KR100819838B1 - Method for treating alpha-melanocyte-stimulating hormone for protecting islet cells for transplantation - Google Patents

Abstract

A method for protecting islet cells during transplantation is provided to increase the transplantation efficiency by efficiently preventing destruction of the islet cells or damage of insulin secretion capacity of the islet cells caused by the islet cell transplantation and improve the transplantation success rate by inducing the stability of the islet cells at an initial stage of the transplantation. A method for treating an alpha-melanocyte-stimulating hormone for protecting islet cells for transplantation is characterized in that the alpha-melanocyte-stimulating hormone is added to a predetermined step during a series of the islet cells transplantation which isolates the islet cells from an animal islet cell supplying source except human and transplants the islet cells into an animal except human, wherein the predetermined step is at least one selected from the group consisting of an islet cell isolation step from the islet cell supplying source, an islet cell culturing step, an in vitro operating step of the islet cell, an islet cell storing step, and an islet cell transplanting step into a donor of the cell and the alpha-melanocyte-stimulating hormone adding step consists of adding the alpha-melanocyte-stimulating hormone to blood monocytes and then adding the alpha-melanocyte-stimulating hormone mixed with the blood monocytes to the islet cells.

Description

METHODS FOR TREATING ALPHA-MELANOCYTE-STIMULATING HORMONE FOR PROTECTING ISLET CELLS FOR TRANSPLANTATION}

1 is a photograph showing reverse transcriptase polymerase reaction (RT-PCR) showing the degree of melanocortin receptor I (MC1R) expression in islets and blood mononuclear cells (PBMCs) of white paper.

2 is a graph showing the survival rate of pancreatic islets according to whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone added.

Figure 3 is a graph showing the high mortality of pancreatic islets according to whether the blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone.

Figure 4 is a graph showing the production amount of nitrite (Nitrite) in the islet cell culture medium according to whether the blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone.

Figure 5 is a graph showing the tumor necrosis factor-alpha (TNF-α) secretion in pancreatic islet cell culture medium according to whether the blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone added.

6 is a graph showing the amount of interleukin 1β (IL-1β) secretion in pancreatic islet cell culture medium according to whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone added.

7 is a graph showing the amount of prostaglandin E ₂ (PGE ₂ ) secretion in islet cell culture medium according to whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone added.

Figure 8 is a graph showing the insulin secretion of pancreatic islets according to whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone.

The present invention is a method for treating transplanted islet cells, characterized in that the addition of alpha melanocyte stimulating hormone at a predetermined stage of the islet cell transplantation process isolating the islets cells from the islet cell source and transplanted to the islet cell recipients It is about.

Diabetes (Diabetes Mellitus) is a disease characterized by hyperglycemic symptoms and complications caused by abnormal insulin secretion of pancreatic β-cells or abnormal receptors in insulin action organs or organs. In order to treat diabetes, mainly insulin injection therapy and exercise therapy and diet are being performed, but there are limitations such as the inability to cure and the risk of complications still exist.

Recently, diabetes treatment through pancreas transplantation and pancreatic islet cell transplantation has been carried out. In the case of pancreas transplantation, there are problems such as absolute shortage of donors, high surgical complications, and difficulty in post-transplant management including continuous administration of immunosuppressive agents. In contrast, pancreatic islet cell transplantation is capable of in vitro manipulation such as in vitro culture and immune regulation of isolated islet cells, and allows long-term cryopreservation to allow long-distance transfer or transplantation time selection. In addition, when the immune response of xenograft is overcome, it is possible to supply infinite islet cells using pigs, etc., and it is easy to transplant without complications due to relatively simple procedure.

However, in order to successfully carry out pancreatic islet transplantation, minimizing pancreatic islet cell damage occurring during the development and isolation of effective pancreatic islet cells, minimizing cell damage caused by nonspecific inflammatory processes during transplantation and resulting engraftment failure, Problems such as overcoming cell damage and securing pancreatic islet cell sources due to the immune response occurring after transplantation should be determined first (Kim Song-cheol, Hanyang Medical Reviews 26 (3), pp.62-69 (2006)).

In particular, the initial inflammatory response after pancreatic islet transplantation causes functional inadequacy or destruction of pancreatic islets due to cell necrosis and apoptosis. Therefore, there is a problem in that a larger amount of pancreatic islets is required to be transplanted than islets actually required (Choi SE et. al ., Transpl Immunol . 13 (1) pp. 43-53 (2004); Tran VV et al ., Diabetes 52 (6) pp. 1423-1432 (2003).

