WO2024041162A1 - Il4、il21、il27至少两种细胞因子处理获得的间充质干细胞及其外泌体和应用 - Google Patents
Il4、il21、il27至少两种细胞因子处理获得的间充质干细胞及其外泌体和应用 Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2304—Interleukin-4 (IL-4)
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- C12N2501/23—Interleukins [IL]
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Definitions
- the present invention relates to technologies in the biomedical field, and in particular refers to mesenchymal stem cells obtained by treating at least two cytokines, IL4, IL21, and IL27, and their exosomes and applications.
- Refractory systemic lupus erythematosus is an autoimmune disease caused by multiple factors. It mostly occurs in women aged 20 to 40 weeks. It has a slow onset and diverse clinical manifestations. Among them, autoantibodies produced due to disorders of the patient's own cellular and humoral immune function can affect multiple organs throughout the body, such as skin, joints, kidneys, nervous system, etc.; common symptoms include arthritis, fever, chest pain, hair loss, Swollen lymph nodes and facial rash, etc. The cause of SLE is still unclear, but it is currently believed to be caused by the interaction of genetic factors, environmental factors, endocrine factors, etc.
- the average incidence rate of SLE is 30-70 per 100,000 people, with a male-to-female ratio of 1:11.9, and an average age of 30.7 years old.
- the mortality and disability rates of patients after onset are relatively high. Once the disease occurs, it will cause a certain financial and psychological burden on the patient and his family.
- In the clinical treatment of refractory systemic lupus erythematosus there are cases where conventional treatments have not completely cured the disease. Existing traditional treatments will also gradually lose their effectiveness as the disease progresses, and body functions will gradually collapse under the long-term chronic inflammatory response. It has a great impact on the patient's quality of life.
- mesenchymal stem cells are a type of pluripotent stem cells that are widely present in various tissues and organs in the body and play a role in tissue repair and immune regulation.
- mesenchymal stem cells In vitro, mesenchymal stem cells have the characteristics of multi-directional differentiation potential, self-renewal, and rapid proliferation. Its biological properties that have attracted widespread attention are mainly its low immunogenicity and strong immunomodulatory ability. Therefore, it has been used in many clinical trials. It is widely used in the field and favored by many experts and scholars in the field.
- autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, psoriasis, Crohn's disease, etc.
- mesenchymal stem cells It has good application scenarios in the treatment of autoimmune diseases and has achieved certain therapeutic effects.
- mesenchymal stem cells can obtain good therapeutic effects.
- Exosomes are a type of small membrane vesicles containing complex RNA, proteins or sugars, usually ranging from 40 to 100 nm in diameter. Exosomes are secreted outside cells in a variety of cell states. As a specifically secreted membrane vesicle, they play an important role in participating in cell communication and cell-cell interactions, and because exosomes only have membrane Structure, no nuclear structure, and less likely to cause safety problems, it has attracted widespread attention in clinical research and academic research. As mesenchymal stem cells, exosomes exert paracrine functions and are of great significance in regulating the body's immune function. However, because ordinary mesenchymal stem cells are difficult to react immediately after entering the body, they can secrete exosomes with immune-regulating functions.
- mesenchymal stem cell activation is a key issue to improve/enhance the therapeutic effect of mesenchymal stem cells.
- the present invention addresses the deficiencies in the existing technology, and its main purpose is to provide mesenchymal stem cells obtained by treatment with at least two cytokines, IL4, IL21, and IL27, and their exosomes and applications.
- the mesenchymal stem cells and their exosomes obtained after pretreatment with the cytokine composition provided by the invention can exert stronger immune regulatory functions and be better used for the treatment of immune diseases.
- the cytokine composition includes at least two cytokines among IL4, IL21, and IL27;
- the method of obtaining the mesenchymal stem cells by the pretreatment is: using a large-scale bioreactor, using the mesenchymal stem cells containing The complete culture medium of the cytokine composition is processed and stimulated in stages to obtain empowered mesenchymal stem cells;
- the pretreatment method to obtain the exosomes is: using a large-scale bioreactor, using The complete culture medium containing the cytokine composition is stimulated in stages, and then replaced with basal medium for starvation treatment. The culture supernatant is collected, and exosomes are obtained by gradient ultracentrifugation.
- the method of staged processing and stimulation is: adding complete culture medium containing different cytokine compositions of corresponding concentrations to the large-scale bioreactor in stages to perform energizing stimulation of mesenchymal stem cells;
- the complete medium is any basic medium for culturing mammalian cells mixed with serum substitute.
- the concentration ranges of the three cytokines IL4, IL21, and IL27 are respectively: IL4: 25-150ng/ml, IL21: 100-300ng/ml, and IL27: 50-250ng/ml.
- the cytokine composition includes IL4 and IL21, or the cytokine composition includes IL21 and IL27, or the cytokine composition includes IL4 and IL27.
- the cytokine composition includes IL4 at a concentration of 30ng/ml and IL21 at a concentration of 200ng/ml; or, the cytokine composition includes IL21 at a concentration of 150ng/ml and IL21 at a concentration of 200ng/ml. IL27; alternatively, the cytokine composition includes IL4 at a concentration of 25 ng/ml and IL27 at a concentration of 75 ng/ml.
- the cytokine composition requires contact culture for 16-36 hours during the pretreatment of mesenchymal stem cells;
- the cytokine composition includes IL4 and IL21, where the IL4 pretreatment time is 10 hours, The IL21 pretreatment time is 12 hours;
- the cytokine composition includes IL21 and IL27, where the IL21 pretreatment time is 10 hours and the IL27 pretreatment time is 12 hours;
- the cytokine composition includes IL4 and IL27, wherein; IL4 pretreatment The time is 6 hours, and the IL27 pretreatment time is 10 hours.
- the starvation treatment time using basal medium is 24 hours.
- mesenchymal stem cells without pretreatment are derived from at least one of bone marrow, fat, placenta, umbilical cord and dental pulp in human tissues; the exosomes are derived from secretion of mesenchymal stem cells.
