Cell conditioned medium, drug for resisting islet cell apoptosis and application

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a cell conditioned culture medium, an islet cell apoptosis resisting medicine and application. The cell conditioned culture medium is prepared according to the following steps: inducing apoptosis of the bone marrow mesenchymal stem cells, culturing the apoptotic bone marrow mesenchymal stem cells in a DMEM / F12 culture medium for 20-30 hours, centrifuging the obtained culture solution at 2000-4000 rpm for 10-15 minutes, and collecting the supernatant, thereby obtaining the cell conditioned culture medium. By means of secreted protein and metabolite high-throughput mass spectrum combined bioinformatics analysis, it is proved that the cell condition medium and the LCN2 protein in the active component iron-loaded form of the cell condition medium inhibit INS-1 cell apoptosis by increasing the concentration of ferric ions in cells, and a new choice is provided for drugs for improving the pancreas islet transplantation curative effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a cell-conditioned medium, a drug for resisting pancreatic islet cell apoptosis and uses thereof. Background Art

[0002] The essence of type 1 diabetes is the destruction of β cells mediated by chronic autoimmune reactions, resulting in an absolute lack of insulin and the resulting hyperglycemia. It has a high incidence rate and serious complications, which not only increases the mortality rate of patients, but also brings a great economic burden to society. At present, the main treatment for type 1 diabetes is subcutaneous injection of exogenous insulin. Although this treatment can control the increase in blood sugar in patients, it is difficult to maintain a stable blood sugar level, and it cannot prevent and reverse the macro- and microvascular lesions and complications caused by diabetes. In 2015, the American Association for Clinical Islet Transplantation confirmed that islet transplantation is a safe and effective method for treating type 1 diabetes with hypoglycemia or severe hypoglycemia, and islet transplantation is the most fundamental treatment method to restore insulin secretion under physiological conditions and maintain normal blood sugar levels. At present, islet transplantation is one of the most effective means of treating diabetes. According to data from the International Islet Transplant Registry, the proportion of insulin-free patients in most transplant centers has exceeded 50% 5 years after islet transplantation, and in some centers it has reached more than 60%. Although the efficacy of islet transplantation has improved significantly in the past two decades, the long-term efficacy of islet transplantation is still not ideal, and there is still much room for improvement in the 5-year insulin weaning rate.

[0003] After entering the body, transplanted islet cells will experience hypoxia, immediate blood-mediated inflammatory response (IBMIR), and persistent immune response, which will lead to the immediate loss of most (80%) transplanted islets after transplantation (24-48 hours). Therefore, the large-scale loss of early transplanted islets is an important factor that reduces the survival rate of the graft, and is also a key issue for the widespread clinical application of islet transplantation. How to reduce the apoptosis of early transplanted islets, improve the function of transplanted islets, and prolong the time of complete insulin independence after transplantation are important issues that need to be urgently addressed in the field of islet transplantation. The current methods to reduce the apoptosis of islet transplants are mainly the following:

[0004] (1) MSCs can inhibit early apoptosis of transplanted pancreatic islets A large number of studies have confirmed that co-culture of mesenchymal stem cells (MSCs) from different tissue sources with pancreatic islet cells can inhibit the early apoptosis of pancreatic islet cells by regulating the expression of apoptosis-related factors such as Bax and Bcl-2 in pancreatic islet cells. The latest studies have shown that MSCs can inhibit the production of reactive oxygen species and glutathione by secreting antioxidant cytokines, and secrete exosomes and IL-6 to inhibit the activation of the oxidative stress-related ERK / Akt pathway; MSCs can also activate the PI3K / Akt / mTOR signaling pathway to increase autophagy, thereby inhibiting the apoptosis of transplanted islets and improving the survival rate of pancreatic islet cells. However, the safety issues caused by the undirected differentiation of MSCs have always been controversial, which means that there are still many scientific and technological problems to be overcome in the clinical application of the combined transplantation of MSCs and pancreatic islet cells.

