阅读说明：本技术 一种免疫抑制或抗炎功能增强型pd-l1阳性间充质干细胞、诱导试剂盒及应用 (Immunosuppressive or anti-inflammatory function enhanced PD-L1 positive mesenchymal stem cell, induction kit and application ) 是由 冉启平 邵文陶 李伟强 王光福 于 2020-04-08 设计创作，主要内容包括：本发明公开一种免疫抑制或抗炎功能增强型PD-L1阳性间充质干细胞、诱导试剂盒及应用,通过如下步骤制备：分离并培养间充质干细胞；采用免疫因子诱导复合剂诱导培养的间充质干细胞,获得免疫抑制或抗炎功能增强型PD-L1阳性间充质干细胞,所述诱导复合剂包括5-20ng/ml IFN-γ和0-20ng/ml TNF-α。本发明提供的间充质干细胞能够高效表达PD-L1受体,同野生型间充质干细胞相比,对胶原诱导的关节炎小鼠和CLP脓毒症模型小鼠均具有更好的治疗疗效,且未见副作用的发生。本发明提供的安全、有效的免疫抑制或抗炎功能增强型间充质干细胞,在自身免疫性疾病和炎性相关疾病的临床治疗中极具应用潜力。(The invention discloses an immunosuppressive or anti-inflammatory function enhanced PD-L1 positive mesenchymal stem cell, an induction kit and application, and the preparation method comprises the following steps: isolating and culturing mesenchymal stem cells; inducing the cultured mesenchymal stem cells by adopting an immune factor inducing complexing agent to obtain the PD-L1 positive mesenchymal stem cells with the immunosuppressive or anti-inflammatory function enhancement function, wherein the inducing complexing agent comprises 5-20ng/ml IFN-gamma and 0-20ng/ml TNF-alpha. The mesenchymal stem cell provided by the invention can efficiently express a PD-L1 receptor, and has better treatment effect on collagen-induced arthritis mice and CLP sepsis model mice compared with wild mesenchymal stem cells, and no side effect is generated. The safe and effective mesenchymal stem cells with the immunosuppressive or anti-inflammatory functions provided by the invention have great application potential in clinical treatment of autoimmune diseases and inflammatory related diseases.)

1. An immunosuppressive or anti-inflammatory function-enhanced PD-L1 positive mesenchymal stem cell, which is prepared by the following steps:

(1) isolating and culturing mesenchymal stem cells;

(2) inducing the cultured mesenchymal stem cells by adopting an immune factor inducing complexing agent to obtain the PD-L1 positive mesenchymal stem cells with the immune suppression or anti-inflammatory function enhancement,

the induction complex agent comprises IFN-y and TNF-alpha, the concentration of the IFN-y is 5-20ng/ml, and the concentration of the TNF-alpha is 0-20 ng/ml.

2. The PD-L1 positive mesenchymal stem cell with enhanced immunosuppressive or anti-inflammatory function as claimed in claim 1, wherein the concentration of IFN-y is 20ng/ml and the concentration of TNF-a is 5-20 ng/ml.

3. The immunosuppression or anti-inflammatory enhancement PD-L1 positive mesenchymal stem cell according to claim 1, wherein the induction complex further comprises an albumin excipient.

4. The PD-L1 positive mesenchymal stem cell with enhanced immunosuppressive or anti-inflammatory function as claimed in claim 1, wherein the induction complexing agent is added when the 3-8 generation mesenchymal stem cell obtained by the step (1) is cultured to 70-80% cell confluency.

5. The PD-L1 positive mesenchymal stem cell with enhanced immunosuppressive or anti-inflammatory function of claim 1, wherein the induction time of step (2) for inducing mesenchymal stem cells by adopting the immune factor induction complexing agent is 18-24 h, and then the induced mesenchymal stem cells are continuously cultured for at least 24h after the induction complexing agent is removed.

6. The PD-L1 positive mesenchymal stem cell with enhanced immunosuppressive or antiinflammatory function as claimed in claim 1, wherein the mesenchymal stem cell is derived from human fat, dental pulp, bone marrow, umbilical cord, placenta or umbilical cord blood.