These nonspecific inflammatory responses are various preinflammatory, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferonγ (IFN-γ). By cytokines (Mandrup-Poulsen T. J. Immunol . 139 pp. 4077-4082 (1987)), and large amounts of cytokines are nitric oxides induced by iNOS in pancreatic islets and in islets. , NO) production, which inhibits aconitase in the Krebs cycle, reducing glucose oxidation, ATP production and insulin production (Eizirik DL et al ., Diabetes Metab Rev. 13 (4) pp. 293-307 (1997); Kang MK et al. , Mech Ageing Dev . 125 (7) pp. 483-490 (2004); Baker MS et al. , J Surg Res. 15; 97 (2) pp. 117-122 (2001); Corbett JA et al ., Biochem J. 299 (Pt 3) pp. 719-724 (1994)).

In addition, prostaglandin E ₂ (Pstagrandin E ₂ , PGE ₂ ) has been reported to inhibit the secretion of glucose-induced insulin in islets or β-cell lines and is known to promote production by IL-1β (Andersson AK et al ., Mol Cell Endocrinol. 220 (1-2) pp. 21-29 (2004).

In order to prevent cell damage caused by nonspecific inflammatory processes occurring in the transplantation process, monoclonal antibodies (infliximab) to tumor necrosis factor-alpha receptors have been developed and used for actual pancreatic islet transplantation (Farney AC. Transplant Proc . (25) 865-866 (1993)), N-monomethyl arginine, which regulates nitric oxide, and 15-deoxypergualin, which controls the function of macrophages, have also been tried (Kaufman DB. Transplant Proc . (24) pp. 1045-). 1047 (1992)) It has not been applied to clinical practice in earnest.

Therefore, if inflammatory cytokines can be controlled, it is possible to induce stable pancreatic islets in the early stages of transplantation and to expect maximum pancreatic islet transplantation effect with a smaller amount of cells, thereby improving the success rate of transplantation.

Accordingly, the present invention was devised to prevent destruction of pancreatic islets or damage to insulin secretion ability generated during islet cell transplantation, and induces stability of early islet cells in the early stages of transplantation, thereby showing sufficient effect even with a small amount of islets. To protect the transplanted pancreatic islet cells, which is characterized by adding alpha melanocyte stimulating hormone to the pancreatic islet cells at a predetermined stage of the islet cell transplantation process, which separates the islets from the islet cell source and transplants them to the islet cell recipient. The purpose is to provide a treatment method.

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In order to achieve the above object, the present invention is alpha-melanocyte stimulating hormone (α-melanocyte-stimulating hormone, referred to as 'α-MSH') when pancreatic islet cell transplantation or destruction of pancreatic islets due to a predetermined inflammatory response Characterized in that it is used to prevent damage to the ability.

Here, α-MSH is a hormone known to be involved in the regulation of various immune responses in humans and rats, and is expressed in the pituitary gland, brain, skin and circulatory tissue as tridecapeptide, which is transferred from propiomelanocortin ( Yoon SW et al., J Biol Chem. 29; 278 (35) pp. 32914-32920 (2003); Lipton JM et al., Ann NY Acad Sci. 20; 885 pp. 173-82 (1999); Sarkar A et al., FEBS α Lett. 23; 553 (3) pp.286-94 (2003)), which interact with cells involved in various immune and inflammatory systems to alter the action of pro-inflammatory cytokines and anti-inflammatory cytokines. Adjust That is, the α-MSH has an anti-inflammatory effect of inhibiting cell destruction caused by inflammatory cytokines such as tumor necrosis factor-alpha and interleukin 1 beta produced by immune cells such as leukocytes and lymphocytes, thereby controlling the inflammatory response. (Yoon SW et al ., J Biol) Chem . . 29; 278 (35) pp. 32914-32920 (2003) and the like).

At this time, the receptor of α-MSH is expressed in various cells, and 5 kinds of melanocortin receptors (melanocortin receptor MC1R to MC5R) are involved in the mechanism in which α-MSH acts on cells, among which melanocortin is involved. Receptor I is expressed on the surface of various inflammatory and immune cells and is known to play an important role in regulating the anti-inflammatory response of α-MSH (Yoon SW et al., J Biol Chem. 29; 278 (35) pp. 32914-32920 (2003); Hill RP et al., Peptides. 26 pp. 1150-1158 (2005)). To date, MC1R expression has been reported in human and mouse pancreatic islets, but no specific relationship between α-MSH and pancreatic islets has been reported.

