CN116064370A

CN116064370A - Method for improving islet beta cell function by gene editing UC-MSC

Info

Publication number: CN116064370A
Application number: CN202211021408.8A
Authority: CN
Inventors: 刘俊; 王旭; 林文龙; 桂智
Original assignee: Shenzhen Woyingda Life Science Co ltd
Current assignee: Shenzhen Woyingda Life Science Co ltd
Priority date: 2022-08-24
Filing date: 2022-08-24
Publication date: 2023-05-05

Abstract

The invention provides a method for promoting or improving insulin secretion of islet beta cells, in particular to a method for improving the functions of islet beta cells by utilizing gene editing UC-MSC, which is characterized in that the islet beta cells are co-cultured with the UC-MSC obtained by utilizing gene editing so as to increase the insulin secretion amount of the islet beta cells and prolong the survival time of the islet beta cells, thereby providing a new choice for treating diabetes.

Description

Method for improving islet beta cell function by gene editing UC-MSC

Technical Field

The invention relates to the crossing field of molecular biology and cell biology, in particular to a method for improving beta cell function by utilizing gene editing UC-MSC.

Background

Mesenchymal stem cells (Mesenchymal Stem Cells, MSC) are multipotent stem cells derived from mesoderm and exist in tissues such as fat, bone marrow, umbilical cord blood and umbilical cord. Compared with adipose and other tissues, umbilical cord blood and umbilical cord serving as medical wastes have great advantages in ethics, cost and extraction convenience as a source way of MSC. The Shetty et al study shows that the MSC content in the Umbilical cord is much higher than that of the Umbilical cord blood, and the success rate of extracting 100% of the MSC (UC-MSC) in the Umbilical cord blood is only 6%. In addition, UC-MSC not only has the differentiation capacity of MSC from other tissues, but also retains the characteristics of some primitive stem cells (embryonic stem cells) and has higher differentiation potential.

Maturation of the crisp technology allows researchers to directly activate and up-regulate endogenous gene expression levels in cells, which provides a new approach to enhancing the nutritional supply and immunomodulatory capacity of MSCs. In 2019, meng et al used CRISPRa technology to up-regulate the expression level of IL10 in bone marrow MSC for treating myocardial infarction of diabetic mice, and the results showed that the therapeutic effect of MSC with gene up-regulated expression was significantly better.

Therefore, the invention provides the MSC which adopts CRISPRa technology to up-regulate NGF genes, and the MSC is used for co-culturing nutrition supply and islet beta cells, so that the expression quantity of insulin is improved.

Disclosure of Invention

In a first aspect of the invention, there is provided a method of promoting insulin secretion by islet beta cells, the method comprising co-culturing islet beta cells with mesenchymal stem cells overexpressing an NGF gene.

Preferably, the method comprises targeting the transcription initiation site of the NGF gene with sgRNA, initiating transcription to activate NGF gene expression.

Preferably, the target site sequence of the sgRNA comprises SEQ ID NO: 5. 8 or 11, or a combination of two or more thereof.

The double-stranded DNA sequence encoding sgRNA is selected from any one or a combination of two or more of the following:

A)SEQ ID NO：6、7；

B)SEQ ID NO：9、10；

C)SEQ ID NO：12、13。

preferably, the method comprises introducing a vector composition into the mesenchymal stem cells to activate NGF gene expression, thereby obtaining mesenchymal stem cells overexpressed by NGF gene.

Preferably, the carrier composition comprises:

a) A vector expressing one or more sgrnas and MS2 binding sites, or one or more vectors expressing sgrnas and MS2 binding sites; wherein, the sgrnas may be the same or different, and the target site thereof is selected from the group consisting of SEQ ID NOs: 5. 8 or 11.

B) A vector expressing fusion protein MS2-P65-HSF 1; the method comprises the steps of,

c) Vector expressing dCas9 protein.

Preferably, the vector composition further comprises other vectors required for packaging the virus.

Preferably, the vector is a bacterial vector, a fungal vector or a viral vector.