7. The immunosuppression or anti-inflammatory enhancement PD-L1 positive mesenchymal stem cell of claim 1, wherein the step (1) of isolating and culturing mesenchymal stem cells comprises: collecting sample tissues for washing; separating the tissue; washing the separated tissue blocks in normal saline, shearing, weighing, transferring into a centrifuge tube, centrifuging, and removing supernatant; adding MSCs culture medium into the centrifuge tube, and culturing for a period of time; when the primary cells are grown to a cell density > 80%, the primary cells are digested and dissociated and subcultured to obtain mesenchymal stem cells.

8. An in vitro culture induction kit of PD-L1 positive mesenchymal stem cells with enhanced immunosuppressive or anti-inflammatory functions as defined in any one of claims 1 to 7, comprising a basal medium for MSCs, an MSCs proliferation culture additive, and an immune factor-inducing complexing agent.

9. A quality evaluation method of PD-L1 positive mesenchymal stem cells with enhanced immunosuppressive or antiinflammatory function as defined in any one of claims 1 to 7, using the positive expression rate of PD-L1 receptor in the mesenchymal stem cells as an index for quality control of the mesenchymal stem cells.

10. A quality evaluation method of the mesenchymal stem cell of claim 9, wherein the positive expression rate of PD-L1 receptor of the mesenchymal stem cell is detected by flow cytometry.

11. A method for assessing the quality of mesenchymal stem cells according to claim 9, wherein the positive expression rate of PD-L1 receptor is at least 30%.

12. Use of the immunosuppressive or anti-inflammatory function-enhanced PD-L1-positive mesenchymal stem cell of any one of claims 1-7 in the preparation of a medicament for the prevention or treatment of an autoimmune disease and/or an inflammatory-related disease.

13. The use of claim 12, wherein the autoimmune disease comprises at least one of rheumatoid arthritis, scleroderma, systemic lupus erythematosus, ankylosing spondylitis, myasthenia gravis, and multiple ulcerative colitis;

the inflammatory-related diseases include at least one of sepsis, degenerative arthritis, neonatal bronchopulmonary dysplastic pneumonia, and the like.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a PD-L1 positive mesenchymal stem cell with an immunosuppressive or anti-inflammatory function enhanced type, an in-vitro induction kit, a quality evaluation method and application thereof.

Background

With the intensive research on Stem Cells, Mesenchymal Stem Cells (MSCs) have been considered as an ideal tool for the repair treatment of diseases associated with tissue damage. MSCs are precursor cells with self-renewal and multipotential differentiation potential, and are widely distributed in tissues such as adult bone marrow, fat, dental pulp, placenta, and umbilical cord. De Miguel et al found in their research (innovative properties of sensory stem cells: advances and applications.) that MSCs not only have strong self-renewal capacity and multi-directional differentiation potential to mesoderm, ectoderm and endoderm, but also have good immunoregulatory capacity and strong anti-inflammatory properties, can effectively inhibit the inflammatory cascade of the body by influencing the activity, proliferation, differentiation and secretion functions of immune cells, can effectively prevent apoptosis of in situ cells of damaged tissues, promote angiogenesis and activate various biological properties of in situ stem cells of tissues, and participate in regeneration and repair of damaged tissues. Therefore, the transplantation of the MSCs not only can balance and correct unbalanced immune response of patients, but also has the functions of tissue regeneration and repair, and the characteristics make the transplantation of the MSCs hopeful to become an ideal strategy for treating injury, inflammation and immune related diseases.