[0005] (2) Anti-apoptotic factors can inhibit the occurrence of early transplanted islet apoptosis During islet transplantation, the islet cell apoptosis cascade initiated by exogenous stimuli, such as the instant blood mediated immune reaction (IBMIR) and inflammatory cytokines, can be inhibited by IL-1β receptor agonists (anakinra) and TNF-α receptor fusion protein (etanercept), thereby improving islet cell survival. In addition, endogenous stimuli such as reactive oxygen species and hypoxia can also induce the activation of the islet cell apoptosis cascade and accelerate the early loss of transplanted islets. The application of new pan-caspase inhibitors, such as F57318, can significantly inhibit the apoptosis of islet transplants and improve long-term efficacy. However, the window period for treatment of these drugs, their homeostasis in the host, whether they are directly toxic to pancreatic β cells or vascular reconstruction, and the half-life and durability of the drugs still need further study.

[0006] (3) Gene therapy provides a new treatment concept for preventing apoptosis of early transplanted islets Before transplantation, in vitro target gene modification of isolated islet cells is one of the important measures to inhibit islet cell apoptosis. Islet cells are cell clusters composed of five types of cells and are called micro-organs with a unique microvascular network. It is reported that the use of different gene therapy methods (adenovirus, adeno-associated virus and lentivirus, etc.) in islet cells to regulate the expression of apoptosis and oxidative stress-related genes, such as Bcl-2, Bcl-XL and adiponectin, can improve the survival rate of transplants. Another study used plasmids to transfect VEGFA into islet cells. The results showed that although VEGFA can significantly improve the level of early vascular reconstruction in transplanted islets, abnormal vascular proliferation was found in the long-term efficacy, leading to the destruction of a large number of islet cells.

[0007] Therefore, finding new, safe and effective substances that inhibit early apoptosis of transplanted islets in the field of islet transplantation is an important way and method to solve the clinical problem of poor long-term efficacy of transplanted islets. Summary of the invention

[0008] Based on the above technical problems, the present invention provides a cell-conditioned medium, a drug for resisting pancreatic islet cell apoptosis and its use. Experiments have shown that the cell-conditioned medium and its active ingredient, the iron-loaded LCN2 protein, can inhibit the apoptosis of INS-1 cells and primary pancreatic islet cells.

[0009] The specific technical solutions provided by the present invention are as follows: In a first aspect, the present invention provides a cell-conditioned medium, which is prepared according to the following steps: Inducing apoptosis of bone marrow mesenchymal stem cells, placing the apoptotic bone marrow mesenchymal stem cells in DMEM / F12 culture medium for 20h~30h, centrifuging the obtained culture solution at 2000 rpm~4000 rpm for 10min~15min, and collecting the supernatant, which is the cell-conditioned medium.

[0010] As a preferred embodiment of the present invention, staurosporine is used to induce apoptosis of bone marrow mesenchymal stem cells, the concentration of staurosporine is 0.1 μM to 0.5 μM, and the treatment time is 1 h to 6 h.

[0011] As a preferred embodiment of the present invention, the apoptosis degree of bone marrow mesenchymal stem cells is 20% to 60%.

[0012] In a second aspect, the present invention provides a kit for culturing bone marrow mesenchymal stem cells in vitro to induce apoptosis, comprising the cell-conditioned medium.

[0013] The third aspect of the present invention provides a use of the cell-conditioned medium in culturing apoptotic bone marrow mesenchymal stem cells.

[0014] In a fourth aspect, the present invention provides a use of the cell-conditioned medium in the preparation of a drug for resisting pancreatic islet cell apoptosis.

[0015] As a preferred embodiment of the present invention, the cell-conditioned medium is used to prepare a drug for resisting islet cell apoptosis in islet transplantation for treating diabetes.

[0016] Further preferably, the drug contains the cell-conditioned medium as the only active ingredient.

[0017] As a preferred embodiment of the present invention, the cell-conditioned medium is used to prepare organ perfusion fluid or preservation fluid.

[0018] Furthermore, the cell-conditioned medium is used to prepare pancreatic organ lavage fluid or preservation fluid.

[0019] In a fifth aspect, the present invention provides an anti-apoptosis drug for pancreatic islet cells, which is prepared by compounding the cell-conditioned medium or the effective active ingredient of the cell-conditioned medium and a pharmaceutically acceptable excipient or carrier, wherein the effective active ingredient of the cell-conditioned medium is LCN2 protein in an iron-loaded form.

