KR100832737B1 - Processes for the preparation of a piglet for xeno-organ transplantation - Google Patents
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Abstract
본 발명은 3 ∼ 7 주령의 돼지에 300 ∼ 500 cGY의 흡수선량으로 방사선을 전신 조사하는 단계를 포함하는, 이종 장기 이식용 돼지의 제조방법 및 상기 제조방법으로 얻어진 이종 장기 이식용 돼지를 제공한다. 또한, 본 발명은 상기 이종 장기 이식용 돼지로부터 적출된 장기를 제공한다.The present invention provides a method for producing a heterologous organ transplanted pig, and a heterologous organ transplanted pig obtained by the method, comprising the step of whole-body irradiation with a radiation dose of 300 to 500 cGY to a pig aged 3 to 7 weeks old. . The present invention also provides an organ extracted from the pig for xenotransplantation.
본 발명에 따른 제조방법은 적혈구 등의 생존에 필요한 혈구의 혈중 농도에는 유의성 있는 변화를 미치지 않으면서 백혈구 및 CD4+ T 및 CD8+ T세포 등의 면역세포만을 선택적으로 감소시킨 이종 장기 이식용 돼지를 제조할 수 있다. 따라서, 본 발명의 제조방법에 의해 얻어지는 돼지는 사람 등의 포유동물에 이식될 수 있는 장기를 제공할 수 있다.The production method according to the present invention can produce a pig for heterologous organ transplantation which selectively reduces only leukocytes and immune cells such as CD4 + T and CD8 + T cells without significantly altering blood levels of blood cells necessary for survival of red blood cells. Can be. Therefore, the pig obtained by the manufacturing method of the present invention can provide an organ which can be transplanted into a mammal such as a human.
돼지, 방사선 조사, 면역세포, 백혈구, 혈소판, 적혈구, 유세포 분석 Swine, irradiation, immune cells, white blood cells, platelets, red blood cells, flow cytometry
Description
도 1은 방사선 조사량에 따른 돼지 생존율의 변화를 분석한 결과를 나타낸다.Figure 1 shows the results of analyzing the change in pig survival rate according to the radiation dose.
도 2a 내지 2c는 방사선 조사 전과 조사 후 혈액속의 적혈구, 백혈구 및 혈소판의 숫자를 분석한 결과를 나타내며, 도 2a는 백혈구 세포, 도 2b는 적혈구 세포, 및 도 2c는 혈소판 수 변화를 각각 나타낸다.2A to 2C show the results of analyzing the numbers of red blood cells, white blood cells and platelets in the blood before and after irradiation, and FIG. 2A shows white blood cells, FIG. 2B shows red blood cells, and FIG. 2C shows changes in platelet count, respectively.
도 3은 유세포 분석기(FACSvantage: Fluorescence Activated Cell Sorting)로 800 cGy의 흡수선량으로 방사선을 조사하기 전과 후의 돼지 혈액 속에 존재하는 돼지 면역세포인 CD4+ T 세포인 헬프 T 세포와 CD8+ T세포인 킬러 T 세포의 변화양상을 나타낸다.FIG. 3 shows helper T cells, CD4 + T cells, and pig T cells, CD8 + T cells, which are pig immune cells present in porcine blood before and after irradiation with an absorbed dose of 800 cGy using a flow cytometer (FACSvantage: Fluorescence Activated Cell Sorting). It shows the change pattern of.
도 4a 내지 4c는 유세포 분석기를 통한 분석에 의해 밝혀진 돼지 혈액 내 존재하는 helper T 면역세포의 방사선 조사에 따른 생체 변화 양상을 분석한 결과를 나타낸다. 도 4a는 800 cGy, 도 4b는 600 cGy, 도 4c는 400 cGy의 흡수선량으로 각각 방사선을 조사한 경우의 분석 결과이다.Figures 4a to 4c shows the results of analyzing the changes in the bio-injury pattern of the helper T immune cells present in porcine blood revealed by analysis by flow cytometry. 4A is 800 cGy, FIG. 4B is 600 cGy, and FIG. 4C is an analysis result when irradiated with an absorbed dose of 400 cGy, respectively.
도 5a 내지 5c는 킬러 T 면역세포의 변화양상을 분석한 결과를 나타낸다. 도 5a는 800 cGy, 도 5b는 600 cGy, 도 5c는 400 cGy의 흡수선량으로 각각 방사선을 조사한 경우의 분석 결과이다.5a to 5c show the results of analysis of changes in killer T immune cells. 5A is 800 cGy, FIG. 5B is 600 cGy, and FIG. 5C is an analysis result when irradiation is performed with absorbed dose of 400 cGy, respectively.
