摘要
移植物抗宿主病（Graft Versus Host Disease，GVHD）是移植物中
的免疫活性细胞识别宿主抗原并对宿主产生的病理损伤状态。去除T
细胞和应用免疫抑制剂等方法虽然可以明显降低GVHD发生率，但是
移植失败率、白血病复发率和感染率均显著增加，未能从根本上解决
这一问题。近年来杀伤细胞抑制性受体（Kill cell Inhibitory Receptor，
KIR）的发现为负性调节NK、T细胞功能提供了良好的途径，也为防
治GVHD提供了新的思路。人与鼠的KIR广泛表达在NK细胞和部分
T细胞上，KIR与相应的MHCⅠ类分子结合后传导抑制性信号抑制
NK、T细胞的杀伤功能和炎性细胞因子的分泌，从而保护靶细胞免受
损伤。本研究试图将能识别受者MHCⅠ类分子的KIR基因转染供者
淋巴细胞并进行移植建立一种既降低急性GVHD又保留移植物抗白
血病（Graft Versus Leukemia GVL）效应的骨髓移植模式，并从杀伤
细胞抑制性受体（KIR）/MHCⅠ识别角度探讨其分子机制，为GVHD
的研究提供理论和实验依据。
本研究采用如下方法：（1）将Prof.Raulet赠送的Ly49A cDNA
测序，测序正确后构建逆转录病毒表达载体pLXSN-Ly49A，双酶切鉴
定正反相。（2）表达载体经PA317细胞包装后转染C57BL/6小鼠的
淋巴细胞；流式细胞仪检测Ly49A受体在转染后淋巴细胞上的表达
率。MTT法检测转染后淋巴细胞对BALB/c小鼠正常成纤维细胞
（H-2~d）和4T_1乳腺癌细胞（H-2~d）的杀伤活性。（3）将H-2~d遗传
背景的EL9611红白血病细胞静脉接种给CB_6F_1~(H-2d/b)小鼠，建立
F；H‘川‘’红白血病模型，为观察半相合异基因骨髓移植后 GVL效应提
供动物模型。K）以C57BL历汁比 V＼鼠为供者，以接种EL96ll红白
血病细胞的BALB儿卜2‘小鼠为受者，预处理条件为全身照射（TBI，
6七。照射9刀Gy人进行脾淋巴细胞混合骨髓细胞移植，建立完全不
相合异基因急性GVHD模型；以亲代C57BL沁卜儿4、鼠为供者，以接
种 EL96ll红白血病细胞的 BALB／CXC57BL／6，FIH”Zd／b（CB6F；）／J’
鼠为受者，预处理条件为全身照射（TBI，加C。照射10．SGy人 进行
脾淋巴细胞混合骨髓细胞移植，建立半相合异基因急性GVHD模型
并在这两个模型基础上考察 Ly49A基因转染的淋巴细胞对 GVHD和
GVL的作用。
本研究结果显示：门）P＊f R川u1Ct赠送的 Ly49A CDNA测序结果
与其报道的序列完全一致。（）Q扔A基因转染的C57BU6小鼠淋巴
细胞24 小时后受体表达率为O6．67t0．35）％，空载体转染组
＊．73L0．85）％，未转染对照组＊石0L0．27）％。Ly49A基因转染的
C57BL／6 ’J＼鼠淋巴细胞对BALB／C ’J＼鼠正常成纤维细胞的杀伤活性降
低了（ZI—25）％，对4TI乳腺癌细胞的杀伤活性无明显改变（P＞0刀5）。
门）CB6FI卜川‘红白血病模型已传10代，静脉接种EL9611细胞数
103个／只即可发病。发病时白血病细胞主要浸润肝、脾和骨髓。小鼠
接种10‘J08个／只EL9611细胞时，生存时间与接种细胞数成线性负相
关。此白血病模型对环磷酚胺、阿糖胞昔等化疗药物敏感。（4）在完
全不相合异基因骨髓移植（C57BL历卜儿一BLAB儿卜川）中，未进行移
植的单纯照射组生存期为（6．50L2．41）天；环磷酚胺治疗组生存期为
（20．90f2．88）天；淋巴细胞混合骨髓细胞移植组生存期为
（17．10L4石5）天；空载体转染的淋巴细胞混合骨髓细胞移植组生存
期为门7．40L5．32）天；Ly49A转染的淋巴细胞混合骨髓细胞移植组
生存期为（35．20L12．52）天，较上述各组生存期明显延长o二0刀00）。
（5）在半刁合异基因移植中（C57BL／6b’bb一CB6FIH”Zd／b），未进年移
植的单纯照射组生存期为 门石0＊3．36）天；环磷酚胺治疗组生存期为
一5 一
（ZI了0L2．87）天；淋巴细胞混合骨髓细胞移植组生存期为（29．4f6．43）
天；空载体转染的淋巴细胞混合骨髓细胞移植组生存期为（29．IL7．39）
天；Ly49A 转染的淋巴细胞混合骨髓细胞移植组生存期为
K5．00f12．38）天，较上述各组生存期明显延长0功．00o人
从上述结果可得出如下结论：门）Ly49A基因转染可抑制淋巴细
胞对BALB／C ’J’鼠正常成纤维细胞的杀伤活性，而对肿瘤细胞的杀伤活
性无明显影响。门）本研究建立的 C57BL伍H“Zb—CB6FI队扣“的急性
GVHD模型和可移植性CB6FIH”2“‘红白血病模型，是探讨半相合骨髓
移植GVHD和GVL作用的较理想模型。（）Ly49A基因转染的淋巴
细胞在半相合及完全异基因骨髓移植模型上均具有一定程度的降低
GVHD并且保留GVL的作用。u）杀伤细
Graft versus host disease (GVHD) is caused by immunocompetent cells
in the allograft, which recognize the hoat's antigens and do harm to the
host?tissues. Deletion of the T cells in the allograft and the use of
immunosuppressive agents will certainly reduce the morbidity of GVHD,
but at the same time, will also contribute to the relapse of leukemia, the
failure of transplantation and the infections. No way has been found yet to
resolve the problem totally. In recent years the discovery of killer cell
inhibitory receptors (KIR) has shed light on the negative regulation of the
functions of NK and T cells and also the resolution of GVHD. KIRs
express on all NK cells and a portion of T cells. When binding with
corresponding MHC I molecules, negative signal is transducted and the
cytotoxity of NK and I cells are abrogated. In this research we planed to
transfect the Ly49AcDNA into the donor's lymphocytes and transplant
them with bone marrow cells in order to find a new way for reducing
GVHD but retaining GVL effect after allogeneic bone marrow
transplantation. The possibility of the detachment of GVHD and GVL
from the aspect of molecular mechanism of the interaction between
KIRIMHC I was also discussed.
The following methods were adopted in this research: (1) Ly49AcDNA
presented by Prof. Raulet was sequenced. The results were compared with
the reports in GenBank. After the confirmation of the correction of the
??
sequence, the expression vector pLXSN-Ly49A was constructed. (2) After
being packaged by PA3 17 cells, spleen lymphocytes of C57BL/6 mice
were transfected. Flowcytometer was used to detect the expression rate of
Ly49A receptor; MTT method was applied to assay the killing effects of
those lymphocytes cells on fibroblast cells and 4T1 cells of BALB/c
origin. (3) The erythroleukemia cells EL9611 H-2d were injected into
CB6F1H2d/b mice to establish the FlH2dIb erythroleukemia model,
providing the test animal model for detecting GVL effect after
halploidentical allogeneic bone marrow transplantation. (4) In the case of
the murine model of fully mismatched allogeneic acute GVHD: the donor
was C57BL/6H2b, the recipient was BALB/cH2d. After the irradiation
(TBI, 60Co 9.OGy) the recipient accepted the transplantation of mixed
spleen cells and bone marrow cells to establish the GVHD model. (5) In
the case of the murine model of haploidentical allogeneic acute GVHD:
the donor was C57BL/6H2b. The recipient was BALB/c×C57BL/6 FlH2d/b
(CB6F1). After the irradiation (TBI, 60Co 1O.5Gy) the recipient accepted
the transplantation of mixed spleen cells and bone marrow cells to
establish the GVHD model. On the basis of these models, the effects of
Ly49A transfected spleen cells on GVHD and GVL post fully mismatched
allogeneic or haploidentical allogeneic bone marrow transplantation were
detected.
Results were shown as the followings: (1) The Ly49AcDNA presented
by Prof. Raulet was the same as that reported in GenBank. (2) The
expression rate of Ly49A receptor was (46.67±0.35)% for lymphocytes
transfected with pLXSN-Ly49A; (18.73±O.85)% for lymphocytes
transfected with pLXSN and (19.60±0.27)% for untransfected control.
The killing effects of Ly49A transfected lymphocytes on 4T1 tumor cells
remained almost the same as that of the untransfected control (P>0.05),
?8 ?
but the kil
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