抗独特型抗体诱导对小鼠移植免疫反应的抑制作用
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摘要
器官移植现已成为挽救和延长患者生命的有效手段，但器官移植术后
的排斥反应成为影响其成功率的主要原因。为了降低排斥反应的发生率，
提高器官移植的成功率，目前需要病人终生使用免疫抑制剂。然而，长期
使用免疫抑制剂除了会引起免疫抑制剂本身的毒副作用（诸如升高血压、
肝肾毒性等不良反应）外，也可引起严重感染、恶性肿瘤的发生，从而影
响器官移植的成功率和受者的长期存活率。若能造成移植宿主对移植抗原
的免疫耐受状态或低免疫应答状态，同时保持对其它抗原免疫应答的反应
能力（包括抗肿瘤免疫和抗感染免疫等），将会避免或减轻这些副作用的
产生，这是目前解决排斥反应的根本途径。我们根据抗独特型抗体的免疫
学机制，运用诱导抗独特型抗体产生的方法，移植前在移植受体体内诱导
其产生与移植物抗原具有相同抗原性的抗独特型的抗体，观察该方法对小
鼠移植排斥反应的抑制作用。
目的：
第四军医人学博上学位论文 中文摘要
本研究通过于移植前在移植受体体内诱导其产生与移植物抗原具有
相同抗原性的抗独特型的抗体（AbZ），观察该方法对小鼠移植免疫耐受
的诱导作用。
方法：
1．抗同种异品系抗体（Ahl）的诱导C57BL／6小鼠作为移植供体，
BALB／C ’J’鼠作为受体。取C57BL／6 ’J’鼠的脾细胞免疫BALB／。小鼠，制
备抗同种异品系抗体…bl卜
2．抗独特型抗体（Ah2）的诱导 取经过上述鉴定的高效价多克隆抗
血清，经饱和硫酸铰盐析法粗提其中的免疫球蛋白，再以DEAE七ephadex
A－50杜层析纯化免疫球蛋白。对Abl和KLH进行交联，联合免疫佐剂
免疫BALB／。小鼠。以夫心ELISA检狈u AbZ。
3．以己诱导出Am的BALB儿小鼠为移植受体，观察AbZ对小鼠移
植物存活时间、单向混合淋巴细胞反应（MLR）、迟发型变态反应（DTH）、
微量淋巴细胞毒等排斥反应指标的影响。
结果：
1．Ahl效价的测定：间接免疫荧光染色和补体依赖细胞毒试验测定
＊hi血清的效价＞1：400。
2．AbZ的检测：用夹心 ELISA检测 AblKLH免疫小鼠的 A45。nm值
为（1．07切．门），较对照组（o．07比D4）高2倍以上。
3．AbZ对皮肤移植物的影响：BALB／c ’J＼鼠移植皮肤后，AbZ诱导组
移植物的存活时间为（26．5士 l、7川，阴性对照组移植物的存活时间为 （ 12
土 2．l）d，CSA组移植物的存活时问为（13．4土 2．6）d。AbZ诱导组移植
物的存活时间与对照组相比较差异显著（P＜0．01人
4．AbZ诱导对心肌移植的影响：心肌组织异位移植后，AbZ诱导组
一5 一
第四车医人学博I：学位论文 中义摘要
移植物存活时间为（31．5士 2．7）d，使用 CsA组移植物存活时间为（14．7
士l．6）d，阴性对照组移植物存活时间为（9．4士2．3）d，AbZ诱导组移植
物存活时间与对照组相比有显著性差异（P＜0刀1）。
5．AbZ对诱导 DTH的影响：AbZ诱导组脚掌的厚度差为（0．43上0．17）
mm，上常 BALB／c ’J’鼠脚掌的厚度差为（1．09士 0．ZI）mm，二者差异显
著（P＜0刀1）。
6．AbZ对混合淋巴细胞培养的影响：AbZ诱导组的人刀加m值为0．24
士0．们，对照组为0．35士0刀4，二者差异显著（P＜0刀1）。
7．AbZ血清对微量淋巴细胞毒的影响：加入AbZ血清的微量淋巳细
胞毒试验着色细胞（损伤细胞）的百分率为 32．5士3．5％，小加 AbZ血清
的对照组着色细胞的百分率为86．7土2．9％，二者比较，差异显著（P＜0刀1）。
结论：
1．C57BL／6小鼠脾细胞免疫BALB／。小鼠，可成功制备抗同种异品
系抗体（Ahl）。
2．Ahl．KLH加弗氏佐剂免疫小鼠可成功地诱导 AbZ产牛。
3．与对照组相比，在受体小鼠体内诱生的AbZ确可延长移植物的存
活时间，说明对排斥反应产生了明显地抑制作用。
4．AbZ诱导对DTH、混合淋巴细胞培养、微量淋巴细胞毒等排斥
反应指标可起到明显的抑制作用。
5．移植前在受体体内诱导产生以移植物抗原为始动抗原的抗独特型
的抗体，叶以有效诱导特异性低反应状态。
Although organ transplantation has become an effective treatment to save lifes and prolong the survival time of patients, the rejection after transplantation is the main cause which affects the final outcome. In order to suppress rejection and increase the survival rate of transplantation, expensive immunosuppressants have to be used in the remainder of patients' life. However, large amount of immunosuppressants can cause not only the side-effects, such as hyperpiesia and hepatorenal toxity, but also severe infection and malignant carcinoma ^specially in a long run. Nowadays the key methods to avoid or alleviate these side-effects are to induce immune tolerance or low immunoreaction state, and sustain the ability of immune response against other antigens (including anticancer, anti-infection and so
on ). According to the theory of immune network, we designed the experiment to induce anti-idiotypic antibodies in recipient which has the same antigenicity as the graft antigens prior to transplantation, and observed the inhibitory effects of anti-idiotypic antibods on the rejection of mice graft.