본 발명은 이종 장기 이식용 돼지의 제조방법, 상기 제조방법으로 얻어진 이종 장기 이식용 돼지, 및 상기 이종 장기 이식용 돼지로부터 적출된 장기에 관한 것이다.The present invention relates to a method for producing a heterologous organ transplantation pig, a heterologous organ transplantation pig obtained by the production method, and an organ extracted from the heterologous organ transplantation pig.
최근의 급격한 출산율 저하 및 고령화 현상과 더불어 골수를 포함한 세포 및 장기 제공자의 절대적인 부족현상은 심각한 사회문제로 대두되고 있다. 또한 헌혈자의 부족 및 인식도의 감소 현상은 날로 심각해지고 있으며 이로 인한 임상 현장에서의 고초는 날로 더해져만 가고 있다. 특히 이식 장기의 부족으로 인해서 고통받고 있는 환자는 날로 증대되고 있으며 이를 해결하고자하는 방법으로서 돼지를 이용한 사람 장기 이식 기술 방법이 개발되고 있다. 실질적으로 인간 장기 대체용 동물로서 주목을 받고 있는 돼지는 장기 제공원으로서 장기의 크기, 번식효율, 사육기술, 인체감염가능성을 고려할 때에 인간에 가장 적합한 동물로서 평가되고 있으며 실제로 선진국에서는 이미 이식용 돼지를 개발하고 있는 단계이다. In addition to the recent rapid decline in fertility and aging, the absolute shortage of cell and organ providers, including bone marrow, has become a serious social problem. In addition, the shortage of blood donors and the decrease in awareness are getting worse day by day, and the suffering in the clinical field is increasing day by day. In particular, the patients suffering from the lack of transplant organs are increasing day by day, as a method to solve this problem has been developed a human organ transplant technique using pigs. Pigs, which are actually attracting attention as a substitute for human organs, are regarded as the most suitable animals for humans in consideration of organ size, breeding efficiency, breeding techniques, and human infection potential. The stage of development.
최근에는 복제 돼지를 생산하는 기술 개발이 이루어지고 있으며 이를 바탕으로 사람에의 생체 이식 시 발생될 수 있는 면역 항원을 유전자 재조합 기술을 이용해 제거하는 시도가 이루어지고 있다. 특히 돼지 배아에서 추출한 섬유아세포 및 EG세포를 이용해 사람 면역 세포에 감작되어 면역억제 반응을 유도하는 돼지 항원 인 GAP 유전자를 발현하지 않은 형질전환 돼지가 보고되고 있다. Recently, the development of technology for producing cloned pigs has been made, and based on this, attempts have been made to remove immune antigens that may be generated when transplanting into humans using genetic recombination technology. In particular, transgenic pigs that do not express the GAP gene, a pig antigen that is sensitized to human immune cells using fibroblasts and EG cells extracted from porcine embryos and induces an immunosuppressive reaction, have been reported.
또한, 면역억제제인 사이클로스포린 A(cyclosporine A)를 투여하여 면역세포의 증식작용을 지닌 사이토카인인 IL-2의 분비억제를 통하여 면역세포의 면역거부 반응을 제어하는 방법이 시도된 바 있다 (Pozzetto et al., Cyclosporine inhibits interleukin-2 production during human cellular immune response to pig antigens. Transplant. Proc. 1994.26.2869-2870). 또한, FK506은 T 면역세포와 다른 면역세포에 발현하고 있는 IL-2 유전자의 발현을 제어하는 전사인인 NFAT의 활성을 제어하는 칼시누린(calcineurin) 억제제로서 개발된 약제로서 사이클로스포린 A와 동일한 면역세포 기능 억제 효능을 지닌 약물로서 미니돼지(miniature swine)의 생체 투여를 통하여 MHC가 다른 동종 돼지의 신장 이식을 시행하여 장기간에 걸친 이식 장기에 대한 면역 관용을 유도시킨 바 있다( Ustuqi et.al., Induction of transplantation tolerance with a short course of tacrolimus(FK506):1. Rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine. Tansplantation 2001. 71(10):1368-79).In addition, a method of controlling the immune rejection response of immune cells has been attempted by administering cyclosporine A, an immunosuppressive agent, by suppressing the secretion of IL-2, a cytokine having an immune cell proliferation effect (Pozzetto et. al., Cyclosporine inhibits interleukin-2 production during human cellular immune response to pig antigens.Transplant.Proc. 1994.26.2869-2870). In addition, FK506 is a drug developed as a calcineurin inhibitor that controls the activity of NFAT, a transcriptional regulator that controls the expression of the IL-2 gene expressed in T immune cells and other immune cells. MHC has induced kidney tolerance of other allogeneic pigs through the in vivo administration of mini swine as a drug having a function of inhibiting function, and induced long-term immune tolerance for transplant organs (Ustuqi et.al., Induction of transplantation tolerance with a short course of tacrolimus (FK506): 1.Rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine.Tansplantation 2001. 71 (10): 1368-79).