According to the present invention, it is preferable to use α-MSH effective for preventing destruction of pancreatic islets or impaired insulin secretion ability due to a predetermined inflammatory response in order to protect pancreatic islets during transplantation.

More preferably, the present invention provides α-MSH to prevent destruction of pancreatic islets or impaired insulin secretion ability due to an inflammatory response induced by an inflammatory cytokine, a physiologically active protein, as the predetermined inflammatory response. It is characterized by using.

The inflammatory cytokines include tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). Tumor necrosis factor-alpha is directly involved in the apoptosis of pancreatic islet cells, induces transplant rejection by increasing adhesion molecules at the site of transplantation, and interleukin 1β stimulates T-cells to cause major immune rejection. Promotes the action of various immune related factors, including IL-2.

The α-MSH may be any type of agent capable of acting on the islet cells such as hormones, chemotherapeutic agents, additives, aqueous solutions, injections, oral medications.

In addition, the present invention is characterized in that the α-MSH is added at a predetermined stage in a series of pancreatic islet cell transplantation procedures in which islet cells are isolated from a pancreatic islet cell source and transplanted into a pancreatic islet cell recipient.
Here, the islet cell source may be a human or an animal other than a human. Preferred animals other than humans are pigs.
The islet cell recipient can also be a human or an animal other than a human.

At this time, the meaning of 'addition' of adding α-MSH at a predetermined stage of the islet cell transplantation process is that the α-MSH can affect the islet cells, that is, the α-MSH can act on the islet cells. This is the case, for example, to mix α-MSH with pancreatic islets and other components so that α-MSH can come into contact with the islets.

Here, a series of pancreatic islet cell transplantation procedures for isolating islets from a pancreatic islet cell source and transplanting them into a pancreatic islet cell recipient may include isolating pancreatic islet cells from a pancreatic islet cell source and islet cell transplantation to a pancreatic islet cell recipient. .

Further, at least one of the steps for isolating the islets from the pancreatic islet cell source and transplanting the islets of the islets to the islet cell recipient may include culturing the isolated islets, in vitro manipulation of the islets, and islet storage. Steps may be included.

And, in the islet islet cell culture step, the in vitro manipulation of the islet cells, pancreatic islet cell storage step, which may be included between the islet cell isolating step from the islet cell source and the islet cell transplantation step to the islet cell recipient, The order of the steps may be reversed, and each step may be performed two or more times.

Furthermore, the step of culturing the pancreatic islets may be cultured at room temperature or 37 ° C., and culturing at a low temperature of 22 ° C. to 24 ° C. may be more preferable to prevent an immune response.

In addition, in the step of culturing the pancreatic islets, in order to take advantage of characteristics such as biofilm stabilization of taurine, role as a free radical scavenger, and osmotic pressure control, culturing pancreatic islet cells with taurine is added to the culture medium. More preferred.

In addition, the in vitro manipulation step may be an manipulation step for overcoming an immune response such as microencapsulation of islet cells.

Here, the step of adding the α-MSH is the step of islet cell separation from the islet cell source, islet cell culture step, in vitro manipulation of the islet cells, islet cell storage step, islet cell transplantation step to the islet cell recipient It can be any one or more.

At this time, it is more preferable to add α-MSH in the islet cell transplantation step to the islet cell recipient. Rather than treating α-MSH when islets are present alone, treating α-MSH at the stage of transplanting or issuing anti-inflammatory and immune responses after transplanting islets is responsible for destruction or It is more effective in preventing impaired insulin secretion.

Further, the step of adding the α-MSH, the first step of adding alpha melanocyte stimulating hormone to blood mononuclear cells, and the step of adding alpha melanocyte stimulating hormone mixed with the blood mononuclear cells to the islet cells Can be configured.

In comparison with the expression level of MC1R in blood mononuclear cells and pancreatic islet cells, the amount of MC1R expression in pancreatic islet cells is lower than that of MC1R expressed in blood mononuclear cells. This may be because the reaction to is weak. Therefore, it may be more effective to induce protection of pancreatic islets by first treating α-MSH to blood mononuclear cells.

In this regard, human dermal fibroblasts have been reported to have lower MC1R expression than other melanin cells, resulting in a lower protective effect of α-MSH than other cells (Hill RP et al ., Peptides 26 pp.1150). -1158 (2005)), Jurkat (T-cell line) cells and macrophages, PMA stimulated macrophage cell line (THP-1) cells, U-937 (monocyte cell line) cells, neutrophils, H4 ( According to the results of analysis of α-MSH binding in glioma cell line), HeLa cells, and NIH 3T3 (fibroblast) cells using MC1R-specific antibodies, the binding rate in macrophages, neutrophils, T-cells, etc. It has been shown to be high, which is reported to be different in the MC1R existing in each cell, and thus also the action of α-MSH (Sarkar A. et al ., FEBS Lett. 23; 553 (3) pp) 286-94 (2003).