[0020] The medicine can be prepared into suitable pharmaceutical preparations, such as oral preparations or injection preparations according to a pharmaceutically acceptable method. Exemplary, oral preparations such as capsules, tablets, granules, etc. Can be selected to add suitable adjuvants or carriers during specific preparation, and the type and amount of adjuvants or carriers are determined according to the properties of the selected preparation type and cell conditioned medium, iron-loaded form of LCN2 protein. Exemplary, optional adjuvants can be suspending agents, suspending agents, wetting agents, thickeners, tackifiers, antioxidants, coating materials, flavoring agents, coloring agents, preservatives, etc.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses high-throughput mass spectrometry combined with bioinformatics analysis of secretory proteins and metabolites to comprehensively study the effects of induced apoptosis on the paracrine function of BMSCs. Combined with the differential analysis of their secretions and their effects on inhibiting early apoptosis of transplanted islets, it is confirmed that cell-conditioned medium and its effective ingredient, the iron-loaded LCN2 protein Holo-LCN2, can inhibit the apoptosis of INS-1 cells by increasing the intracellular iron ion concentration, providing new potential targets and theoretical basis for the research and development of targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of detecting apoptosis of bone marrow mesenchymal stem cells in each group; A~B, flow cytometry detection of stem cell apoptosis level and its statistical graph; C, RT-qPCR detection of Bcl-2 / BAX mRNA expression level in bone marrow mesenchymal stem cells in each group; Figure 2 The results of pre-treatment of INS-1 cell lines in each group; A, CCK8 method to identify the activity of INS-1 cells; B~C, flow cytometry to identify the apoptosis level of INS-1 cells and its statistical graph; D~E, Western Blot to detect the expression level of Bcl-2 / BAX protein in INS-1 cells in each group and its statistical graph; Figure 3 The results of AO / PI staining after pretreatment of primary islet cells in each group; A, AO and PI dye staining of cells in each group; B, statistical graph of apoptosis rate of islet cells in each group; Figure 4These are the results of Western Blot after pretreatment of primary islet cells in each group; A, Bcl-2 / BAX expression blot; B, Bcl-2 / BAX protein expression level statistical chart; Figure 5 The blood glucose level of each group was detected; A, schematic diagram of animal experiment; B, weight change of rats 7 days after transplantation; C, blood glucose level of rats 7 days after transplantation; D, results of intraperitoneal glucose tolerance test (IPGTT) of rats in each group 7 days after transplantation; E, area under the curve of IPGTT blood glucose level of rats in each group on POD7; F, weight change of rats 14 days after transplantation; G, blood glucose level of rats 14 days after transplantation; H, results of intraperitoneal glucose tolerance test (IPGTT) of rats in each group on POD7; I, area under the curve of IPGTT blood glucose level of rats in each group on POD7; Figure 6 These are the immunohistochemical staining results of insulin in each group of tissues; A, staining diagram; B, statistical diagram of insulin-positive area; (scale bar: 400μm) Figure 7 The immunofluorescence staining results of Bcl-2 and insulin in each group of tissues; A, staining diagram; B, Bcl-2 ＋ / Insulin ratio; (Scale bar: 200 μm) Figure 8 A is the effect of LCN2 protein in iron-loaded form (Holo-LCN2) on INS-1 cell apoptosis; A and B are the expression levels of Bcl-2 / BAX proteins related to cell apoptosis in each group and their statistical graphs; C is the mRNA expression level of Bcl-2 / BAX related to cell inflammation in each group; D is the effect of Holo-LCN2 on intracellular iron levels after pretreatment of each group of INS-1; E~F, are the expression levels of ACSL4, GPX4 and COX2 proteins related to ferroptosis in each group of INS-1 cells detected by Western Blot after pretreatment of each group of INS-1 with Holo-LCN2; G, is the mRNA expression level of ACSL4, GPX4 and COX2 proteins related to ferroptosis in each group of cells after pretreatment of INS-1 with Holo-LCN2. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0024] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The essence of type 1 diabetes is the destruction of β cells mediated by chronic autoimmune reactions, resulting in an absolute lack of insulin and the resulting hyperglycemia. It has a high incidence rate and serious complications, which not only increases the mortality rate of patients, but also brings a great economic burden to society.