- the at least two cytokines IL4, IL21 and IL27 are treated to obtain Mesenchymal stem cells and their exosomes have enhanced anti-SLE therapeutic activity.
- mesenchymal stem cells and their exosomes obtained by treating at least two cytokines of IL4, IL21, and IL27 is used to prepare drugs for treating systemic and immune inflammation caused by SLE.
- the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solutions, the cytokine composition provided by the present invention is based on the expression of inflammatory microenvironment cytokines in the body during the pathogenesis of SLE patients.
- human mesenchymal stem cells can be stimulated to have better anti-inflammatory activity against SLE; compared with human mesenchymal stem cells or their exosomes obtained by conventional culture methods, the mesenchymal stem cells and exosomes provided by the present invention Its exosomes can have better anti-inflammatory environmental activity and can be used to treat immune-inflammatory responses caused by SLE, reduce the damage of inflammatory responses to systemic tissues, and slow down the progression of the disease; specifically manifested in: reducing the proliferation of T/B lymphocytes ratio, reduce the proliferation ratio of T helper lymphocytes, and increase the proliferation ratio of T regulatory lymphocytes; the protection degree of human mesenchymal stem cells and their exosomes obtained by the culture method of the present invention on the disease model is better than that of human mesenchymal stem cells obtained by conventional methods. The degree of protection of stem cells and their exosomes in disease models.
- Figure 1 is a diagram showing the changes in serum IL4, IL21 and IL27 in the SLE model mouse of the present invention during the development of the disease;
- Figure 2 is a graph showing the therapeutic effects of human umbilical cord mesenchymal stem cells and their exosomes on serum dsDNA and ANA in SLE mice before and after pretreatment of the present invention
- Figure 3 shows the impact of the biological properties of human umbilical cord mesenchymal stem cells and exosomes before and after pretreatment of the present invention (A is the expression of IDO in different groups, B is the expression of PGE2 in different groups, C is the expression of different groups Different expression of VEGF);
- Figure 4 shows the effects of different cytokine concentrations of the present invention on cytokines expressed by human umbilical cord mesenchymal stem cells and their exosomes (A is the expression of IDO in different groups, B is the expression of PGE2 in different groups);
- Figure 5 is a diagram showing the influence of in vitro immunosuppressive functions of human umbilical cord mesenchymal stem cells and their exosomes after pretreatment with different cytokine compositions of the present invention
- Figure 6 is a diagram showing the influence of the combination of factors of the present invention on the in vitro immunosuppressive function of human umbilical cord mesenchymal stem cells and their exosomes under different treatment times;
- Figure 7 is a diagram showing the effects of the combination of cytokines at different concentrations of the present invention on the in vitro immunosuppressive function of human umbilical cord mesenchymal stem cells and their exosomes;
- Figure 8 is a graph showing the therapeutic effects on human umbilical cord mesenchymal stem cells and their exosomes after pretreatment with different cytokine compositions in the SLE model of the present invention.
- the present invention is a kind of mesenchymal stem cells and exosomes obtained by treating at least two cytokines, IL4, IL21 and IL27, which pretreat mesenchymal stem cells with a cytokine composition.
- the mesenchymal stem cells and their exosomes are then obtained, and the cytokine composition includes at least two cytokines among IL4, IL21, and IL27;
- the pretreatment method for obtaining the mesenchymal stem cells is: using a large-scale biological reaction
- the mesenchymal stem cells are treated and stimulated in stages using a complete culture medium containing a cytokine composition to obtain empowered mesenchymal stem cells;
- the method of obtaining the exosomes through this pretreatment is: using a large-scale biological reaction
- the mesenchymal stem cells are treated and stimulated in stages using a complete medium containing a cytokine composition, and then replaced with a basic medium for starvation treatment, the culture supernatant is collected, and exosomes are obtained by gradient ultracentrifugation.
- This staged method of processing stimulation is to add different cytokines containing corresponding concentrations in stages.
- the complete medium of the composition is put into a large-scale bioreactor to perform energizing stimulation of mesenchymal stem cells; the complete medium is any basic medium for culturing mammalian cells mixed with serum substitute.
- the basal culture medium can use Dulbecco's modified Eagle's medium (DMEM), a mixture of Dulbecco's modified Eagle's medium and F12 (DMEM/F12), or RPMI 1640.
- DMEM Dulbecco's modified Eagle's medium
- DMEM/F12 Dulbecco's modified Eagle's medium and F12
- RPMI 1640 Dulbecco's modified Eagle's medium
- the concentration ranges of the three cytokines IL4, IL21, and IL27 are respectively: IL4: 25-150ng/ml, IL21: 100-300ng/ml, and IL27: 50-250ng/ml.
- the cytokine composition includes IL4 and IL21, or the cytokine composition includes IL21 and IL27, or the cytokine composition includes IL4 and IL27.
- the cytokine composition includes IL4 at a concentration of 30ng/ml and IL21 at a concentration of 200ng/ml; alternatively, the cytokine composition includes IL21 at a concentration of 150ng/ml and IL27 at a concentration of 200ng/ml; alternatively, the cell
- the factor composition included IL4 at a concentration of 25 ng/ml and IL27 at a concentration of 75 ng/ml.
- the cytokine composition requires contact culture for 16-36 hours during the pretreatment of mesenchymal stem cells; the cytokine composition includes IL4 and IL21, where the pretreatment time for IL4 is 10 hours and the pretreatment time for IL21 is 12 hours; The cytokine composition includes IL21 and IL27, wherein the pretreatment time for IL21 is 10 hours, and the pretreatment time for IL27 is 12 hours; the cytokine composition includes IL4 and IL27, wherein the pretreatment time for IL4 is 6 hours, and the pretreatment time for IL27 is 6 hours. The time is 10 hours.
- the starvation treatment time using basal medium is 24 hours.
- the unpretreated mesenchymal stem cells are derived from at least one of bone marrow, fat, placenta, umbilical cord and dental pulp in human tissues; the exosomes are derived from secreted by mesenchymal stem cells.
- the mesenchymal stem cells and their exosomes obtained by treating at least two cytokines of IL4, IL21 and IL27 have the effect of enhancing anti-SLE therapeutic activity.