In one embodiment of the present invention, the vector is any vector that can deliver genetic material into cells in the prior art, such as phage vectors, baculovirus vectors, herpesvirus vectors, poxvirus vectors, RNA virus vectors, bovine papillomavirus vectors, epstein barr virus vectors, adenovirus vectors, lentiviral vectors, or retrovirus vectors.

In one embodiment of the invention, after the vector composition is introduced into a mesenchymal stem cell, the vector expressing the sgRNA and the MS2 binding site transcribes into the sgRNA and the RNA with a stem-loop structure capable of specifically binding to the MS2 protein, and forms a complex of the "genomic DNA-dCAS9-sgRNA-MS2 binding site-MS2-P65-HSF1" structure with the other two vectors (the vector expressing the fusion protein MS2-P65-HSF1, the vector expressing the dCAS9 protein). Thereby initiating transcription and activating NGF gene expression.

Preferably, the mesenchymal stem cells are derived from a human or non-human animal.

Preferably, the mesenchymal stem cells are derived from umbilical cord, bone marrow, cord blood or fat.

In one embodiment of the invention, the mesenchymal stem cells are derived from umbilical cord of a human or non-human animal.

Preferably, the mesenchymal stem cells can be obtained by screening and extracting umbilical cord, bone marrow, cord blood or fat of human or non-human animals, or purchasing the finished product.

Preferably, the non-human animal is a non-human mammal, including but not limited to wild animals, zoo animals, economic animals, pets, laboratory animals, and the like. Preferably, the non-human mammal includes, but is not limited to, a pig, cow, sheep, horse, donkey, fox, raccoon dog, marten, camel, dog, cat, rabbit, mouse (e.g., rat, mouse, guinea pig, hamster, gerbil, dragon cat, squirrel) or monkey, and the like.

Preferably, the method further comprises the step of culturing the mesenchymal stem cells introduced into the carrier composition.

Preferably, the culture of mesenchymal stem cells employs a culture medium comprising fetal bovine serum, more preferably a DMEM culture medium comprising fetal bovine serum, wherein the culture medium preferably comprises fetal bovine serum of 1% -20%, more preferably 2% -10%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

Preferably, the medium further comprises a label (e.g., hygromycin) and an antibiotic (e.g., puromycin).

In one embodiment of the invention, the method for obtaining mesenchymal stem cells overexpressed by NGF gene comprises infecting mesenchymal stem cells with the above-described vector composition for 12-48 hours (preferably 24-32 hours, e.g., 12, 15, 18, 20, 24, 28, 32, 35, 40, 45, 48), and exchanging culture medium containing the marker and/or the antibiotic.

In one embodiment of the invention, the method for obtaining mesenchymal stem cells overexpressing NGF gene comprises inoculating mesenchymal stem cells into DMEM medium (10% fetal bovine serum added), and culturing the above-described vector composition for 12-48 hours (preferably 24-32 hours, e.g., 12, 15, 18, 20, 24, 28, 32, 35, 40, 45, 48) with replacement of antibiotic-containing DMEM medium (10% fetal bovine serum, 400. Mu.g/mL Hygromycin (Hygromycin) and 5. Mu.g/mL Puromycin (Puromycin)) for 2-5 days (preferably 3 days) when the cell density grows to 50% -90% (preferably 60% -80%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%).

Preferably, the islet β cells and mesenchymal stem cells overexpressed by NGF genes are present at 1: (3-7), for example, the ratio may be 1: (3, 4, 5, 6 or 7).

In one embodiment of the invention, the mesenchymal stem cells overexpressing the islet beta cells and NGF genes are expressed at 1:5, co-culturing in proportion.

The co-culture may be carried out in a DMEM medium, and may be high sugar (5.5 mM or more glucose, preferably 10 to 40mM, more preferably 20 to 30mM, for example, 10, 15, 20, 25, 30, 35, 40mM glucose), low sugar (5.5 mM or less glucose, preferably 1 to 5.5, for example, 1, 2, 3, 4, 5, 5.5mM glucose), or the like. Further preferred, the medium is a DMEM medium comprising fetal bovine serum, wherein preferably the fetal bovine serum comprises 1% -20%, further preferably 2% -10%, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

In one embodiment of the invention, the co-culture is performed in DMEM medium with 2% fetal bovine serum under high sugar conditions.

