preparation of RNA: collecting 50ml of peripheral blood of a volunteer, separating lymphocyte (PBMC) by using lymphocyte separation liquid, and preparing total RNA by using a TRIzol reagent;

s10133: RT-PCR: using total RNA as a template, using a primer 5 and a primer 6 as primers, preparing an FGF1 gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit, and purifying by using an ES Spin column PCR product purification kit;

s10134: pDisplay vector digested by restriction endonuclease and PCR product

s10136: transfecting an allelopathic bacterium: transfecting DH5 alpha competent bacteria with the pD-FGF1 product;

Further, in S102, the method includes the following steps:

s1023: and then changing the culture medium every 3-4 days according to the color of the culture medium, and after the primary cells reach 60-80% of the covered culture dish, giving a 1:2, passage;

s1025: directly using, or storing in liquid nitrogen for later use.

Of course, the present embodiment is not limited to the umbilical cord-derived MSCs, but includes MSCs derived from placenta, bone marrow, dental pulp, fat, and the like.

Further, in the above S103, the method includes the steps of:

s1032, 1 microgram of each 4 groups of plasmids, namely pDIL22, pDIGF 1 and pDIL35EBI3 + pDIL35IL12A, are uniformly mixed;

s1034, adding about 100ul of DNA-Lipofectamine LTX compound into each hole of cell, and slightly shaking the culture plate back and forth, left and right to mix evenly;

s1036 fluorescence microscope (Olympus) observation result shows that the positivity of FGF1, IL22 and IL35 proteins is about 55%.

On the other hand, the MSCd12235 cells of this example were used in the treatment of diabetes.

Establishment of T1DM and T2DM rat animal models and experimental treatment

Male SD rats, after 6 weeks of high-fat high-sugar feeding, were modeled on type 2 diabetes mellitus (T2 DM) by a single intraperitoneal injection of 2% STZ solution at 35mg/kg, and after 6 weeks of ordinary feed feeding, were modeled on type 1 diabetes mellitus (T1 DM) by a single intraperitoneal injection of 2% STZ solution at 60 mg/kg.

Animal and feed is 6-7 weeks old clean male SD rat with body weight of 180-200g; the high-fat high-sugar feed formula comprises 10% of lard, 20% of cane sugar, 2.5% of cholesterol, 0.5% of cholate and 67% of basal feed.

Streptozotocin (STZ, sigma usa), glucometer (roche germany).

Establishing and grouping rat animal models, adaptively feeding rats for 3 days.

(1) 10 patients were fed with high-fat and high-sugar diet at the beginning of the experiment, i.e., week 0, and fed for 12 hours after fasting for 6 weeks, and then 2% STZ solution (pH 4.4) was intraperitoneally injected at a single dose of 35mg/kg to establish a type 2 diabetes model (T2 DM group);

(2) 10 rats were fed with ordinary feed only, fed with 2% STZ (pH 4.4) solution at 60mg/kg for a single intraperitoneal injection after fasting for 12 hours for 6 weeks, and type 1 diabetes was established (T1 DM group).

After 3 days of molding, the blood sugar of the rat tail vein is measured, and the random blood sugar is more than 16.7mmol/L and is selected into the experiment.

Another 10 SD rats with matched sex, week age and body weight are taken as a normal control group (CONT) and fed with common feed without any treatment.

The tail vein blood glucose was measured by glucometer at

weeks

0, 4 and 6 of the experiment and at weeks 1, 4 and 6 after the model.

Results the blood glucose levels in the DM group were significantly higher than those in the CONT group (P < 0.01) at 1, 4, and 8 weeks after the model was established, but the differences between the T1DM and the T2DM groups were not statistically significant (P > 0.05), as detailed in Table 1.