[0026] After entering the body, transplanted islet cells will experience hypoxia, immediate blood-mediated inflammatory response (IBMIR), and persistent immune response, which will lead to the immediate loss of most (80%) transplanted islets after transplantation (24-48 hours). Therefore, the early loss of large amounts of transplanted islets is an important factor in reducing graft survival and is also a key issue in the widespread clinical application of islet transplantation. Currently, there are several main methods to reduce islet transplant apoptosis:

[0027] (1) MSCs can inhibit early apoptosis of transplanted pancreatic islets Mesenchymal stem cells from different tissue sources can be co-cultured with pancreatic islet cells to inhibit early apoptosis of pancreatic islet cells by regulating the expression of apoptosis-related factors such as Bax and Bcl-2 in pancreatic islet cells. However, the safety issues caused by the undirected differentiation of MSCs have always been controversial, which means that there are still many scientific and technological problems to be overcome in the clinical application of MSCs and pancreatic islet cell co-transplantation.

[0028] (2) Anti-apoptotic factors can inhibit the occurrence of early transplanted islet apoptosis During islet transplantation, the islet cell apoptosis cascade initiated by exogenous stimuli, such as immediate blood-mediated inflammatory responses and inflammatory cytokines, can be inhibited by IL-1β receptor agonists (anakinra) and TNF-α receptor fusion proteins (etanercept), thereby improving islet cell survival. In addition, endogenous stimuli such as reactive oxygen species and hypoxia can also induce the activation of the islet cell apoptosis cascade and accelerate the early loss of transplanted islets. The use of new pan-caspase inhibitors, such as F57318, can significantly inhibit the apoptosis of islet transplants and improve long-term efficacy. However, the window period for treatment of these drugs, their homeostasis in the host, whether they are directly toxic to islet β cells or vascular reconstruction, and the half-life and durability of the drugs still need further study.

[0029] (3) Gene therapy provides a new treatment concept for preventing apoptosis of early transplanted islets Before transplantation, in vitro target gene modification of isolated islet cells is one of the important measures to inhibit islet cell apoptosis. Another study used plasmids to transfect VEGFA into islet cells. The results showed that although VEGFA can significantly improve the early vascular reconstruction level of transplanted islets, in the long-term efficacy, abnormal vascular proliferation was found to cause a large number of islet cell damage.

[0030] Based on this, the present invention provides a cell-conditioned medium, which is prepared according to the following steps: Induce bone marrow mesenchymal stem cells to apoptosis, place the apoptotic bone marrow mesenchymal stem cells in DMEM / F12 culture medium and culture for 20h~30h, centrifuge the obtained culture medium at 2000 rpm~4000 rpm for 10min~15min, and collect the supernatant, which is the cell-conditioned medium.

[0031] On this basis, the present invention also provides the use of cell-conditioned medium in culturing apoptotic bone marrow mesenchymal stem cells or in preparing islet transplantation to treat diabetes and resist islet cell apoptosis.

[0032] Through experiments, the present invention identified that one of the effective active ingredients in the cell-conditioned medium is LCN2 protein in an iron-loaded form.

[0033] The present invention uses high-throughput mass spectrometry combined with bioinformatics analysis of secretory proteins and metabolites to study the effect of induced apoptosis on the paracrine function of BMSCs. Combined with the differential analysis of its secretions and its effect on inhibiting the early apoptosis of transplanted islets, it is confirmed that cell-conditioned medium and its effective active ingredient, iron-loaded LCN2 protein Holo-LCN2, inhibit the apoptosis of INS-1 cells by increasing the intracellular iron ion concentration, providing a new potential target and theoretical basis for the research and development of targeted drugs.