- the application of exosomes is used to prepare drugs for the treatment of systemic and immune inflammation caused by SLE.
- IL4, IL21, and IL27 cytokines were purchased from R&D Systems.
- the serum-free culture system is: MSC XF Medium (BI, Israel) + 2% platelet lysate + MSC XF Supplement Mix (BI, Israel).
- P2 generation refers to: primary cells are expanded and cultured at 3000-5000 cells/m2 in a cell factory. After accumulating a certain number, they are transferred to a cylindrical cell expansion bioreactor, and the cells are mixed with microcarriers for culture. Cell amplification is carried out at a culture concentration of 10,000-12,000 cells/ml, and the culture medium is replenished in time through the perfusion system; after 24 days of culture, the treatment stage with cytokine compositions is entered.
- cytokines are added to the cell culture medium in stages and at corresponding concentrations in a combination manner, and the original culture medium is gradually replaced with the perfusion system. Each factor in the combination is used in a corresponding order to stimulate human umbilical cord mesenchymal stem cells. After the corresponding time, replace the next cytokine or replace the basal medium for starvation treatment.
- Pretreatment refers to the following process. In the process of culturing human umbilical cord-derived mesenchymal stem cells, the cell culture medium of a cytokine composition containing at least two cytokines among IL4, IL21, and IL27 is mixed with human umbilical cord-derived mesenchymal stem cells. Contact culture of mesenchymal stem cells.
- the "basic culture medium” can use any commercially available cell culture medium suitable for mammalian species, such as: DMEM, MEM, DMEM/F12; in this example, MSC is used XF Medium (BI, Israel).
- the cytokine composition in order to target the pathological characteristics of SLE disease, includes IL4 and IL21, which is called cytokine composition 1; the cytokine composition includes IL21 and IL27, which is called cytokine composition 2; The factor composition includes IL4 and IL27, which is called cytokine composition 3; the cytokine concentration range is as follows: IL4: 25-150ng/ml, IL21: 100-300ng/ml, IL27: 50-250ng/ml.
- the contact culture time between the pretreatment medium containing the cytokine composition and the cells is:
- Cytokine composition 1 is IL4 pretreatment time is 10 hours, IL21 pretreatment time is 12 hours; cytokine composition 2 is IL21 pretreatment time is 10 hours, IL27 pretreatment time is 12 hours; cytokine composition 3 is The pretreatment time for IL4 is 6 hours, and for IL27, it is 10 hours.
- the mesenchymal stem cells treated with the pretreatment medium of the cytokine composition still need to be starved for 24 hours before they can be digested, prepared into cell preparations, stored and transported to the patient for use.
- the culture supernatant after starvation treatment and centrifuge it for 10 minutes at 200g to remove most of the cell debris; then, use a hollow fiber column as a filter carrier for tangential flow concentration, and adjust the pressure.
- the force is between 40-100 psi, impurities greater than 250 kD are removed and the liquid is concentrated 50 to 100 times. In this embodiment, the concentration factor is 50 times.
- the concentrated solution is subjected to ultrafast gradient centrifugation.
- the specific method is as follows:
- the in vitro cytokine secretion test mainly examines the normal human umbilical cord mesenchymal stem cells, human umbilical cord mesenchymal stem cells pretreated with different cytokine concentrations and pretreated with different cytokine compositions under in vitro culture conditions and within the same culture time.
- the cytokine concentration in the culture supernatant of human umbilical cord mesenchymal stem cells examine different cytokine concentrations or different cytokine combinations Effects of chemicals on the expression of cytokines in human umbilical cord mesenchymal stem cells.
- exosomes from human umbilical cord mesenchymal stem cells before and after pretreatment were obtained by starving the pretreated mesenchymal stem cells after replacing the basal medium, and then collecting the culture supernatant for laddering. centrifuge to obtain concentrated mesenchymal stem cell exosomes with enhanced anti-SLE activity, and identify them through nanoparticle size analyzer and western blot detection; "pretreatment time” refers to the contact culture time.
- Figure 4 shows that when using different cytokine concentrations of individual components to express human umbilical cord mesenchymal stem cells and their exosomes, the preferred concentration of IL4 is 45ng/ml, the preferred concentration of IL21 is 200ng/ml, and the preferred concentration of IL27 is 75ng. /ml.
- Co-culture of mesenchymal stem cells with human peripheral blood mononuclear cells in vitro is an important experimental detection method for detecting the immunosuppressive ability of mesenchymal stem cells in vitro.
- human peripheral blood mononuclear cells are labeled with CFSE and activated with CD3 and CD28 monoclonal antibodies, and then combined with ordinary human umbilical cord mesenchymal stem cells or their exosomes, pretreated human umbilical cord mesenchymal stem cells or their exosomes, respectively.
- Exosomes (combination types include: cytokine composition 1, cytokine composition 2, and cytokine composition 3) were co-cultured for 72-80 hours, and the proliferation of PBMC was detected by flow cytometry.
- the results of Figure 6 show that in the pretreatment, the pretreatment time for cytokine composition 1 is IL4 for 10 hours and the pretreatment time for IL21 is 12 hours; the pretreatment time for cytokine composition 2 is IL21 for 10 hours and the pretreatment time for IL27 is 10 hours.
- the pretreatment time for cytokine composition 3 is 6 hours for IL4 and 10 hours for IL27.
- cytokine composition 1 are 30ng/ml IL4 and 200ng/ml IL21; the optimal concentration of cytokine composition 2 They are 150ng/ml IL21 and 200ng/ml IL27; the preferred concentrations of cytokine composition 3 are 25ng/ml IL4 and 75ng/ml IL27.
- MRL/lpr mice are used as SLE mouse models. After MRL/lpr mice are raised in the laboratory to 12 weeks of age, the mice will begin to naturally develop symptoms. From the 10th week, serum cytokines (IL4, IL -18, IL27) changes, and detect mouse serum antinuclear antibody (ANA) and anti-dsDNA antibody (dsDNA) concentrations.
