阅读说明：本技术 一种hIL-12p40-MSCs的制备方法及其应用 (Preparation method and application of hIL-12p40-MSCs ) 是由 孙艳 迟作华 余功旺 刘爱梅 陈立基 李金花 于 2020-07-03 设计创作，主要内容包括：本发明公开一种hIL-12p40-MSCs(人IL-12p40基因修饰的MSCs)制备方法及其应用。制备方法包括(1)分离培养鉴定hMSCs；(2)克隆扩增hIL-12p40基因；(3)构建重组hIL-12p40慢病毒表达载体；(4)慢病毒的包装；(5)hIL-12p40慢病毒感染MSCs；(6)MSCs表达慢病毒介导的外源性hIL-12p40的鉴定。本发明制备的hIL-12p40基因修饰的MSCs具有抑制细胞免疫的生物活性,且可用于制备治疗细胞免疫异常增强相关的再生障碍性贫血、移植排斥反应及自身免疫性疾病等疾病的药物,具有较好的市场前景。(The invention discloses a preparation method and application of hIL-12p40-MSCs (human IL-12p40 gene modified MSCs). The preparation method comprises (1) separating, culturing and identifying hMSCs; (2) cloning and amplifying hIL-12p40 gene; (3) constructing a recombinant hIL-12p40 lentivirus expression vector; (4) packaging of lentiviruses; (5) hIL-12p40 lentivirus infects MSCs; (6) MSCs express lentivirus-mediated identification of exogenous hIL-12p 40. The hIL-12p40 gene modified MSCs prepared by the invention have biological activity of inhibiting cellular immunity, can be used for preparing medicines for treating diseases such as aplastic anemia, transplant rejection, autoimmune diseases and the like related to cellular immune abnormality enhancement, and have better market prospect.)

1. A preparation method of hIL-12p40-MSCs is characterized by comprising the following steps:

(1) separating, culturing and identifying hMSCs;

(2) cloning and amplifying hIL-12p40 gene;

(3) constructing a recombinant hIL-12p40 lentivirus expression vector;

(4) packaging of lentiviruses;

(5) hIL-12p40 lentivirus infects MSCs;

(6) MSCs express lentivirus-mediated identification of exogenous hIL-12p 40.

2. The method of claim 1, wherein the source of said hIL-12p40-MSCs comprises human umbilical cord, fat, bone marrow, amniotic fluid, placenta.

3. The method of claim 1, wherein the hIL-12p40-MSCs have the nucleotide sequence of SEQ NO 1 (NM-002187.2) in the hIL-12p40 gene.

4. The method of claim 3, wherein the hIL-12p40-MSCs have an expression cassette comprising: the EF-1 alpha promoter and the hIL-12p40 coding gene are arranged in sequence from the 5 '-3' direction.

5. The method of claim 1, wherein the hIL-12p40-MSCs are prepared by introducing hIL-12p40 gene into MSCs using lentiviral vector in step (3) to allow the hIL-12p40 gene to be stably expressed in the MSCs.

6. The method for preparing hIL-12p40-MSCs according to claim 1, wherein in step (5) the hMSCs are grown in logarithmic growth phase within 6 passages of lentivirus infection.

7. The method of claim 1, wherein the slow virus infecting hMSCs of step (5) have an MOI value in the range of 20 to 50.

8. The hIL-12p40-MSCs prepared by the preparation method according to any one of claims 1-7 are used for preparing medicines for inhibiting cellular immunity.

9. The use of hIL-12p40-MSCs prepared by the preparation method according to any one of claims 1-7 in the preparation of medicaments for treating cell-mediated immunity-enhanced diseases such as aplastic anemia, transplant rejection, autoimmune diseases and the like.

Technical Field

The invention relates to the field of biological treatment, in particular to a preparation method and application of MSCs (mesenchymal stem cells) capable of stably expressing hIL-12p40 gene and continuously inhibiting cellular immunity.