TABLE 1 blood sugar monitoring table for rats of each group after molding (X + -S, mmol/L)

Group of	n	1 week after molding	4 weeks after molding	8 weeks after molding
					CONT	10	5.74±0.45	5.77±0.32	5.71±0.12
T1DM	10	28.17±3.13	24.89±3.14	24.01±2.12
					T2DM	10	27.11±1.10	23.32±3.13	23.98±4.42

Experimental treatment of diabetic rats:

1) Grouping of diabetic rats: (1) blank control (Contr) group, (2) MSC cell (MSC) group, (3) MSCd12235 cell (MSCD) group; each group had 5 rats.

2) Treatment of diabetic rats: the cell concentration of MSCd12235 was adjusted to 5 X109/ml with physiological saline, 0.5ml of the corresponding test substance was injected into the tail vein of each group of diabetic rats, and physiological saline was injected into the blank control group.

3) And (3) blood sugar detection: the tail vein blood glucose was measured every 3 days by glucometer for each group of rats to day 27.

4) And (3) test results: the blood glucose levels in the control rats were consistently at a high steady level, and in T1DM rats, the blood glucose levels were slightly higher than in T2DM rats, further surface animal models were successful. MSC cells showed better hypoglycemic effect (P < 0.01) in both T1DM and T2DM rats, but were still unable to restore normal blood glucose levels (around 6, see figure 3).

The gene recombinant MSCd12235 (MSCd) cells (see figure 3) have similar blood sugar reducing effects on both T1DM and T2DM rats, and have very obvious difference compared with the MSC cell group (P < 0.01).

In conclusion, MSC has certain curative effect on T1DM and T2DM, but only improves symptoms and cannot achieve the curative effect; that is, the therapeutic effect of MSC on diabetes is yet to be improved. IL-35 has certain curative effect on T1DM, IL-22 has certain curative effect on T2DM, FGF1 intracerebroventricular injection has good curative effect on both T1DM and T2 DM; however, IL-35 and IL-22 can only be used for one type of diabetes, and the intracerebroventricular injection of FGF1 is difficult.

The invention displays (expresses) IL-35 which has known therapeutic activity on T1DM, IL-22 which is effective on T2DM and FGF1 which is effective on both T1DM and T2DM on the surface of MSC cells which are effective on both T1DM and T2DM, thereby generating gene recombinant FGF1IL22IL35DMSC (MSCd 12235, MSC displayed FGF1IL22 IL-35) cells which simultaneously display IL-35, IL-22 and FGF1. The MSCd12235 cells which simultaneously display IL-35, IL-22 and FGF1 on the surface not only retain the function of MSC for treating diabetes, but also provide support equivalent to solid phase for the factors by anchoring IL-35, IL-22 and FGF1 on the surface of the MSC, so that the stability of the factors is improved, the half life period is prolonged, and the MSCd12235 cells are more effective than free factors. MSCd12235 was effective against both T1DM and T2DM and was more efficacious than MSC cells.

Of course, the present invention is not limited to MSCs, but may be other stem cells (such as ipscs, embryonic stem cells, etc.) or lymphocytes (such as T cells, NK cells, DC cells, etc.).

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

Sequence listing

<110> Shenzhen Hanke Lishenengineering Limited

<120> preparation method and application of MSC-based gene recombinant cell

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 687

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaccccgc agcttctcct ggcccttgtc ctctgggcca gctgcccgcc ctgcagtgga 60

aggaaagggc ccccagcagc tctgacactg ccccgggtgc aatgccgagc ctctcggtac 120

ccgatcgccg tggattgctc ctggaccctg ccgcctgctc caaactccac cagccccgtg 180

tccttcattg ccacgtacag gctcggcatg gctgcccggg gccacagctg gccctgcctg 240

cagcagacgc caacgtccac cagctgcacc atcacggatg tccagctgtt ctccatggct 300

ccctacgtgc tcaatgtcac cgccgtccac ccctggggct ccagcagcag cttcgtgcct 360

ttcataacag agcacatcat caagcccgac cctccagaag gcgtgcgcct aagccccctc 420

gctgagcgcc agctacaggt gcagtgggag cctcccgggt cctggccctt cccagagatc 480

ttctcactga agtactggat ccgttacaag cgtcagggag ctgcgcgctt ccaccgggtg 540

gggcccattg aagccacgtc cttcatcctc agggctgtgc ggccccgagc caggtactac 600

gtccaagtgg cggctcagga cctcacagac tacggggaac tgagtgactg gagtctcccc 660

gccactgcca caatgagcct gggcaag 687

<210> 2

<211> 708

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggtcagcg ttccaacagc ctcaccctcg gcatccagca gctcctctca gtgccggtcc 60

agcatgtgtc aatcacgcta cctcctcttt ttggccaccc ttgccctcct aaaccacctc 120

agtttggcca gggtcattcc agtctctgga cctgccaggt gtcttagcca gtcccgaaac 180

ctgctgaaga ccacagatga catggtgaag acggccagag aaaaactgaa acattattcc 240

tgcactgctg aagacatcga tcatgaagac atcacacggg accaaaccag cacattgaag 300

acctgtttac cactggaact acacaagaac gagagttgcc tggctactag agagacttct 360

tccacaacaa gagggagctg cctgccccca cagaagacgt ctttgatgat gaccctgtgc 420

cttggtagca tctatgagga cttgaagatg taccagacag agttccaggc catcaacgca 480

gcacttcaga atcacaacca tcagcagatc attctagaca agggcatgct ggtggccatc 540

gatgagctga tgcagtctct gaatcataat ggcgagactc tgcgccagaa acctcctgtg 600

ggagaagcag acccttacag agtgaaaatg aagctctgca tcctgcttca cgccttcagc 660

acccgcgtcg tgaccatcaa cagggtgatg ggctatctga gctccgcc 708

<210> 3

<211> 536

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggccgccc tgcagaaatc tgtgagctct ttccttatgg ggaccctggc caccagctgc 60

ctccttctct tggccctctt ggtacaggga ggagcagctg cgcccatcag ctcccactgc 120

aggcttgaca agtccaactt ccagcagccc tatatcacca accgcacctt catgctggct 180

aaggaggcta gcttggctga taacaacaca gacgttcgtc tcattgggga gaaactgttc 240

cacggagtca gtatgagtga gcgctgctat ctgatgaagc aggtgctgaa cttcaccctt 300

gaagaagtgc tgttccctca atctgatagg ttccagcctt atatgcagga ggtggtgccc 360

ttcctggcca ggctcagcaa caggctaagc acatgtcata ttgaaggtga tgacctgcat 420

atccagagga atgtgcaaaa gctgaaggac acagtgaaaa agcttggaga gagtggagag 480

tcaaagcaat tggagaactg gatttgctgt ttatgtctct gagaaatgcc tgcatt 536

<210> 4

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggctgaag gggaaatcac caccttcaca gccctgaccg agaagtttaa tctgcctcca 60

gggaattaca agaagcccaa actcctctac tgtagcaacg ggggccactt cctgaggatc 120

cttccggatg gcacagtgga tgggacaagg gacaggagcg accagcacat tcagctgcag 180

ctcagtgcgg aaagcgtggg ggaggtgtat ataaagagta ccgagactgg ccagtacttg 240

gccatggaca ccgacgggct tttatacggc tcacagacac caaatgagga atgtttgttc 300

ctggaaaggc tggaggagaa ccattacaac acctatatat ccaagaagca tgcagagaag 360

aattggtttg ttggcctcaa gaagaatggg agctgcaaac gcggtcctcg gactcactat 420

ggccagaaag caatcttgtt tctccccctg ccagtctctt ctgat 465

Claims

1. The preparation method of the gene recombinant cell based on the MSC is characterized by comprising the following steps:

s101: cloning genes encoding IL-35EBI3, IL-35IL12A, IL-22 and FGF1 into a eukaryotic expression vector pDisplay with a transmembrane region structure to form IL-35EBI3, IL-35IL12A, IL-22 and FGF1 cell membrane surface display plasmids pD-IL35EBI3, pD-IL35IL12A, pD-IL22 and pD-FGF1;

s102: preparing hUC-MSC cells;

s103: transfecting the pD-IL35EBI3, pD-IL35IL12A, pD-IL22 and pD-FGF1 plasmids in the step S101 in an equal mixing manner to obtain the hUC-MSC cells in the step S102 to generate MSCd12235 cells;

the surface of the MSCd12235 cell membrane displays pD-IL35EBI3, pD-IL35IL12A, pD-IL22 and pD-FGF1 proteins.

2. The method of claim 1, wherein the step of S101 comprises:

s1011: constructing IL-35EBI3 and IL-35IL12A membrane surface display plasmids pD-IL35EBI3 and pD-IL35IL12A;

s1012: constructing an IL-22 membrane surface display plasmid pD-IL22;

s1013: FGF1 membrane surface display plasmid pD-FGF1 was constructed.

3. The method of preparing an MSC-based recombinant gene cell according to claim 2, wherein the S1011 comprises:

s10111: primer design

1) Primer 1 IL35EBI3upSacII GACGTCCCGCGGatgacccgcagcttcctg;

2) Primer 2 IL35EBI3dnSalI GACGTCGTCGACCttgcaccaggcttgtggca;

3) Primer 3 IL35IL12AupBglII GACGTCAGATCTatggtcgtccaacacagcc;

4) Primer 4 IL35IL12AdnSalI GACGTCGTCGACggcgagctcagatagcccat;

s10112: preparation of RNA: collecting 50ml of peripheral blood of a volunteer, separating lymphocyte PBMC by using lymphocyte separating medium, and preparing total RNA by using a TRIzol reagent;

S10113：RT- PCR：

s10114: pDisplay vector digested by restriction enzyme and PCR product

s10115: connection of

s10116: transfecting an infected bacterium:

s10117: selecting bacterial colonies and verifying:

selecting positive bacterial colony, small amount culturing, extracting plasmid DNA, primary selecting with SacI/SalI and BglII/SalI restriction enzyme digestion, and sequencing to confirm; storing the recombinant pD-IL35EBI3 and pD-IL35IL12A bacteria;

4. The method of preparing an MSC-based recombinant cell according to claim 2, wherein the S1012 comprises:

s10121: primer design

1) Primer 5 IL22upBglII GACGTCAGATCTatggccgctgcagaatct;

2) Primer 6 IL22dnSalI GACGTCGTCGACaatgcaggcattctcagga;

s10122: preparation of RNA:

collecting 50ml of peripheral blood of a volunteer, separating lymphocyte PBMC by using lymphocyte separating medium, and preparing total RNA by using a TRIzol reagent;

S10123：RT- PCR

preparing an Il-22 gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit and purifying by using an ES Spin column PCR product purification kit by using total RNA as a template and a primer 5 and a primer 6 as primers;

s10124: pDisplay vector digested by restriction endonuclease and PCR product

s10127: selecting bacterial colonies and verifying: selecting positive bacterial colonies, carrying out small-scale culture, extracting plasmid DNA, carrying out primary selection by BglII/SalI restriction enzyme digestion, finally carrying out sequencing confirmation, and preserving the pD-IL22 recombinant bacteria;

5. The method of claim 2, wherein the step S1013 comprises:

s10131: primer design

1) Primer 7 FGF1upBglII GACGTCAGATCTatggctgaagggaaaatcaccc;

2) A primer 8 FGF1dnSalI GACGTCGTCGACatcagagagaactggcaggg;

s10132: preparation of RNA: collecting 50ml of peripheral blood of a volunteer, separating lymphocyte PBMC by using lymphocyte separating medium, and preparing total RNA by using a TRIzol reagent;

s10133: RT-PCR: using total RNA as a template, using a primer 7 and a primer 8 as primers, preparing an FGF1 gene fragment by using an Invitrogen SuperScript IV one-step RT-PCR kit, and purifying by using an ES Spin column PCR product purification kit;

s10134: pDisplay vector digested by restriction endonuclease and PCR product

2) Digesting FGF1 gene segment by BglII/SalI restriction enzyme, and respectively purifying by EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain FGF1 gene BglII/SalI segment enzyme digestion gene products;

s10137: selecting bacterial colonies and verifying: selecting positive bacterial colonies, carrying out small-scale culture, extracting plasmid DNA, carrying out primary selection by BglII/SalI restriction enzyme digestion, finally carrying out sequencing confirmation, and preserving the pD-FGF1 recombinant bacteria;

6. The method for preparing an MSC-based gene recombinant cell according to claim 1, wherein the step of S102 comprises the steps of:

s1021: the umbilical cord is taken from a healthy pregnant woman in full-term caesarean section;

s1022: collecting umbilical cord under aseptic condition, cutting into small segments of 1cm each, removing arteriovenous, cutting into pieces in glass bottle, placing in 15ml centrifuge tube, adding 8ml IV collagenase for digestion, digesting for 3 hr, filtering with 100 mesh filter screen or centrifuging at 500rpm for 3-5 min, discarding undigested tissue blocks, transferring supernatant to 15ml centrifuge tube of 5ml each, adding PBS to 10ml, centrifuging at 3000-4000rpm at high speed, discarding supernatant, transferring precipitate to culture dish, culturing at 37 deg.C and 5 CO ₂ Incubating in a saturated humidity incubator, half replacing liquid for 48 hours, completely replacing liquid after 4 days, and removing suspended cells;

s1024: identification and use criteria of umbilical cord mesenchymal stem cells: using the 3 rd-10 th generation cells, when the number of the cells reaches 10 ⁸ Washing with PBS for 2 times, adding 0.05% Ttypsin-EDTA solution, digesting, adding culture medium, and making into 1-3 × 10 ⁶ The cell suspension of (2), detected by a flow cytometer, shows: CD14 or CD11b, CD79a or CD19, CD34, CD45, CD109, HLA-DR negative<2 percent; the positive rates of CD29, CD44, CD73, CD90 and CD105 are all>95 percent, the EB virus, the cytomegalovirus, the HIV virus, the hepatitis B virus, the mycoplasma, the bacterial culture and the fungal culture are all negative;

s1025: directly using, or storing in liquid nitrogen for use.

7. The method for preparing an MSC-based recombinant cell according to claim 1, wherein the step of S103 comprises the steps of:

s1031 preparation of cells: taking each well of a 6-well plate as an example, the MSC cells are suspended in 2ml of culture solution one day before transfection, and transfection is carried out when the cell density is 50-80% full;

s1032, 1 mu g of each of 4 groups of plasmids consisting of pD-IL22, pD-FGF1, pD-IL35EBI3 and pD-IL35IL12A are mixed uniformly;

s1034, adding 100 mu l of DNA-Lipofectamine LTX compound into each hole of cell, and slightly shaking the culture plate back and forth and left and right to mix evenly;

S1035:37℃,5%CO ₂ incubating in an incubator, taking transfected MSCd12235 cells after 24 hours, respectively adding mouse anti-human FGF1, IL22 and IL35 monoclonal antibodies, incubating at 37 ℃ for 1 hour, washing for 2 times, adding a FITC fluorescent labeled rabbit anti-mouse secondary antibody, incubating at 30 ℃ for 30 minutes, washing for 2 times, and suspending in 50 mu l of physiological saline;

s1036 fluorescence microscope observation result shows that the positive rate of FGF1, IL22 and IL35 proteins is 55%.

8. Use of the MSCd12235 cells of any of claims 1 to 7 in the manufacture of a medicament for the treatment of diabetes.