[0034] 1. Establishment of rat BMSCs induced with apoptosis BMSCs were extracted from rats and cultured until P3. BMSCs were treated with 0.2 μM staurosporine (STS) for 2 hours to make the apoptosis degree of BMSCs reach 40%. Apoptosis-induced BMSCs (BMSCs-STS) were constructed ( Figure 1 A~C). The conditioned medium of BMSCs-STS and BMSCs were collected and recorded as BMSCs-STS CM and BMSCs CM, respectively. The specific method is as follows:

[0035] BMSCs CM: When the degree of fusion of BMSCs reached 90%, the culture medium was discarded, the cells were washed three times with PBS buffer, and then replaced with DMEM / F12 medium, and cultured in the incubator for 24 hours. The culture medium was then collected and centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was retained and filtered with a 0.22-μm sterile filter to obtain BMSCs CM.

[0036] BMSCs-STS CM: When the degree of fusion of BMSCs reached 90%, BMSCs were treated with 0.2 μM STS for 2 h, and then the cells were washed three times with PBS buffer and replaced with DMEM / F12 medium. The culture medium was continued in the incubator for 24 h, and then the culture medium was collected and centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was retained and filtered with a 0.22 μm sterile filter to obtain BMSCs CM.

[0037] It should be noted that in the process of obtaining BMSCs CM and BMSCs-STS CM, the concentration of staurosporine used to induce apoptosis of bone marrow mesenchymal stem cells is 0.1-0.5 μM, and the treatment time can be selected to be 1-6 hours; the bone marrow mesenchymal stem cells are placed in DMEM / F12 medium for 20 hours to 30 hours, and the obtained culture fluid is centrifuged at 2000-4000 rpm at 4°C for 10-15 minutes. Within this range, BMSCs CM and BMSCs-STS CM with the expected effects of the present invention can be obtained, and the above examples are not special restrictions on the conditions for obtaining BMSCs CM and BMSCs-STS CM.

[0038] 2. Detection of the protective effect of BMSCs-STS CM on INS-1 and primary pancreatic islet cells against apoptosis under in vitro simulated transplant injury conditions (1) CCK8 assay to detect the protective effect of BMSCs-STS CM on INS-1 apoptosis INS-1 cells were cultured at 5×10 4 / ml density 100μl / well inoculated in 96-well plates and cultured overnight. The cells were divided into 6 groups, including blank control group, STZ injury group, BMSCs CM pretreatment group, BMSCs-STS CM pretreatment group, BMSCs CM pretreatment + STZ injury group, BMSCs-STS CM pretreatment + STZ injury group. After 24h of pretreatment, 3mm streptozotocin (STZ) was added to the BMSCs CM pretreatment group and the BMSCs-STS CM pretreatment group for injury treatment for 24h. Subsequently, 10ul CCK8 solution was added to each well and cultured for 1h. The OD value of the microplate reader was measured. 450nm Measure the absorbance of each well.

[0039] The relative activity of cells can be calculated based on the absorbance value. Figure 2 A) Under STZ injury and non-injury conditions, the cell activity of the BMSCs CM pretreatment group and the BMSCs-STS CM pretreatment group was better. At the same time, the therapeutic effect of BMSCs-STS CM in reversing STZ injury was better than that of BMSCs CM.

[0040] (5) Western Blot reveals the molecular mechanism of BMSCs-STS CM's protective effect on INS-1 apoptosis INS-1 cells were cultured to the logarithmic growth phase, and the cells were grouped into blank control group, STZ injury group, BMSCs CM pretreatment + STZ injury group, and BMSCs-STS CM pretreatment + STZ injury group. After culture at 37°C, the cells were collected and RIPA lysis buffer and protease inhibitors were added. Cytoplasmic proteins were electrophoresed using 12.5% ​​PAGE gel and then transferred to PVDF membrane to detect the protein expression levels of Bcl-2 and BAX related to cell apoptosis.

[0041] The results show that ( Figure 2 D~E), the Bcl-2 / BAX level in the BMSCs-STS CM pretreatment + STZ injury group was higher than that in the BMSCs CM pretreatment + STZ injury group, proving that BMSCs-STS CM pretreatment can achieve better anti-apoptosis effect.