Aims:
To induce the of anti-idiotypic antibodies in recipient which have the same antigenicity as the graft antigen prior to transplantation, and then to investigate their suppression effects on immune rejection in mice.
Methods:
1. Induction of isotypic heterostrain antibody(Abl) : BALB/c mice were immunized with C57BL/6 splenocytes to raise antibody(Abl) between the two difference strains.
2. Induction of anti-idiotypic antibody (Ab2) : Immunoglobulin was purified grossly from polyclonal antisemm which had been identified with high titer, by saturated ammonium sulfate salting out. Then Immunoglobulin was further purified by DEAE-Sephadex A-50 column. In the experiment, Abl was cross-linked to KLH, Abl-KLH plus freund's adjuvant were used to induce anti-idiotypic antibody (Ab2) in BALB/c mice, and then Sandwich ELISA was used to detect Ab2.
3. The BALB/c mice in which Ab2 had been induced were used as transplantation recipient,the survival time of transplanted graft > delayed-type hypersensitivity^ mixture lymphocytes culture and microlymphocytotoxicity were investigated to observe the effect of Ab2 induction on mice immunoreaction.
Results:
1. Determine the titer of Abl: By using indirect immuno fluorescent staining and complement dependent cytotoxicity test, the titer of Abl was > 1: 400.
2. Determine the titer of Ab2: By using sandwich ELISA , the A^onm of the mice after immuned with Abl-KLH was 1.07 ?.1 3, over two times than that of the contol group which was 0.07 + 0.04.
3. Influence of Ab2 induction on skin graft: The survival time of skin graft of Ab2 induction group is ( 26.5 ?1 .7 ) d, while ( 1 1 .2 ?2. 1 ) d in control group, ( 1 3.4 ?.6 ) d in CsA treated group, the difference was significant (P < 0.01).
4. Influence of Ab2 induction on cardiac tissue transplantation: The survival time of transplantated cardiac tissue in Ab2 induction group is (31.5 ?.7) d, while ( 9.4 ?2.3 ) d in control group, ( 14.7 + 1.6) d in CsA treated group, the difference was significant (P < 0.01).
5. Influence of Ab2 induction on delayed-type hypersensitivity: To compare the thickness of soles, the Ab2 induction group is ( 0.43 ?.17 )mm , while the control group was ( 1 .09 ?0.2 1 ) mm, the difference was significant
6. Influence of Ab2 induction on mixed lymphocytes culture: Asvonm in Ab2 induction group was 0.24?.03,the control group was 0.35 + 0.04, the difference was significant (P < 0.01).
7. Influence of Ab2 serum on microlymphocytotoxicity: the positive cell rate of the group containing Ab2 serum was 32. 5 ?. 5 %, the control
group was 86.7 ?.9%, the difference was significant (P < 0.01). Conclusions:
1. Isotypic heterostrain antibody(Abl) can be successfully raised by immunizing BALB/c mice with C57BL/6 mice splenocytes.