On the other hand, in order to induce the protection of pancreatic islet cells by first treating the α-MSH to blood mononuclear cells as described above, adding the α-MSH to the blood mononuclear cells, (a) culturing blood mononuclear cells, (b) adding alpha melanocyte stimulating hormone to the blood mononuclear cells in the culture, and adding alpha melanocyte stimulating hormone in the mixed state with the blood mononuclear cells to the islet cells, (c) the It is preferable to mix pancreatic islet cells with a culture solution containing blood mononuclear cells in a state in which alpha melanocyte stimulating hormone is added.

At this time, the mixed culture of the pancreatic islet cells and the blood mononuclear cells in order to create an in vitro environment that induces the damage of pancreatic islet cells by various inflammatory cells and toxic substances secreted by the pancreatic islets may be two types of cells. Direct contact method can be used to make direct contact, but indirect contact method, ie, physical contact between two kinds of cells is excluded and only the medium is shared so that soluble substances secreted from blood mononuclear cells can come into contact with the islets. It is more preferable to use a method to make it possible.

The concentration of α-MSH added to the blood mononuclear cells in the culture may be nM units, in particular 10 to 500 nM. Herein, in order to increase the survival rate of pancreatic islets and to effectively suppress the production of various inflammatory cytokines, the concentration of α-MSH is preferably 50 to 500 nM. This is because, when the concentration of α-MSH is 10 nM or less, the α-MSH shows little effect of inhibiting nitrogen oxide production, and when the concentration of 50-500 nM is effective, the production of nitrogen oxide is suppressed.

In particular, when 50-500 nM of α-MSH were treated in the islet culture group, there was no effect on the survival rate and nitric oxide production of the islets, but the same concentration of α-MSH was added to the blood mononuclear cells first. When the α-MSH is added to the pancreatic islet cells by culturing to obtain a pre-culture mixture of α-MSH and blood mononuclear cells and mixing them with the islets, the survival rate of the islet cells damaged by the blood mononuclear cells is Increased, and the production of various inflammation related cytokines is effectively suppressed. Therefore, by adding α-MSH to the blood mononuclear cells and mixing them first, and then adding a mixture of the blood mononuclear cells and α-MSH to the islet cells, a portion of the islets are removed from the destruction of the islets and the impaired insulin secretion ability. I can protect it.

Next, a more specific embodiment of the present invention will be described.

[ Example ]

One. Islet cells  blood Mononuclear  Isolation and Cultivation

(1) Isolation and Culture of Islet Cells

Lewis white papers around 300 g in weight were used as a source of pancreatic islet cells. First, in order to obtain pancreatic islet cells, the pancreas of the white paper was isolated and subjected to tissue digestion at 37 ° C. for 20 minutes using 1000 U / mL collagenase XI (collagenase XI, Sigma, St. Louis, MO, USA). Digested pancreatic tissue was mixed with 27% Ficoll (Sigma) solution and then superimposed 18% and 13% Ficoll solution and centrifuged at 2,000 × g for 20 minutes to separate only pancreatic islet cells.

Isolated pancreatic islets were cultured in RPMI-1640 (Sigma) medium containing 10% fetal bovine serum (FBS; Invitrogen, Grand Island, NY, USA).

(2) Isolation of Blood Mononuclear Cells

Lewis white papers around 300 g in weight were used as a source of blood mononuclear cells. 10 ml of blood was collected from the portal vein of the white paper, superimposed on Ficoll-Histopaque (Sigma), and centrifuged at 1,800 rpm for 25 minutes to separate blood mononuclear cells.

(3) Mixed culture of pancreatic islets and blood mononuclear cells

Isolated pancreatic islets and blood mononuclear cells were cultured using Transwell ^™ (Corning, New York, USA) to share only culture media without physical contact (Tran VV et al ., Diabetes 52 (6) pp. 1423-). 1432 (2003).

First, trypsin-EDTA (Invitrogen) treatment on a 24-well plate to process 5x10 ⁵ islet cells isolated into single cells and transfer them to Transwell ^TM . After the addition, ⁵ x 10 ⁵ blood mononuclear cells were added thereto and cultured. RPMI1640 was used as a medium, and 10 ng / mL phorbol myristic acid (PMA; Sigma) and 1 ㎍ / mL ionomycin (Sigma) were used to induce activation of blood mononuclear cells.