돼지를 이용해 사람의 심근세포를 추출하여 돼지의 심장에의 이식을 시행하여 돼지 생체 조직에서의 사람 심근세포의 증식과 분화를 분석한 결과 약 7개월 이상의 장기간에 걸처 사람 세포의 생착이 관찰한 연구결과가 보고된 바 있다 ( Husnain et.al., Myolbast transplantatin for cardiac repair using transient immunosuppression. Basic Appl. Myol.13(1):45-52, 2003).Human cardiomyocytes were extracted from pigs and transplanted into pig heart to analyze the proliferation and differentiation of human cardiomyocytes in pig tissues. Results have been reported (Husnain et.al., Myolbast transplantatin for cardiac repair using transient immunosuppression. Basic Appl. Myol. 13 (1): 45-52, 2003).
최근에는 양이나 돼지를 이용한 사람의 줄기세포를 생체 이식한 연구가 진행 되고 있으며 이러한 대 동물 생체 내에서 줄기세포의 다양한 특성 및 기능 분석 연구가 진행되고 있다. 특히 양을 이용해 사람의 조혈 줄기세포를 분리하여 자궁을 통하여 임신 태아 복강 내에 세포이식을 시행하여 장기간에 걸친 조혈세포의 재생능력을 분석하는 연구결과가 보고되고 있으며, 또한 사람의 신경줄기세포(human neural stem cells, NSC)를 복간에 이식하여 조혈세포로의 분화를 유도하는 연구가 진행된 바 있다(Almeida-Porada et.al., In vivo haematopoietic potential of human neural stem cells. Br. J. Haematol. 2005. 130(2):276-83. McNiece et. al., Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential. Exp. Hematol. 2002. 30(6):612-6.)Recently, studies have been conducted on transplanting human stem cells using sheep or pigs in vivo, and researches on various characteristics and functions of stem cells in animals have been conducted. In particular, studies have been conducted to analyze hematopoietic stem cell regeneration ability over a long period of time by isolating human hematopoietic stem cells using sheep and carrying out cell transplantation through the uterus through the uterus to human neural stem cells. Stem cells (NSC) have been studied in the abdominal cavity to induce differentiation into hematopoietic cells (Almeida-Porada et.al., In vivo haematopoietic potential of human neural stem cells.Br. J. Haematol. 2005. 130 (2): 276-83. McNiece et. Al., Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential.Exp. Hematol. 2002. 30 (6): 612-6. )
한편, 방사선은 백혈병과 같은 조혈질환의 치료 방법으로서 이용되고 있으며 1968년부터 시행되어 사람의 골수이식의 효능을 증진할 수 있는 가장 중요한 치료 방법 중 하나이다. 고용량의 항암화학요법과 방사선 조사를 통하여 골수 및 혈액 속에 존재하는 백혈병 세포의 제거를 통하여 환자의 성공적인 치료를 유도하기 위한 방법으로서 현재에도 유용하게 이용되고 있다. 그러나, 전신 방사선 조사는 백혈병세포를 제거함과 동시에 정상적인 혈액 세포의 파괴도 유도하는 한계를 가지고 있다.Radiation has been used as a method for treating hematopoietic diseases, such as leukemia, and has been implemented since 1968. It is one of the most important treatment methods for enhancing the efficacy of bone marrow transplantation in humans. As a method for inducing successful treatment of patients through the removal of leukemia cells present in the bone marrow and blood through high dose chemotherapy and irradiation, it is still usefully used. However, systemic irradiation has the limitation of removing leukemia cells and inducing destruction of normal blood cells.
최근, 마우스의 면역세포에 의한 면역거부반응을 억제하는 수단으로서, 방사선 조사를 이용한 예가 보고된 바 있다. 즉, 마우스의 동종 이식 연구에서 치사량(120 cGY) 또는 반치사량(750 cGY)의 방사선을 조사한 후 동종세포의 생체 이식 을 시도한 예가 보고된 바 있다 (Mohiuddin MM et.al., Long-term survival of cardiac xenografts in fully xenogeneic bone marrow chimeras. Ann Thorac Surg. 2001 Sep;72(3):740-5). Recently, an example of using irradiation has been reported as a means of suppressing immune rejection by immune cells in mice. In other words, in the allogeneic transplantation study of mice, an attempt was made to transplant allogeneic cells after irradiation with a lethal dose (120 cGY) or a half lethal dose (750 cGY) (Mohiuddin MM et.al., Long-term survival of cardiac xenografts in fully xenogeneic bone marrow chimeras.Ann Thorac Surg. 2001 Sep; 72 (3): 740-5).