2. Abl-KLH with freund's adjuvant can induce anti-idiotypic antibody (Ab2) in BALB/c mice successfully.
3. Compared with those in cont
的排斥反应成为影响其成功率的主要原因。为了降低排斥反应的发生率，
提高器官移植的成功率，目前需要病人终生使用免疫抑制剂。然而，长期
使用免疫抑制剂除了会引起免疫抑制剂本身的毒副作用（诸如升高血压、
肝肾毒性等不良反应）外，也可引起严重感染、恶性肿瘤的发生，从而影
响器官移植的成功率和受者的长期存活率。若能造成移植宿主对移植抗原
的免疫耐受状态或低免疫应答状态，同时保持对其它抗原免疫应答的反应
能力（包括抗肿瘤免疫和抗感染免疫等），将会避免或减轻这些副作用的
产生，这是目前解决排斥反应的根本途径。我们根据抗独特型抗体的免疫
学机制，运用诱导抗独特型抗体产生的方法，移植前在移植受体体内诱导
其产生与移植物抗原具有相同抗原性的抗独特型的抗体，观察该方法对小
鼠移植排斥反应的抑制作用。
目的：
第四军医人学博上学位论文 中文摘要
本研究通过于移植前在移植受体体内诱导其产生与移植物抗原具有
相同抗原性的抗独特型的抗体（AbZ），观察该方法对小鼠移植免疫耐受
的诱导作用。
方法：
1．抗同种异品系抗体（Ahl）的诱导C57BL／6小鼠作为移植供体，
BALB／C ’J’鼠作为受体。取C57BL／6 ’J’鼠的脾细胞免疫BALB／。小鼠，制
备抗同种异品系抗体…bl卜
2．抗独特型抗体（Ah2）的诱导 取经过上述鉴定的高效价多克隆抗
血清，经饱和硫酸铰盐析法粗提其中的免疫球蛋白，再以DEAE七ephadex
A－50杜层析纯化免疫球蛋白。对Abl和KLH进行交联，联合免疫佐剂
免疫BALB／。小鼠。以夫心ELISA检狈u AbZ。
3．以己诱导出Am的BALB儿小鼠为移植受体，观察AbZ对小鼠移
植物存活时间、单向混合淋巴细胞反应（MLR）、迟发型变态反应（DTH）、
微量淋巴细胞毒等排斥反应指标的影响。
结果：
1．Ahl效价的测定：间接免疫荧光染色和补体依赖细胞毒试验测定
＊hi血清的效价＞1：400。
2．AbZ的检测：用夹心 ELISA检测 AblKLH免疫小鼠的 A45。nm值
为（1．07切．门），较对照组（o．07比D4）高2倍以上。
3．AbZ对皮肤移植物的影响：BALB／c ’J＼鼠移植皮肤后，AbZ诱导组
移植物的存活时间为（26．5士 l、7川，阴性对照组移植物的存活时间为 （ 12
土 2．l）d，CSA组移植物的存活时问为（13．4土 2．6）d。AbZ诱导组移植
物的存活时间与对照组相比较差异显著（P＜0．01人
4．AbZ诱导对心肌移植的影响：心肌组织异位移植后，AbZ诱导组
一5 一
第四车医人学博I：学位论文 中义摘要
移植物存活时间为（31．5士 2．7）d，使用 CsA组移植物存活时间为（14．7
士l．6）d，阴性对照组移植物存活时间为（9．4士2．3）d，AbZ诱导组移植
物存活时间与对照组相比有显著性差异（P＜0刀1）。
5．AbZ对诱导 DTH的影响：AbZ诱导组脚掌的厚度差为（0．43上0．17）
mm，上常 BALB／c ’J’鼠脚掌的厚度差为（1．09士 0．ZI）mm，二者差异显
著（P＜0刀1）。
6．AbZ对混合淋巴细胞培养的影响：AbZ诱导组的人刀加m值为0．24
士0．们，对照组为0．35士0刀4，二者差异显著（P＜0刀1）。
7．AbZ血清对微量淋巴细胞毒的影响：加入AbZ血清的微量淋巳细
胞毒试验着色细胞（损伤细胞）的百分率为 32．5士3．5％，小加 AbZ血清
的对照组着色细胞的百分率为86．7土2．9％，二者比较，差异显著（P＜0刀1）。
结论：
1．C57BL／6小鼠脾细胞免疫BALB／。小鼠，可成功制备抗同种异品
系抗体（Ahl）。
2．Ahl．KLH加弗氏佐剂免疫小鼠可成功地诱导 AbZ产牛。
3．与对照组相比，在受体小鼠体内诱生的AbZ确可延长移植物的存
活时间，说明对排斥反应产生了明显地抑制作用。
4．AbZ诱导对DTH、混合淋巴细胞培养、微量淋巴细胞毒等排斥
反应指标可起到明显的抑制作用。
5．移植前在受体体内诱导产生以移植物抗原为始动抗原的抗独特型
的抗体，叶以有效诱导特异性低反应状态。
Although organ transplantation has become an effective treatment to save lifes and prolong the survival time of patients, the rejection after transplantation is the main cause which affects the final outcome. In order to suppress rejection and increase the survival rate of transplantation, expensive immunosuppressants have to be used in the remainder of patients' life. However, large amount of immunosuppressants can cause not only the side-effects, such as hyperpiesia and hepatorenal toxity, but also severe infection and malignant carcinoma ^specially in a long run. Nowadays the key methods to avoid or alleviate these side-effects are to induce immune tolerance or low immunoreaction state, and sustain the ability of immune response against other antigens (including anticancer, anti-infection and so
on ). According to the theory of immune network, we designed the experiment to induce anti-idiotypic antibodies in recipient which has the same antigenicity as the graft antigens prior to transplantation, and observed the inhibitory effects of anti-idiotypic antibods on the rejection of mice graft.