- serum cytokines IL4, IL -18, IL27
- ANA mouse serum antinuclear antibody
- dsDNA anti-dsDNA antibody
- ordinary human umbilical cord mesenchymal stem cells or their exosomes pretreated human umbilical cord mesenchymal stem cells or their exosomes (combination types include: cytokine composition 1, cytokine composition 2 , the immunosuppressive functional properties of the cytokine composition 3) are used in the treatment of SLE.
- the "SLE model treatment experiment” is to use MRL/lpr gene mice (these genetic mice will have disease characteristics similar to clinical systemic lupus erythematosus when they are 12 weeks old, and are a good disease research model for SLE) for 12 weeks.
- 1 ⁇ 106 human umbilical cord mesenchymal stem cells before and after pretreatment were injected into the tail vein at 10 years of age. cells for treatment, or tail vein injection of human umbilical cord mesenchymal stem cell exosomes with a total protein content of 200 ⁇ g before and after pretreatment for treatment, and the mice were sacrificed on the 14th day after modeling, and their visceral tissues were taken for pathological sections.
- the control group was subjected to H&E staining to determine the therapeutic effect.
- Exosomes released by treated human umbilical cord mesenchymal stem cells also show the same biological properties, indicating that unpretreated mesenchymal stem cells can exert limited immunomodulatory functions; however, in SLE disease models, cytokine combinations Pretreated human umbilical cord mesenchymal stem cells or their exosomes with substance 1, cytokine composition 2, and cytokine composition 3 show an immunosuppressive ability that is superior to that of ordinary umbilical cord mesenchymal stem cells or their exosomes. .
- the cytokine composition provided by the present invention has an enhanced effect on the use of human umbilical cord mesenchymal stem cells or their exosomes in the treatment of SLE.
- the present invention provides mesenchymal stem cells cultured with a cytokine composition and their exosomes, which have good anti-inflammatory activity against SLE. It has been experimentally verified that compared with ordinary existing mesenchymal stem cells and their exosomes, mesenchymal stem cells and their exosomes treated with a cytokine composition containing at least two cytokines among IL4, IL21, and IL27 are more effective. Exosomes have significantly improved therapeutic effects on SLE and can effectively reduce the secretion levels of dsDNA and ANA in animal models.
- anti-SLE activity refers to the effect of mesenchymal stem cells and their exosomes on T/B lymphocytes after being treated with a cytokine composition containing at least two cytokines among IL4, IL21, and IL27. Better immunosuppressive function.
- the main realization mechanism of the "anti-SLE activity” is: by using the corresponding cytokine composition to pretreat mesenchymal stem cells in vitro, and being exposed to the cytokines that mainly function in the disease microenvironment in advance. , so that it begins to activate under in vitro culture, shortening the activation time into the body, and at the same time enhancing its functionality for the special microenvironment, thus improving the anti-activity and therapeutic effect of the corresponding disease.