[0042] (6) Flow cytometry reveals the molecular mechanism of BMSCs-STS CM's protective effect on INS-1 apoptosis INS-1 was cultured to the logarithmic growth phase, and the cells were grouped as in the Western Blot experiment. The cells of each group (including the supernatant) were collected in a flow cytometry tube, washed once by centrifugation with pre-cooled PBS, and 500 μL of Binding Buffer diluted to 1× with ddH2O was added to resuspend the precipitate. Then 5 μL of Annexin V-APC and 10 μL of 7-AAD were added to each tube, mixed and incubated at room temperature in the dark for 5 min, and then detected by the flow cytometry machine.

[0043] The results show that ( Figure 2 B~C), the proportion of early apoptotic + late apoptotic cells in the BMSCs-STS CM pretreatment + STZ injury group was lower than that in the BMSCs CM pretreatment + STZ injury group, proving that BMSCs-STS CM pretreatment can achieve better anti-apoptotic effects.

[0044] (7) AO / PI staining Primary rat pancreatic islet cells were extracted by density gradient centrifugation and stained after 24 hours of in vitro culture. The cells were divided into blank control group, BMSCs CM treatment group, and BMSCs-STS CM treatment group. 5 μl AO dye and 10 μl PI dye were added to 1.5 ml complete culture medium and stained for 20 minutes before observation under a fluorescence microscope.

[0045] The results show that ( Figure 3 ), the PI staining of cells in the BMSCs-STS CM treatment group and the BMSCs CM treatment group was lighter than that in the blank control group, and the PI staining of the BMSCs-STS CM treatment group was lighter, which proved that BMSCs CM pretreatment can better inhibit the apoptosis of primary pancreatic islet cells.

[0046] (8) Western Blot reveals the molecular mechanism of BMSCs-STS CM's protective effect on primary pancreatic islet cell apoptosis Primary rat pancreatic islet cells were extracted by density gradient centrifugation and stained after 24 hours of in vitro culture. The cells were grouped and stained with AO / PI. After 24 hours of in vitro culture, RIPA lysis buffer and protease inhibitors were added. Cytoplasmic proteins were analyzed by 10% SDS-PAGE and transferred to PVDF membrane to detect the protein expression levels of Bcl-2 and BAX related to cell apoptosis.

[0047] The results show that ( Figure 4 ), the Bcl-2 / BAX level in the BMSCs-STS CM pretreatment group was higher than that in the BMSCs CM pretreatment group, proving that BMSCs-STS CM pretreatment can achieve better antigen-generation pancreatic islet cell apoptosis effect.

[0048] 3. Establishment of in vivo islet transplantation model and detection of the protective effect of BMSCs-STS on transplanted islet apoptosis (1) Experimental groups SPF SD rats weighing 100-150g were fasted for 12h and weighed. STZ was injected intraperitoneally at 50mg / kg, and random blood glucose and body weight were measured every 2 days. Rats with blood glucose values ​​greater than 16.7mM / L for three consecutive times were selected for transplantation. The experimental groups were modeling group, islet cell transplantation group (Islets), BMSCs and islet cell combined transplantation group (Islets+BMSCs), BMSCs-STS and islet cell combined transplantation group (Islets+BMSCs-STS), Figure 5 A.

[0049] (2) Collection of materials On the 7th and 14th days after transplantation (post operative day, POD), after the small animals were successfully anesthetized by an anesthesia machine, physiological saline was perfused through the renal artery until the effluent was clear. The kidneys and surrounding tissues were carefully separated, paying attention to maintaining the integrity of the renal capsule, and the tissues were fixed in 4% paraformaldehyde solution.

[0050] (3) Detection indicators 1) Detection of graft function in each group The blood glucose and body weight of rats were continuously monitored after transplantation. The SD rats in each group were fasted for 6 h on the 7th and 14th days after transplantation. The body weight and blood glucose were measured before the start. The amount of glucose to be injected was calculated based on the concentration of 2 g / kg and injected into the rats intraperitoneally. The blood glucose levels of the rats were tested at 0, 15, 30, 60, 90 and 120 min.