As a control group, only islet cells were cultured alone, and in the experimental group, 50 nM α-MSH was added to blood mononuclear cells and cultured by mixing α-MSH treated blood mononuclear cells first cultured for 2 hours. Was used.

2. α- MSH Involved in the functioning of MC1R of Islet cells  And blood In monocytes  Expression

After culturing pancreatic islets and blood mononuclear cells, each was washed with PBS (phosphate buffered saline) and treated with 1 mL of TRIzol (Isothiocyanate and phenol, Invitrogen) solution per 5-10 × 10 ⁶ / ml of islets. Cells were destroyed by incubation at room temperature for minutes. After 200 μl of chloroform was added to the cells destroyed as described above, the reaction tube was shaken vigorously for about 15 seconds by hand, and then left at room temperature for 2 to 3 minutes. Then, the phenol component was removed by centrifugation at 12,000 × g for 15 minutes at 4 ° C., and only the upper RNA was carefully collected. 500 μl of isopropyl alcohol (isoprophylalcohol) was added to the collected RNA, and the tube was inverted and left at room temperature for 10 minutes. This was centrifuged at 12,000 × g for 10 minutes to remove the supernatant, then treated with 1 ml of 75% ethyl alcohol and mixed uniformly, followed by centrifugation at 7,500 × g for 5 minutes. The supernatant was removed, washed and suspended in 20 μl of diethyl pyrocarbonate (Sigma) aqueous solution to separate total RNA.

First strand cDNA was synthesized using ImProm-II ^TM reverse transcriptase (Promega, Madison, Wis.) From 0.5-1 μg of total RNA isolated. Polymerase chain reaction (PCR) consists of 1 μl cDNA, 0.2 μM of each primer, 1 × PCR buffer (Takara, Otsu Shiga, Japan), 10 mM dNTP mix (Takara) and 1 unit Ex Taq DNA polyerase (Takara). ) Was mixed and performed in a PCR system (MJ Research, SanFrancisco, CA, USA). The amplification program was repeated 30 times for 45 seconds at 94 ° C, 45 seconds at 53 ° C, and 1 minute at 72 ° C. The final PCR product was confirmed by staining with Ethidium bromide after electrophoresis on a 1.8% agarose gel (agarose gel). MC1R primers used in the reaction were 5'-TCTGGGGAATGTCAGAGACC-3 '(forward) and 5'-TAGTGGTCTCCAGCACGATG-3' (reverse), and GAPDH primers were 5'-TCATGACCACAGTCCATGCCCA-3 '(forward) and 5'-GGGAFTTGCTGTTGAAGTCAC-3 Produced by '(reverse) to proceed with the reaction.

As a result, expression of MC1R was observed in both pancreatic cells and blood mononuclear cells of the white paper. FIG. 1 is a photograph showing the results of reverse transcriptase polymerase reaction (RT-PCR) showing the degree of melanocortin receptor I (MC1R) expression in pancreatic islets and blood mononuclear cells (PBMCs) of white paper, wherein 'PBMCs' are blood mononuclear cells. The cell, 'Islets', refers to pancreatic islets. Figure 1 shows that the degree of MC1R expression in blood mononuclear cells is significantly higher than the degree of MC1R expression in pancreatic islets.

3. Pancreatic islet cells  Test ( apoptosis Rate measurement

In order to measure the mortality rate of pancreatic islets, phospholipids phosphatidylserine (PS) was detected with Annexin V. When apoptosis occurs, the PS accumulates on the extracellular surface, where it is detected by binding to annexin V with fluorescent material. 5 μl of Annexin V (BD Bioscience Pharmingen, San Jose) was isolated from the cultured cells of blood mononuclear cells and pancreatic islets and cultured 5 × 10 ⁵ pancreatic islet cells for 48 hours, followed by centrifugation. , CA, USA) and 10 μl of Propidium Iodide (PI; Sigma) were stored in the cow for 15 minutes, and then stored in the cow using a fluorescence activated cell sorter (FACS, Becton Dickinson Facs caliber, San Jose, California, USA). Mortality was analyzed.

Figure 3 is a graph showing the high mortality rate of pancreatic islets according to whether or not mixed blood mononuclear cells (PMBC) culture and alpha melanocyte stimulating hormone, '+' in the presence of blood mononuclear cells or α-MSH, ' -'. '*' Indicates P <0.05 when compared with the first bar, and '**' indicates P <0.05 when compared with the second bar.