The way that the MSCs promote the regeneration and repair of damaged tissues by inhibiting inflammation is proved, and the theoretical basis is laid for the cell transplantation based on the MSCs to treat inflammation and immune-related diseases. The mechanisms by which MSCs interact with inflammation/immunity in the various pathological processes that regulate immune responses are being increasingly studied. Although the exact functions of the known endogenous MSCs in vivo are lacked, the in vitro properties and potential clinical application values of the MSCs are proved, but in order to obtain better clinical curative effect, the MSCs with specific function enhancement aiming at specific diseases need to be obtained through related intervention means according to in vitro and in vivo action mechanisms of the MSCs. Most studies are currently focused on the differentiation potential and immunomodulatory capacity of the population of MSCs expanded in vitro, but it is generally accepted that MSCs-based cell therapy exerts therapeutic effects not only because MSCs provide a cellular source for tissue remodeling, but also modulate the body's inflammatory microenvironment and "enhance/activate" the tissue repair capacity of other in situ tissue cells. Although cell replacement is an important component of MSCs in the treatment of certain diseases, published studies indicate that MSCs are mainly treated by exerting their immunomodulatory or anti-inflammatory effects, and that this effect is achieved by stimulating MSCs by the inflammatory microenvironment and then functionally transforming them to regulate and balance the inflammatory response of the body, and exerting clinical therapeutic effects. In short, MSCs do not have anti-inflammatory and immunomodulatory capacity under natural conditions, and only under the action of inflammatory mediators, functionally transformed MSCs are capable of producing large amounts of immunomodulatory proteins, cell mobilization factors, anti-inflammatory factors and growth factors, thereby promoting tissue repair by activating tissue resident stem cells.

In addition, although MSCs can affect both innate and adaptive immunity. However, the immunomodulatory capacity of MSCs is not constitutive, i.e. natural MSCs are poorly immunomodulatory or anti-inflammatory, and their acquisition of immunomodulatory or anti-inflammatory function requires their "energization" by induction of inflammatory cytokines (e.g. cytokines in the inflammatory microenvironment). However, during the occurrence and development of inflammatory diseases, the number and types of inflammatory cytokines greatly vary in different stages of the disease, and these changes seriously affect the efficacy of MSCs in activating the immunomodulatory or anti-inflammatory functions, so that the immune microenvironment of the body of a patient becomes an important determinant for finally determining the immunomodulatory or anti-inflammatory functions of MSCs and the clinical efficacy of MSCs, which is also an important reason for the great difference in clinical efficacy among some patients who have MSCs transplanted and treated the same disease. Therefore, if the MSCs can be directly induced into the MSCs with immunoregulation function or enhanced anti-inflammatory function in vitro, the clinical curative effect of the MSCs can be obviously improved, and the MSCs cell preparation product with high drug-forming property can be obtained.

Research shows that the immunosuppressive or anti-inflammatory action of the MSCs depends on the induction of IFN-gamma in the microenvironment, and the use of IFN-gamma receptor neutralizing antibodies can eliminate the T cell proliferation inhibiting effect of the MSCs. After the MSCs cultured and amplified in vitro are pretreated by IFN-gamma, the MSCs can up-regulate the mRNA expression of immunosuppressive factors such as IL-10, IDO, TSG2 and the like through an STAT signal path, increase the secretion of the immunosuppressive factors, play the functions of immunosuppression and anti-inflammation on the treatment of immune and inflammatory related diseases, and achieve the clinical treatment effect. In addition, in recent years, the immune checkpoint PD-1/PD-L1 signal plays an important role in the negative regulation of the body immunity, can participate in tumor immune evasion, is an important treatment target point of tumor immunotherapy, and plays a key role in the occurrence, development and treatment of autoimmune diseases or inflammatory related diseases. Therefore, if the expression of the MSCs immunosuppressive receptor PD-L1 can be increased while the MSCs are induced to highly express and secrete the immunosuppressive factors, and the immune response of the organism can be balanced through the channel, the MSCs cell preparation product with enhanced immune function can be obtained.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a PD-L1 positive mesenchymal stem cell with enhanced immunosuppressive or anti-inflammatory functions, which is prepared by the following steps: (1) isolating and culturing mesenchymal stem cells; (2) inducing the cultured mesenchymal stem cells by adopting an immune factor inducing complexing agent to obtain the PD-L1 positive mesenchymal stem cells with the immunosuppressive or anti-inflammatory function enhancement function, wherein the inducing complexing agent comprises IFN-gamma and TNF-alpha, the concentration of the IFN-gamma is 5-20ng/ml, and the concentration of the TNF-alpha is 0-20 ng/ml.