[0051] The results showed that the blood sugar level in the BMSCs-STS and islet cell combined transplantation group (Islets+BMSCs-STS) returned to normal levels faster than in the other two groups ( Figure 5 C, G), the body weight of rats in each transplantation group increased steadily, and there was no significant difference in the body weight growth of rats in each transplantation group ( Figure 5 B, F). Intraperitoneal glucose tolerance tests were performed 7 and 14 days after transplantation. The area under the curve (AUC) of blood glucose levels was significantly lower than that of the diabetic model group. Rats in the Islets+BMSCs-STS group showed the best improvement in glucose tolerance ( Figure 5 D, E, H, I).

[0052] 2) Immunohistochemical staining of insulin in the sampled tissue Immunohistochemical staining was used to observe the insulin secretion of transplanted islet cells, and the staining area of ​​each group was statistically analyzed.

[0053] The results showed that the insulin-positive area under the renal capsule was the largest in the Islets+BMSCs-STS group ( Figure 6 ).

[0054] The above results all indicate that BMSCs-STS can better ensure the survival of islet transplants compared with BMSCs, enabling them to exert better insulin secretion function.

[0055] 9) Perform immunofluorescence staining of Bcl-2 and insulin on the tissues collected By immunofluorescence staining and counting, Bcl-2 ＋ / Insulin area.

[0056] Bcl-2 in the Islets+BMSCs-STS group under the renal capsule ＋ / Insulin ratio is the largest ( Figure 7 ). It was proved that BMSCs-STS can better alleviate the apoptosis of pancreatic islet transplantation compared with BMSCs.

[0057] 4. Iron-loaded LCN2 protein (Holo-LCN2) can alleviate apoptosis of INS-1 cells Proteins in the conditioned medium of BMSCs-STS and BMSCs were extracted using lysis buffer, and the protein concentration was determined using a BCA kit. After pretreatment of the samples, LC-MS / MS was used to separate and fractionate the samples. Proteins with fold change >1.2 and P value <0.005 were considered to be differentially expressed proteins between the conditioned medium groups of the two groups. After analysis, one of the active ingredients, the iron-loaded LCN2 protein (Holo-LCN2), was selected to detect its protective effect on INS-1 cell apoptosis.

[0058] (10) Western Blot reveals the molecular mechanism of Holo-LCN2's protective effect on INS-1 apoptosis INS-1 cells were cultured to the logarithmic growth phase and divided into STZ injury group, Fe 3+ Pretreatment + STZ injury group, non-iron loaded LCN2 (Apo-LCN2) pretreatment + STZ injury group, Holo-LCN2 pretreatment + STZ injury group. After culture at 37°C, cells were collected and RIPA lysis buffer and protease inhibitors were added. Cytoplasmic proteins were analyzed by 10% SDS-PAGE and transferred to PVDF membrane to detect the protein expression levels of Bcl-2 and BAX related to cell apoptosis.

[0059] The results show that ( Figure 8 A~B), using Fe alone 3+ Pretreatment with Apo-LCN2 and Holo-LCN2 could not increase the expression level of Bcl-2 / BAX. Only Holo-LCN2 could increase the expression level of Bcl-2 / BAX and alleviate cell apoptosis, which proved that Holo-LCN2 was the active ingredient in BMSCs-STS CM.

[0060] (11) RT-qPCR reveals the molecular mechanism of Holo-LCN2's protective effect on INS-1 apoptosis INS-1 cells were cultured to the logarithmic growth phase, and the cells were grouped as in the Western Blot experiment. The cells were collected, lysed with Trizol lysis buffer, and the cell RNA was extracted to detect the mRNA expression levels of Bcl-2 and BAX, which are related to cell inflammation.

[0061] The results show that ( Figure 8 C), using Fe alone3+ Pretreatment with Apo-LCN2 and Holo-LCN2 could not increase the expression level of Bcl-2 / BAX. Only Holo-LCN2 could increase the expression level of Bcl-2 / BAX and alleviate cell apoptosis, which proved that Holo-LCN2 was the active ingredient in BMSCs-STS CM.

[0062] (3) Calcein staining reveals the effect of Holo-LCN2 on intracellular iron levels after pretreatment with INS-1 INS-1 cells were cultured at 5×10 4 / ml density 100μl / well inoculated in 96-well plates and cultured overnight, the cells were grouped as in Western Blot experiments. Each group of cells was stained with 0.5μM Calcein, and then the fluorescence intensity was detected at an emission wavelength of 490nm and an absorption wavelength of 510nm using a fluorescence microplate reader, and statistical analysis was performed.