그러나, 마우스와 같은 소동물과 달리, 인간 장기 제공원으로서 바람직한 동물인 돼지를 방사선 조사에 의해 면역 거부반응을 억제시킨 보고는 아직 없다. 또한, 면역 억제를 위해 중대 동물에 속하는 돼지에 방사선을 조사할 경우, 면역세포 뿐만 아니라, 생존에 필요한 적혈구 등의 혈구세포도 파괴됨으로써 돼지가 사망하는 결과가 초래되게 된다.However, unlike small animals such as mice, there have been no reports of suppressing immune rejection by irradiation of pigs, which are animals preferred as human organ donors. In addition, when radiation is irradiated to pigs belonging to a serious animal for immune suppression, not only immune cells but also blood cells such as red blood cells necessary for survival are destroyed, resulting in the death of pigs.
본 발명자들은 면역세포만이 선택적으로 억제된 이종 장기 이식용 돼지를 개발하고자 연구를 거듭한 결과, 어린 돼지(piglet)에 특정 흡수선량으로 전신 방사선 조사(total body irradiation, TBI)를 실시할 경우, 적혈구 등의 생존에 필요한 혈구의 혈중 농도에는 유의성 있는 변화를 미치지 않으면서 백혈구 및 CD4+ T 및 CD8+ T세포 등의 면역세포만을 선택적으로 감소시킬 수 있다는 것을 발견하였다.The present inventors conducted a study to develop a pig for heterogeneous organ transplantation, in which only immune cells were selectively suppressed, and when piglets were subjected to total body irradiation (TBI) at a specific absorbed dose, It has been found that only white blood cells and immune cells such as CD4 + T and CD8 + T cells can be selectively reduced without significant change in blood concentration of blood cells necessary for survival of red blood cells and the like.
따라서, 본 발명은 방사선을 전신 조사하는 단계를 포함하는 이종 장기 이식용 돼지의 제조방법을 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a method for producing a pig for xenotransplantation, comprising the step of irradiating the whole body with radiation.
또한, 본 발명은 상기 제조방법으로 얻어진 이종 장기 이식용 돼지를 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a pig for xenotransplantation obtained by the production method.
또한, 본 발명은 상기 이종 장기 이식용 돼지로부터 적출된 장기를 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide an organ extracted from the pig for xenotransplantation.
본 발명의 일 태양에 따라, 3 ∼ 7 주령의 돼지에 300 ∼ 500 cGY의 흡수선량으로 방사선을 전신 조사하는 단계를 포함하는, 이종 장기 이식용 돼지의 제조방법이 제공된다.According to one aspect of the present invention, there is provided a method of producing a pig for xenotransplantation, comprising the step of whole-body irradiation with an absorbed dose of 300-500 cGY to pigs 3-7 weeks old.
본 발명의 다른 태양에 따라, 상기 제조방법으로 얻어진 이종 장기 이식용 돼지가 제공된다.According to another aspect of the present invention, there is provided a pig for xenotransplantation obtained by the production method.
본 발명의 또 다른 태양에 따라, 상기 이종 장기 이식용 돼지로부터 적출된 장기가 제공된다.According to another aspect of the present invention, an organ extracted from the pig for xenotransplantation is provided.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 명세서에서, "이종 장기 이식용 돼지"라 함은 인간 등을 포함한 포유동물에 심장, 신장, 폐, 간, 췌장 등의 장기를 이식할 수 있는 돼지로서, 혈액분석학적으로(hematologically) 백혈구 및 CD4+ T 및 CD8+ T세포 등의 면역세포의 혈중 농도는 유의성 있게 감소되나, 적혈구의 혈중 농도는 유의성 있는 변화가 없는 돼지를 말한다.As used herein, the term “homologous xenotransplantation pig” refers to a pig capable of transplanting organs such as heart, kidney, lung, liver, and pancreas to a mammal, including humans, and the like. Blood levels of immune cells, such as CD4 + T and CD8 + T cells, are significantly reduced, but blood levels of erythrocytes do not change significantly.