Background

Interleukin (IL-12), a cytokine produced by activated monocytes, antigen presenting cells and B-lymphocytes, has important immunomodulatory effects. Usually, two subunits of IL-12P40 and P35 form a heterodimeric molecule, i.e., the intact IL-12P70, via disulfide bonds. IL-12P70 can promote proliferation of natural killer cell (NK cell) and cytotoxic T Cell (CTL), stimulate T cell and NK cell to generate cytotoxic cytokines such as IFN-gamma, induce development and differentiation of Th1 cell, and enhance cellular immune response function of organism. In addition, one member of IL-23 and IL-12 heterodimer cytokine family is composed of IL-23P19 and IL-12P40 subunits, acts on Th17 cells through interaction with the receptors (composed of IL-12 receptor beta 1 and IL-23 receptor 2 subunits), plays an important role in the proliferation and stabilization of Th17 cells, promotes Th17 cells to generate inflammatory factors such as IL-17A, IL-17F and IL-22, further activates immune cells such as T cells and the like, forms a cascade effect of immune response, and causes autoimmune diseases. The P40 monomer or homodimer alone has no biological function, but P40 can competitively antagonize the biological activities of IL-12P70 and IL-23 by competing for binding to IL-12 betaR on the surface of immune cells. Studies show that the level of IL-12 family members including IL1-2 and IL-23 is increased when aplastic anemia, transplant rejection and autoimmune diseases are treated, and abnormal activation of cellular immunity is mediated, which is an important mechanism for the pathogenesis of the diseases. While inhibition of the biological functions of IL-12 and IL-23 may be helpful in treating the above diseases.

MSCs (mesenchyme stem cells, MSCs) are adult stem cells with self-renewal ability derived from the mesoderm. MSCs have wide application, and are also ideal gene carrier cells besides being used as important tissue engineering cells for treating bone and cartilage diseases. MSCs have various advantages as gene delivery vectors: 1. has rich source, and can be separated and extracted from bone marrow, umbilical cord, cord blood, fat, amniotic fluid, placenta, etc. The isolated culture method is simple and has strong amplification capacity. MSCs express no or low HLA molecules, are poorly immunogenic and survive longer in allogenic species. MSCs themselves can modulate immune function by the action of secreted cytokines (e.g., TGF-. beta., IL-10) or expressed surface ligands. MSCs also have the ability to migrate in vivo to the site of pathological injury.

Key issues in the production of genetically modified cell products are the efficiency of gene transfer and expression in the cell. Existing methods for gene transfection of suspension cells include electroporation, viral transfection and transient transfection of liposomes. Among them, viral transfection is the most effective method. Lentivirus vectors (Lentivirus vectors) derived from immunodeficiency viruses can integrate genes into genomes of dividing cells or non-dividing cells with high transfer efficiency, so that transferred genes are stably and long-term expressed in the cells, and the Lentivirus vectors are widely applied to clinical gene therapy, particularly blood cell gene modification products. Starting from the second generation lentiviral vectors, the vectors are safer and have lower cytotoxicity and immunogenicity due to the removal of numerous accessory independent factors.

Disclosure of Invention

The invention aims to utilize the effects of IL-12P40 monomer or homodimer and MSCs on inhibiting cellular immunity, and provides a preparation method of hIL-12P40-MSCs, wherein the generated MSCs can continuously and stably express hIL-12P40 and have biological activity of inhibiting cellular immunity.

The invention realizes the aim through the following technical scheme:

a method for preparing hIL-12p40-MSCs comprises the following steps:

(1) separating, culturing and identifying hMSCs;

(2) cloning and amplifying hIL-12p40 gene;

(3) constructing a recombinant hIL-12p40 lentivirus expression vector;

(4) packaging of lentiviruses;

(5) hIL-12p40 lentivirus infects MSCs;

(6) identification of exogenous hIL-12p40 mediated by expression of lentiviruses by MSCs;

further, the source of MSCs comprises human umbilical cord, fat, bone marrow, amniotic fluid, placenta.

