韩文玲 论文题目：新的CC家族趋化因子UCK–1及其变异体的克隆、表达和功能研究 作者简介：韩文玲，女，1970年09月出生，1996年09月师从于北京大学(医学部)马大龙教授，于1999年07月获博士学位。
摘 要
细胞因子是由机体细胞合成并分泌的小分子多肽类因子，它们参与多种细胞的增殖、分化，在免疫和炎症反应中发挥重要作用。细胞因子包括白细胞介素、集落刺激因子、干扰素、肿瘤坏死因子、生长因子、趋化因子等。趋化因子是一类具有趋化作用的小分子多肽，能吸引免疫细胞到免疫应答局部。绝大多数趋化因子都有4个保守的半胱氨酸，根据其结构不同，可分为CXC、CC、CX3C和C 4个亚家族，C代表半胱氨酸，X代表任一氨基酸。趋化因子在机体的免疫防御和调节、血管生成、HIV感染等方面发挥重要作用。在临床应用方面，已有多种基因工程的细胞因子药物得以成功应用。
细胞因子在机体的生理和病理过程中发挥重要作用，同时具有重要的应用前景，因此克隆和研究新的细胞因子具有重要的理论意义和应用价值。国际上随着现代分子生物学技术的迅速发展和人类基因组计划的趋于完成，越来越多的细胞因子得以发现。我国目前在这一领域的研究还比较落后。
为了有所突破，本文首次通过一种新的途径来发现新的细胞因子。过去的研究证明，人髓性白血病细胞系U937细胞在有丝分裂原PHA刺激下，能产生IL-1、IL-8、GM-CSF等多种细胞因子，而IL-10是一种广谱的细胞因子合成抑制剂，能制抑IL-1、GM-CSF等多种已知细胞因子的产生，同时被IL-10所抑制的可能还有其它未被发现的新细胞因子。基于这些发现，我们利用IL-10的细胞因子合成抑制活性，把SSH技术和EST拼接方法有机结合起来，以PHA刺激的U937细胞为tester，以PHA刺激同时IL-10抑制的U937细胞为driver进行减数杂交，研究IL-10抑制表达的新基因，其中有可能有新的细胞因子候选基因。
通过这一技术路线，我们发现了6个新的EST片段，挑选其中4个进行Northern
Blot分析，其中3个能被IL-10所抑制，包括以后被证实的新的CC家族趋化因子和D-E-A-Dbox家族RNA解旋酶，说明SSH是一种高效的差异表达基因筛选方法，同时证明我们的实验设计是合理的。
通过EST拼接和RT-PCR扩增，得到了上述新趋化因子的全长cDNA序列，Northern
Blot分析表明该分子大小与预期的一致，且其在PHA刺激的U937细胞中的表达可被IL-10部分抑制。此全长cDNA由534个碱基组成，有一个编码99个氨基酸的完整开放读码框架，起始密码ATG周边序列符合kozak规律（CGCATGG），3′非翻译区有相对少见的加尾信号ATTAAA和polyA尾巴。编码的蛋白质为疏水性碱性蛋白，分子量为10.9kDa，含有两个连续半胱氨酸的CC家族趋化因子的特征性结构。NR数据库检索发现为新基因，与已发现的CC家族趋化因子没有明显同源性，只与线虫的Amino acid permease表现为低水平的同源性。基因定位的STS数据库查询提示其基因定位于16号染色体上。手工排列进一步发现，其在蛋白质水平上与定位于16号染色体上的2个CC家族趋化因子TARC和STCP-1有较低水平的同源性。因此，认为这是一个新的CC家族趋化因子，命名为UCK-1(U937-derived chemokine-1，U937细胞来源的趋化因子1，后经国际命名委员会批准为CKLF1, chemokine-like facter1，趋化素样因子1)，UCK-1在GenBank中的登录号为AF096895。RT-PCR和DNA序列分析发现UCK-1存在多种不同的变异体，命名为UCK-2、UCK-3、UCK-4，分别编码152、67和120个氨基酸，均有连续两个半胱氨酸的特征性结构，与UCK-1有相同的C末端和N末端，在GenBank中的登录号分别为AF135380、AF135381和AF145216。
为了研究UCK-1的体内外生物学活性，我们构建了UCK-1的真核表达载体pCDI-UCK-1和原核表达载体pMTY4-UCK-1。与空载体转染上清相比，瞬时转染pCDI-UCK-1的COS-7细胞上清对正常人外周血中性粒细胞、淋巴细胞、单核细胞和U937细胞、k562细胞及DMSO刺激的HL-60细胞等多种人类白血病细胞有明显的剂量依赖性趋化活性；同时，对小鼠腹腔巨噬细胞和脾脏淋巴细胞有明显的趋化作用。此外，COS-7细胞表达的重组UCK-1对骨髓低密度造血干细胞有明显的促增殖活性，在半固体培养基中，能明显促进集落的形成。在集落形成实验中，UCK-1刺激形成的集落细胞体积大，较单纯GM-CSF刺激的细胞存活时间长，这些现象尚未在其它趋化因子中发现。目前，尚需进一步的实验证明UCK-1刺激的骨髓低密度造血干/祖细胞属于哪一分化阶段，但这种明显的造血刺激活性，提示UCK-1对造血障碍性疾病可能有一定的治疗应用前景。
实验研究发现，DTT能够完全阻断COS-7细胞表达的UCK-1的生物学活性，说明二硫键对于UCK-1的功能是必需的。根据蛋白质的结构特点，连续两个半胱氨酸之间不能形成二硫键，因而推测UCK-1可能通过链间二硫键形成二聚体或多聚体发挥作用。MS2-UCK-1融合蛋白在大肠杆菌中获得高效表达，且存在较大量的双体形式，初步证明UCK-1的活性形式为共价双体。
体内实验发现，真核表达质粒pCDI-UCK-1裸质粒直接注射小鼠骨骼肌后，可在局部趋化吞噬细胞（后来实验证明，注射局部有淋巴细胞、单核细胞和中性粒细胞浸润，与体外实验相符）；同时引起骨骼肌细胞核内移，提示UCK-1能够以直接或间接方式促进骨骼肌细胞再生（后来实验证明，UCK-1能够促进肌母细胞增殖），提示其可能用于治疗退行性肌病变。
对UCK-1变异体的活性研究发现，COS-7细胞表达的UCK-2能明显促进小鼠骨髓细胞的大量增殖，而UCK-3的作用不明显。Pc/Gene分析发现，UCK-2和UCK-4为膜结合型趋化因子（细胞表达定位实验已证明此结论），但UCK-2转染上清仍有较强的生物学活性，提示一些蛋白酶能够降解膜结合型UCK-2使其活性形式释放到上清中，与目前已发现的唯一一种膜结合型趋化因子Fractalkine相似。
对32种胚胎组织、成人组织及肿瘤组织的单链cDNA文库进行RT-PCR分析发现，UCK-1及其变异体在胚胎和肿瘤组织中高表达，在正常成人组织中低表达，表达带行亦较单一。EST同源性比较的结果同样表明：与UCK-1部分DNA序列高度同源或完全相同的EST片段，绝大部分来源于胚胎和肿瘤组织，间接证明了UCK-1基因表达的组织分布，提示UCK-1及其变异体可能在肿瘤发生和胚胎发育过程中起一定作用。
综上所述，本文首次通过一种新的技术路线，在国际上首次成功克隆了新的具有独特结构特点和功能特性的CC家族趋化因子UCK-1及其变异体UCK-2、3、4。体内、外实验证明其具有广谱的趋化活性、造血刺激活性和骨骼肌刺激活性。在肿瘤和胚胎组织中，UCK-1及其变异体特征性的高表达模式，提示其在肿瘤发生和胚胎发育中可能发挥重要作用。这些新的发现对促进趋化因子的深入研究，具有重要的理论意义和潜在的应用价值。同时，本文所采用的技术路线可加以推广，结合其它的细胞因子产生细胞和细胞因子合成抑制剂，以发现更多的具有自主知识产权的细胞因子。
Abstract
Cytokines consist
of a broad group of small proteins that play a crucial role in the immune and
inflammatory responses. In general, cytokines are induced transiently upon cell
activation. Cytokines include interleukins, interferons, tumor necrosis
factors, growth factors and chemokines. Chemokines are a family of small,
structurally related proteins. They play a major role in recruitment and
activation of leukocytes in vivo.
Chemokines share a conserved four-cysteine motif in their amino acid sequences
and can be divided into four families based on the position of the first two cysteine
residues: CXC, CC, C and CX3C. (C refers to cysteine and X is any amino acid.)
Cytokines are
important factors both in physiological and pathological conditions; at the same
time, some of them can be successfully used in clinical application. So it
would be valuable to clone and study novel cytokines. With the rapid
development of modern molecular biology and the complement of Human Genome
Project, many novel cytokines have been cloned, but few were discovered in our
country. In order to make a breakthrough in this field, we devised a strategy
to find novel cytokines.
It has been known
for a long time that U937 cells, a human promonocytic cell line, can produce
many kinds of cytokines and that IL-10 can inhibit expression of cytokines.
Based on these two ideas, we used the suppression subtractive hybridization
(SSH) to isolate cDNAs that could be inhibited by IL-10 in PHA-stimulated U937
cells. There may be fragments of novel cytokine cDNAs in the subtracted
library. In the SSH procedure, U937 cells stimulated with PHA were used as
tester, while the U937 cells with the same treatment in presence of IL-10 were
used as driver.
Using this method,
we cloned 6 novel EST fragments after running a BLASTN search against the
public database of ESTs (dbEST). Expression of 4 potentially interesting cDNA
clones was studied using Northern Blot analysis and 3 of them could be
inhibited by IL-10, including a novel CC family chemokine and a novel D-E-A-D
box family RNA helicase. These results verify that SSH is an effective method
for screening differentially expressed genes and our strategy is reasonable.
A number of human
ESTs were initially identified using an EST fragment obtained from the
PHA-stimulated U937 subtracted library after running a dbESTs database search.
These ESTs were retrieved and a contig was assembled from overlapping ESTs
using EST Assembly Machine. RT-PCR and DNA sequencing verified that the EST
extension was right. Northern Blot analysis demonstrated that it could be
inhibited by IL-10 and that its mRNA was 0.6Kb long, which was in accord with
the expected one. The full-length cDNA has 534bp nucleotides and a single open
reading frame encoding 99 amino acid residues with an ATG initiation at
nucleotides 96-98, whose surrounding sequence (GCGATGG) matches Kozak’s rule.
There is a poly A tail and a single polyadenylation site signal of a rare type
(ATTAAA) that is also found in human Eotaxin and TARC in the 3’ untranslated
region. It was a novel sequence after search of NR database. The open reading
frame of the novel cDNA encodes a highly basic and hydrophobic polypeptide of
total 99 amino acids with a calculated molecular mass of 10.9 KDa. The deduced
protein contains no putative N-glycosylation site. It was found that its amino
acid sequence had only limited homology with that of the permease of ceanorhabcitis elegans and shared no
obvious homology with other proteins. The protein possesses some unique
structural characteristics: 1). It contains three cysteine residues, the last
two of which are organized in a pattern characteristic for the CC chemokine
subfamily; 2). It shares no significant homology with other chemokines of CC
family by BLAST searching. However, hand-operated alignment of it with TARC and
STCP-1, two of the members of CC chemokines located on chromosome 16, indicated
positoning of identical key residues near the CC motif. So we designated it as
a novel CC chemokine UCK-1(U937 derived chemokine-1,which was modified as
CKLF1, chemokine-like factor 1 by HUGD Nomenclature Committee). Its accession
number in GenBank is AF096895.
By RT-PCR
technique, we amplified the full cDNA sequence of UCK-1 from PHA-stimulated
U937 cell cDNA library. In addition, three other bands were found. After
cloning and sequencing, we named them UCK-2, UCK-3 and UCK-4. Open reading
frame analysis showed UCK-2,3,4 encoded 152, 67 and 120 amino acids
respectively. They share conserved amino- and carboxyl-terminal ends with
UCK-1. All of them maintain the CC motif of chemokines. According to the amino
acid numbers of the longest isoforms UCK-2, it appears to be that UCK-1, 3, 4
are the RNA splicing forms of UCK-2 at 27-79, 27-111 and 80-111 amino acid
positions and their accession number in GenBank is AF135380, AF135381 and
AF145216 respectively.
To investigate the
bioactivity of UCK-1, we constructed a mammalian expression vector pCDI-UCK-1
and a prokaryotic expression vector pMTY4-UCK-1. Separated cultures of COS-7
cells were transfected with pCDI-UCK-1 and the control vector pCDI. The
supernatants from cell cultures were collected for bioassay. The supernatants
from the pCDI-UCK-1 transfected cells had chemotactic effect on human
neutrophils, lymphocytes and monocytes compared with the control supernatants
in a dose-dependent manner. In addition, it can also attract U937, K562,
DMSO-treated HL-60 cells and mouse macrophages, lymphocytes. The recombinant
UCK-1 in the supernatants of COS-7 cell culture was analyzed for its effect on
the proliferation of human bone marrow cells of low density. The supernatants
contained UCK-1 have obvious enhancing effect compared to the control
supernatants. In the semi-solid culture, UCK-1 can promote the colony formation
of hematopoietic progenitor cells. These cells were bigger than those
stimulated with GM-CSF and could live a longer time. There were no such reports
on other chemokines. Further experiments are necessary to verify which kind of
progenitors UCK-1 can stimulate, but this enhancing activity suggests that it
may be used in clinical application.
The activity of
UCK-1 could be prevented completely by DTT. This indicates that the covalent
bond is indispensable for UCK-1 activity. According to the protein structure
characteristics, two adjacent cysteines can’t form disulfide bridge, so the
active form of UCK-1 maybe dimer or multimer form. The MS2-UCK-1 fusion protein
has been expressed in E. Coli. with higher level, and SDS-PAGE showed that
there was a dimer form of UCK-1 in the expressed proteins.
The chemotactic
properties of recombinant UCK-1were further analyzed after naked plasmid DNA
administration in vivo. It arose
macrophage infiltration in the injection site. (Further experiments verified
that UCK-1 could attract monocytes, neutrophils and lymphocytes, which is in
agreement with the chemotactic effect of UCK-1 in vitro.) Except for infiltration of more cells in UCK-1 expressed
muscle, there were also morphological changes. Central nuclei were visible
within the muscle fibres in cross sections. The existence of such nucleus
changes indicated that the muscle were ongoing regeneration and UCK-1 could
induce satellite cell activation directly or indirectly.(Later results have
confirmed that UCK-1 could stimulate the proliferation of myoblast cells
directly.)
As for the
activity of the UCK-1 isoforms, the supernatants of COS-7 cell transfected with
UCK-1 and UCK-2 eukaryotic expression plasmids can stimulate the proliferation
of mouse bone marrow cells in vitro
and UCK-3 had no obvious activity for the same cells. Computer analysis showed
UCK-2 and UCK-4 contained highly hydrophobic regions and might be transmembrane
proteins. (Subcellular localization analysis of UCK-2 and UCK-4 has validated
that they are transmembrane proteins.) Although UCK-2 is membrane-bound
protein, part of its active form can be secreted into the supernatant, which is
similar with human CX3C chemokine Fractalkine.
RT-PCR analysis
with multiple Tissue cDNA Panel Kit revealed that UCK-1 and its variants had
relatively higher expression level in tumor and fetal tissues, less expression
level in normal adult tissues, which indicates that UCK-1 and its variants may
play an important role in tumor and fetal development.
In summary, we
cloned a novel CC chemokine UCK-1 with unique structural and functional
characteristics and found its several isoforms UCK-2, 3 and 4. The UCK-1 is a
potent chemoattractant for a variety of cells, and also a stimulator for bone
marrow cells. The higher expression level in tumor and fetal tissues indicates
that UCK-1 and its isoforms may play important roles in the embryonic
development and tumorigenesis. All these findings are helpful for further
understanding of chemokines and UCK-1 as well as its isoforms, which may have
important significance for research and development of biotechnology.