본 발명에 따라, 어린(약 3 ∼ 7 주령) 돼지에 특정 흡수선량으로 전신 방사선 조사(total body irradiation, TBI)를 실시할 경우, 적혈구 등의 생존에 필요한 혈구의 혈중 농도에는 유의성 있는 변화를 미치지 않으면서 백혈구 및 CD4+ T 및 CD8+ T세포 등의 면역세포만을 선택적으로 감소시키는 혈액분석학적 양상(hematological profile)을 나타낸다. 특히, 특정 흡수선량으로 전신 방사선 조사를 실시함으로써, 돼지와 같은 중대(中大)형 동물이 면역거부반응을 최소화할 수 있다는 것은 매우 놀라운 것이며, 따라서, 본 발명의 제조방법에 의해 얻어지는 돼지는 사람 등의 포유동물에 이식될 수 있는 장기를 제공할 수 있다.According to the present invention, when whole body irradiation (TBI) is applied to a young (about 3 to 7 week old) pig at a specific absorbed dose, there is no significant change in the blood concentration of blood cells necessary for survival of red blood cells and the like. Hematological profile that selectively reduces only leukocytes and immune cells such as CD4 + T and CD8 + T cells. In particular, it is surprising that a medium-sized animal such as a pig can minimize an immune rejection reaction by performing whole-body irradiation at a specific absorbed dose. Therefore, a pig obtained by the manufacturing method of the present invention is a human. Organs can be provided that can be transplanted into a mammal, for example.
본 발명은 3 ∼ 7 주령, 바람직하게는 4 ∼ 6 주령, 더욱 바람직하게는 약 5 주령의 돼지에 300 ∼ 500 cGY의 흡수선량으로 방사선을 전신 조사하는 단계를 포함하는, 이종 장기 이식용 돼지의 제조방법을 제공한다. The present invention relates to a pig for heterologous organ transplantation comprising the step of whole-body irradiation with an absorbed dose of 300-500 cGY to pigs 3-7 weeks old, preferably 4-6 weeks old, more preferably about 5 weeks old. It provides a manufacturing method.
본 발명의 제조방법에 있어서, 상기 방사선의 흡수선량은 300 ∼ 500 cGY이며, 바람직하게는 약 400 cGY 이다. 상기 방사선은 의료 분야에서 널리 사용되는 방사선을 채용할 수 있으며, 예를 들면 X-선, γ-선, 전자빔 등을 사용할 수 있다.In the production method of the present invention, the absorbed dose of the radiation is 300 to 500 cGY, preferably about 400 cGY. The radiation may employ a radiation widely used in the medical field, for example X-rays, γ-rays, electron beams and the like can be used.
본 발명의 제조방법에 있어서, 상기 방사선 조사는 전신 조사(total body irradiation)로 수행되며, 방사선 전신 조사방법은 의료 분야에서 통상적으로 사용되는 방법으로 실시할 수 있으며, 별도로 제한되지 않는다. 예를 들어, 방사선 조사를 위해 전신을 고정시킨 후 방사선 조사기의 중앙에 위치시켜, 배면과 복면에 각각 방사선을 조사하여 방사선 전신 조사를 실시할 수 있다.In the manufacturing method of the present invention, the irradiation is carried out by total body irradiation (total body irradiation), the radiation whole body irradiation method may be carried out by a method commonly used in the medical field, it is not limited separately. For example, the whole body may be fixed for irradiation, and then positioned at the center of the radiation irradiator, and radiation may be irradiated to the back and the back surface, respectively, to perform radiation whole body irradiation.
본 발명은 상기 제조방법으로 얻어진 이종 장기 이식용 돼지를 포함하며, 또한, 상기 이종 장기 이식용 돼지로부터 적출된 장기를 포함한다. 상기 적출된 장기는 상기에서 열거한 심장, 신장, 폐, 간, 췌장 등을 모두 포함하나, 이에 제한되는 것은 아니다.The present invention includes a pig for heterologous organ transplantation obtained by the production method, and also includes an organ extracted from the pig for heterologous organ transplantation. The extracted organs include, but are not limited to, all of the above listed heart, kidney, lung, liver, pancreas, and the like.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.
실시예Example . .
생후 5∼7주령의 돼지를 마취하고, 분당 차병원 방사선실에서 800 cGy, 600 cGy, 400 cGy의 흡수선량으로 방사선을 조사한 다음, 방사선 조사전과 방사선 조사후의 돼지의 말초혈액을 항응고제가 포함된 튜브로 2-5ml 혈액을 채취하여 돼지 면역세포의 변화 양상 및 혈액세포의 변화를 분석하였다.Anesthetized 5-7 week old pigs and irradiated with absorbed doses of 800 cGy, 600 cGy and 400 cGy at Cha Hospital Radiation Bundang, and then the peripheral blood of pigs before and after irradiation with tubes containing anticoagulant. 2-5ml blood was collected and analyzed for changes in porcine immune cells and changes in blood cells.