In the group treated with α-MSH, the mortality rate of pancreatic islets was decreased due to the mixed culture with blood mononuclear cells. In other words, the death rate was 38.2% when only islet cells were cultured alone. The death rate was 56.8% when PMA / Ionomycin-stimulated blood mononuclear cells were cultured together. However, after treatment with α-MSH in PMA / Ionomycin-stimulated blood mononuclear cells, the mortality rate was 43.2% (p = 0.018).

4. Pancreatic islet cells Survival rate  Measure

MTT assay was performed to observe the survival rate of pancreatic islets according to the mixed culture of blood mononuclear cells and pancreatic islets. Isolates only pancreatic islets from a mixed culture of blood mononuclear cells and pancreatic islets, and 0.5 mg / mL MTT (3- (4,5-dimethylthiazol-2-yl)-, for 5 × 10 ⁴ islet cells per 96 well plates. After incubation for 2-4 hours by treatment with 5-diphenyletrazolium bromide (Sigma), the medium was removed, and the absorbance was measured at 570 nm by processing 100 μl of DMSO (Sigma) and 100 μl / well of glycin buffer (Spectra MAS PLUS). And relative survival rates were compared.

Figure 2 is a graph showing the survival rate of pancreatic islets according to whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone added, '+' in the presence of blood mononuclear cells or α-MSH, '-in the absence '. '*' Indicates P <0.05 when compared with the first bar, and '**' indicates P <0.05 when compared with the second bar.

Compared to the case of the islet culture alone, pancreatic islet cells mixed with blood mononuclear cells showed a 53.8% survival rate, in which α-MSH was first treated with blood mononuclear cells and then mixed with the islets. In this case, the survival rate was 85.8%, indicating that destruction of pancreatic islets is reduced by treating α-MSH in blood mononuclear cells first and then incubating with pancreatic islets (p = 0.011).

5. Measurement of Nitric Oxide (NO) Formation

In order to check the degree of NO production, the cell supernatant obtained from the mixed culture of blood mononuclear cells and pancreatic islets was put into a 96-well plate, and Griess Reagent (p-aminobenzene sulfonamide, N- (1-naphthyl) ethylene-diamine in O-phosphoic acid; Promega). After inducing the reaction in the dark for 10 minutes, the absorbance was measured at 540 nm to compare the amount of NO produced.

Figure 4 is a graph showing the production of nitrite (Nitrite) in the pancreatic islet cell culture medium according to whether or not blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone added, '+ in the presence of blood mononuclear cells or α-MSH 'And'-'in absence. '*' Indicates P <0.05 when compared with the first bar, and '**' indicates P <0.05 when compared with the second bar.

 Nitrite production in the control group cultured only with islets was 3.1 ± 0.34μM, but the production increased rapidly to 7.6 ± 0.7μM when the islets were mixed with mononuclear cells. However, in the pancreatic islet cell group mixed with α-MSH treated blood mononuclear cells, nitrite production was reduced to 4.2 ± 0.4 μM (p = 0.031). On the other hand, when only α-MSH was treated in the islet cells, there was no statistical difference between the control group and 3.3 ± 0.09μM. The above results show that α-MSH alone does not affect the nitrite production of pancreatic islets.

6. Measurement of secretion level of inflammatory cytokines

In order to analyze the effect of α-MSH on the production of inflammatory cytokines, cell supernatants obtained from the mixed culture of blood mononuclear cells and pancreatic islets were collected and measured for TNF-α and IL-1β secretion. In order to perform ELISA, an ELISA kit (R & D systems, Inc., Minneapolis, MN, USA) for each cytokine was used. In a 96-well plate attached with antibodies to each cytokine, 50 µl of cell culture solution (5 × 10 ⁵ cells / 24well plate, 48 hours of incubation) and a standard solution were reacted for 2 hours, and then washed 4 times. Then, 100 μl of HRP-bound secondary antibody was added and incubated at room temperature for 1 hour. After the same washing process, 100 μl of substrate was treated to induce color development for 30 minutes. Finally, 50 μl of stop solution (1M H ₂ SO ₄ ) was treated, and then absorbance was measured at 450 nm to compare the cytokine secretion ability of each group.

FIG. 5 is a graph showing tumor necrosis factor-alpha (TNF-α) secretion in pancreatic islet cell culture medium according to whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone are added. The hour is marked with '+' and the absence is marked with '-'. '*' Indicates P <0.05 when compared with the first bar, and '**' indicates P <0.05 when compared with the second bar.