Further, the concentration of the IFN-gamma is 20ng/ml, and the concentration of the TNF-alpha is 5-20 ng/ml.

Further, the induction complexing agent also comprises an albumin excipient.

Further, when the 3-8 generation mesenchymal stem cells obtained by the step (1) are proliferated and cultured to 70-80% of cell fusion degree, the induction complexing agent is added.

Further, the induction time of inducing the mesenchymal stem cells by adopting the immune factor induction complexing agent in the step (2) is 18-24 hours, then withdrawing the induction complexing agent to continuously culture the induced mesenchymal stem cells for at least 24 hours, preferably withdrawing the induction complexing agent to continuously culture for 24-96 hours.

Further, the mesenchymal stem cells are derived from human fat, dental pulp, bone marrow, umbilical cord, placenta, or umbilical cord blood.

Further, the step (1) of isolating and culturing mesenchymal stem cells comprises: collecting sample tissues for washing; separating the tissue; washing the separated tissue blocks in normal saline, shearing, weighing, transferring into a centrifuge tube, centrifuging, and removing supernatant; adding MSCs culture medium into the centrifuge tube, and culturing for a period of time; when the primary cells are grown to a cell density > 80%, the primary cells are digested and dissociated and subcultured to obtain mesenchymal stem cells.

The invention also provides an in vitro culture induction kit of the PD-L1 positive mesenchymal stem cells with the immunosuppressive or anti-inflammatory function enhanced type, which comprises a basic culture medium of MSCs, an MSCs proliferation culture additive and an immune factor induction complexing agent.

The invention also provides a quality evaluation method of the PD-L1 positive mesenchymal stem cell with the immunosuppression or anti-inflammatory function enhancement function, wherein the positive expression rate of a PD-L1 receptor in the mesenchymal stem cell is used as a quality control index of the mesenchymal stem cell.

Further, the positive expression rate of the PD-L1 receptor of the mesenchymal stem cells was detected by flow cytometry.

Further, the positive expression rate of the PD-L1 receptor is at least 30%.

The invention also provides application of the PD-L1 positive mesenchymal stem cell with the immunosuppressive or anti-inflammatory function enhanced in preparation of a medicament for preventing or treating autoimmune diseases and/or inflammatory related diseases.

Further, the autoimmune disease comprises at least one of rheumatoid arthritis, scleroderma, systemic lupus erythematosus, ankylosing spondylitis, myasthenia gravis, multiple ulcerative colitis and the like; the inflammatory-related diseases include at least one of sepsis, degenerative arthritis, neonatal bronchopulmonary dysplastic pneumonia, and the like.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

the invention provides an IFN-gamma combined TNF-a in vitro induction method and a kit, and induces and obtains MSCs which can highly express PD-L1 immunosuppressive receptors and can greatly secrete immunosuppressive factors such as IL-10 and IDO (not limited to the IDO detected by the invention, for example, COX2, TSG6 and the like). In addition, the PD-L1 positive MSCs with enhanced immune function can be simply and conveniently detected by cell flow to express PD-L1, namely, the quality evaluation of the anti-inflammatory or immunosuppressive function of the MSCs cell medicine preparation can be carried out, and standardized indexes and schemes are provided for the quality control of the MSCs cell medicine preparation with enhanced immune function.

According to the characteristic that the mesenchymal stem cells are non-constitutive (i.e. non-naturally occurring) and can be endowed with the immunosuppressive or anti-inflammatory functions only after being induced by immune factors such as IFN-gamma, TNF-alpha and the like, the invention develops an in vitro induction kit and establishes a preparation process for inducing the mesenchymal stem cells with the immunosuppressive or anti-inflammatory functions enhanced by applying the kit. Meanwhile, a new method and a new scheme for detecting the expression of the mesenchymal stem cell immune checkpoint receptor PD-L1 by using flow cytometry to control the quality of the functional cell are provided and established. In addition, the invention further proves that compared with wild mesenchymal stem cells, the mesenchymal stem cells with enhanced functions have better treatment effect on collagen-induced arthritis mice and CLP sepsis model mice, and no side effect is generated. The invention provides a safe and effective mesenchymal stem cell with an immunosuppressive or anti-inflammatory function enhanced function, and has great application potential in clinical treatment of autoimmune diseases and inflammatory related diseases.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the result of flow detection of mesenchymal stem cell surface marker molecules.