실시예Example 1. 방사선 조사에 따른 돼지 생존율 1.Swin Survival Rate by Irradiation
생후 5∼7주령의 란드레이스(Landrace)계 돼지에 X-Ray 방사선기(Linac 2100CD, Varian. USA)를 방사선 전신 조사를 실시하였다. 방사선 조사 전에 케타민과 럼푼으로 돼지를 마취하고, 돼지의 전 체적을 측정하였다. 방사선 조사는 각각 800 cGy (n=2), 600 cGy (n=2), 및 400 cGy (n=4)의 3개의 군으로 나누어 실시하였으며, 방사선 조사 후 사육장에서 사육하면서 방사선 조사에 따른 돼지의 생존율 변화를 관찰하였다. Landrace pigs, 5-7 weeks of age, were irradiated with an X-ray radiograph (Linac 2100CD, Varian. USA). Pigs were anesthetized with ketamine and lumps before irradiation and the whole volume of the pigs was measured. Irradiation was divided into three groups of 800 cGy (n = 2), 600 cGy (n = 2), and 400 cGy (n = 4), respectively. Survival change was observed.
도 1은 각각의 조사량에 따른 돼지의 생존율 변화를 그래프로 나타낸 것이다. 800 cGy의 흡수선량으로 방사선을 조사한 경우 조사 2주령에서 1 마리가 사망하였으며 그 후 4주째에 나머지 한 마리도 사망하였다. 600 cGy의 흡수선량으로 방사선을 조사한 경우에도 유사한 결과를 얻었으며 방사선 조사 후 약 4 주령에서 모든 돼지가 사망하였다. 이에 반해, 400 cGy의 흡수선량으로 방사선을 조사한 경우 2주령에서 한 마리가 사망하였고 5주에도 한 마리가 사망하였으나 나머지 두 마리 의 돼지는 7주 이상 생존하였다. 1 is a graph showing the change in survival rate of pigs at each dose. Irradiation with an absorbed dose of 800 cGy resulted in one death at 2 weeks of age and the other at 4 weeks. Similar results were obtained when irradiated with absorbed dose of 600 cGy, and all pigs died about 4 weeks after irradiation. On the other hand, irradiation with 400 cGy absorbed dose resulted in one death at two weeks of age and one death at five weeks, but the remaining two pigs survived for more than seven weeks.
실시예Example 2. 방사선 조사에 따른 돼지 혈구세포의 변화 분석 2. Analysis of Changes in Porcine Blood Cells Following Irradiation
실시예 1에서 방사선 조사된 돼지로부터 혈액을 채취하여 방사선 조사에 따른 혈액 내의 혈액세포인 백혈구, 적혈구 및 혈소판의 변화를 분석하였다. 구체적으로, 방사선 조사 전 및 조사 후의 소정의 시간에 채취한 혈액(1 ml)을 채취하여 혈액 1 ㎕에 존재하는 백혈구, 적혈구 및 혈소판의 숫자를 혈구카운터로서 측정하였다. Blood was collected from the irradiated pigs in Example 1 and analyzed for changes in leukocytes, erythrocytes and platelets, which are blood cells in the blood following irradiation. Specifically, the blood (1 ml) collected before the irradiation and at the predetermined time after the irradiation was taken and the number of white blood cells, red blood cells and platelets present in 1 µl of blood was measured as a blood count counter.
도 2a 내지 2c는 방사선 조사 전과 조사 후 혈액속의 적혈구, 백혈구 및 혈소판의 숫자를 분석한 결과를 나타내며, 도 2a는 백혈구 세포, 도 2b는 적혈구 세포, 및 도 2c는 혈소판 수 변화를 각각 나타낸다. 도 2a에서 알 수 있는 바와 같이 모든 흡수선량에서 백혈구가 크게 감소함으로써 면역이 크게 저하되는 것을 확인할 수 있다. 이에 반해, 도 2b에서 알 수 있는 바와 같이 800 cGy를 조사한 경우, 조사 10 일후부터 급격한 적혈구 감소의 빈혈 현상이 관찰되었고, 600 cGy 및 400cGy를 조사한 경우 적혈구는 크게 변화하지 않았다. 또한, 도 2c에서 알 수 있는 바와 같이, 800 cGy, 600 cGy, 및 400 cGy의 흡수선량 모두에서 흡수선량 의존적으로 혈소판의 감소가 관찰되었으나, 400cGy의 흡수선량으로 방사선을 조사한 경우에는 2 주까지에도 약 50% 이상을 유지하고 있어 감소폭이 매우 작았다. 따라서, 사망률 및 혈구 분석 결과에 근거할 때, 적어도 600 cGy의 흡수선량 미만으로 방사선을 조사한 경우, 생존에 필요한 적혈구 수준 및 혈소판 수준을 유지하면서, 면역력 만을 선택적으로 감소시킬 수 있을 것으로 판단된다.2A to 2C show the results of analyzing the numbers of red blood cells, white blood cells and platelets in the blood before and after irradiation, and FIG. 2A shows white blood cells, FIG. 2B shows red blood cells, and FIG. 2C shows changes in platelet count, respectively. As can be seen in Figure 2a it can be seen that the immunity is greatly reduced by a significant decrease in white blood cells in all the absorbed dose. On the other hand, when irradiated with 800 cGy as can be seen in Figure 2b, the anemia phenomenon of rapid red blood cell reduction was observed from 10 days after irradiation, red blood cells did not change significantly when irradiated with 600 cGy and 400cGy. Also, as can be seen in FIG. 2C, platelet reduction was observed in all absorbed dose-dependent doses of 800 cGy, 600 cGy, and 400 cGy, but even when irradiated with an absorbed dose of 400 cGy, even up to 2 weeks. The reduction was very small since it was maintained at about 50% or more. Therefore, based on the mortality and blood cell analysis results, if irradiated at an absorbed dose of at least 600 cGy, it is believed that only immune power can be selectively reduced while maintaining the level of erythrocytes and platelets necessary for survival.
실시예Example 3. 방사선 조사에 따른 돼지 면역세포의 특성 3. Characteristics of Porcine Immune Cells by Irradiation
실시예 1에서 방사선 조사된 돼지로부터 혈액을 채취하여 방사선 조사에 따른 면역세포(CD4+T 세포 및 CD8+ T 세포)의 변화를 분석하였다. 구체적으로, 방사선 조사 전 및 조사 후 1주부터 7주까지 혈액(1 ml)을 매주 채취하여 면역세포 분석용 샘플로 이용하였다. 항응고제가 함유된 튜브에 돼지의 귀 정맥에서 채취한 혈액 3-5 ml를 이용해 유세포 분석용 세포로 면역염색을 실시하였다. 혈액속의 단핵구는 Ficoll(슈크로스의 중합체)과 Hypaque(디크리조에이트나트륨: sodium ditrizoate)의 중합체인 Ficoll-Hypaque를 이용하여 분리하였다. 혈액을 Ficoll-Hypaque 위에 중층하여 2000 rpm으로 20분간 원심분리하여 중간층의 단핵구 부분을 채취한 후, PBS(Phosphate Buffer Saline)를 넣은 코니컬튜브에 채취된 단핵구를 넣고 1300rpm으로 5분간 2회 원심분리하여, 코니컬튜브 바닥에 모인 백혈구가 함유된 단핵구 세포만을 분리하였다. Blood was collected from the irradiated pigs in Example 1 and analyzed for changes in immune cells (CD4 + T cells and CD8 + T cells) following irradiation. Specifically, blood (1 ml) was collected weekly from 1 week to 7 weeks before and after irradiation, and used as a sample for immune cell analysis. The tube containing anticoagulant was immunostained with cells for flow cytometry using 3-5 ml of blood collected from a pig's ear vein. Monocytes in the blood were separated using Ficoll (polymer of sucrose) and Ficoll-Hypaque, a polymer of Hypaque (sodium ditrizoate). The blood was layered on Ficoll-Hypaque and centrifuged at 2000 rpm for 20 minutes to collect monocytes in the middle layer. Then, the monocytes were collected into conical tubes containing PBS (Phosphate Buffer Saline) and centrifuged twice for 5 minutes at 1300 rpm. Thus, only monocyte cells containing leukocytes collected at the bottom of the conical tube were isolated.
분리된 단핵구 세포 1x106 세포를 돼지 면역세포 항원인 CD3의 항체인 항 CD3 항체(BD bioscience, USA)와 돼지 헬퍼(helper) T 세포 항원인 CD4에 대한 항 CD4 항체(BD bioscience, USA), 돼지 cytotoxic T세포 항원인 CD8에 대한 항 CD8 항체(BD bioscience, USA)로 96 웰 플레이트에서 면역염색을 30분 동안 실시하였다. 염색되지 않은 항체를 제거하기 위해서 면역 염색용 완충액인 Staining Media(PBS(-)에 2% 우태아혈청(FBS:fetal bovine serum, GIBCO)와 0.05%의 소듐 아지드(sodium azide)를 첨가한 용액)으로 2회 세척하였다. The isolated monocyte 1x10 6 cells were treated with anti-CD3 antibody (BD bioscience, USA), an antibody of the porcine immune cell antigen CD3, and anti-CD4 antibody (BD bioscience, USA), porcine, against the swine helper T cell antigen, CD4. Immunostaining was performed for 30 minutes in 96-well plates with anti-CD8 antibody against CD8, a cytotoxic T-cell antigen (BD bioscience, USA). To remove unstained antibody, 2% fetal bovine serum (GBSCO) and 0.05% sodium azide (FBS) were added to Staining Media (PBS (-)), an immunostaining buffer. ) Twice.
도 3은 유세포 분석기(FACSvantage: Fluorescence Activated Cell Sorting)로 800 cGy의 흡수선량으로 방사선을 조사하기 전과 후의 돼지 혈액 속에 존재하는 돼지 면역세포인 CD4+ T 세포인 헬프 T 세포와 CD8+ T세포인 킬러 T 세포의 변화양상을 나타낸다. 도 3에서 알 수 있는 바와 같이, 1 주후부터 CD3+CD4+ 헬프 T 세포와 CD3+CD8+ 킬러 T세포는 방사선 조사 전에 비해서 크게 감소하였고 이러한 현상은 2 주후에도 지속되었다. 이는 방사선 조사를 통하여 돼지 면역세포인 CD4+ T세포와 CD8+ T 면역세포를 직접적으로 생체에서 제거된다는 것을 나타낸다.FIG. 3 shows helper T cells, CD4 + T cells, and pig T cells, CD8 + T cells, which are pig immune cells present in porcine blood before and after irradiation with an absorbed dose of 800 cGy using a flow cytometer (FACSvantage: Fluorescence Activated Cell Sorting). It shows the change pattern of. As can be seen in Figure 3, after one week CD3 + CD4 + help T cells and CD3 + CD8 + killer T cells were significantly reduced compared to before irradiation and this phenomenon persisted even after two weeks. This indicates that the irradiation of pig immune cells, CD4 + T cells and CD8 + T immune cells directly removed from the body.
도 4a 내지 4d는 유세포 분석기를 통한 분석에 의해 밝혀진 돼지 혈액 내 존재하는 helper T 면역세포의 방사선 조사에 따른 생체 변화 양상을 분석한 결과를 나타낸다. 800 cGy와 600 cGy의 방사선 조사에 따른 면역세포의 변화(각각 도 4a 및 4b)는 조사 후 일주 후부터 급격한 감소현상이 관찰되었으며, 400 cGy의 흡수선량으로 조사된 돼지도 역시 면역세포의 감소가 관찰되었으나(도 4c 참조).Figures 4a to 4d shows the results of analyzing the changes in the bio-injury pattern of the helper T immune cells present in the pig blood revealed by analysis by flow cytometry. Changes in immune cells following irradiation of 800 cGy and 600 cGy (Figs. 4A and 4B, respectively) were observed. After one week of irradiation, the decrease of immune cells was observed. (See FIG. 4C).
도 5a 내지 5d는 상기와 동일한 방법으로 킬러 T 면역세포의 변화양상을 분석한 결과를 나타낸다. 800 cGy의 방사선을 조사한 경우, 킬러 T 면역세포의 비율이 급격히 감소하였으며, 400 cGy의 흡수선량으로 조사된 돼지도 역시 면역세포의 감소가 관찰되었으나(도 5c 참조).5a to 5d show the results of analyzing changes in killer T immune cells in the same manner as described above. When irradiated at 800 cGy, the proportion of killer T immune cells was drastically reduced, and the pigs irradiated with an absorbed dose of 400 cGy were also found to have reduced immune cells (see FIG. 5C).
본 발명에 따른 제조방법은 적혈구 등의 생존에 필요한 혈구의 혈중 농도에 는 유의성 있는 변화를 미치지 않으면서 백혈구 및 CD4+ T 및 CD8+ T세포 등의 면역세포만을 선택적으로 감소시킨 이종 장기 이식용 돼지를 제조할 수 있다. 따라서, 본 발명의 제조방법에 의해 얻어지는 돼지는 사람 등의 포유동물에 이식될 수 있는 장기를 제공할 수 있다.The production method according to the present invention produces a pig for heterologous organ transplantation which selectively reduces only leukocytes and immune cells such as CD4 + T and CD8 + T cells without significantly changing the blood concentration of blood cells necessary for survival of red blood cells. can do. Therefore, the pig obtained by the manufacturing method of the present invention can provide an organ which can be transplanted into a mammal such as a human.
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