- the design focus of the present invention is that the mesenchymal stem cells and their exosomes obtained after pretreatment with the cytokine composition provided by the present invention can exert stronger immune regulatory functions and be better used for the treatment of immune diseases. , and at the same time lays the foundation for providing personalized cell therapy programs for different states of the disease; the present invention provides a set of practical clinical stem cell therapy programs for precise treatment, which can better target different SLE patients with different degrees of disease severity. Precise and precise treatment can effectively improve the treatment effect and reduce the clinical symptoms of the disease.
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Abstract
本发明公开一种IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体和应用，涉及生物医学技术领域，该间充质干细胞及其外泌体由细胞因子组合物对间充质干细胞进行预处理后获得，预处理获得该间充质干细胞的方式为：对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，获得赋能后的间充质干细胞；预处理获得该外泌体的方式为：对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，然后更换为基础培养基饥饿处理。
Description
本发明涉及生物医学领域技术，尤其是指一种IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体和应用。
难治性系统性红斑狼疮(SLE)是一种多因素引起的自身免疫性疾病，好发于20至40周龄女性，发病缓慢，临床表现多样。其中，由于患者自身细胞、体液免疫功能存在障碍所产生的自身抗体，会累及全身多处脏器，如：皮肤、关节、肾脏、神经系统等；常见症状包括关节炎、发烧、胸痛、脱发、淋巴肿大及脸部红疹等等。造成SLE的病因尚不明确，目前认为可能与遗传因素、环境因素、内分泌因素等共同相互作用下导致的。在我国，SLE人群的平均发病率在30-70人/每10万人，其中男女患病比例为1:11.9，平均患病年龄在30.7岁，患者发病后致死率和致残率较高，一旦发病对患者本人、家庭都会造成一定的经济和心理负担。而难治性系统性红斑狼疮在临床治疗上，常规疗法没有完全治愈的案例出现，现有传统疗法也会随疾病进程逐渐失去应有的疗效，身体机能在长期慢性炎症反应下逐渐崩解，对患者的生活质量有极大的影响。
间充质干细胞，是一类具有多能性干细胞，广泛存在于机体内的各种组织脏器中，起到组织修复及免疫调节作用。在体外，间充质干细胞具有多向分化潜能、能自我更新、增殖速度快的特点。而其被广泛关注的生物学特性主要是具备较低的免疫原性、较强的免疫调节能力，因此，在众多临床试验
中被大量使用，得到领域内众多专家学者的青睐。在间充质干细胞应用的临床试验中，有一大部分涉及治疗自身免疫性疾病，如：系统性红斑狼疮、类风湿性关节炎、银屑病、克罗恩病等，说明间充质干细胞在治疗自身免疫性疾病上具有较好的应用场景，并且也已取得一定的治疗效果。但，并不是所有参与使用间充质干细胞治疗的临床试验患者都能得到较好的治疗效果，普通间充质干细胞在治疗自身免疫性疾病上存在一定的个体差异性，往往并非所有的受试者都能得到较好的预期疗效，这主要是因为普通间充质干细胞本身的异质性所导致的。用于常规临床治疗所制备的普通间充质干细胞虽然经过了数次传代扩增，但其细胞群内仍存在各种功能性状不一的细胞亚群，而间充质干细胞在针对自身免疫性疾病上主要是通过旁分泌途径发挥免疫调节功能，临床治疗时使用的普通间充质干细胞由于细胞异质性，在注射至体内后，需要经过体内微环境的刺激后，才会逐渐形成功能统一、性状相似的细胞亚群，之后才会发挥治疗效果。而细胞在注射至体内后其代谢周期相对固定，且有一大部分细胞会被截留在肺部并不会游走全身，而细胞需要接收体内刺激后才能发挥功能占用了大部分代谢周期内的时间后，能发挥治疗效果的时间就会大大减少，从而导致不同患者间的治疗效果参差不齐。
外泌体是一类包含了复杂RNA、蛋白质或糖类的小膜泡，通常直径范围在40-100nm之间。外泌体在多种细胞状态下均会被分泌至细胞外，作为一种特异性分泌的膜泡，在参与细胞通讯、细胞间相互作用上发挥着重要功能，且由于外泌体仅有膜结构，没有核结构，不容易产生安全性问题的特点，受到临床研究及学术研究的广泛关注。而外泌体作为间充质干细胞发挥旁分泌功能，在调节机体免疫功能上具有重要意义，但因为普通间充质干细胞难以在进入机体后第一时间进行反应，能分泌具有调节免疫功能的外泌体是十分有
限的。因此，更需要一种技术方式使得间充质干细胞接受相应炎症环境信号刺激，迅速、大量产生相应的具有对应炎症环境调节功能的外泌体。
综上，解决间充质干细胞活化问题，是提高/增强间充质干细胞治疗效果的关键问题，已有研究证明，间充质干细胞在体外接受相应细胞因子刺激后会出现相应的生物学特性的改变，且会随细胞因子类型、浓度、刺激程度产生不同的效果，因此，提供一种用于治疗SLE的间充质干细胞及其外泌体具有重要的研究意义。
发明内容
有鉴于此，本发明针对现有技术存在之缺失，其主要目的是提供一种IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体和应用，其通过使用本发明提供的细胞因子组合物预处理后得到的间充质干细胞及其外泌体可发挥更强的免疫调节功能，更好的用于免疫性疾病的治疗。
为实现上述目的，本发明采用如下之技术方案：