In a second aspect of the invention, there is provided a sgRNA for constructing a mesenchymal stem cell over-expressed by NGF gene, the target site sequence of the sgRNA comprising the sequence of SEQ ID NO: 5. 8 or 11, or a combination of two or more thereof.

A)SEQ ID NO：6、7；

B)SEQ ID NO：9、10；

C)SEQ ID NO：12、13。

in a third aspect of the invention there is provided a vector or vector combination comprising the sgrnas described above.

In a fourth aspect of the present invention, there is provided a carrier composition comprising:

a) A vector expressing one or more sgrnas and MS2 binding sites, or one or more vectors expressing sgrnas and MS2 binding sites; wherein, the sgrnas may be the same or different, and the target site thereof is selected from the group consisting of SEQ ID NOs: 5. 8 or 11. Preferably a carrier or a combination of carriers as described in the third aspect.

c) Vector expressing dCas9 protein.

In a fifth aspect of the invention there is provided a cell comprising the sgRNA, vector or vector combination described above, or the vector composition described above.

Preferably, the cells are microbial cells (e.g., bacteria, fungi, viruses), and may be plant cells, animal cells, or human cells.

In one embodiment of the invention, the cells include, but are not limited to, E.coli, mesenchymal stem cells, HEK293.

In a sixth aspect of the invention there is provided a use of a sgRNA, vector or vector combination as defined above, or a vector composition as defined above, or a cell as defined above, said use comprising:

a) The application in preparing the mesenchymal stem cells with the over-expressed NGF genes;

b) Use in promoting insulin secretion by islet beta cells;

c) Use in inhibiting islet beta cell apoptosis;

d) The application of prolonging the survival time of islet beta cells or improving the survival rate of islet beta cells;

e) Use in the treatment of diabetes;

f) Application in preparing medicine for treating diabetes; or alternatively, the first and second heat exchangers may be,

g) Use in lowering blood glucose.

In a seventh aspect of the invention, a method for preparing mesenchymal stem cells overexpressed by NGF gene is provided.

Preferably, the method of preparation comprises targeting the transcription initiation site of the NGF gene with sgRNA, initiating transcription to activate NGF gene expression.

A)SEQ ID NO：6、7；

B)SEQ ID NO：9、10；

C)SEQ ID NO：12、13。

Preferably, the carrier composition comprises:

c) Vector expressing dCas9 protein.

In one embodiment of the invention, the preparation method comprises inoculating mesenchymal stem cells into DMEM medium (10% fetal bovine serum added), and when the cell density grows to 50% -90% (preferably 60% -80%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), transfecting the above-mentioned vector composition into cells for 12-48 hours (preferably 24-32 hours, such as 12, 15, 18, 20, 24, 28, 32, 35, 40, 45, 48), and culturing for 2-5 days (preferably 3 days) with replacement of the DMEM medium (10% fetal bovine serum, 400. Mu.g/mL Hygromycin (Hygromycin) and 5. Mu.g/mL Puromycin (Puromycin)).

According to an eighth aspect of the present invention, there is provided mesenchymal stem cells obtained by the above-described preparation method.

According to a ninth aspect of the present invention, there is provided a medicament comprising the sgRNA, the vector or vector combination, the vector composition, the mesenchymal stem cells obtained by the cells or the preparation method, or a mixed system of co-culturing the islet beta cells and the mesenchymal stem cells overexpressed by NGF genes, and pharmaceutically acceptable auxiliary materials.

In a tenth aspect of the invention, there is provided a method of inhibiting apoptosis of pancreatic islet beta cells, the method comprising co-culturing pancreatic islet beta cells with mesenchymal stem cells overexpressing NGF genes.

In an eleventh aspect of the invention, there is provided a method of prolonging survival time of islet beta cells or increasing survival rate of islet beta cells, the method comprising co-culturing islet beta cells with mesenchymal stem cells overexpressing NGF genes.

In a twelfth aspect of the invention, there is provided a method of treating diabetes comprising administering to an individual the vector composition described above, sgRNA, cells, the drug described above, or a mixed system of the islet beta cells described above co-cultured with mesenchymal stem cells overexpressing NGF genes.

In a thirteenth aspect of the invention, there is provided a method of lowering blood glucose comprising administering to an individual the above-described vector composition, sgRNA, cells, the above-described drug or a mixed system of the above-described islet β cells co-cultured with mesenchymal stem cells overexpressing NGF genes.

The invention activates and up-regulates the expression level of NGF gene by gene editing technology, enhances the nutrition effect and micro-environment regulation capability of UC-MSC, and achieves the purpose of improving the functions of islet beta cells by co-culturing with islet beta cells.

The "NGF gene" as used herein refers to Nerve growth factor, which refers to nerve growth factor.

"MS2 gene" stands for Multiple sclerosis, susceptibility to,2, multiple sclerosis-sensitive 2 gene.

"P65 gene" means RELA pro-to-oncogene, NF-kB debug, RELA protooncogene, NF-kB subunit gene.

"HSF1 gene" represents heat shock transcription factor 1, heat shock transcription factor 1 gene.

The expression of the corresponding gene is improved compared with the original cell by the over-expression. For example, in one embodiment of the present invention, the term "mesenchymal stem cell overexpressed by NGF gene" means a mesenchymal stem cell before gene editing, and the expression level of NGF gene is increased as compared with a mesenchymal stem cell after gene editing. Of course, the term "over-expression" as used herein mainly refers to the content or degree of expression, and is not necessarily limited to the operation performed by gene editing or any editing principle or means.

The "HEK293" described herein represents human embryonic kidney cells 293.

The "dCas9" described in the present invention is a protein formed by mutating the Cas9 cleavage site, which has only the ability to bind to genome and sgRNA, but does not have the ability to cleave DNA.

The number of the "plurality" is more than 2, including but not limited to 2 to 50, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.

The "activation" of the present invention may be to activate a gene which is not expressed originally, to be expressed, or to increase the expression level of a gene which is expressed in a low amount originally.

The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

The "pharmaceutically acceptable auxiliary materials" in the present invention include, but are not limited to, one or more of carriers, excipients, diluents, wetting agents, fillers, binders, lubricants, disintegrants, antioxidants, buffers, suspending agents, solubilizers, thickeners, stabilizers, flavoring agents, preservatives, etc.

The "drug" as described herein may be administered by any suitable route, such as by gastrointestinal (e.g., oral) or parenteral (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.) route.

The "drug" according to the present invention may be any suitable dosage form, such as a parenteral or parenteral dosage form, and preferably includes, but is not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder mists, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, drop pills, gels and the like.

The various dosage forms of the medicament can be prepared according to the conventional production method in the pharmaceutical field.

The "individual" as used herein may be a human or non-human mammal, which may be a wild animal, zoo animal, economic animal, pet animal, laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, a pig, cow, sheep, horse, donkey, fox, raccoon dog, marten, camel, dog, cat, rabbit, mouse (e.g., rat, mouse, guinea pig, hamster, gerbil, dragon cat, squirrel) or monkey, and the like.

All combinations of items to which the term "and/or" is attached "in this description shall be taken to mean that the respective combinations have been individually listed herein. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".

The term "comprising" or "including" as used herein is an open reading frame, and when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: the structure diagram of the modified lentiviral vector is shown in the specification, wherein the lentiviral vectors are Lenti-dCAs9-VP64-T2A-BlastR, lenti-MS2-P65-HSF1-T2A-hygroR and Lenti-sgRNA2.0-PuroR respectively.

Fig. 2: bsmBI cleavage site and the sticky ends formed by single cleavage of Lenti-sgRNA2.0-PuroR by BsmBI.

Fig. 3: relative expression levels of NGF genes after independent and co-transfection of 3 sgrnas of NGF genes into HEK293 cells. RT-PCR analysis of different sgRNA activation efficiencies. qPCR analysis of the activation efficiency of different sgRNAs on NGF genes.

Fig. 4: relative expression levels of NGF genes in uninfected lentivirus Lenti-dCAs9-SAM/sgRNA and post-infection UC-MSC.

Fig. 5A: UC-MSC and infection UC-MSC and islet beta cells co-cultured for 24 hours affect the expression quantity of the insulin gene.

Fig. 5B: UC-MSC and infection UC-MSC co-cultured with islet beta cells for 24h affect insulin secretion.

Fig. 6: UC-MSC and infection UC-MSC co-cultured with islet beta cells for 24 hours affect apoptosis-related gene expression in islet beta cells.

Detailed Description

The method and application of the present invention will now be described with reference to the accompanying drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.

The lentiviral vectors used in the examples, lenti-dCAs9-VP64_blast (# 61425), lenti-MS2-P65-HSF1_hygro (# 61426) and Lenti-sgRNA2.0 (# 61427) were all from ADDGNE.

PGK-Puror used in the examples was purchased from SBI (System Biosciences) company, catalog number: CD810A-1.

Example 1: lenti-dCAS9-SAM/sgRNA2.0 vector construction

1. The Lenti-dCAS9-VP64_blast and Lenti-MS2-P65-HSF1_hygro vectors replaced the EF1a promoter with a CMV strong promoter.

The CMV promoter primer with NheI and BsiWI endonuclease sites is designed, and the primer sequence is as follows:

NheI-CMV-F：5’GCTAGCCCCGTTACATAACTTACGG3’(SEQ ID NO：1)；

CMV-BsiWI-R：5’CGTACGGATCTGACGGTTCACTAAA3’(SEQ ID NO：2)；

amplifying NheI-CMV-BsiWI DNA fragment from the existing plasmid carrying CMV promoter, cloning the DNA fragment onto pEasy vector to construct pEasy-CMV vector, sequencing, screening positive monoclonal, and extracting plasmid for standby. The NheI-BsiWI double cleavage was performed with the Lenti-dCAs9-VP64_blast, lenti-MS2-P65-HSF1_hygro and pEasy-CMV vectors, and the cleavage system was shown in Table 1.

Table 1: enzyme cutting system

The digested products were separated by 1% agarose gel electrophoresis at 37℃overnight, and DNA fragments of 12820bp (Lenti-dCAS 9-VP 64_blast), 10471bp (Lenti-MS 2-P65-HSF1_hygro) and 521bp (CMV) were recovered and purified, respectively. The 12820bp and 10471bp fragments were ligated with the 521bp fragment with T4 ligase, respectively. The reaction product is transformed into stbl3 escherichia coli competent, coated on LB solid plates containing Amp, cultured overnight at 37 ℃, and positive clones are screened, thus being modified lentiviral vectors Lenti-dCAs9-VP64-T2A-BlastR and Lenti-MS2-P65-HSF1-T2A-hygroR, as shown in figure 1.

2. Lenti-sgRNA2.0 vector modification, wherein EF1a-BleoR was modified to PGK-PuroR.

The PGK-PuroR primer with BamHI and EcoRI endonuclease sites was designed and the sequence was:

BamHI-PGK-F:5'-GGATCCGGGTAGGGGAGGCGCTTTTC-3' (SEQ ID NO: 3), and PuroR-EcoRI-R:5'-GAATTCTCAGGCACCGGGCTTGCGGG-3' (SEQ ID NO: 4), using the plasmid with PGK-Puror as a template, amplifying by DNA high-fidelity polymerase to obtain BamHI-PGK-Puror-EcoRI fragment, connecting to a blunt end vector to form a pEasy-PGK-Puror vector, screening for correct cloning, and extracting the plasmid for standby. BamHI-EcoRI double digested Lenti-sgRNA2.0 and pEasy-PGK-PuroR, the digestion system is shown in Table 2.