At this time, the change of TNF-α, an inflammatory cytokine, was 70.2 ± 5.9 pg / mL in the control group that cultured only islet cells, but 122.5 ± 8.4pg / mL when the pancreatic cells were mixed with mononuclear cells. appear. On the other hand, the blood mononuclear cells treated with α-MSH first and then mixed with the islet cells showed 77.6 ± 12.7 pg / mL, similar to the control group (p = 0.045).

FIG. 6 is a graph showing the amount of interleukin 1β (IL-1β) secretion in pancreatic islet cell culture medium according to whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone were added. In the presence of blood mononuclear cells or α-MSH, + 'And'-'in absence. '*' Indicates P <0.05 when compared with the first bar, and '**' indicates P <0.1 when compared with the second bar.

Interleukin 1β was 18.1 ± 7.6 pg / mL in the control group in which only the islet cells were cultured alone, but increased to 185.4 ± 43.6pg / mL in the mixed culture of blood mononuclear cells and pancreatic islets. However, the production of interleukin 1β was 130.9 ± 26.1 pg / mL in the islet cells mixed with the α-MSH-treated blood mononuclear cells, which was slightly decreased compared with the simple culture of the blood mononuclear cells and the pancreatic islets. This suggests that α-MSH inhibits the production of various inflammatory cytokines.

7. PGE ₂ Generation of

In order to analyze the effect of α-MSH on PGE ₂ production, cell supernatants obtained from the mixed culture of blood mononuclear cells and pancreatic islets were collected to measure the amount of PGE ₂ secreted. At this time, PGE ₂ ELISA kit (R & D systems) was used. Into a 96-well plate to which the antibody to PGE ₂ is attached, add 50 µl of cell culture solution (5 × 10 ⁵ cells / 24well plate, 48 hours incubation) and standard solution for 2 hours, and wash 4 times. In addition, 100 μl of HRP-bound secondary antibody was added and incubated at room temperature for 1 hour. After the same washing process, 100 μl of substrate was treated to induce color development for 30 minutes. Finally, 50 μl of stop solution (1M H ₂ SO ₄ ) was treated, and then absorbance was measured at 450 nm to compare and analyze PGE ₂ secretion ability of each group.

FIG. 7 is a graph showing the amount of prostaglandin E ₂ (PGE ₂ ) secretion in pancreatic islet cell culture medium depending on whether blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone are added. In the presence of blood mononuclear cells or α-MSH, + 'And'-'in absence. '*' Indicates that P <0.05 when compared to the first bar.

The amount of prostaglandin E ₂ in the control group was 1382 ± 353.3 pg / ml, and increased to 2523.8 ± 213 pg / ml in the mixed culture of blood mononuclear cells and pancreatic islets (p <0.05), and blood treated with α-MSH In the mixed culture of mononuclear cells and pancreatic islets, it appeared to be 2065 ± 265.4 pg / ml, which showed a small decrease but was not statistically significant (p> 0.05).

8. Islet cells  Insulin Secretion Capacity Analysis

Insulin assay ELISA kit (SHIBAYAGI co., Ltd., Japan) was used to determine the insulin secretion ability of pancreatic islet by collecting the cell culture supernatant by dividing a-MSH-treated and non-ischemic groups. The culture supernatant was diluted in steps (1/10, 1/50, 1/100, 1/500) to determine the optimal assay concentration, and then treated with 100 μl / well of biotin-conjugated anti-insulin in a 96-well plate. After that, 10 μl of cell culture solution and standard solution were added and reacted for 2 hours. After the reaction, the plate was washed four times with a washing solution to completely remove water. Then, 100 μl of HRP-coupled streptoavidin solution was added thereto and incubated for 30 minutes. In the same manner, 100 μl of substrate chromogen reagent was treated for 30 minutes, and the reaction was stopped using 100 μl of stop solution (1M H ₂ SO ₄ ), and absorbance was measured at 450 nm. The standardized curve was used to quantitatively compare the amount of insulin produced in each group.

FIG. 8 is a graph showing insulin secretion of pancreatic islets according to whether or not blood mononuclear cells (PMBC) mixed culture and alpha melanocyte stimulating hormone are added. In the absence of blood mononuclear cells or α-MSH, FIG. -'. '*' Indicates P <0.05 when compared with the first bar, and '**' indicates P <0.05 when compared with the second bar.