一种IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其由细胞因子组合物对间充质干细胞进行预处理后获得该间充质干细胞及其外泌体，所述细胞因子组合物包括IL4、IL21、IL27中的至少两种细胞因子；所述预处理获得该间充质干细胞的方式为：采用大规模生物反应器，对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，获得赋能后的间充质干细胞；所述预处理获得该外泌体的方式为：采用大规模生物反应器，对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，然后更换为基础培养基饥饿处理，收集培养上清，梯度超速离心获得外泌体。
作为一种优选方案：所述分阶段处理刺激的方法为：分阶段添加含有对应浓度之不同细胞因子组合物的完全培养基至大规模生物反应器中，对间充质干细胞进行赋能刺激；所述完全培养基为培养哺乳动物细胞的任一基础培养基混合血清替代物。
作为一种优选方案：所述IL4、IL21、IL27三种细胞因子的浓度范围分别为：IL4：25-150ng/ml、IL21：100-300ng/ml、IL27：50-250ng/ml。
作为一种优选方案：所述细胞因子组合物包括IL4、IL21，或者，该细胞因子组合物包括IL21、IL27，或者，该细胞因子组合物包括IL4、IL27。
作为一种优选方案：所述细胞因子组合物包括浓度为30ng/ml的IL4和浓度为200ng/ml的IL21；或者，该细胞因子组合物包括浓度为150ng/ml的IL21和浓度为200ng/ml的IL27；或者，该细胞因子组合物包括浓度为25ng/ml的IL4和浓度为75ng/ml的IL27。
作为一种优选方案：所述细胞因子组合物对间充质干细胞进行预处理过程中需接触培养16-36小时；所述细胞因子组合物包括IL4、IL21，其中IL4预处理时间为10小时，IL21预处理时间为12小时；该细胞因子组合物包括IL21、IL27，其中IL21预处理时间为10小时，IL27预处理时间为12小时；该细胞因子组合物包括IL4、IL27，其中；IL4预处理时间为6小时，IL27预处理时间为10小时。
作为一种优选方案：所述使用基础培养基饥饿处理时间为24小时。
作为一种优选方案：未经预处理的间充质干细胞来源于人体组织中骨髓、脂肪、胎盘、脐带和牙髓中的至少一种；该外泌体来源于间充质干细胞所分泌。
作为一种优选方案：所述IL4、IL21、IL27至少两种细胞因子处理获得
的间充质干细胞及其外泌体，具有抗SLE治疗活性增强的作用。
所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体的应用，用于制备治疗SLE引起的全身性、免疫性炎症药物。
本发明与现有技术相比具有明显的优点和有益效果，具体而言，由上述技术方案可知，本发明提供的细胞因子组合物是根据SLE患者发病进程中机体内炎症微环境细胞因子的表达特点筛选出来的，可刺激人间充质干细胞在针对SLE上具有更好的抗炎症活性；相比常规培养方法获得的人间充质干细胞或其外泌体，经本发明提供的间充质干细胞及其外泌体可以有更好的抗炎症环境活性，以用于治疗SLE引起的免疫炎症反应，降低炎症反应对全身组织的破坏，缓解疾病进展速度；具体表现在：降低T/B淋巴细胞增殖比例、降低T辅助淋巴细胞增殖比例、上调T调节淋巴细胞增殖比例；经本发明培养方法获得的人间充质干细胞及其外泌体对疾病模型的保护程度要优于常规方法获得的人间充质干细胞及其外泌体对疾病模型的保护程度。
为更清楚地阐述本发明的结构特征和功效，下面结合附图与具体实施例来对其进行详细说明。
图1为本发明之SLE模型小鼠疾病发展期间体内血清中IL4、IL21及IL27的变化图；
图2为本发明之预处理前后人脐带间充质干细胞及其外泌体的治疗效果在SLE小鼠的血清dsDNA及ANA的表现图；
图3为本发明之预处理前后人脐带间充质干细胞及其外泌体生物学特性的影响(A为不同组别IDO的表达情况、B为不同组别PGE2的表达情况、C为不同组别VEGF的表达情况)；
图4为本发明之不同细胞因子浓度对人脐带间充质干细胞及其外泌体表达的细胞因子的影响(A为不同组别IDO的表达情况、B为不同组别PGE2的表达情况)；
图5为本发明之不同细胞因子组合物预处理后人脐带间充质干细胞及其外泌体的体外免疫抑制功能的影响图；
图6为本发明之在不同处理时间下因子组合对人脐带间充质干细胞及其外泌体的体外免疫抑制功能的影响图；
图7为本发明之在不同浓度下细胞因子组合对人脐带间充质干细胞及其外泌体的体外免疫抑制功能的影响图；
图8为本发明之SLE模型下不同细胞因子组合物预处理后对人脐带间充质干细胞及其外泌体的疗效图。
本发明如图1至图8所示，一种IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其由细胞因子组合物对间充质干细胞进行预处理后获得该间充质干细胞及其外泌体，该细胞因子组合物包括IL4、IL21、IL27中的至少两种细胞因子；该预处理获得该间充质干细胞的方式为：采用大规模生物反应器，对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，获得赋能后的间充质干细胞；该预处理获得该外泌体的方式为：采用大规模生物反应器，对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，然后更换为基础培养基饥饿处理，收集培养上清，梯度超速离心获得外泌体。
该分阶段处理刺激的方法为：分阶段添加含有对应浓度之不同细胞因子
组合物的完全培养基至大规模生物反应器中，对间充质干细胞进行赋能刺激；该完全培养基为培养哺乳动物细胞的任一基础培养基混合血清替代物。
基础培养基可使用达尔伯克氏改良伊格尔氏培养基(DMEM)、达尔伯克氏改良伊格尔氏培养基和F12的混合物(DMEM/F12)、RPMI 1640。
该IL4、IL21、IL27三种细胞因子的浓度范围分别为：IL4：25-150ng/ml、IL21：100-300ng/ml、IL27：50-250ng/ml。
该细胞因子组合物包括IL4、IL21，或者，该细胞因子组合物包括IL21、IL27，或者，该细胞因子组合物包括IL4、IL27。
该细胞因子组合物包括浓度为30ng/ml的IL4和浓度为200ng/ml的IL21；或者，该细胞因子组合物包括浓度为150ng/ml的IL21和浓度为200ng/ml的IL27；或者，该细胞因子组合物包括浓度为25ng/ml的IL4和浓度为75ng/ml的IL27。
该细胞因子组合物对间充质干细胞进行预处理过程中需接触培养16-36小时；该细胞因子组合物包括IL4、IL21，其中IL4预处理时间为10小时，IL21预处理时间为12小时；该细胞因子组合物包括IL21、IL27，其中IL21预处理时间为10小时，IL27预处理时间为12小时；该细胞因子组合物包括IL4、IL27，其中；IL4预处理时间为6小时，IL27预处理时间为10小时。
该使用基础培养基饥饿处理时间为24小时。
该未经预处理的间充质干细胞来源于人体组织中骨髓、脂肪、胎盘、脐带和牙髓中的至少一种；该外泌体来源于间充质干细胞所分泌。
该IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，具有抗SLE治疗活性增强的作用。
该的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其
外泌体的应用，用于制备治疗SLE引起的全身性、免疫性炎症药物。
本发明中，所用试剂和试验材料均可从市售渠道获取，其中生物材料来源如下：
IL4、IL21、IL27细胞因子均购自R&D Systems公司。
实施例一：
细胞因子组合物预处理的人脐带间充质干细胞及其外泌体的制备方法：
第一：人脐带间充质干细胞制备方法：
人脐带间充质干细胞的分离及培养：
在医学伦理委员会的监督下，从健康供体获取新鲜脐带进行华通氏胶的分离，对华通氏胶进行剪碎，采用贴壁法培养，在无血清培养系统下进行原代细胞的获取和扩增，该无血清培养系统为：MSCXF Medium(BI，以色列)+2％血小板裂解物+MSCXF Supplement Mix(BI，以色列)。
对原代细胞进行扩增并于“P2代”进行冻存做为种子细胞：
“P2代”指：原代细胞在细胞工厂中，按3000-5000cells/m2进行扩增培养，积累一定数量后，转入圆柱形细胞扩增生物反应器中，将细胞与微载体混合培养，以10000-12000cells/ml为培养浓度进行细胞扩增，并通过灌流系统及时补充培养液；在培养24后，进入使用细胞因子组合物处理阶段。
第二：细胞因子组合物预处理人脐带间充质干细胞：
将不同细胞因子按组合方式，分阶段、以对应浓度添加入细胞培养基中，并随灌流系统，逐步替换原有培养基，组合内每种因子的使用按相应顺序刺激人脐带间充质干细胞相应时间后，即更换下一种细胞因子或更换基础培养基进行饥饿处理。
“预处理”是指如下过程，在培养人脐带来源的间充质干细胞的过程中，将添加有IL4、IL21、IL27中至少2种细胞因子的细胞因子组合物的细胞培养基与人脐带来源的间充质干细胞接触培养。
该“基础培养基”可以使用市售任意哺乳动物适用的细胞培养基，如：DMEM、MEM、DMEM/F12；本实施例中使用MSCXF Medium(BI，以色列)。
在本说明书中，为针对SLE疾病病理特点，该细胞因子组合物包括IL4、IL21，称为细胞因子组合物1；该细胞因子组合物包括IL21、IL27，称为细胞因子组合物2；该细胞因子组合物包括IL4、IL27，称为细胞因子组合物3；细胞因子浓度范围如下IL4：25-150ng/ml、IL21：100-300ng/ml、IL27：50-250ng/ml。
在本说明书中，含有细胞因子组合物的预处理培养基与细胞接触培养时间：
细胞因子组合物1为IL4预处理时间为10小时，IL21预处理时间为12小时；细胞因子组合物2为IL21预处理时间为10小时，IL27预处理时间为12小时；细胞因子组合物3为IL4预处理时间为6小时，IL27预处理时间为10小时。
在本说明书中，经细胞因子组合物的预处理培养基处理后的间充质干细胞，仍需进行24小时的饥饿处理后，方可进行消化，制备为细胞制剂，存储运输至患者处使用。
第三：外泌体制备方法：
收集饥饿处理后的培养上清液，在200g的离心力下，离心10min去除大部分细胞碎片；之后，使用中空纤维柱为过滤载体进行切向流浓缩，调整压
力在40-100psi之间，去除大于250kD的杂质并将液体浓缩50至100倍，本实施例中，浓缩倍数为50倍。
将浓缩液进行超速梯度离心，具体方法如下：
4℃下，10,000g离心30min，保留上清；将上清于4℃下，10,0000g离心90min，保留沉淀；将沉淀加入20ml D-PBS在4℃下，100,000g，离心90min，保留离心沉淀，即获得分离后的外泌体；使用1ml D-PBS溶解并混匀离心沉淀后，于-80℃下保存备用。
效果实施例一：
预处理前后人脐带间充质干细胞及其外泌体的生物学特性对比
在本说明书中，为进一步阐明细胞因子组合物对人脐带间充质干细胞的生物学特性影响，进行了体外细胞因子分泌检测。其中，体外细胞因子分泌检测主要考察在体外培养的条件下，相同培养时间内，普通人脐带间充质干细胞、不同细胞因子浓度预处理的人脐带间充质干细胞和不同细胞因子组合物预处理的人脐带间充质干细胞(组合类型包括：细胞因子组合物1、细胞因子组合物2、细胞因子组合物3)的培养上清中的细胞因子浓度，考察不同细胞因子浓度或不同细胞因子组合物对人脐带间充质干细胞表达细胞因子的影响。
具体的是，在细胞融合度达到80％以上是，添加对应浓度或不同组合的含有细胞因子的完全培养基刺激间充质干细胞10h-24h,然后收集培养上清检测间充质干细胞细胞因子表达情况，同时更换为基础培养基再饥饿处理24h检测外泌体中细胞因子表达情况。
预处理前后的人脐带间充质干细胞外泌体的获取方式为，经过预处理后的间质干细胞在更换基础培养基后对其饥饿处理，然后收集培养上清进行梯
度离心，获得浓缩后的抗SLE活性增强的间质干细胞的外泌体，并通过纳米粒径分析仪以及western blot检测鉴定；“预处理时间”是指接触培养时间。
图3和图4结果表明，使用ELISA法检测IDO、PGE2、VEGF蛋白表达量发现，预处理后人脐带间充质干细胞的培养上清及外泌体中，IDO、PGE2、VEGF的浓度明显上升。
图4表明，采用单独组分的不同细胞因子浓度对人脐带间充质干细胞及其外泌体的表达时，IL4优选浓度为45ng/ml、IL21优选浓度为200ng/ml、IL27优选浓度为75ng/ml。
效果实施例二：
预处理前后人脐带间充质干细胞及其外泌体的体外免疫抑制试验
间充质干细胞在体外与人外周血单个核细胞共培养，是检测间充质干细胞在体外免疫抑制能力的重要实验检测方法。本发明中，将人外周血单个核细胞标记CFSE且经过CD3、CD28单抗激活后，分别与普通人脐带间充质干细胞或其外泌体、预处理后的人脐带间充质干细胞或其外泌体(组合类型包括：细胞因子组合物1、细胞因子组合物2、细胞因子组合物3)进行共培养72-80小时，流式检测PBMC的增殖情况。
图5、图6和图7结果表明：经过80小时的共培养后，经过细胞因子组合物预处理后的人脐带间充质干细胞或其外泌体的免疫抑制效果明显优于普通人脐带间充质干细胞或其外泌体的免疫抑制效果。从流式检测结果上看，细胞因子组合物1、细胞因子组合物2、细胞因子组合物3预处理后的人脐带间充质干细胞或其外泌体共培养的CD3+总T细胞的增殖比例明显下降，增殖下降比例明显高于与普通人脐带间充质干细胞或其外泌体共培养的CD3+总T细胞。从抑制百分比统计，与因子组合预处理后的人脐带间充质干细胞或其
外泌体共培养的CD3+总T细胞的抑制效果好，抑制比例明显高于与普通人脐带间充质干细胞或其外泌体共培养的CD3+总T细胞的抑制比例。
图6结果表明，所述预处理中，细胞因子组合物1为IL4预处理时间为10小时，IL21预处理时间为12小时；细胞因子组合物2为IL21预处理时间为10小时，IL27预处理时间为12小时；细胞因子组合物3为IL4预处理时间为6小时，IL27预处理时间为10小时。
图7和图8结果表明，通过体外免疫抑制实验及SLE模型治疗实验，得出细胞因子组合物1的优选浓度为30ng/ml的IL4、200ng/ml的IL21；细胞因子组合物2的优选浓度为150ng/ml的IL21、200ng/ml的IL27；细胞因子组合物3的优选浓度为25ng/ml IL4、75ng/ml IL27。
效果实施例三：
不同细胞因子组合物预处理的人脐带间充质干细胞或其外泌体治疗SLE小鼠模型