Table 2: enzyme cutting system

Plasmid(s)	1μg
		BamHI	1μl
EcoRI	1μl
		10×CutSmart Buffer	5μl
ddH ₂ O	Make up to 50. Mu.l

The enzyme-digested product is separated by 1% agarose gel electrophoresis after being incubated overnight at 37 ℃, DNA fragments with the sizes of 8351bp (Lenti-sgRNA2.0) and 1146bp (PGK-PuroR) are purified and recovered, T4 ligase is connected, stbl3 escherichia coli is converted by the ligation product, positive clones are screened, and the modified lentiviral plasmid Lenti-sgRNA2.0-PuroR is obtained, and the legend is shown in figure 1.

3. sgRNA design and vector construction

sgRNA design of NGF genes using chopchopop (version 3) online sgRNA design tool, the sgRNA sequences are shown in table 3.

Table 3: sgRNA sequences and positions

Based on the cohesive ends obtained by single cleavage of Lenti-sgRNA2.0-Puror by the endonuclease BsmBI (FIG. 2), cohesive ends of the sgRNA insert vector (FIG. 2) were designed and synthesized by commercial company.

Single-stranded oligodeoxyribonucleic acid synthesized by commercial companies is synthesized into double strands. The method comprises the following steps: dissolving the synthesized freeze-dried powder into 10 mu M, taking 5 mu L of complementary strands, adding into a 0.2ml PCR tube, and putting into a PCR instrument, wherein the reaction procedure is that the temperature is 37 ℃ for 10min;95 ℃ for 5min; the temperature was lowered by 1.5℃per minute to room temperature.

Double-stranded sgRNA phosphorylation. T4 Polynucleotide kinase the synthesized double stranded sgRNA was phosphorylated, and the reaction system is shown in Table 4.

Table 4: reaction system

Double-stranded sgRNA	1μl
		10× T4 PNK buffer	1μl
T4 PNK	0.25μl
		ddH ₂ O	Final volume	10. Mu.l

Storing at 37 deg.C for 1h at-20 deg.C.

Construction of sgRNA vector. The endonuclease BsmBI single enzyme cuts the Lenti-sgRNA2.0-PuroR, the enzyme cutting system is shown in Table 5.

Table 5: enzyme cutting system

Lenti-sgRNA2.0-PuroR	1μg
			10×CutSmart Buffer	5μl
BsmBI	0.5μl
		ddH ₂ O	Make up to 50. Mu.l
Final volume	50μl

37℃for 20min. The digested product was separated by 1% agarose gel, and the large fragment was recovered by purification. T4 ligase connects double-stranded sgRNA and Lenti-sgRNA2.0-PuroR, the connection product is converted into stbl3 escherichia coli competent, and positive clones are screened by sequencing.

Example 2: identification of expression efficiency of dCAS9-SAM/sgRNA activating NGF gene

HEK293 was inoculated into 6-well cell culture plates in DMEM supplemented with 10% fetal bovine serum and 4mM L-glutamine at 37℃with 5% CO ₂ The incubator was cultured until the cell density was 70%. Transfection reagent Lipofectamine 3000 cotransfection of Lenti-dCAs9-VP64-T2A-BlastR, lenti-MS2-P65-HSF1-T2A-hygroR and Lenti-sgRNA2.0-Puror prepared in example 1, transfection systems are shown in Table 6.

Table 6: transfection system

Standing at room temperature for 15min at 37deg.C with 5% CO ₂ Culturing in an incubator for 24 hours, replacing fresh culture medium, culturing for 48 hours, and collecting cells. Invitrogen TRIzol RNA the extraction kit is used for extracting total RNA, and the extraction process is described in the product specification. The quality of the extracted RNA samples was identified by 1% agarose gel electrophoresis.