Fig. 2 shows the mesenchymal stem cell three-line differentiation result.

FIG. 3 shows the immunoblot assay results of different concentrations of TNF- α in combination with IFN- γ induced expression of IDO1 and PD-L1 by MSCs.

FIG. 4 shows the result of quantitative PCR detection of different concentrations of TNF-alpha in combination with IFN-gamma to induce expression of PD-L1 by MSCs.

FIG. 5 shows the result of quantitative PCR detection of different concentrations of TNF- α in combination with IFN- γ induced expression of IDO1 by MSCs.

FIG. 6 shows the results of flow assays of TNF- α and IFN- γ alone or in combination inducing expression of PD-L1 by MSCs.

FIG. 7 is a histopathological examination result of negative control group mice and CIA mice injected intravenously with PBS, WT-MSCs and T + I-MSCs.

FIG. 8 shows the results of the imaging examination of negative control mice and CIA mice injected intravenously with PBS, WT-MSCs and T + I-MSCs.

FIG. 9 is a comparison of survival rates of CLP sepsis mice in sham-operated control group, PBS-treated control group, WT-MSCs group, and T + I-MSCs group.

FIG. 10 is a comparison of serum concentrations of inflammatory factors TNF- α, IL-1 β, IL-6 and IL-10 in control and treatment groups of mice.

Detailed Description

The invention provides an in vitro induction kit of immunosuppressive or anti-inflammatory function-enhanced MSCs (mesenchymal stem cells) with IFN-gamma and TNF-alpha as main factor components, a preparation process flow for inducing MSCs to efficiently express PD-L1 immune checkpoint membrane receptors and secrete and express IDO and other immunosuppressive factors, a quality control standard and a scheme, and a preclinical effectiveness and safety of the immunosuppressive or anti-inflammatory function-enhanced MSCs on treatment of immune or inflammation related diseases are evaluated by simultaneously applying a rheumatoid arthritis model mouse and a CLP model sepsis mouse.

The research of the invention finds that although IFN-gamma alone can up-regulate the expression of hUC-MSCs cellular immunosuppressive factor IDO and immune checkpoint receptor PD-L1 mRNA, IFN-gamma alone cannot up-regulate the expression of immune checkpoint receptor PD-L1 at the protein level. While TNF- α alone did not up-regulate the mRNA expression of IDO and PD-L1, nor their protein expression, it is interesting that TNF- α in combination with IFN- γ treatment of the hhc-MSCs significantly up-regulated the protein expression of IDO and PD-L1, enhancing immunomodulatory and anti-inflammatory functions of the hhc-MSCs.

The invention innovatively provides a method for inducing and treating hUC-MSCs by using TNF-alpha and IFN-gamma, namely, the immunosuppressive factor IDO is remarkably up-regulated, the expression of an immune checkpoint receptor PD-L1 can be stimulated, the immune regulation and anti-inflammatory capability of the hUC-MSCs are improved from different angles, and the treatment curative effect of the hUC-MSCs on immune and inflammation related diseases is enhanced.

Meanwhile, the invention creatively uses the positive rate expressed by the immune checkpoint receptor PD-L1 as a quality control index for judging the judgment of the anti-inflammatory function of immunosuppressive or anti-inflammatory function enhanced MSCs, and provides a simple and operable quality control standard for further researching and developing new cell medicine for anti-inflammatory MSCs.

The invention innovatively provides that the hUC-MSCs with the immunosuppressive or anti-inflammatory functions obtained by the invention can be used for treating sepsis, and the death rate and inflammatory reaction of sepsis can be remarkably reduced. The clinical symptoms of arthritis of mice can also be remarkably improved by treating the arthritis induced by the collagen. Meanwhile, according to the induction process flow of the anti-inflammatory MSCs, the anti-inflammatory MSCs induction kit which can be simply popularized and applied is developed.

The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature.