KR100324549B1 - Novel cytokine stimulating B cell proliferation and preparation method thereof - Google Patents
Novel cytokine stimulating B cell proliferation and preparation method thereof Download PDFInfo
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- KR100324549B1 KR100324549B1 KR1019990020373A KR19990020373A KR100324549B1 KR 100324549 B1 KR100324549 B1 KR 100324549B1 KR 1019990020373 A KR1019990020373 A KR 1019990020373A KR 19990020373 A KR19990020373 A KR 19990020373A KR 100324549 B1 KR100324549 B1 KR 100324549B1
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Abstract
본 발명은 B 임파구의 성장을 유도하는 새로운 생쥐 적혈백혈병 (erythroleukemia) 세포주 LK1, 이 세포주의 배양 상등액으로부터 분리 정제된 사이토카인 (cytokine), 그의 분리 정제 방법, 사이토카인 유전자의 클로닝과 이를 이용하여 재조합 사이토카인 (히스타민 분비 인자)을 제조하는 방법 및 상기 사이토카인을 유효성분으로 하는 B 임파구 증식 유도제에 관한 것으로, LK1 세포주로부터 분비되는 사이토카인은 히스타민 분비 인자로서, 본 발명에 의해 기존의 히스타민 분비 유도 기능 외에도 B 임파구 증식을 유도하는 새로운 기능이 있음이 밝혀졌으며, 다른 백혈병 세포주에서 생산되는 사이토카인에 비해 30배 이상 높은 B 임파구 증식 유도율을 나타내므로 효과적인 B 임파구 증식 유도제로 이용될 수 있다. 아울러, 본 발명의 새로운 LK1 세포주는 적혈백혈병 세포주와 B 임파구 간의 상호작용을 연구하는 데 이용될 수 있다.The present invention relates to a new mouse erythroleukemia cell line LK1, which induces the growth of B lymphocytes, cytokine isolated from the culture supernatant of this cell line, its purification method, cloning of cytokine gene and recombination using the same. The present invention relates to a method for producing a cytokine (histamine secretion factor) and a B lymphocyte proliferation inducer using the cytokine as an active ingredient, wherein the cytokine secreted from the LK1 cell line is a histamine secretion factor and induces histamine secretion according to the present invention. In addition to the function, it has been found that there is a new function to induce B lymphocyte proliferation, and since it shows a 30-fold higher induction rate of B lymphocyte proliferation than cytokines produced in other leukemia cell lines, it can be used as an effective B lymphocyte proliferation inducer. In addition, the new LK1 cell line of the present invention can be used to study the interaction between red blood leukemia cell line and B lymphocytes.
Description
본 발명은 B 임파구의 성장을 유도하는 새로운 생쥐 적혈백혈병 세포주 LK1, 이 세포주의 배양 상등액으로부터 분리 정제된 사이토카인, 그의 분리 정제 방법, 사이토카인 유전자의 클로닝과 이를 이용하여 재조합 사이토카인 (히스타민 분비 인자)을 제조하는 방법 및 상기 사이토카인을 유효성분으로 하는 B 임파구 증식 유도제에 관한 것이다.The present invention relates to a new mouse erythroleukemia cell line LK1 which induces the growth of B lymphocytes, a purified cytokine isolated from the culture supernatant of this cell line, its purification method, the cloning of the cytokine gene and the recombinant cytokine (histamine secretion factor) using the same. ) And B lymphocyte proliferation inducer comprising the cytokine as an active ingredient.
생쥐 적혈백혈병 (erythroleukemia) 세포주는 적혈구 (erythroid) 세포주의 성장과 분화를 연구하는 데 유용하며, 여러 가지 서로 다른 생쥐 적혈백혈병 세포주가 확립되어 있다. 이들 세포주는 TGF-β (transforming growth factor), EPO (erythropoietin), c-kit, HMBA (N,N'-hexamethylene-bis-acetamide), DMSO (dimethylsulfoxide) 등의 여러 가지 요인들에 의해 분화가 유도되는 것으로 알려져 있다 (Krystal, G.,et al., J. Exp. Med. 180: 851, 1994). 상기 적혈백혈병세포주는 면역 세포를 포함한 다른 여러 가지 세포들의 성장에 중요한 역할을 수행하는 사이토카인 (cytokine)들을 생산하는데, 이러한 생쥐 적혈벽혈병 세포주의 성장과 분화에는 EPO와 GM-CSF (granulocyte-macrophage colony stimulating factor)가 매우 중요한 작용을 한다 (Miyazaki, Y.,et al., Leukemia, 11: 1941, 1997). 이처럼 생쥐 적혈백혈병 세포주는 다른 면역세포들과 상호작용하여 여러 가지 세포반응에 영향을 미치지만, 이들 세포들간의 상호작용에 대해서는 아직 구체적으로 밝혀진 것이 없다.Mouse erythroleukemia cell lines are useful for studying the growth and differentiation of erythroid cell lines, and several different mouse erythroleukemia cell lines have been established. These cell lines induce differentiation by a number of factors, including transforming growth factor (TGF-β), erythropoietin (EPO), c-kit, HMBA (N, N'-hexamethylene-bis-acetamide), and dimethylsulfoxide (DMSO). (Krystal, G., et al ., J. Exp. Med. 180: 851, 1994). The erythroid leukemia cell line produces cytokines, which play an important role in the growth of various other cells, including immune cells. The growth and differentiation of these mouse erythropoietic cell lines involves EPO and GM-CSF (granulocyte-macrophage). colony stimulating factor) plays a very important role (Miyazaki, Y., et al ., Leukemia, 11: 1941, 1997). As such, the mouse erythroleukemia cell line interacts with other immune cells and affects various cellular responses, but the interaction between these cells has not been identified yet.
한편 B 임파구 (B lymphocyte) 분화 단계에 관한 연구는 B 임파구의 배중심 (germinal center, GC)과 기억 B 임파구 (memory B lymphocyte) 형성에 관련하는 인자에 대한 연구가 주종을 이루고 있으며, 원형 (naive) B 임파구가 어떠한 과정에 의해 배중심이나 기억 B 임파구로 분화되는지, 그 분화 과정에 어떠한 분자 (CD40, IL-4 등)가 관련하는지 등에 대한 연구들이 현재 활발히 진행되고 있다. 여러 연구 결과 B 임파구 수용체를 통해 다른 신호전달 (NF-κB 등)이 이루어짐으로써 세포신호가 결정되어 B 임파구 항원에 의한 활성화가 유도된다는 것이 밝혀졌다. 이러한 B 임파구의 활성화에 작용하는 사이토카인들로는 IL-2, IL-4, IL-5, IL-6, IL-10 등이 알려져 있는데, B 임파구의 분화와 증식에 대하여 보다 진전된 연구를 수행하기 위해서는 B 임파구의 활성화에 관계하는 새로운 사이토카인에 대한 연구가 필요하다.Meanwhile, the study on the differentiation stage of B lymphocytes is mainly studied on factors related to germinal center (GC) and memory B lymphocyte formation of B lymphocytes. There is currently a lot of research into how B lymphocytes differentiate into germinal centers or memory B lymphocytes and which molecules (CD40, IL-4, etc.) are involved in the differentiation process. Several studies have shown that different signaling (NF-κB, etc.) occurs through B lymphocyte receptors to determine cellular signals that induce activation by B lymphocyte antigens. Cytokines that act on the activation of B lymphocytes are known to be IL-2, IL-4, IL-5, IL-6, IL-10, etc., and to conduct further research on the differentiation and proliferation of B lymphocytes. To do this, new cytokines involved in the activation of B lymphocytes are required.
이에 본 발명자들은 새로운 생쥐 적혈백혈병 세포주를 확립하고 B 임파구와의 상호작용에 관한 연구를 수행한 결과, 분화과정의 초기 단계에 있는 적혈구 전구세포의 특성을 갖고 있는 새로운 생쥐 적혈백혈병 세포주를 확립하여 이를 LK1이라 명명하고 그 특성을 분석하였으며, 이 세포로부터 분비되는 배양 상등액으로부터 B 임파구의 성장을 유도하는 사이토카인 단백질을 분리 정제하였다. 또한 LK1에서 분비되는 사이토카인이 히스타민 분비 인자로서, 기존의 세포주에서 분비되는 사이토카인에 비해 30배 이상 뛰어난 B 임파구 증식 유도 효과를 보임을 밝혀냄으로써 히스타민 분비 인자를 B 임파구 증식 유도제로 사용하는 새로운 용도를 알아내고, 상기 사이토카인 유전자를 클로닝하여 재조합 사이토카인 (히스타민 분비 인자)을 제조하는 방법을 제공함으로써 본 발명을 완성하였다.Therefore, the present inventors established a new mouse erythroleukemia cell line and conducted a study on the interaction with B lymphocytes. As a result, we established a new mouse erythroleukemic cell line having the characteristics of erythrocyte progenitor cells in the early stage of differentiation process. The LK1 was named and analyzed, and the cytokine protein which induces the growth of B lymphocytes was isolated and purified from the culture supernatant secreted from these cells. In addition, the use of histamine secretion factor as a B lymphocyte proliferation inducer by revealing that the cytokine secreted by LK1 is a histamine secretion factor and shows an effect of inducing B lymphocyte proliferation more than 30 times that of the cytokine secreted by the existing cell line. The present invention was completed by providing a method for producing a recombinant cytokine (histamine secretion factor) by cloning the cytokine gene.
본 발명의 목적은 B 임파구의 성장을 유도하는 새로운 생쥐 적혈백혈병 세포주 LK1, 이 세포주의 배양 상등액으로부터 분리 정제된 사이토카인, 그의 분리 정제 방법, 사이토카인 유전자의 클로닝과 이를 이용하여 재조합 사이토카인 (히스타민 분비 인자)을 제조하는 방법 및 상기 사이토카인을 유효성분으로 하는 B 임파구 증식 유도제를 제공하는 것이다.An object of the present invention is a new mouse erythroleukemia cell line LK1 which induces the growth of B lymphocytes, a purified cytokine isolated from the culture supernatant of this cell line, its purification method, the cloning of the cytokine gene and the recombinant cytokine (histamine using the same). Secretion factor) and a method for producing B lymphocyte proliferation inducer comprising the cytokine as an active ingredient.
도 1은 LK1 세포의 형태를 나타낸 것이고, 1 shows the morphology of LK1 cells,
A :100배율 광학현미경으로 관찰한 것 A: observed with a 100x optical microscope
B :김사 (Giemsa) 염색하여 400배율 광학현미경으로 관찰한 것 B: Kimsa (Giemsa) dyeing and observed with a 400x optical microscope
C :전자염색하여 투과전자현미경으로 관찰한 것 C: Electrostained and observed by transmission electron microscope
D :광학현미경으로 관찰한 염색체 분석 결과를 나타낸 것 D shows the result of chromosome analysis observed by optical microscope
도 2는 LK1 세포의 Sca-1과 Ter-119 발현을 나타낸 세포형광 분석 결과이고, Figure 2 is a cell fluorescence analysis showing the expression of Sca-1 and Ter-119 in LK1 cells,
도 3은 LK1 세포로부터 사이토카인과 MER-5 유전자가 발현된 것을 역전사 중합효소 연쇄반응 (RT-PCR)으로 확인한 것이고, 3 is a reverse transcriptase polymerase chain reaction (RT-PCR) confirmed that the expression of the cytokine and MER-5 gene from LK1 cells,
A :LK1 세포로부터 여러 가지 사이토카인 유전자가 발현된 것을 보여주는 것 A: showing the expression of various cytokine genes from LK1 cells
B :LK1 세포와 대조군인 MEL-1, DS19로부터 MER-5 유전자가 발현된 것을 보여주는 것 B: Expression of MER-5 gene from LK1 cells and MEL-1, DS19 control group
도 4는 여러 가지 사이토카인들에 의한 LK1 세포의 증식 결과를 나타내는것이고, Figure 4 shows the results of proliferation of LK1 cells by various cytokines,
도 5는 LK1 배양 상등액이 면역세포의 증식에 미치는 영향을 나타내는 세포형광 분석결과이고, 5 is a cell fluorescence assay showing the effect of LK1 culture supernatant on the proliferation of immune cells,
위 : CD3 T 세포군에 대한 증식 효과를 나타내는 것Stomach: proliferative effect on CD3 T cell population
아래 : CD45R B 임파구에 대한 증식 효과를 나타내는 것Below: showing the proliferative effect on CD45R B lymphocytes
도 6은 LK1 세포의 배양 상등액이 생쥐 비장 B 임파구의 증식 (-■-)과 IL-6의 생산 (-□-)에 미치는 영향을 나타낸 것이고, Figure 6 shows the effect of the culture supernatant of LK1 cells on the proliferation (-■-) and IL-6 production (-□-) of mouse spleen B lymphocytes,
도 7은 LK1 세포 배양 상등액을 모노-큐 (Mono-Q) 칼럼 크로마토그래피로 정제 분리한 분획들이 생쥐 비장 B 임파구의 증식에 미치는 영향을 나타내는 SDS-PAGE 결과이고, 7 is SDS-PAGE showing the effects of the fractions purified by mono-Q column chromatography on LK1 cell culture supernatants on the proliferation of mouse splenic B lymphocytes.
A :1차 용출액의 결과 A: Result of the primary eluate
B :2차 용출액의 결과 B: Result of the Secondary Eluate
C :3차 용출액의 결과 C: Result of tertiary eluate
도 8a는 히스타민 분비 인자를 암호화하는 사이토카인 유전자가 삽입된 발현 벡터 pMAL-c2를 나타낸 것이고, Figure 8a shows an expression vector pMAL-c2 inserted with a cytokine gene encoding histamine secretion factor,
도 8b는 재조합 히스타민 분비 인자 (사이토카인)가 분리된 것을 보여주는 SDS-PAGE 결과이고, 8B is an SDS-PAGE result showing that recombinant histamine secretion factor (cytokine) was isolated,
레인 1 : 단백질 사이즈 마커 (PM)Lane 1: protein size marker (PM)
레인 2 : 말토스 결합 단백질 (MBP)Lane 2: maltose binding protein (MBP)
레인 3 : 말토스 결합 단백질과 히스타민 분비 인자 (HRF)의 융합 단백질Lane 3: fusion protein of maltose binding protein and histamine secretion factor (HRF)
도 9a는 재조합 히스타민 분비 인자 (사이토카인)가 B 임파구 증에 미치는 영향을 나타낸 것이고, Figure 9a shows the effect of recombinant histamine secretion factor (cytokine) on B lymphocytes,
도 9b는 말토스 결합 단백질과 히스타민 분비 인자를 끓이거나 히스타민 분비 인자의 항체로 처리하기 전후 B 임파구 증식에 미치는 영향을 나타낸 것이다. Figure 9b shows the effect on B lymphocyte proliferation before and after boiling maltose binding protein and histamine secretion factor or treatment with antibodies of histamine secretion factor.
상기 목적을 달성하기 위하여, 본 발명에서는 B 임파구 증식을 유도하는 새로운 생쥐 적혈백혈병 (erythroleukemia) 세포주 LK1을 제공한다.In order to achieve the above object, the present invention provides a new mouse erythroleukemia cell line LK1 inducing B lymphocyte proliferation.
또한 본 발명은 상기 생쥐 적혈백혈병 세포주 LK1이 분비하는, B 임파구 증식 유도 사이토카인 (cytokine)을 제공한다.The present invention also provides a B lymphocyte proliferation-inducing cytokine (cytokine) secreted by the mouse erythroleukemia cell line LK1.
특히, B 임파구 증식을 유도하는 사이토카인은 히스타민 분비 인자 (histamine releasing factor, HRF)인 것을 특징으로 하는 사이토카인을 제공한다.In particular, the cytokine that induces B lymphocyte proliferation provides a cytokine, which is a histamine releasing factor (HRF).
더불어 본 발명은 상기 LK1 세포의 배양 상등액을 투석하고 FPLC 시스템을 사용해 모노-큐 칼럼 크로마토그래피로 분리하는 과정을 염화나트륨의 용출 (elution) 농도를 달리하여 3회 반복하는 것을 특징으로 하는 상기 사이토카인 (히스타민 분비 인자)을 분리 정제하는 방법을 제공한다.In addition, the present invention is characterized in that the cytokine (3) by repeating the culture supernatant of the LK1 cells and separating by mono-Q column chromatography using a FPLC system by varying the elution concentration of sodium chloride ( A method for separating and purifying histamine secretion factor) is provided.
또한 본 발명은 상기 사이토카인 (히스타민 분비 인자)의 유전자를 포함하는 재조합 발현 벡터 pMAL-c2-thr-HRF를 제공한다.The present invention also provides a recombinant expression vector pMAL-c2-thr-HRF comprising the gene of the cytokine (histamine secretion factor).
또한 상기 재조합 발현 벡터 pMAL-c2-thr-HRF로 대장균을 형질전환시켜 얻은 형질전환체E. coliJM109/pMAL-c2-thr-HRF를 제공한다.Also provided is a transformant E. coli JM109 / pMAL-c2-thr-HRF obtained by transforming Escherichia coli with the recombinant expression vector pMAL-c2-thr-HRF.
또한 상기 형질전환체를 배양하여 서열번호 1의 아미노산 서열을 갖는 재조합 히스타민 분비 인자 (사이토카인)를 제조하는 방법을 제공한다.It also provides a method of culturing the transformant to produce a recombinant histamine secretion factor (cytokine) having the amino acid sequence of SEQ ID NO: 1.
또한 본 발명은 상기 생쥐 적혈백혈병 세포주 LK1이 분비하는 사이토카인 또는 상기 형질전환체로부터 제조된 재조합 히스타민 분비 인자 (사이토카인)를 유효성분으로 함유하는 B 임파구 증식 유도제를 제공한다.The present invention also provides a B lymphocyte proliferation inducer containing a cytokine secreted by the mouse erythroleukemia cell line LK1 or a recombinant histamine secretion factor (cytokine) prepared from the transformant.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
생쥐의 간세포인 BNL-CL2를 발브씨 (Balb/C) 생쥐에 피하주사하여 종양이 나타나면, 생쥐로부터 비장 세포를 분리하여 배양한다. 배양 후 약 2-3주 정도 경과하면, 조혈세포 성장인자를 첨가하지 않아도 성장률이 급격하게 빨라지고 계속적으로 자라는 크고 둥근 세포군이 나타나며, 이 세포군을 최소 희석 (limiting dilution)에 의해 클로닝하여 단일세포를 얻어 이를 LK1이라 명명하고, 이를 한국과학기술연구원 부설 생명공학연구소 유전자은행에 1999년 4월 22일자로 기탁하였다 (수탁번호 : KCTC 0604BP).BNL-CL 2 , a mouse liver cell, is injected subcutaneously into Balb / C mice, and when tumors appear, splenocytes are separated from the mice and cultured. About 2-3 weeks after the incubation, the growth rate is rapidly increased and continuous rounded large round cell group appears without adding hematopoietic growth factor. Cloning this cell group by limiting dilution yields single cells. This was named LK1, and it was deposited on 22 April 1999 to the Korea Institute of Science and Technology Biotechnology Research Institute Gene Bank (Accession Number: KCTC 0604BP).
상기 LK1 세포주의 형태학적 특징을 알아보기 위해, LK1 세포주를 원심 분리한 후 김사 (Giemsa) 염색하여 광학현미경으로 관찰하고 또한 전자염색하여 전자투과현미경 (TEM; Transmission Electron Microscope)으로 관찰한다. 광학현미경 관찰에 의하면, LK1 세포는 핵이 단일 엽(葉)을 가지고 있는 것으로 보아 이 세포가 성숙된 다형핵세포 (polymorpho nuclear cell, PMNC)가 아니라는 것을 알 수 있고, 핵, 세포질 및 핵인 (nucleoli)의 비율이 크다는 것으로부터 아직 분화 중에 있는 세포라는 것을 알 수 있다. 또한 세포질이 비교적 염기성 (basophilia)인 것으로 나타나, 이 세포가 일종의 폡타이드 물질들을 분비하고 있다는 것을 알 수 있다. 이 세포의 세포질의 중심 부분에는 골지 (Golgi) 구역이라 불리는 밝은 지역이 나타난다. 또한 세포질 중심부분에 잘 발달된 골지체와 많은 활면소포체 (SER; smooth endoplasmic reticulum)가 있고 세포질 주변부위에 조면소포체 (RER)와 미토콘드리아 (mitochondria)가 있다. 핵 주변으로부터 DNA 단편화(fragmentation)가 시작되고 핵 부분이 넓어지는 (widening) 등, 이 세포는 세포예정사 과정 (apoptotic process)의 초기단계에 있는 것으로 보인다. 따라서 이 세포는 분화 과정 중 생쥐 적혈백혈병 세포의 초기단계에 있는 적혈구 전구세포로 생각된다.In order to determine the morphological characteristics of the LK1 cell line, centrifugation of the LK1 cell line, followed by Gimessa staining, followed by optical microscopy, and electrostaining, was performed by transmission electron microscopy (TEM). Microscopic observations indicate that LK1 cells have a single lobe in the nucleus, indicating that they are not mature polymorpho nuclear cells (PMNCs). The large ratio of) shows that the cells are still in differentiation. In addition, the cytoplasm appears to be relatively basic (basophilia), indicating that the cells secrete some sort of pentide. In the central part of the cytoplasm of these cells a bright area called the Golgi zone appears. In addition, there are well-developed Golgi apparatus and many smooth endoplasmic reticulum (SER) in the central part of the cytoplasm, and rough endoplasmic reticulum (RER) and mitochondria. The DNA appears to be in the early stages of the apoptotic process, beginning with DNA fragmentation from the periphery of the nucleus and widening of the nucleus. Thus, these cells are thought to be erythroid progenitors in the early stages of mouse erythroleukemic cells during differentiation.
한편 상기 LK1 세포주의 염색체를 광학현미경으로 관찰한 바에 따르면, LK1 세포의 염색체는 4배체 (tetraploidy, 82.5±4.3)로 나타나고 이미 분열이 이루어진 염색체에서 다배수체 (polyploidy)가 형성되는 비정상적인 특징들이 나타난다.On the other hand, the chromosome of the LK1 cell line was observed under an optical microscope, and the chromosome of the LK1 cell appeared as a tetraploid (tetraploidy, 82.5 ± 4.3), and abnormal features in which polyploidy was formed were already formed on the chromosome that had already been divided.
LK1 세포의 다른 특성을 알아보기 위하여, 여러 가지 세포 표면 분자들에 대한 항체를 이용하여 면역염색한 결과, LK1 세포는 CD3, CD4, CD25, CD45R 및 CD11b에 의해서는 염색되지 않으며, 따라서 상기 세포는 B 임파구나 T 세포 또는 단핵구/대식세포가 아니라는 것을 알 수 있다. 또한 호염구 (basophil)를 특이적으로 염색시키는 알시안 블루 (alcian blue)나 단핵구/대식세포를 특이적으로 염색시키는 비특이적 에스터라제 (nonspecific esterase)에 의해서도 염색되지 않는다. 반면 LK1 세포는 적혈구 계통 세포 표면에 특이적으로 존재하는 항원에 대한 항체인 Ter-119와 미분화된 조혈세포 표면에 공통적으로 발현되는 Sca-1에 의해 면역염색되며, 대조군으로서 사용한 DS19와 MEL-1 세포의 세포형광 분석 결과가 LK1 세포와 유사한 것으로 밝혀져, LK1 세포가 생쥐 적혈백혈병 세포 분화 초기 단계의 적혈구 전구세포라는 사실을 뒷받침해준다.To characterize the different characteristics of LK1 cells, immunostaining with antibodies to various cell surface molecules revealed that LK1 cells were not stained by CD3, CD4, CD25, CD45R and CD11b. It can be seen that they are not B lymphocytes or T cells or monocytes / macrophages. It is also not stained by alkian blue, which specifically stains basophils, or by nonspecific esterases, which specifically stain monocytes / macrophages. In contrast, LK1 cells are immunostained by Ter-119, an antibody to antigens specific to the surface of erythroid cells, and Sca-1, which is commonly expressed on the surface of undifferentiated hematopoietic cells. Cell fluorescence analysis of the cells was found to be similar to LK1 cells, supporting the fact that LK1 cells are erythroid progenitors in the early stages of mouse erythroleukemia cell differentiation.
LK1 세포의 특성을 더 규명하기 위하여, LK1 세포의 유전자를 역전사 중합효소 연쇄반응으로 증폭하여 분석한다.To further characterize LK1 cells, the genes of LK1 cells are analyzed by amplification by reverse transcriptase polymerase chain reaction.
우선 LK1 세포로부터 RNA를 얻어 내고 역전사효소로 외가닥 cDNA를 합성하여, 각각의 사이토카인 특이적인 시발체를 이용하여 역전사 중합효소 연쇄반응 (RT-PCR; RetroTranscription Polymerase Chain Reaction)을 실시하고 다시 중합효소 연쇄반응 (PCR; Polymerase Chain Reaction)을 실시한다. 증폭된 유전자를 전기영동한 결과에 의하면, LK1 세포에서 어떠한 자극 없이도 IL-5, IL-10, IFN-γ, TNF-α 및 TGF-β가 발현되고, 대조군으로 사용한 MEL-1과 DS19에서와 마찬가지로 LK1 세포에서도 MER-5가 발현된다.First, RNA was obtained from LK1 cells, and a single-stranded cDNA was synthesized using reverse transcriptase. Then, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using each cytokine-specific primer, followed by polymerase chain reaction. (PCR; Polymerase Chain Reaction). Electrophoresis of the amplified genes revealed that IL-5, IL-10, IFN-γ, TNF-α, and TGF-β were expressed in the LK1 cells without any stimulation, and were used in MEL-1 and DS19 as controls. Likewise, MER-5 is expressed in LK1 cells.
여러 가지 사이토카인들에 의한 LK1 세포의 증식을 측정하기 위해 LK1 세포에 여러 가지 사이토카인을 가한 후 배양한다. 그 결과 LK1 세포는 IL-1, IL-2, IL-3 및 TNF-α에 의해서 증식이 유도된 반면, IL-4, IL-6 및 IFN-γ에 의해서는 증식에 아무 영향도 받지 않음을 알 수 있다.To measure the proliferation of LK1 cells by various cytokines, various cytokines are added to LK1 cells and then cultured. As a result, LK1 cells were induced to proliferate by IL-1, IL-2, IL-3 and TNF-α, whereas IL-4, IL-6 and IFN-γ were not affected by proliferation. Able to know.
한편, LK1 세포가 적혈구로 분화될 수 있는지 알아보기 위하여, 적혈구 전구세포 (proerythroblast)로부터 적혈구가 분화되는 것을 유도하는 것으로 알려진 DMSO (dimethylsulfoxide)와 HMBA(N, N'- Hexamethylene-bis-acetamide)를 이 세포에 가하여 배양하고, 여기에 최종분화된 적혈구를 특이적으로 염색시키는 것으로 알려진 벤지딘 디히드로클로라이드 (benzidine dihydrochloride)를 첨가한다. 그 결과 LK1 세포는 벤지딘 디히드로클로라이드에 의해 염색되지 않으며, 따라서 LK1 세포는 적혈구로 분화되지 않는다는 것을 알 수 있다. 실제로 생쥐 적혈백혈병 세포들은 염색체 이상 (Vodenicharov, M. D.,et al., DNA and Cell Biol., 15: 287, 1996)이나 유전적 결함 (Quang, C. T.,et al., Oncogene, 11: 1229, 1995) 또는 MafB (Sieweke, M. H.,et al., Cell, 85: 49, 1996)나 NOS (nitric oxide syntase, Rafferty,et al., Blood, 88: 1070, 1996) 등에 의해 적혈구로 분화하는 능력을 잃게 된다는 사실이 알려져 있다.In order to determine whether LK1 cells can be differentiated into erythrocytes, dimethylsulfoxide (DMSO) and HMBA (N, N'-hexamethylene-bis-acetamide), which are known to induce differentiation of erythrocytes from erythrocyte proerythroblasts, The cells are added and cultured, and benzidine dihydrochloride, which is known to specifically stain final differentiated red blood cells, is added thereto. As a result, it can be seen that LK1 cells are not stained by benzidine dihydrochloride, so that LK1 cells do not differentiate into red blood cells. Indeed, mouse erythroleukemic cells may have chromosomal abnormalities (Vodenicharov, MD, et al. , DNA and Cell Biol., 15: 287, 1996) or genetic defects (Quang, CT, et al. , Oncogene, 11: 1229, 1995). Or MafB (Sieweke, MH, et al. , Cell, 85: 49, 1996) or NOS (nitric oxide syntase, Rafferty, et al. , Blood, 88: 1070, 1996) or the like The fact is known.
생쥐 적혈백혈병 세포들은 적혈구 분화과정을 연구하는데 있어서 매우 유용한 것으로 알려져 있지만, 아직까지 이 세포와 면역세포간의 상호작용에 대하여 알려져 있는 것들이 거의 없다. 따라서 LK1 세포가 어떠한 면역세포군을 증가시키고 이 세포군의 성장에 미치는지, 특히 B 임파구의 증식에 미치는 영향을 조사한다.Mouse erythroleukemia cells are known to be very useful for studying erythrocyte differentiation, but little is known about the interaction between these cells and immune cells. Therefore, the effect of LK1 cells on the growth and growth of these cell populations, in particular on the proliferation of B lymphocytes, is investigated.
우선 LK1 세포를 무혈청 배지로 세척하고 무혈청-무단백질 배지에서 배양한 후 원심 분리하여 얻은 배양 상등액을 생쥐의 비장 세포에 처리하고 배양한다. 그 결과, LK1 세포는 CD3 T 임파구의 증식을 약간 감소시킨 반면, CD45R B 임파구의 증식을 현저히 증가시키며, 이러한 B 임파구 특이적인 증식 유도 효과는 기존의 적혈백혈병 세포주의 사이토카인에 비해 약 30배 이상에 달한다. 이로부터 LK1 세포가 B 임파구를 선택적으로 증가시킨다는 것을 알 수 있다.First, LK1 cells are washed with serum-free medium, cultured in serum-free protein medium, and centrifuged, and the culture supernatant is treated with spleen cells of mice and cultured. As a result, LK1 cells slightly reduced the proliferation of CD3 T lymphocytes, while significantly increasing the proliferation of CD45R B lymphocytes. Such B lymphocyte-specific proliferation-inducing effects were approximately 30-fold higher than that of the cytokine of erythroleukemic cell lines. To reach It can be seen from this that LK1 cells selectively increase B lymphocytes.
한편 상기 비장 세포로부터 B 임파구를 분리하고 LK1 세포의 배양 상등액의 농도를 달리하면서 처리했을 때, B 임파구의 증식과 B 임파구로 부터 생성되는 IL-6의 양은 처리해 준 LK1 세포의 농도에 의존하여 증가한다. 이러한 증식 유도 효과가 LK1 세포로부터 배양 상등액 내로 분비되는 단백질에 의한 것인지를 확인하기 위하여, 상기 LK1 세포의 배양 상등액을 끓이거나 트립신 처리한 후 B 임파구에 대한 증식 유도 효과를 조사한 결과, 배양 상등액을 끓이거나 트립신 처리한 경우에는 증식 효과는 나타나지 않는 것으로 보아, LK1 세포로부터 분비된 단백질들에 의해 B 임파구의 증식이 유도된다는 결론을 내릴 수 있다. 또한 IL-4, IL-6 등 기존의 B 임파구 증식을 유도하는 사이토카인들의 항체를 처리하여도 LK1 세포의 증식 유도 효과가 저해되지 않으며, 따라서 LK1 세포의 경우 IL-4, IL-6와 같은 기존에 알려진 사이토카인이 아닌 새로운 사이토카인에 의해 B 임파구의 증식이 유도된다는 것을 알 수 있다.Meanwhile, when B lymphocytes were isolated from the spleen cells and treated with different culture supernatant concentrations of LK1 cells, proliferation of B lymphocytes and the amount of IL-6 produced from B lymphocytes increased depending on the concentration of LK1 cells treated. do. In order to confirm whether the proliferation inducing effect is caused by the protein secreted from the LK1 cells into the culture supernatant, the culture supernatant of the LK1 cells was examined after boiling or trypsin treatment, and the proliferation induction effect on B lymphocytes was examined. It is concluded that proliferation of B lymphocytes is induced by proteins secreted from LK1 cells, since no proliferative effect is seen in the case of or by trypsin treatment. In addition, treatment with cytokines that induce B lymphocyte proliferation, such as IL-4 and IL-6, does not inhibit the proliferation-inducing effect of LK1 cells. It can be seen that the proliferation of B lymphocytes is induced by new cytokines other than the previously known cytokines.
새로운 B 임파구 성장인자를 LK1 세포의 배양 상등액으로부터 분리정제하기 위해, 상기 배양상등액을 농축, 투석 (dialysis) 및 원심 분리한 후, FPLC (fast protein liquid chromatography) 시스템을 사용하여 Mono-Q 컬럼 크로마토그래피를 실시한다. 1차 정제과정으로 용출된 분획들 중 생쥐 비장의 B 임파구의 증식을 유도하는 분획들만을 다시 투석하고 용출농도를 바꾸어가며 다시 FPLC를 시행하여 사이토카인을 2차 정제한다. 최종적으로 다시 원하는 분획만을 모아 바로 이전의 정제과정에서와 같은 용출농도로 FPLC로 정제한다. 최종 용출액으로 SDS-PAGE를 실시한 결과, 23kDa의 단백질이 분리, 정제되며, B 임파구 증식에 대해서 높은 활성을 나타내는 것을 확인한다.To isolate and purify the new B lymphocyte growth factor from the culture supernatant of LK1 cells, the culture supernatant was concentrated, dialysis and centrifuged, followed by Mono-Q column chromatography using a fast protein liquid chromatography (FPLC) system. Is carried out. Among the fractions eluted by the primary purification process, only the fractions that induce the proliferation of B lymphocytes in the mouse spleen are redialysis, the elution concentration is changed, and FPLC is performed again to purify cytokines. Finally, only the desired fractions are collected and purified by FPLC at the same elution concentration as in the previous purification. SDS-PAGE was carried out with the final eluate to confirm that 23 kDa protein was isolated and purified, showing high activity against B lymphocyte proliferation.
최종 정제과정을 거친 분자량 23 kDa인 단백질을 이용하여 아미노 말단의 아미노산 서열을 분석한 결과 아미노 말단은 서열번호 2의 아미노산 서열을 갖는 것으로 밝혀졌는데, 이 아미노산 서열을 기존에 알려진 아미노산의 서열과 비교한 결과 상기 단백질은 히스타민 분비 인자인 것으로 판명되었다. 이로써 이미 알려져있는 히스타민의 분비를 유도하는 고유의 기능 외에도, B 임파구의 증식을 유도하는 새로운 기능이 있음을 밝혀내었다.The amino acid sequence of the amino terminus was analyzed using a protein having a final molecular weight of 23 kDa, and the amino terminus was found to have an amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence was compared with a known amino acid sequence. Results The protein was found to be a histamine secretion factor. In addition to the inherent function of inducing the secretion of histamine, which is already known, it has revealed a new function of inducing proliferation of B lymphocytes.
상기 단백질을 대량으로 얻기 위하여, 발현 벡터로 pMAL-c2를 사용하여 재조합 발현 벡터를 제조한다. pMAL-c2 (NEB; 800-64S)는 세포질에서 말토스 결합 단백질 (MBP; maltose binding protein)과 원하는 단백질을 융합하여 발현함으로써 융합 단백질을 쉽게 분리할 수 있는 장점을 가지고 있다.In order to obtain the protein in large quantities, a recombinant expression vector is prepared using pMAL-c2 as the expression vector. pMAL-c2 (NEB; 800-64S) has the advantage of easily separating fusion proteins by fusion and expression of maltose binding protein (MBP) and a desired protein in the cytoplasm.
먼저 인간 cDNA 라이브러리로부터 서열번호 3의 시발체 A와 서열번호 4의 시발체 B를 이용하여 중합효소 연쇄반응을 통해 히스타민 분비 인자를 암호화하는 서열번호 38의 유전자를 증폭하고, 발현 벡터 pMAL-c2에 삽입시킨다. 이때 융합 단백질로부터 말토스 결합 단백질을 분리하기 위하여 서열번호 5의 트롬빈 분리 부위 (thrombin cleavage site)를 삽입한다. 제조된 재조합 발현 벡터 pMAL-c2-thr-HRF로 E.coli(JM109)를 형질전환시켜 형질전환체 E. coli JM109/pMAL-c2-thr-HRF를 한국과학기술연구원 부설 생명공학연구소 유전자은행에 1999년 5월 25일자로 기탁하였다 (수탁번호 : KCTC 0617BP).First, the gene of SEQ ID NO: 38 encoding histamine secretion factor is amplified by polymerase chain reaction using primer A of SEQ ID NO: 3 and primer B of SEQ ID NO: 4 from the human cDNA library, and inserted into the expression vector pMAL-c2. . At this time, the thrombin cleavage site of SEQ ID NO: 5 is inserted to separate the maltose binding protein from the fusion protein. E. coli (JM109) was transformed with the prepared recombinant expression vector pMAL-c2-thr-HRF, and the transformant E. coli JM109 / pMAL-c2-thr-HRF was transferred to the Korea Institute of Science and Technology. Deposited May 25, 1999 (Accession No .: KCTC 0617BP).
상기 형질전환체에 IPTG (Isopropylthio-β-D-galactoside)를 첨가하여 배양한 대장균 형질전환체는 원심 분리하여 침전시킨 뒤 초음파 분쇄하고, 여기서 얻은 상등액을 아밀로스 칼럼으로 정제한다. 정제된 융합 단백질은 투석하여 분리하고, 트롬빈으로 처리하여 융합 단백질의 말토오즈 결합 단백질과 히스타민 분비 인자를 분리한다. 이 용액을 다시 투석한 후, DEAE-세파로스 칼럼으로 히스타민 분비 인자를 분리 및 정제한다. 이후 분리된 히스타민 분비 인자에 OBDG (Octyl-β-D-glucopyranoside)를 첨가하고 폴리마이신 B 칼럼에 통과시켜 내독소 (endotoxin)를 제거한다.Escherichia coli transformants cultured by adding IPTG (Isopropylthio-β-D-galactoside) to the transformants are centrifuged and precipitated, followed by ultrasonic grinding, and the supernatant obtained therefrom is purified by an amylose column. Purified fusion proteins are isolated by dialysis and treated with thrombin to separate the maltose binding proteins and histamine secretion factors of the fusion proteins. After dialysis of this solution, the histamine secretion factor is separated and purified by DEAE-Sepharose column. Then, OBDG (Octyl-β-D-glucopyranoside) is added to the separated histamine secretion factor and passed through a polymycin B column to remove endotoxin.
위와 같이 분리된 재조합 히스타민 분비 인자가 B 임파구의 증식에 미치는 효과를 알아보기 위하여, 생쥐 B 임파구를 분리하고 재조합 히스타민 분비 인자를 농도를 달리하여 가하고 배양한다. 그 결과 재조합 히스타민 분비 인자의 농도에 의존적으로 B 임파구의 증식이 유도된다. 한편 대조군인 말토스 결합 단백질을 처리했을 때에도 고농도에서는 B 임파구 증식이 다소 유도되었지만, 재조합 히스타민 분비 인자에 의한 증식 효과에 비하면 훨씬 낮았다. 말토스 결합 단백질만을 처리한 경우에도 B 임파구가 약간 증가한 것은 형질전환체로부터 재조합 히스타민 분비 인자를 분리 및 정제하는 과정 중에 내독소가 완전히 제거되지 않고 남아 B 임파구의 증식을 자극하는 항원으로 작용하기 때문으로 여겨진다. 재조합 히스타민 분비 인자와 말토스 결합 단백질을 끓이거나 히스타민 분비 인자에 대한 항체로 처리한 경우, B 임파구의 증식 효과는 나타나지 않거나 중화되어, 결국 B 임파구의 증식을 유도하는 것은 히스타민 분비 인자임을 알 수 있다.In order to determine the effect of the recombinant histamine secretion factor isolated on the proliferation of B lymphocytes, the mouse B lymphocytes are isolated and the recombinant histamine secretion factor is added at different concentrations and cultured. The result is proliferation of B lymphocytes depending on the concentration of recombinant histamine secretion factor. On the other hand, B lymphocyte proliferation was slightly induced at high concentrations even when the maltose binding protein was treated as a control, but was much lower than the proliferative effect by recombinant histamine secretion factor. Even if only maltose binding protein was treated, B lymphocytes increased slightly because endotoxin was not completely removed during the process of separating and purifying recombinant histamine secretion factor from the transformant, which acts as an antigen that stimulates the proliferation of B lymphocytes. Is considered. When recombinant histamine and maltose binding proteins are boiled or treated with an antibody against histamine secretion factor, the proliferative effect of B lymphocytes is not shown or neutralized, and thus, it can be seen that it is histamine secretory factor that induces the proliferation of B lymphocytes. .
이하 본 발명을 실시예에 의하여 더욱 상세하게 설명한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
단, 하기 실시예들은 본 발명을 예시하는 것으로 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.
<실시예 1> LK1 세포주의 확립Example 1 Establishment of LK1 Cell Line
1) LK1 세포1) LK1 cells
생쥐의 간 (liver) 세포인 BNL-CL2를 발브씨 (Balb/C) 생쥐에 피하주사하면 약 4주 후 주사 부위에 커다란 종양이 나타나고, 비장은 정상에 비하여 2-3배 정도 크기가 부풀었다. 종양이 발달한 생쥐로부터 비장 세포를 분리하여 10% 우태아혈청이 포함된 RPMI 1640 배지에서 배양하였고, 1주일에 2회씩 신선한 배지로 갈아주며 계대배양하였다. 배양 후 약 2-3주 정도 경과하면, 다른 조혈세포 성장인자를 첨가하지 않아도 성장률이 급격하게 빨라지고 계속적으로 자라는 크고 둥근 세포군이 나타났다. 이 세포군을 배양하여 최소 희석 (limiting dilution)에 의해 동일한 세포특성을 지니고 있는 단일세포클론을 얻고 LK1 세포로 명명하였다.Subcutaneous injection of BNL-CL 2 , a liver cell of mice, to Balb / C mice shows a large tumor at the injection site after about 4 weeks, and the spleen is 2-3 times larger than normal. It was. Spleen cells were isolated from tumor-developed mice and cultured in RPMI 1640 medium containing 10% fetal calf serum, and subcultured twice a week with fresh medium. About 2-3 weeks after the incubation, the growth rate was rapidly increased and continued to grow, even without adding other hematopoietic growth factor. This cell group was cultured to obtain single cell clones having the same cellular characteristics by limiting dilution and named LK1 cells.
2) LK1 세포 분석2) LK1 Cell Analysis
LK1 세포의 형태학적 특징을 알아보기 위해, LK1 세포를 광학현미경 및 전자투과현미경 (TEM; Transmission Electron Microscope)으로 관찰하였다 (도 1참조).In order to determine the morphological characteristics of LK1 cells, LK1 cells were observed under a light microscope and a transmission electron microscope (TEM) (see FIG. 1 ).
(1) 김사염색 (광학현미경 관찰)(1) Kimsa Dyeing (Optical Microscopy)
LK1세포를 인산완충용액 (phosphate buffered saline, PBS)으로 1회 세척하고 1,000 rpm 에서 1분간 사이토스핀 (cytospin) 원심 분리기로 원심 분리하였다.이를 즉시 건조한 후 메틸알콜 원액으로 2-5분 동안 고정시켜 재차 공기 중에서 건조시킨 다음, 4% 김사용액 (Gurr R66 Giemsa, 독일)을 만들어 약 12분 동안 실온에서 염색한 후 증류수로 세척하고 건조하여 광학현미경으로 관찰하였다 (도 1B참조).LK1 cells were washed once with phosphate buffered saline (PBS) and centrifuged with a cytospin centrifuge at 1,000 rpm for 1 min. The cells were immediately dried and fixed in methyl alcohol stock for 2-5 minutes. After drying again in air, a 4% seaweed solution (Gurr R66 Giemsa, Germany) was made, dyed at room temperature for about 12 minutes, washed with distilled water, dried and observed with an optical microscope (see FIG. 1B ).
(2) 전자염색 (전자투과현미경 관찰)(2) Electrostaining (electron transmission microscope observation)
배양된 세포를 2% PFCHO (Paraformaldehyde)와 2.5% GCHO (Glutaldehyde)로 제조된 1차 고정액에서 4℃로 4시간 동안 고정시켰다. 이후 0.1M 인산염 완충용액으로 1시간씩 3회 세척하고, 0.1M 인산염 완충용액으로 조성된 1% 오스뮴산 (osmic acid, OsO4)에서 상온으로 1시간 동안 2차 고정시킨 후, 에폰 812 (Epon 812)를 이용하여 초박절편 (ultra-thin section)을 제작하기 위하여 조직내의 모든 틈새를 포매제로 채워 넣는 포매과정 (embedding)을 거친 후, 액체상태의 포매제를 절편제작이 용이한 고체상태로 전환시키는 중합 (polymerization) 과정을 수행하였다. 상기와 같이 처리된 LK1 세포를 100nm 이하의 은색 (silver color)의 절편을 만들고 포화 초산우라늄 (uranyl acetate)과 구연산납 (lead citrate)으로 2중으로 전자염색하여 투과전자현미경으로 관찰하였다 (도 1C참조).The cultured cells were fixed at 4 ° C. for 4 hours in a primary fixer prepared with 2% PFCHO (Paraformaldehyde) and 2.5% GCHO (Glutaldehyde). After washing three times with 0.1M phosphate buffer for 1 hour, and fixed in 1% osmic acid (osmic acid, OsO 4 ) composed of 0.1M phosphate buffer solution at room temperature for 1 hour, and then Epon 812 (Epon 812) by embedding all the gaps in the tissue with embedding to produce ultra-thin sections, and then converting the liquid sieve into a solid that is easy to fabricate. A polymerization process was performed. LK1 cells treated as described above were made into sections of silver color of 100 nm or less, and double-stained with saturated uranium acetate and lead citrate and observed by transmission electron microscope (see FIG. 1C ). ).
광학현미경에 의한 관찰에 의하면, LK1 세포는 핵이 단일 엽(葉)을 가지고 있어 이 세포가 성숙된 다형핵세포 (polymorpho nuclear cell, PMNC)가 아니라는것을 알 수 있고, 핵, 세포질 및 핵인 (nucleoli)의 비율이 크다는 것으로부터 아직 분화 중에 있는 세포라는 것을 알 수 있다. 또한 세포질이 비교적 염기성 (basophilia)인 것으로 나타났는데, 이는 이 세포가 일종의 폡타이드 물질들을 분비하고 있다는 것을 의미한다. 세포질의 중심 부분에서는 골지 (Golgi) 구역이라 불리는 밝은 지역이 발견되었는데 이는 투과전자현미경에 의한 분석결과와 잘 일치한다. 세포질 중심부분에서는 잘 발달된 골지체와 많은 활면소포체 (SER; smooth endoplasmic reticulum)가 관찰되고 세포질 주변 부위에 조면소포체 (RER)와 미토콘드리아 (mitochondria)가 관찰되었다. 또한 핵주변으로부터 DNA 단편화 (fragmentation)가 시작되고 핵부분이 넓어지는 (widening) 등, 핵은 세포예정사 과정 (apoptotic process)의 초기단계에 있는 것처럼 보여진다. 따라서 이 세포는 분화과정 중 생쥐 적혈백혈병 세포의 초기단계에 있는 적혈구 전구세포로 생각된다.Observation by light microscopy shows that LK1 cells have a single lobe in the nucleus, which is not a mature polymorpho nuclear cell (PMNC). The large ratio of) shows that the cells are still in differentiation. It has also been shown that the cytoplasm is relatively basic (basophilia), which means that the cells secrete some sort of cactide. In the central part of the cytoplasm, a bright area called the Golgi area was found, which is in good agreement with the results of transmission electron microscopy. In the central cytoplasm, well-developed Golgi bodies and many smooth endoplasmic reticulum (SER) were observed, and rough endoplasmic reticulum (RER) and mitochondria were observed in the periplasm. The nucleus also appears to be in the early stages of the apoptotic process, with DNA fragmentation starting around the nucleus and widening of the nucleus. Thus, these cells are thought to be erythrocyte progenitors in the early stages of mouse erythroleukemic cells during differentiation.
(3) 염색체 분석(3) chromosome analysis
LK1 세포를 1㎍/㎖ 의 콜세미드 (colcemid)로 3-6시간 처리하여 세포분열단계를 중기 (metaphase)에 정지시킨 후 HBSS (Hank's balanced salt solution, Sigma사)로 3회 세척하였다. 여기에 고정액으로 75mM 염화칼륨 (KCl) 5㎖를 처리하고 37℃에서 10분간 방치한 후, 600 rpm에서 5분간 원심 분리하여 상등액을 제거했다. 여기에 카르노이 (Carnoy) 용액 (메탄올:빙초산=3:1) 8㎖을 천천히 가하고 4℃에서 30분간 두어 고정시켰다. 고정이 완료되면 4% 김사용액으로 염색하고, 염색체를 광학현미경으로 관찰하였다 (도 1D참조).LK1 cells were treated with 1 μg / ml of colsemid for 3-6 hours to stop the cell division step in metaphase and washed three times with HBSS (Hank's balanced salt solution, Sigma). 5 ml of 75 mM potassium chloride (KCl) was treated as a fixed solution, and the mixture was left at 37 ° C for 10 minutes, and then centrifuged at 600 rpm for 5 minutes to remove the supernatant. To this was added 8 ml of a Carnoy solution (methanol: glacial acetic acid = 3: 1) slowly and placed at 4 ° C for 30 minutes to fix. When the fixation was completed, the cells were stained with 4% seaweed solution, and the chromosomes were observed under an optical microscope (see FIG. 1D ).
LK1 세포의 염색체는 4배체 (tetraploidy, 82.5±4.3)로 나타났고 이미 분열이 이루어진 염색체에서 다배수체 (polyploidy)가 형성되는 비정상적인 특징들이 나타났다.The chromosome of LK1 cells was tetraploid (82.5 ± 4.3), and abnormal features of polyploidy were formed in the already cleaved chromosome.
(4) 면역염색 (세포형광 분석)(4) immunostaining (cell fluorescence analysis)
LK1 세포의 특성을 알아보기 위하여 하기에서와 같이 여러 가지 세포 표면 분자들에 대한 항체를 이용하여 면역염색하였다.To characterize the LK1 cells, immunostaining was performed using antibodies to various cell surface molecules as follows.
우선 LK1 세포를 1×106세포수로 조정하여 염색 완충용액 (20mM HEPES, 3% 우태아혈청, 0.1% NaN3가 포함된 인산염 완충용액, pH 7.4)으로 1회 세척하였다. 여기에 FITC (fluoresceinated isothiocyanate) 또는 PE (phycoerythrin) 형광물질이 결합된 CD3, CD4, CD8, CD11b, CD25, CD45R, Sca-1, Ter-119 등의 항체를 가하여 0℃에서 30분간 반응시키고, 상기 염색 완충용액으로 2회 세척한 후 세포형광 분석기 (flow cytometer)로 분석하였다 (도 2참조). 또한 알시안 블루 (alcian blue)와 비특이적 에스터라제 (nonspecific esterase)를 사용하여 상기와 같은 방법으로 반응시켰다. 대조군으로는 이미 알려져 있는 생쥐 적혈백혈병 세포인 DS19와 MEL-1 세포를 선택하여 상기와 같은 방법으로 염색하여 세포형광 분석을 실시하였다.First, LK1 cells were adjusted to 1 × 10 6 cells and washed once with staining buffer (20 mM HEPES, 3% fetal bovine serum, phosphate buffer containing 0.1% NaN 3 , pH 7.4). To this was added an antibody such as CD3, CD4, CD8, CD11b, CD25, CD45R, Sca-1, Ter-119, or a combination of FITC (fluoresceinated isothiocyanate) or PE (phycoerythrin) phosphor, and reacted at 0 ° C. for 30 minutes. After washing twice with staining buffer solution it was analyzed by a flow cytometer (see Fig. 2 ). Also, alkian blue and nonspecific esterase were used to react in the same manner as described above. As a control group, DS19 and MEL-1 cells, which are known as mouse erythroleukemia cells, were selected and stained by the same method as described above.
LK1 세포는 CD3, CD4, CD8, CD25, CD45R 및 CD11b에 의해서는 염색되지 않았으며, 또한 호염구 (basophil)를 특이적으로 염색시키는 알시안 블루나 단핵구/대식세포를 특이적으로 염색시키는 비특이적 에스터라제에 의해서도 염색되지 않았다. 따라서 LK1 세포는 B 임파구나 T 세포 또는 단핵구/대식세포가 아니라는 것을 알 수 있었다. 반면 LK1 세포는 적혈구 계통 세포 표면에 특이적으로 존재하는 항원에 대한 항체인 Ter-119와 미분화된 조혈세포 표면에 공통적으로 발현되는 Sca-1에 의해 염색되었으며, 이것은 상기 광학현미경 및 투과전자현미경의 분석에 의한 LK1 세포가 분화과정 중 초기 단계에 있는 적혈구 전구세포라는 결과와 일치한다. 또한 대조군으로서 사용한 DS19와 MEL-1 세포의 세포형광 분석 결과가 LK1 세포와 유사한 것으로 밝혀져, LK1 세포가 생쥐 적혈백혈병 세포 분화 초기 단계의 적혈구 전구세포라는 사실을 뒷받침해준다.LK1 cells were not stained by CD3, CD4, CD8, CD25, CD45R, and CD11b, and also nonspecific esterars that specifically stain alcy blue or monocytes / macrophages that specifically stain basophil. It was not stained even by the agent. Thus, it was found that LK1 cells are not B lymphocytes or T cells or monocytes / macrophages. On the other hand, LK1 cells were stained by Ter-119, an antibody to antigens specifically present on the surface of erythroid cells, and Sca-1, which is commonly expressed on the surface of undifferentiated hematopoietic cells. The analysis indicated that LK1 cells are erythroid progenitors in the early stages of differentiation. In addition, cell fluorescence analysis of DS19 and MEL-1 cells used as a control group was found to be similar to LK1 cells, supporting the fact that LK1 cells are erythroid progenitor cells in the early stages of mouse erythroleukemia cell differentiation.
<실시예 2> LK1 유전자의 발현Example 2 Expression of LK1 Gene
LK1 세포의 특성을 더 규명하기 위하여, 역전사 중합효소 연쇄반응으로 LK1 세포의 유전자를 증폭하여 분석하였다.To further characterize LK1 cells, the genes of LK1 cells were amplified and analyzed by reverse transcriptase polymerase chain reaction.
LK1 세포로부터 RNA를 얻기 위해 2 × 106의 LK1 세포를 인산염 완충용액으로 1회 세척한 후 RNA 분리용액 (RNAzol B, TEL-TEST) 500㎕를 각 시료에 넣고 부드럽게 피펫팅하여 세포를 깨고 50㎕의 클로로포름을 넣고 잘 섞은 후 얼음에서 5분간 방치하였다. 이를 12,000 rpm, 4℃에서 15분 동안 원심 분리한 후 상등액을취하고 동량의 이소프로필 알콜을 넣어 얼음에서 20분 동안 방치하였다. 다시 12,000 rpm, 4℃에서 20분 동안 원심 분리한 후 침전물을 80% 에탄올로 세척하고 건조시킨 후에 0.1% 디에틸 피로카보네이트 (diethyl pyrocarbonate)가 포함된 물을 20㎕ 넣고 잘 녹였다. 이와 같이 얻은 RNA로부터 MMLV (murine moloney leukemia virus) 역전사효소 (Promega)로 외가닥 cDNA를 합성하였고 각각의 사이토카인 특이적인 시발체 (표 1 참조)를 이용하여 하기 표 2의 반응 혼합물로 역전사 중합효소 연쇄반응 (RT-PCR; Reverse Transcription - Polymerase Chain Reaction)을 실시하였다.In order to obtain RNA from LK1 cells, 2 × 10 6 LK1 cells were washed once with phosphate buffer solution, and 500 µl of RNA isolation solution (RNAzol B, TEL-TEST) was added to each sample and gently pipetted to break the cells. After the addition of chloroform and mixed well and left for 5 minutes on ice. The mixture was centrifuged at 12,000 rpm and 4 ° C. for 15 minutes, and then the supernatant was removed and the same amount of isopropyl alcohol was left to stand on ice for 20 minutes. After centrifugation at 12,000 rpm and 4 ° C. for 20 minutes, the precipitate was washed with 80% ethanol and dried, and 20 μl of water containing 0.1% diethyl pyrocarbonate was dissolved. Single stranded cDNA was synthesized from the RNA thus obtained by murine moloney leukemia virus (MMLV) reverse transcriptase (Promega), and reverse transcriptase polymerase chain reaction was carried out with the reaction mixture of Table 2 below using respective cytokine-specific primers (see Table 1). (RT-PCR; Reverse Transcription-Polymerase Chain Reaction) was performed.
상기 혼합물을 42℃에서 30분간 반응시키고 90℃에서 5분간 반응시킨 후 반응을 정지시켰다. 이 반응물은 다시 하기 표 3의 혼합물로 중합효소 연쇄반응 (PCR; Polymerase Chain Reaction)을 실시하였다.The mixture was reacted at 42 ° C. for 30 minutes and at 90 ° C. for 5 minutes to stop the reaction. This reactant was further subjected to polymerase chain reaction (PCR) with a mixture of Table 3 below.
상기 혼합물을 95℃에서 5분간 방치하여 다른 효소활성을 저해시킨 후, 95℃에서 1분 30초 (denaturation), 55℃에서 1분 (hybridization), 72℃에서 2분 (synthesis) 동안 반응시키는 과정을 30회 반복하고 마지막으로 95℃에서 1분 30초, 55℃에서 1분, 72℃에서 5분간 반응을 실시하였다. 이후 최종 반응물 중 10 ㎕를 취하여 1% 아가로스 겔에서 30분간 100V에서 전기영동을 실시하였다 (도 3참조). 한편, 대조군으로서 MEL-1과 DS19를 사용하여 상기 방법에 의해 유전자를 증폭하여 전기영동을 실시하였다.The mixture was left at 95 ° C. for 5 minutes to inhibit other enzymatic activity, followed by reaction for 1 minute 30 seconds at 95 ° C., hybridization at 55 ° C., and synthesis at 72 ° C. for 2 minutes (synthesis). The reaction was repeated 30 times, and finally, the reaction was conducted at 95 ° C. for 1 minute 30 seconds, at 55 ° C. for 1 minute, and at 72 ° C. for 5 minutes. 10 μl of the final reaction was then taken and subjected to electrophoresis at 100 V for 30 minutes on a 1% agarose gel (see FIG. 3 ). On the other hand, using the MEL-1 and DS19 as a control group by amplifying the gene by the above method was performed electrophoresis.
그 결과 LK1 세포는 다른 자극이 없어도 IL-5, IL-10, IFN-γ, TNF-α 및 TGF-β 를 발현하였다 (도 3A참조). 또한 생쥐 적혈백혈병 세포에서 과발현되고 이 세포의 분화에 중요한 역할을 하는 것으로 알려진 MER-5 유전자도, 대조군으로 사용한 MEL-1과 DS19에서와 마찬가지로 LK1 세포에서 발현된 것을 확인할 수 있었다 (도 3B참조).As a result, LK1 cells expressed IL-5, IL-10, IFN-γ, TNF-α and TGF-β without any other stimulus (see FIG. 3A ). In addition, the MER-5 gene, which is overexpressed in mouse erythroleukemia cells and known to play an important role in the differentiation of these cells, was also expressed in LK1 cells as in MEL-1 and DS19 used as controls (see FIG. 3B ). .
<실시예 3> LK1 세포의 증식 및 분화Example 3 Proliferation and Differentiation of LK1 Cells
1) LK1 세포의 증식1) Proliferation of LK1 Cells
여러 가지 사이토카인들이 LK1 세포의 증식에 미치는 영향을 측정하기 위해, 96개의 웰에 각 웰 당 2×104개의 LK1 세포를 넣고 여러 가지 사이토카인을 가한 후 3일 동안 배양하였다. 사이토카인으로는 IL-1, IL-2, IL-3, IL-4, IL-6, TNF-α, IL-1+TNF-α 그리고 IFN-γ를 사용하였다. 3일의 배양기간 중 68시간 되는 때에 앨리 등의 방법 (Alley, M.C. et al., Cancer Res. 48, 589, 1988)에 따라 3,4,5-디메틸 티아졸-2,5-디페닐 테트라졸리움 브로마이드 (MTT, Sigma사)를 0.5㎎/웰 씩 가하고 4시간 동안 배양하였다. 그 후 배양액을 원심 분리하여 상등액은 버리고, 산성화시킨 이소프로필 알콜 (0.04N HCl의 이소프로판올 용액)을 100㎕씩 각 웰에 가하여 생존한 세포로부터 형성된 포르마잔 (formazan)을 용출시켰다. 이후 엘라이자 리더 (Titertek multiskan Mcc/340)로 540 nm에서의 광학 밀도 (O.D.)를 측정하였다 (도 4참조). 그 결과 LK1 세포는 IL-1, IL-2, IL-3, TNF-α 및 IL-1+TNF-α에 의해서 증식이 유도된 반면, IL-4, IL-6 및 IFN-γ에 의해서는 증식에 별다른 영향을 받지 않았다.To measure the effects of various cytokines on the proliferation of LK1 cells, 2 × 10 4 LK1 cells were added to each well in 96 wells and cultured for 3 days after adding various cytokines. As cytokines, IL-1, IL-2, IL-3, IL-4, IL-6, TNF-α, IL-1 + TNF-α and IFN-γ were used. 3,4,5-dimethyl thiazole-2,5-diphenyl tetra according to the method of Ally et al. (Alley, MC et al., Cancer Res. 48, 589, 1988) at 68 hours of the three-day incubation period. Zolium bromide (MTT, Sigma) was added at 0.5 mg / well and incubated for 4 hours. The culture solution was then centrifuged to discard the supernatant, and 100 μl of acidified isopropyl alcohol (isopropanol solution of 0.04 N HCl) was added to each well to elute formazan formed from the surviving cells. The optical density (OD) at 540 nm was then measured with an ELISA reader (Titertek multiskan Mcc / 340) (see FIG. 4 ). As a result, LK1 cells were proliferated by IL-1, IL-2, IL-3, TNF-α and IL-1 + TNF-α, whereas IL-4, IL-6 and IFN-γ There was no significant effect on proliferation.
2) LK1 세포의 분화2) Differentiation of LK1 Cells
한편 LK1 세포가 적혈구로 분화될 수 있는지 알아보기 위하여, 적혈구 전구세포 (proerythroblast)로부터 적혈구가 분화되는 것을 유도하는 2% DMSO (dimethylsulfoxide)와 200mM HMBA (N, N'-Hexamethylene- bis-acetamide)를 LK1에 가하여 배양하였다. 0.2% 벤지딘 디히드로클로라이드는 최종분화된 적혈구를 특이적으로 염색시키기 때문에, 상기 배양물에 벤지딘 디히드로클로라이드를 첨가하였으나 염색되지 않았다. 따라서 LK1 세포는 적혈구로 분화되지 않는 것을 알 수 있는데, 이러한 특징은 일반적으로 염색체 이상, 유전적 결합, MafB 또는 NOS에 의해 적혈구로 분화되는 능력을 읽게 된 적혈백혈병 세포주의 공통적인 특징으로서 LK1이 새롭게 확립된 적혈백혈병 세포주임을 나타낸다.In order to determine whether LK1 cells can be differentiated into erythrocytes, 2% DMSO (dimethylsulfoxide) and 200 mM HMBA (N, N'-Hexamethylene- bis-acetamide), which induce the differentiation of erythrocytes from erythrocyte proerythroblasts, It was added to LK1 and incubated. Since 0.2% benzidine dihydrochloride specifically stains the final differentiated erythrocytes, benzidine dihydrochloride was added to the culture but not stained. Thus, it can be seen that LK1 cells do not differentiate into erythrocytes, which is a common feature of erythroleukemia cell lines that generally read the ability to differentiate into erythrocytes by chromosomal aberrations, genetic binding, and MafB or NOS. Indicates an established erythroleukemia cell line.
<실시예 4> LK1 세포가 B 임파구 증식에 미치는 영향Example 4 Effect of LK1 Cells on B Lymphocyte Proliferation
LK1 세포를 무혈청 배지로 3회 세척하고 5×106세포/㎖의 세포수로 무혈청-무단백질 배지 (Sigma사)에 현탁하여 24시간 동안 배양한 후 2,000 rpm에서 10분간 원심 분리하여 배양 상등액을 회수하였다. 그후 LK1 세포의 배양 상등액을 생쥐의비장 세포에 처리하고 4일 동안 배양하여 LK1 세포 배양액이 면역세포군의 증식에 미치는 영향을 조사하였다 (도 5참조). 그 결과 LK1 세포는 CD3 T 임파구의 증식을 약간 감소시킨 반면, CD45R B 임파구들을 현저히 증가시키고 있다. 따라서 LK1 세포가 B 임파구를 선택적으로 증가시킨다는 것을 알 수 있었다.LK1 cells were washed three times with serum-free medium, suspended in serum-free protein medium (Sigma) at 5 × 10 6 cells / ml, and cultured for 24 hours, followed by incubation for 10 minutes at 2,000 rpm. The supernatant was recovered. Thereafter, the culture supernatant of LK1 cells was treated to spleen cells of mice and cultured for 4 days to examine the effect of LK1 cell culture on the proliferation of immune cell populations (see FIG. 5 ). As a result, LK1 cells slightly reduced proliferation of CD3 T lymphocytes, while significantly increasing CD45R B lymphocytes. Thus, it was found that LK1 cells selectively increase B lymphocytes.
한편 상기 비장 세포로부터 B 임파구를 분리하고 LK1 세포의 배양 상등액의 농도를 달리하면서 처리했을 때, B 임파구의 증식 (-■-)과 B 임파구로부터 생성되는 인터루킨-6의 양 (-□-)은 처리해 준 LK1 세포의 농도에 의존하여 증가하는 것으로 나타났다 (도 6참조). 구체적으로 생쥐 적혈백혈병 세포주를 첨가하지 않은 배지나 MEL-1나 DS19 등의 생쥐 적혈백혈병 세포주의 배양 상등액과 비교할 때, LK1 세포의 배양 상등액은 B 임파구 증식 유도율을 약 30배 정도 증가시켰다.Meanwhile, when B lymphocytes were isolated from the spleen cells and treated at different concentrations of the culture supernatant of LK1 cells, the proliferation of B lymphocytes (-■-) and the amount of interleukin-6 produced from B lymphocytes (-□-) It appeared to increase depending on the concentration of LK1 cells treated (see FIG. 6 ). Specifically, the culture supernatant of LK1 cells increased B lymphocyte proliferation induction rate by about 30 times as compared with the culture supernatant of the mouse erythroleukemia cell line or MEL-1 or DS19.
이러한 증식 유도 효과가 LK1 세포로부터 배양 상등액 내로 분비되는 단백질에 의한 것인지를 확인하기 위하여, 상기 LK1 세포의 배양 상등액 일부를 10 분간 끓이고, 또 다른 일부는 트립신 5% 농도로 37℃에서 24시간 동안 처리한 후 B 임파구에 대한 증식 유도 효과를 조사하였다. 그 결과 배양 상등액을 끓이거나 트립신 처리한 경우에 증식 유도 효과는 나타나지 않았으며, 결국 LK1 세포로부터 분비된 단백질들에 의해 B 임파구의 증식이 유도된다는 결론을 내릴 수 있었다. 또한 IL-4, IL-6 등 기존의 B 임파구 증식을 유도하는 사이토카인들의 항체를 처리하여도 LK1 세포의 증식 유도 효과가 저해되지 않았다. 따라서 LK1 세포의 경우 IL-4, IL-6와 같은 기존에 알려진 사이토카인이 아닌 새로운 사이토카인에 의해 B 임파구의 증식이 유도된다는 것을 알 수 있었다.In order to confirm that this proliferation induction effect is caused by the protein secreted from the LK1 cells into the culture supernatant, a portion of the culture supernatant of the LK1 cells is boiled for 10 minutes, and another portion is treated with trypsin 5% concentration at 37 ° C. for 24 hours. We investigated the effect of proliferation on B lymphocytes. As a result, when the culture supernatant was boiled or trypsin-treated, no proliferation inducing effect was observed, and it was concluded that proliferation of B lymphocytes was induced by proteins secreted from LK1 cells. In addition, the treatment of cytokine-induced B lymphocyte proliferation, such as IL-4 and IL-6, did not inhibit the proliferation-inducing effect of LK1 cells. Therefore, in the case of LK1 cells, it was found that the proliferation of B lymphocytes is induced by new cytokines other than previously known cytokines such as IL-4 and IL-6.
<실시예 5> 사이토카인의 정제 및 분석Example 5 Purification and Analysis of Cytokines
새로운 B 임파구 성장인자를 LK1 세포의 배양 상등액으로부터 하기와 같은 방법에 의해 분리, 정제하였다.The new B lymphocyte growth factor was isolated and purified from the culture supernatant of LK1 cells by the following method.
1) 사이토카인의 정제1) Purification of Cytokines
상기 LK1 세포의 배양 상등액 1.5 ℓ를 아미콘 디아플로 막 (Amicon Diaflo membrane, YM-10)을 이용하여 약 10㎖로 농축하고, 50mM 인산염 완충용액 (Tris-HCl, pH 7.8) 2 ℓ로 4℃에서 6시간마다 4회 투석 (dialysis)하였다. 투석액은 2,000 rpm으로 10분간 원심 분리하여 침전물을 제거한 후, FPLC 시스템 (Phamarcia사, LKB)을 사용하여 미리 상기와 같은 50mM 인산염 완충용액으로 평형화한 모노-큐 (Mono-Q) 칼럼 크로마토그래피로 분리하였다. 세척용액으로는 50mM 인산염 완충용액 (pH 7.8)을 사용하여 칼럼을 통과한 용액의 흡광도가 280 nm에서 0이 될 때까지 세척하였다. 용출용액으로는 1M 염화나트륨 용액(NaCl, pH 7.8)을 사용하여, 0M에서 1M가지 농도를 선형 (linear)으로 변화시켜가며 시간당 60㎖씩 용출시켰다. 용출액 중의 단백질 함량은 280 nm에서의 최대 흡광도가 1.0이 되도록 하여 각각 1㎖씩의 분획으로 수집하였다. 상기 정제과정으로 얻은 1차 용출액은 200-400 mM 염화나트륨 농도구간에 해당하는 22-24번 분획들에서, 비록 단백질양은 많지 않지만 생쥐의 비장 B 임파구의 증식을 유도하는 활성이 가장 높은 것으로 나타났다. 이 분획들을 12% SDS-변성 폴리아크릴 아미드 겔 전기영동 (SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis)을 실시하여 실버 염색(Silver staining)한 결과, 23 KDa의 단백질이 주요 밴드로 나타났다 (도 7A참조).1.5 l of the culture supernatant of the LK1 cells was concentrated to about 10 ml using an Amicon Diaflo membrane (YM-10), and 4 ° C to 2 l of 50 mM phosphate buffer (Tris-HCl, pH 7.8). Dialysis was performed 4 times every 6 hours at. The dialysate was centrifuged at 2,000 rpm for 10 minutes to remove precipitates, and then separated by Mono-Q column chromatography previously equilibrated with the 50 mM phosphate buffer solution using the FPLC system (Phamarcia, LKB). It was. The washing solution was washed with 50 mM phosphate buffer (pH 7.8) until the absorbance of the solution passed through the column became zero at 280 nm. As the elution solution, 1M sodium chloride solution (NaCl, pH 7.8) was used to elute 60ml per hour, varying the concentration from 1M to 1M in a linear form. The protein content in the eluate was collected in fractions of 1 ml each so that the maximum absorbance at 280 nm was 1.0. The primary eluate obtained by the above purification process was found to have the highest activity in inducing the proliferation of spleen B lymphocytes in mice, although the amount of protein was 22-24, corresponding to the concentration range of 200-400 mM sodium chloride. Silver fractions of these fractions were subjected to 12% SDS-modified polyacrylamide gel electrophoresis (SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis), indicating that 23 KDa protein was the main band ( FIG. 7A). Reference).
1차 정제과정으로 용출된 분획들 중 생쥐 비장의 B 임파구의 증식을 유도하는 분획들만을 다시 투석하고 용출농도를 바꾸어가며 다시 FPLC를 시행하여 사이토카인을 2차 정제하였다. 그 결과 200-300 mM 염화나트륨 농도구배에 해당하는 16번 분획이 B 임파구 증식에 대한 활성을 가지고 있는 것으로 나타났다 (도 7B참조). 이 분획들을 12% SDS-PAGE로 분리하고 실버 염색하여, 브래드포드 (Bradford) 시약을 사용하고 소 혈청 알부민 (bovine serum albumin)을 표준으로 하여 단백질을 정량한 결과, 역시 16번 분획에서 분자량이 23kDa인 주요 밴드가 나타났다.Among the fractions eluted by the first purification process, only the fractions that induce the proliferation of B lymphocytes in the mouse spleen were redialysis, and the elution concentration was changed again to perform FPLC to purify cytokines for the second time. As a result, fraction 16 corresponding to the 200-300 mM sodium chloride concentration gradient was found to have activity for B lymphocyte proliferation (see FIG. 7B ). The fractions were separated by 12% SDS-PAGE and silver stained to quantify the protein using Bradford reagent and bovine serum albumin as standard, and the molecular weight was 23kDa in fraction 16 as well. The main band appeared.
최종적으로 15-17번 분획을 모아 바로 이전의 정제과정에서와 같은 용출농도로 FPLC로 정제하고 SDS-PAGE를 실시한 결과, 20번 분획에서 23kDa의 단백질이 분리 정제되었고 B 임파구 증식에 대해서 높은 활성을 나타내었다 (도 7C참조).Finally, 15-17 fractions were collected, purified by FPLC at the same elution concentration as in the previous purification process, and subjected to SDS-PAGE. 23 kDa protein was isolated and purified from fraction 20, and high activity against B lymphocyte proliferation was obtained. (See FIG. 7C ).
2) 사이토카인의 아미노산 서열 분석2) Amino Acid Sequence Analysis of Cytokines
최종 정제과정을 거친 분자량 23 kDa인 단백질을 12% SDS-PAGE로 분리한 후 PVDF 막 (polyvinylidene difluoride membrane)으로 옮겨, 단백질 서열분석기 (automatic protein sequencer)로 에드만 분해 (Edman degradation) 방법에 의해 아미노 말단의 아미노산 서열을 분석하였다. 그 결과 아미노 말단은 서열번호 2의 아미노산 서열을 갖는 것으로 밝혀졌는데, 이 아미노산 서열을 기존에 알려진 아미노산의 서열과 비교한 결과 상기 단백질은 히스타민 분비 인자인 것으로 판명되었다. 즉, 이미 알려져 있는 상기 아미노산 서열을 갖는 단백질이 히스타민의 분비를 유도하는 고유의 기능 외에도, B 임파구의 증식을 유도하는 새로운 기능을 갖고 있음을 밝혀낸 것이다.After final purification, the protein having a molecular weight of 23 kDa was separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane, which was then purified by an Edman degradation method using an automatic protein sequencer. The terminal amino acid sequence was analyzed. As a result, the amino terminus was found to have the amino acid sequence of SEQ ID NO: 2, and the protein was found to be a histamine secretion factor by comparing this amino acid sequence with a sequence of known amino acids. In other words, the protein having the amino acid sequence already known has a new function of inducing the proliferation of B lymphocytes, in addition to the intrinsic function of inducing histamine secretion.
<실시예 6> 재조합 히스타민 분비 인자 (사이토카인)의 발현 및 정제Example 6 Expression and Purification of Recombinant Histamine Secretion Factor (Cytokines)
1) 재조합 히스타민 분비 인자의 발현1) Expression of Recombinant Histamine Secretion Factor
상기 단백질을 대량으로 얻기 위하여 발현 벡터로 pMAL-c2를 사용하여 재조합 발현 벡터를 제작하였다. pMAL-c2 (NEB; 800-64S)는 세포질에서 말토스 결합 단백질 (MBP; maltose binding protein)과 원하는 단백질을 융합하여 발현하므로 융합 단백질을 쉽게 분리할 수 있는 장점을 가지고 있다.To obtain a large amount of the protein, a recombinant expression vector was constructed using pMAL-c2 as an expression vector. pMAL-c2 (NEB; 800-64S) has the advantage of easily separating the fusion protein because it is expressed by fusing maltose binding protein (MBP) with the desired protein in the cytoplasm.
인간 cDNA 라이브러리 (library)로부터 서열번호 3의 시발체 A와 서열번호 4의 시발체 B를 이용하여 중합효소 연쇄반응을 통해 상기 사이토카인의 히스타민 분비 인자를 암호화하는 서열번호 38의 유전자를 증폭하였다. 이를 제한효소EcoRI,SalI를 이용하여 발현 벡터 pMAL-c2에 삽입시켰다. 또한 융합 단백질로부터 말토스 결합 단백질을 분리하기 위하여 서열번호 5의 트롬빈 분리 부위 (thrombin cleavage site)를 제한효소EcoRI의 인지부위에 삽입하였다 (도 8a참조). 제조된 재조합 발현 벡터 pMAL-c2-thr-HRF로 E.coli(JM109)를 형질전환시켜 얻은 형질전환체를 배양한 후, 600 nm에서의 흡광도가 0.8이 되었을 때 1mM의 IPTG (isopropylthio-β-D-galactoside)를 첨가하여 20℃에서 24시간 동안 배양하였다.The gene of SEQ ID NO: 38 encoding the histamine secretion factor of the cytokine was amplified by polymerase chain reaction using primer A of SEQ ID NO: 3 and primer B of SEQ ID NO: 4 from a human cDNA library. This was inserted into the expression vector pMAL-c2 using the restriction enzymes Eco RI, Sal I. In addition, a thrombin cleavage site of SEQ ID NO: 5 was inserted at the recognition site of restriction enzyme Eco RI to separate maltose binding protein from the fusion protein (see FIG. 8A ). After culturing the transformant obtained by transforming E. coli (JM109) with the prepared recombinant expression vector pMAL-c2-thr-HRF, when the absorbance at 600 nm was 0.8, 1 mM IPTG (isopropylthio-β- D-galactoside) was added and incubated at 20 ° C. for 24 hours.
2) 재조합 히스타민 분비 인자 (사이토카인)의 정제2) Purification of Recombinant Histamine Secretion Factor (Cytokines)
상기 배양된 대장균 형질전환체를 10,000rpm으로 1분간 원심분리하여 침전시킨 뒤 20mM 인산염 완충용액 (pH 7.5)에서 초음파 분쇄하고, 여기서 얻은 상등액을 아밀로스 칼럼 (Biolabs사, #800-21L)으로 정제하였다. 정제된 융합 단백질은 50mM 인산염 완충용액 (pH 7.5)에서 투석하여 분리하고, 트롬빈 (Sigma사, T-6634)으로 처리하여 융합 단백질의 말토스 결합 단백질과 히스타민 분비 인자를 분리하였다. 이 용액을 다시 10mM 인산염 완충용액 (pH 8.0)에서 투석한 후, DEAE-세파로스 칼럼 (Pharmacia Biotech사, 17-0710-01)으로 히스타민 분비 인자를 분리 및 정제하였다. 이후 분리된 히스타민 분비 인자에 상온에서 30분 동안 100 mM OBDG (Octyl-β-D-glucopyranoside, Sigma사, O-9882)를 첨가하고 폴리마이신 B 칼럼 (Polymyxin B, Sigma사, P1411)으로 통과시켜 내독소 (endotoxin)를 제거하였다 (도 8b참조).The cultured E. coli transformants were precipitated by centrifugation at 10,000 rpm for 1 minute and then ultrasonically pulverized in 20 mM phosphate buffer (pH 7.5), and the supernatant obtained therefrom was purified by an amylose column (Biolabs, # 800-21L). . Purified fusion proteins were isolated by dialysis in 50 mM phosphate buffer (pH 7.5) and treated with thrombin (Sigma, T-6634) to separate maltose binding proteins and histamine secretion factors of the fusion proteins. The solution was again dialyzed in 10 mM phosphate buffer (pH 8.0), and then histamine secretion factors were isolated and purified by DEAE-Sepharose column (Pharmacia Biotech, 17-0710-01). Then, 100 mM OBDG (Octyl-β-D-glucopyranoside, Sigma, O-9882) was added to the isolated histamine secretion factor at room temperature for 30 minutes and passed through a polymyxin B column (Polymyxin B, Sigma, P1411). Endotoxin was removed (see FIG. 8B ).
<실시예 7> 재조합 히스타민 분비 인자 (사이토카인)에 의한 B 임파구의 증식Example 7 Proliferation of B Lymphocytes by Recombinant Histamine Secretion Factor (Cytokines)
1) 생쥐 비장 B 임파구의 분리1) Isolation of Mouse Spleen B Lymphocytes
발브씨 생쥐의 비장으로부터 적혈구 (red blood cell, RBC)를 제거하기 위해, 생쥐의 비장을 37℃에서 적혈구 용출용액 (Sigma사)으로 5분간 처리하고 무혈청 배지로 2회 세척하였다. 또한 상기 용액을 Thy-1, Thy-2 항체와 함께 얼음에서 30분 동안 방치하고 2회 세척한 후, 여기에 토끼의 보체 (complement)를 가하여 37℃에서 1시간 동안 방치한 후 다시 2회 세척하여 세파덱스 칼럼 (Sephadex G-10, Pharmacia사)에 통과시켜 부착세포와 파괴된 T 세포들을 제거하였다. 이후 세포를 72, 65, 50%로 불연속 중충된 퍼콜 (Percoll, Sigma사) 위에 중층하고 4℃에서 2,000×g으로 30분간 원심 분리하여, 65-72% 사이의 층을 회수하여 B 임파구로 사용하였다. 이때 B 임파구 특이적인 B220 항체를 이용하여 세포형광 분석을 실시한 결과, 상기 분리된 B 임파구의 순도는 95% 이상이었다.In order to remove red blood cells (RBCs) from the spleens of Valv mice, the spleens of mice were treated with red blood cell elution solution (Sigma) at 37 ° C. for 5 minutes and washed twice with serum-free medium. In addition, the solution was left for 30 minutes on ice with Thy-1 and Thy-2 antibodies and washed twice, followed by adding a rabbit complement to the solution at 37 ° C. for 1 hour and then washing again. The cells were passed through a Sephadex column (Sephadex G-10, Pharmacia) to remove adherent cells and broken T cells. Cells were then layered on 72, 65 and 50% of discretely impregnated percol (Percoll, Sigma) and centrifuged at 2,000 × g for 30 minutes at 4 ° C, recovering between 65-72% of the layers and using them as B lymphocytes. It was. In this case, cell fluorescence analysis was performed using B lymphocyte specific B220 antibody, and the purity of the isolated B lymphocyte was 95% or more.
2) 재조합 히스타민 분비 인자 (사이토카인)에 의한 B 임파구의 증식2) Proliferation of B lymphocytes by recombinant histamine secretion factor (cytokine)
상기에서 분리된 생쥐 B 임파구를 20% 소태아혈청이 포함된 RPMI 1640 배지에 5×106세포/㎖로 현탁하여, 96개의 웰 배양 플레이트에 웰 당 100㎕씩 넣고 상기 실시예 6에서 얻은 재조합 히스타민 분비 인자 (사이토카인)를 농도별로 20㎕씩 가하여 37℃, 5% CO2에서 68시간 동안 배양하였다. 그 다음 0.5 μCi의3H-티미딘을 50㎕씩 각 웰에 가하고 같은 조건에서 4시간 더 배양한 후, 다중 세포수확기 (multiple cell harvester, Inotech사)로 유리 섬유 여과지에 배양액을 회수하여 액체 신틸레이션 계수기 (liquid scintillation counter, Beckman사)로3H-티미딘의 함입량을 측정하여 B 임파구의 증식을 조사하였다.The mouse B lymphocytes isolated above were suspended at 5 × 10 6 cells / ml in RPMI 1640 medium containing 20% fetal bovine serum, and 100 μl per well was put into 96 well culture plates. 20 μl of histamine secretion factor (cytokine) was added to each concentration and incubated at 37 ° C. and 5% CO 2 for 68 hours. Then, 50 μl of 0.5 μCi 3 H-thymidine was added to each well and incubated for 4 hours under the same conditions. Then, the culture solution was recovered on a glass fiber filter paper using a multiple cell harvester (Inotech) and liquid scintillation was performed. Proliferation of B lymphocytes was examined by measuring the incorporation of 3 H-thymidine with a liquid scintillation counter (Beckman).
그 결과 재조합 히스타민 분비 인자 (사이토카인)의 농도에 의존적으로 B 임파구의 증식이 유도되는 것으로 나타났으며 (도 9a참조), 500 ㎍/㎖ 이상은 포화농도로서 더 이상 B 임파구 증식이 유도되지 않았다.As a result, it was shown that B lymphocyte proliferation was induced depending on the concentration of recombinant histamine secretion factor (cytokine) (see FIG. 9A ), and the B lymphocyte proliferation was no longer induced as a saturation concentration. .
한편 대조군인 말토스 결합 단백질을 처리했을 때에도 고농도에서는 B 임파구 증식이 다소 유도되었지만, 재조합 히스타민 분비 인자에 의한 증식 효과에 비하면 훨씬 낮았다. 말토스 결합 단백질만을 처리한 경우에도 B 임파구가 약간 증가한 것은 형질전환체로부터 재조합 히스타민 분비 인자를 분리 및 정제하는 과정 중에 내독소가 완전히 제거되지 않고 남아 B 임파구의 증식을 자극하는 항원으로 작용하기 때문으로 생각되었다. 이것을 확인하기 위해서 재조합 히스타민 분비 인자와 말토스 결합 단백질 각각의 일부를 10분간 끓이고 다른 일부는 히스타민 분비 인자에 대한 항체로 처리하였는데, 이 경우 B 임파구의 증식 효과는 나타나지 않거나 중화되어 (도 9b참조), 결국 히스타민 분비 인자에 의해 B 임파구의 증식이 유도되는 것을 확인할 수 있었다.On the other hand, B lymphocyte proliferation was slightly induced at high concentrations even when the maltose binding protein was treated as a control, but was much lower than the proliferative effect by recombinant histamine secretion factor. Even if only maltose binding protein was treated, B lymphocytes increased slightly because endotoxin was not completely removed during the process of separating and purifying recombinant histamine secretion factor from the transformant, which acts as an antigen that stimulates the proliferation of B lymphocytes. Was thought of as. To confirm this, a portion of each of the recombinant histamine and maltose binding proteins was boiled for 10 minutes and the other part was treated with an antibody against histamine secretion factor, in which case the proliferative effect of B lymphocytes was not shown or neutralized (see FIG. 9B ). In the end, it was confirmed that B lymphocyte proliferation was induced by histamine secretion factor.
이상 살펴본 바와 같이, 본 발명의 새로운 생쥐 적혈백혈병 세포주 LK1으로부터 분리 정제된 사이토카인 및 사이토카인 유전자를 발현시켜 얻은 재조합 히스타민 분비 인자는 B 임파구 증식을 유도하는 효과를 갖고 있으며, 상기 생쥐 적혈백혈병 세포주 LK1은 적혈백혈병 세포주와 B 임파구 간의 상호작용을 연구하는데 있어 좋은 모델로 이용할 수 있다.As described above, the recombinant histamine secretion factor obtained by expressing purified cytokine and cytokine genes isolated from the new mouse erythroleukemia cell line LK1 of the present invention has an effect of inducing B lymphocyte proliferation, and the mouse erythroleukemia cell line LK1. May be a good model for studying the interaction between erythroleukemia cell lines and B lymphocytes.
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