TakaRa PrimeScript RT reagent Kit reverse transcription kit cDNA was synthesized (see product description for details).

cDNA samples were subjected to qPCR and RT-PCR analysis using ABI-7500. Untransfected HEK293 cells served as negative control, labeled CN, beta-actin served as internal reference, and primer sequences are shown in table 7.

Table 7: primer sequences

The relative expression of the gene is 2 ^-ΔΔC The method is specifically as follows:

the beta-actin gene is used as an internal reference, and the Ct values of the cell samples of CN and NGF_sgRNA1 are respectively expressed as Ct (beta-actin_CN) and Ct (beta-actin_NGF_sgRNA 1), and the Ct values of the two samples to be detected are respectively expressed as Ct (CN) and Ct (NGF_sgRNA 1).

ΔCt(CN)＝Ct(CN)-Ct(β-actin_CN)；

ΔCt(NGF_sgRNA1)＝Ct(NGF_sgRNA1)-Ct(β-actin_NGF_sgRNA1)；

ΔΔCt＝ΔCt(NGF_sgRNA1)-ΔCt(CN)；

The relative expression level of NGF in NGF_sgRNA1 transfected cells is 2 of the CN sample ^-ΔΔCt 。

The results showed that the efficiency of 3 sgrnas synergistically activating NGF genes was significantly higher than the activation efficiency of 3 sgrnas alone (fig. 3).

Example 3: lentivirus package and expression analysis

The lentivirus package adopts a four-plasmid system, PEI is used as a transfection reagent, HEK293 cells are transfected together, liquid is changed after 12 hours, the supernatant is collected after 72 hours, and the concentrated virus is purified by a purification column. The virus titer was determined by qPCR. Wherein, the sgrnas are 3 sgrnas to act synergistically.

1. dCAS9-SAM/sgRNA activation of NGF Gene in UC-MSC

The P3 generation UC-MSC was inoculated into DMEM medium containing 10% fetal bovine serum, and when the cell density was 80%, the lentivirus Lenti-dCAs9-VP64-T2A-BlastR prepared in example 1, lenti-MS2-P65-HSF1-T2A-hygroR and Lenti-sgRNA2.0-Puror co-infected cells. 24h after infection, the DMEM medium containing the antibiotics (10% foetal calf serum, 400. Mu.g/ml hygromycin (hygromycin) and 5. Mu.g/ml Puromycin (Puromycin)) was changed for three days. UC-MSC without lentivirus served as a parallel control.

2. NGF Gene expression level analysis in UC-MSC

The UC-MSC infected with the 4d is collected, the total RNA is extracted by Invitrogen TRIzol RNA extraction kit, and the quality of the extracted RNA sample is identified by 1% agarose gel electrophoresis.

cDNA samples were subjected to qPCR analysis using ABI-7500. The uninfected UC-MSC served as a parallel control, the beta-actin served as an internal reference, and the primer sequences are shown in Table 7.

The relative expression of the gene is 2 ^-ΔΔC And (5) analyzing by a method.

The results showed that NGF gene expression in UC-MSC infected with lentivirus Lenti-dCAs9-SAM/sgRAN was 15.55 times that of uninfected UC-MSC (see FIG. 4).

Example 4: improvement of islet beta cell function by gene editing UC-MSC

After 4d of lentivirus infection, UC-MSC and islet beta cells were co-cultured in DMEM high-sugar medium (25 mM glucose, 2% fetal bovine serum) using a transwell cell co-culture system, with UC-MSC being the upper chamber and islet beta cells being the lower chamber. Uninfected UC-MSC were co-cultured with islet beta cells as a parallel control. The results were as follows:

1. effects on insulin Gene expression and secretion

After 24h co-culture, islet beta cells and culture supernatant were collected. Islet beta cells were subjected to extraction of total RNA using Invitrogen TRIzol RNA extraction kit, and 1% agarose gel electrophoresis to identify the quality of the extracted RNA samples. TakaRa PrimeScript RT reagent Kit reverse transcription kit cDNA was synthesized, and analyzed by qPCR, and beta-actin was used as an internal reference. The relative expression of the gene is 2 ^-ΔΔC And (5) analyzing by a method.

The supernatant was collected and assayed for insulin secretion using the Abcam insulin ELISA kit.

The results showed that the relative expression of the insulin genes was 9.36.+ -. 1.17 and insulin secretion was 12.208.+ -. 0.914. Mu.g/l for islet beta cells co-cultured with infected UC-MSCs, which were significantly higher than for islet beta cells co-cultured with uninfected UC-MSCs (FIG. 5).

2. Effects on beta cell apoptosis

To analyze the effect of upregulation of gene NGF in UC-MSC cells on islet apoptosis, the present invention examined changes in expression levels of apoptosis-related genes bcl2, bax, bad, caspase-9 and survivin in islet β cells after 24h co-culture with infected UC-MSC. After 24h co-culture, islet beta cells were collected. Islet beta cells were subjected to extraction of total RNA using Invitrogen TRIzol RNA extraction kit, and 1% agarose gel electrophoresis to identify the quality of the extracted RNA samples. TakaRa PrimeScript RT reagent Kit reverse transcription kit cDNA was synthesized, and analyzed by qPCR, and beta-actin was used as an internal reference. The relative expression of the gene is 2 ^-ΔΔC And (5) analyzing by a method.

The results showed that the expression levels of pro-apoptotic genes (bax, bad and Caspase-9) and apoptosis-inhibiting genes (bcl 2 and survivin) were increased in islet beta cells co-cultured with infected UC-MSC, but the amount of the increase in expression of the apoptosis-inhibiting genes was significantly higher than that of the pro-apoptotic genes (FIG. 6). Indicating that the up-regulated expression of NGF gene in UC-MSC can inhibit the apoptosis of human beta cells under high sugar environment.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. A method for promoting insulin secretion from islet beta cells, comprising co-culturing islet beta cells with mesenchymal stem cells overexpressing NGF genes.

2. The method of claim 1, comprising targeting the transcription initiation site of the NGF gene with sgRNA, initiating transcription to activate NGF gene expression.

3. The method of claim 2, wherein the target site sequence of the sgRNA comprises SEQ ID NO: 5. 8 or 11, or a combination of two or more thereof.

4. A method according to any one of claims 1 to 3, wherein the method comprises introducing a vector composition into a mesenchymal stem cell to activate NGF gene expression to obtain an NGF gene overexpressed mesenchymal stem cell;

preferably, the carrier composition comprises:

a) A vector expressing one or more sgrnas and MS2 binding sites, or one or more vectors expressing sgrnas and MS2 binding sites;

c) A vector expressing dCas9 protein;

preferably, the vector is a viral vector.

5. The method of any one of claims 1-4, wherein the mesenchymal stem cells are derived from umbilical cord, bone marrow, cord blood or fat of a human or non-human animal.

6. The method of any one of claims 1-5, wherein islet β cells and NGF gene overexpressed mesenchymal stem cells are present at 1: (3-7) Co-culturing in a ratio.

7. An sgRNA for constructing mesenchymal stem cells over-expressed by NGF genes, wherein the target site sequence of the sgRNA comprises the sequence of SEQ ID NO: 5. 8 or 11, or a combination of two or more thereof.

8. A vector or vector composition comprising the sgRNA of claim 7;

preferably, the carrier composition comprises:

c) A vector expressing dCas9 protein;

preferably, the vector is a viral vector.

9. A method for preparing mesenchymal stem cells overexpressed by NGF genes, comprising targeting the transcription initiation site of NGF genes with the sgrnas of claim 7, initiating transcription to activate NGF gene expression;

preferably, comprises introducing the vector composition of claim 8 into a mesenchymal stem cell to activate NGF gene expression, thereby obtaining a mesenchymal stem cell over-expressed by NGF gene.

10. A method of inhibiting apoptosis of islet beta cells, prolonging survival time of islet beta cells, or increasing survival rate of islet beta cells, comprising co-culturing islet beta cells with mesenchymal stem cells overexpressing NGF genes.