利用MRL/lpr小鼠作为SLE小鼠模型，MRL/lpr小鼠在实验室饲养到12周龄后，小鼠会开始自然发病，从第10周开始检测SLE小鼠血清细胞因子(IL4、IL-18、IL27)变化，检测小鼠血清抗核抗体(ANA)、抗dsDNA抗体(dsDNA)浓度。在本发明中，利用普通人脐带间充质干细胞或其外泌体、预处理后的人脐带间充质干细胞或其外泌体(组合类型包括：细胞因子组合物1、细胞因子组合物2、细胞因子组合物3)的免疫抑制功能特性，应用于治疗SLE上。
“SLE模型治疗实验”为在MRL/lpr基因鼠(该基因鼠在12周龄时，会出现与临床上系统性红斑狼疮相似的疾病特点，是一种SLE良好的疾病研究模型)达到12周龄时尾静脉注射1×106个预处理前后的人脐带间充质干细
胞进行治疗，或尾静脉注射总蛋白含量为200μg的预处理前后的人脐带间充质干细胞外泌体进行治疗，并于造模后第14天处死小鼠取其内脏组织进行病理切片，与对照组一同进行H&E染色，判断治疗效果。
由图2、图5至图8表明，结果发现，普通人脐带间充质干细胞或其外泌体在对SLE有一定的治疗效果，说明普通人脐带间充质干细胞或其外泌体能够接受到体内炎性因子的刺激后发挥相应的免疫抑制作用，但由于普通人脐带间充质干细胞未经过特定细胞因子预处理，并不能在进入体内后立刻发挥作用，因此治疗作用效果有限，而未处理的人脐带间充质干细胞所释放的外泌体也表现出同样的生物学特性，说明未经过预处理的间充质干细胞能发挥的免疫调节功能有限；然而在SLE疾病模型中细胞因子组合物1、细胞因子组合物2、细胞因子组合物3预处理后的人脐带间充质干细胞或其外泌体表现出了优于普通人脐带间充质干细胞或其外泌体的免疫抑制能力。
因此，这一结果与本说明书中在体外实验中得出的结论相符，本发明提供的细胞因子组合物对人脐带间充质干细胞或其外泌体用于治疗SLE具有增强作用。
由图2、图5至图8表明，本发明提供了含有细胞因子组合物培养得到的间充质干细胞及其外泌体，其针对SLE具有良好的抗炎活性。经实验验证，相比于普通现有的间充质干细胞及其外泌体，经过含有IL4、IL21、IL27中的至少2种细胞因子的细胞因子组合物处理后的间充质干细胞及其外泌体，在针对SLE上有明显改善的治疗效果，可以有效降低dsDNA及ANA在动物模型上的分泌水平。
其中：“抗SLE活性”是指经过含有IL4、IL21、IL27中至少2种细胞因子的细胞因子组合物处理后间充质干细胞及其外泌体对T/B淋巴细胞具有
更好地免疫抑制功能。
在本发明中，所述“抗SLE活性”的主要实现机制为：通过在体外提前使用相应的细胞因子组合物对间充质干细胞进行预处理，提前接触疾病微环境下主要发挥功能地细胞因子，使之在体外培养下便开始活化，缩短进入机体内的活化时间，同时增强其针对特殊微环境的功能性，从而提高了对应疾病的对抗活性及治疗效果。
图8结果表明，使用本说明书中的预处理后的间充质干细胞或外泌体进行治疗后，出现明显的治疗效果。
本发明的设计重点在于，通过使用本发明提供的细胞因子组合物预处理后得到的间充质干细胞及其外泌体可发挥更强的免疫调节功能，更好的用于免疫性疾病的治疗，同时为针对疾病的不同状态提供个性化的细胞治疗方案打下基础；本发明为精准治疗提供了一套切实可行的临床干细胞治疗方案，可以更好的对不同SLE病人的疾病严重不同程度进行针对性的精准治疗，有效提高治疗效果，降低疾病临床症状。
以上所述，仅是本发明的较佳实施例而已，并非对本发明的技术范围作任何限制，故凡是依据本发明的技术实质对以上实施例所做的任何细微修改、等同变化与修饰，均仍属于本发明技术方案的范围内。
Claims (10)
- 一种IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；由细胞因子组合物对间充质干细胞进行预处理后获得该间充质干细胞及其外泌体，所述细胞因子组合物包括IL4、IL21、IL27中的至少两种细胞因子；所述预处理获得该间充质干细胞的方式为：采用大规模生物反应器，对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，获得赋能后的间充质干细胞；所述预处理获得该外泌体的方式为：采用大规模生物反应器，对间充质干细胞使用含有细胞因子组合物的完全培养基进行分阶段处理刺激，然后更换为基础培养基饥饿处理，收集培养上清，梯度超速离心获得外泌体。
- 根据权利要求1所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；所述分阶段处理刺激的方法为：分阶段添加含有对应浓度之不同细胞因子组合物的完全培养基至大规模生物反应器中，对间充质干细胞进行赋能刺激；所述完全培养基为培养哺乳动物细胞的任一基础培养基混合血清替代物。
- 根据权利要求1所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；所述IL4、IL21、IL27三种细胞因子的浓度范围分别为：IL4：25-150ng/ml、IL21：100-300ng/ml、IL27：50-250ng/ml。
- 根据权利要求1所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；所述细胞因子组合物包括IL4、IL21，或者，该细胞因子组合物包括IL21、IL27，或者，该细胞因子组合物包括IL4、IL27。
- 根据权利要求4所述的IL4、IL21、IL27至少两种细胞因子处理获得 的间充质干细胞及其外泌体，其特征在于；所述细胞因子组合物包括浓度为30ng/ml的IL4和浓度为200ng/ml的IL21；或者，该细胞因子组合物包括浓度为150ng/ml的IL21和浓度为200ng/ml的IL27；或者，该细胞因子组合物包括浓度为25ng/ml的IL4和浓度为75ng/ml的IL27。
- 根据权利要求4所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；所述细胞因子组合物对间充质干细胞进行预处理过程中需接触培养16-36小时；所述细胞因子组合物包括IL4、IL21，其中IL4预处理时间为10小时，IL21预处理时间为12小时；该细胞因子组合物包括IL21、IL27，其中IL21预处理时间为10小时，IL27预处理时间为12小时；该细胞因子组合物包括IL4、IL27，其中；IL4预处理时间为6小时，IL27预处理时间为10小时。
- 根据权利要求1所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；所述使用基础培养基饥饿处理时间为24小时。
- 根据权利要求1所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；未经预处理的间充质干细胞来源于人体组织中骨髓、脂肪、胎盘、脐带和牙髓中的至少一种；该外泌体来源于间充质干细胞所分泌。
- 根据权利要求1所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体，其特征在于；具有抗SLE治疗活性增强的作用。
- 一种如权利要求1-9任意一项所述的IL4、IL21、IL27至少两种细胞因子处理获得的间充质干细胞及其外泌体的应用，其特征在于：用于制备治疗SLE引起的全身性、免疫性炎症药物。
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