Furthermore, the hIL-12p40 gene has a nucleotide sequence shown in SEQ NO 1(NM _ 002187.2). The expression frame of the hIL-12 gene is composed of: the EF-1 alpha promoter and the hIL-12p40 coding gene are arranged in sequence from the 5 '-3' direction. Furthermore, in the step (3), a lentiviral vector is adopted to introduce the hIL-12p40 gene into the MSCs, so that the hIL-12p40 gene is stably expressed in the MSCs.

Further, in the step (5), the slow virus infects hMSCs in logarithmic growth phase within 6 generations.

Further, the MOI value of the lentivirus infected hMSCs in the step (5) is in the range of 20-50.

The invention also comprises the application of the hIL-12p40-MSCs in inhibiting cellular immunity.

The invention also comprises the application of the hIL-12p40-MSCs in preparing medicaments for treating cell immunity enhanced diseases such as aplastic anemia, transplant rejection, autoimmune diseases and the like.

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

the invention utilizes the IL-12P40 monomer or homodimer and the function of MSCs in inhibiting cellular immunity, and utilizes MSCs as ideal gene carrying vectors and lentiviruses to realize the function of stable transfection, thereby preparing MSCs for stably expressing hIL-12P40 genes. The prepared hIL-12p40 gene modified MSCs have biological activity of inhibiting cellular immunity, can be used for preparing medicines for treating diseases such as aplastic anemia, transplant rejection, autoimmune diseases and the like related to abnormal enhancement of cellular immunity, and have better market prospect. .

Drawings

FIG. 1 shows human umbilical cord mesenchymal stem cells (hUMSCs) isolated and cultured by the adherence method.

Wherein, 1A is fresh umbilical cord tissue, 1B is migration of MSCs from the umbilical cord tissue block, and 1C is morphological observation of isolated culture generation 3 hUMSCs.

FIG. 2 shows the construction of hIL-12p 40-MSCs.

Wherein, 2A is an electrophoretogram of hIL-12p40 gene cloned by an RT-PCR method; FIG. 2B is an electrophoretogram of purified empty vector pSin; 2C is an electrophoresis picture of an empty vector and a constructed pSin-EF-1 alpha-hIL-12 p40 lentivirus plasmid. FIG. 2D is the colony PCR detection hIL-12p40 gene electrophoresis diagram. 2E is the sequencing alignment.

FIG. 3 shows hIL-12p40-MSCs expressing hIL-12p 40.

ELISA method for detecting hIL-12p40-MSCs expression hIL-12p40 level. MSCs as blank control group; LV-MSCs are negative control group; hIL-12p40-MSCs were the experimental group.

FIG. 4 shows hIL-12p40-MSCs suppressing cellular immunity.

Wherein, 4A is the effect of CCK8 method for detecting hIL-12p40-MSCs culture supernatant on PBMCs proliferation; 4B, detecting the influence of hIL-12p40-MSCs culture supernatant on the secretion of the effector cell factor IFN-gamma by the PBMCs by an ELISA method; 4C is a flow cytometry image for detecting the influence of hIL-12p40-MSCs culture supernatant on the expression of a T cell activating molecule CD25 by flow; 4D is hIL-12p40-MSCs culture supernatant on T cell activating molecule CD25 expression effect statistical map Medium is blank control group 1, using RPMI-1640 culture Medium to culture PBMCs; MSCs are blank control group 2, and PBMCs are cultured by MSCs culture supernatant; LV-MSCs are used as a negative control group, and PBMCs are cultured by using LV-MSCs culture supernatant; hIL-12p40-MSCs were used as experimental groups, and PBMCs were cultured using hIL-12p40-MSCs culture supernatant.

Detailed Description

The present invention will be further explained with reference to the following specific examples and the accompanying drawings, but the examples do not limit the scope of the present invention in any way. The following examples are given by way of illustration and are given in the art of routine experimentation and reagent testing.