In the control group, the insulin secretion was 522.6 ± 85ng / ml, but the insulin secretion was reduced to 277.5 ± 18.6ng / ml when mixed with blood mononuclear cells. However, in the presence of α-MSH, the insulin secretion was 471.0 ± 62.9 ng / ml in the mixed culture of blood cells and pancreatic islets, which was higher than in the absence of α-MSH (p = 0.044). When α-MSH was added to the islet cell culture group, insulin secretion was 509.5 ± 21.0 ng / ml, which was not statistically significant with the control group. This result suggests that the treatment of α-MSH protects pancreatic islet cells from impaired insulin secretion ability of pancreatic islets caused by mixed culture with blood mononuclear cells.

Tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide, which are known as representative inflammation-related cytokines, are mixed with blood mononuclear cells with pancreatic islets, resulting in a marked increase in production (secretion) and a decrease in insulin secretion. Although it may worsen the destruction of insulin and impaired insulin secretion ability, the addition of α-MSH decreases the production of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide, or narrows the decrease in insulin secretion. Could.

As described above, embodiments of the present invention have been described in detail, but since the embodiments have been described so that those skilled in the art to which the present invention pertains may easily implement the present invention, The technical spirit of the present invention should not be limitedly interpreted.

Therefore, according to the present invention, by preventing the destruction of pancreatic islets or impaired insulin secretion ability by various inflammatory cytokines and nitric oxides, it is possible to reduce the inflammatory response and the accompanying immune response occurring during transplantation of pancreatic islets. It works. As a result, it is possible to ultimately increase the efficiency of transplantation, and induce stability of early islet cells in the early stages of transplantation, thereby achieving the purpose of transplantation even with a smaller amount of islets, thereby improving the success rate of transplantation.

Claims (10)

delete delete Transplanted pancreatic islet, which is characterized by adding alpha melanocyte stimulating hormone at a predetermined stage in a series of pancreatic islet cell transplantation procedures, which isolated from islet cells of non-human animal islets and transplanted to recipients of islet cells. Treatment for cell protection. The method of claim 3, wherein Adding the alpha melanocyte stimulating hormone, Any one of the following steps: islet cell isolation from an islet cell source other than human, islet cell culture step, in vitro manipulation of the islet cell, islet cell storage step, and islet cell transplantation to a pancreatic islet cell recipient other than human The treatment method for the protection of transplanted pancreatic islet cells, characterized in that above. The method according to claim 3 or 4, Adding the alpha melanocyte stimulating hormone, First adding alpha melanocyte stimulating hormone to blood mononuclear cells, Treatment method for the protection of transplanted pancreatic islet cells, characterized in that it comprises the step of adding alpha melanocyte stimulating hormone mixed with the blood mononuclear cells to the islet cells. The method of claim 5, wherein Adding the alpha melanocyte stimulating hormone to the blood mononuclear cells, (a) culturing blood mononuclear cells, (b) adding alpha melanocyte stimulating hormone to the blood mononuclear cells in the culture, Adding the alpha melanocyte stimulating hormone to the pancreatic islet cells in a mixed state with the blood mononuclear cells, Treatment method for the protection of transplanted pancreatic islet cells, characterized in that the pancreatic islet cells are mixed with a culture medium containing blood mononuclear cells in the alpha melanocyte stimulating hormone added state. The method of claim 6, The concentration of the alpha melanocyte stimulating hormone added to the blood mononuclear cells in the culture method for the protection of transplanted islet cells, characterized in that 50 to 500nM. A method for the protection of transplanted pancreatic islet cells, characterized in that the addition of alpha melanocyte stimulating hormone in any one or more of the steps of culturing the pancreatic islets, in vitro manipulation of the pancreatic islets, and storing the pancreatic islets. The method of claim 8, Adding the alpha melanocyte stimulating hormone, First adding alpha melanocyte stimulating hormone to blood mononuclear cells, Treatment method for the protection of transplanted pancreatic islet cells, characterized in that it comprises the step of adding alpha melanocyte stimulating hormone mixed with the blood mononuclear cells to the islet cells. The method of claim 9, Adding the alpha melanocyte stimulating hormone to the blood mononuclear cells, (a) culturing blood mononuclear cells, (b) adding alpha melanocyte stimulating hormone to the blood mononuclear cells in the culture, Adding the alpha melanocyte stimulating hormone to the pancreatic islet cells in a mixed state with the blood mononuclear cells, Treatment method for the protection of transplanted pancreatic islet cells, characterized in that the pancreatic islet cells are mixed with a culture medium containing blood mononuclear cells in the alpha melanocyte stimulating hormone added state.

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