[0063] Calcein is a specific Fe 3+ Dye, with Fe 3+ After complexation, the fluorescence is quenched. According to the fluorescence intensity analysis, it can be concluded that ( Figure 8 D) Fe alone 3+ Neither the pretreatment with Apo-LCN2 nor the 3+ After being transferred into cells, only Holo-LCN2 can transfer Fe 3+ into cells.

[0064] (4) Western Blot Holo-LCN2 affects the intracellular iron level of INS-1 cells and changes in the level of cellular iron death INS-1 cells were cultured to the logarithmic growth phase, and the cells were grouped as in the Western Blot experiment. After culture at 37°C, the cells were collected and RIPA lysis buffer and protease inhibitors were added. Cytoplasmic proteins were analyzed by 10% SDS-PAGE and transferred to PVDF membranes to detect the protein expression levels of ACSL4, GPX4, and COX2 related to cell ferroptosis.

[0065] According to the above protein expression levels, Figure 8 E~F), Holo-LCN2 pretreatment 3+ After being transferred into the cells, it does not cause cell ferroptosis, but on the contrary can reduce the level of cell ferroptosis.

[0066] (5) RT-qPCR revealed changes in cellular ferroptosis levels after Holo-LCN2 affected intracellular iron levels in INS-1 cells INS-1 cells were cultured to the logarithmic growth phase, and the cells were grouped as in the above Western Blot experiment. The cells were collected, lysed with Trizol lysis buffer, and the cell RNA was extracted to detect the mRNA expression levels of ACSL4, GPX4, and COX2 related to ferroptosis.

[0067] According to the mRNA expression levels of the above proteins, Figure 8 G), Holo-LCN2 pretreatment 3+ After being transferred into the cells, it does not cause cell ferroptosis, but on the contrary can reduce the level of cell ferroptosis.

[0068] In summary, Holo-LCN2 increases intracellular Fe 3+ Ion concentration inhibits the apoptosis of INS-1 cells.

[0069] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cell-conditioned medium, characterized in that It is prepared according to the following steps: Inducing apoptosis of bone marrow mesenchymal stem cells, culturing the apoptotic bone marrow mesenchymal stem cells in DMEM / F12 culture medium for 20h~30h, centrifuging the obtained culture solution at 2000 rpm~4000 rpm for 10min~15min, and collecting the supernatant, which is the cell-conditioned medium.

2. The cell-conditioned medium according to claim 1, characterized in that Staurosporine was used to induce apoptosis of bone marrow mesenchymal stem cells. The concentration of staurosporine was 0.1 μM~0.5 μM and the treatment time was 1 h~6 h.

3. The cell-conditioned medium according to claim 2, characterized in that The degree of apoptosis of bone marrow mesenchymal stem cells is 20%~60%.

4. A kit for in vitro culture of bone marrow mesenchymal stem cells to induce apoptosis, characterized in that: It comprises the cell-conditioned medium according to claim 1.

5. Use of the cell-conditioned medium according to claim 1 in culturing apoptotic bone marrow mesenchymal stem cells.

6. Use of the cell-conditioned medium according to claim 1 in preparing a drug for preventing pancreatic islet cell apoptosis.

7. The use according to claim 6, characterized in that The cell conditioned medium is used for preparing a drug for resisting islet cell apoptosis in islet transplantation for treating diabetes.

8. The use according to claim 6 or 7, characterized in that The drug has the cell-conditioned medium as the only active ingredient.

9. The use according to claim 6, characterized in that: The cell-conditioned medium is used for preparing organ perfusion fluid or preservation fluid.

10. A drug for preventing pancreatic islet cell apoptosis, characterized in that: The invention is prepared by compounding the cell-conditioned medium or the effective active ingredient of the cell-conditioned medium according to claim 1 and a pharmaceutically acceptable adjuvant or carrier, wherein the effective active ingredient of the cell-conditioned medium is LCN2 protein in an iron-loaded form.

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