Aims:
To induce the of anti-idiotypic antibodies in recipient which have the same antigenicity as the graft antigen prior to transplantation, and then to investigate their suppression effects on immune rejection in mice.
Methods:
1. Induction of isotypic heterostrain antibody(Abl) : BALB/c mice were immunized with C57BL/6 splenocytes to raise antibody(Abl) between the two difference strains.
2. Induction of anti-idiotypic antibody (Ab2) : Immunoglobulin was purified grossly from polyclonal antisemm which had been identified with high titer, by saturated ammonium sulfate salting out. Then Immunoglobulin was further purified by DEAE-Sephadex A-50 column. In the experiment, Abl was cross-linked to KLH, Abl-KLH plus freund's adjuvant were used to induce anti-idiotypic antibody (Ab2) in BALB/c mice, and then Sandwich ELISA was used to detect Ab2.
3. The BALB/c mice in which Ab2 had been induced were used as transplantation recipient,the survival time of transplanted graft > delayed-type hypersensitivity^ mixture lymphocytes culture and microlymphocytotoxicity were investigated to observe the effect of Ab2 induction on mice immunoreaction.
Results:
1. Determine the titer of Abl: By using indirect immuno fluorescent staining and complement dependent cytotoxicity test, the titer of Abl was > 1: 400.
2. Determine the titer of Ab2: By using sandwich ELISA , the A^onm of the mice after immuned with Abl-KLH was 1.07 ?.1 3, over two times than that of the contol group which was 0.07 + 0.04.
3. Influence of Ab2 induction on skin graft: The survival time of skin graft of Ab2 induction group is ( 26.5 ?1 .7 ) d, while ( 1 1 .2 ?2. 1 ) d in control group, ( 1 3.4 ?.6 ) d in CsA treated group, the difference was significant (P < 0.01).
4. Influence of Ab2 induction on cardiac tissue transplantation: The survival time of transplantated cardiac tissue in Ab2 induction group is (31.5 ?.7) d, while ( 9.4 ?2.3 ) d in control group, ( 14.7 + 1.6) d in CsA treated group, the difference was significant (P < 0.01).
5. Influence of Ab2 induction on delayed-type hypersensitivity: To compare the thickness of soles, the Ab2 induction group is ( 0.43 ?.17 )mm , while the control group was ( 1 .09 ?0.2 1 ) mm, the difference was significant
6. Influence of Ab2 induction on mixed lymphocytes culture: Asvonm in Ab2 induction group was 0.24?.03,the control group was 0.35 + 0.04, the difference was significant (P < 0.01).
7. Influence of Ab2 serum on microlymphocytotoxicity: the positive cell rate of the group containing Ab2 serum was 32. 5 ?. 5 %, the control
group was 86.7 ?.9%, the difference was significant (P < 0.01). Conclusions:
1. Isotypic heterostrain antibody(Abl) can be successfully raised by immunizing BALB/c mice with C57BL/6 mice splenocytes.
2. Abl-KLH with freund's adjuvant can induce anti-idiotypic antibody (Ab2) in BALB/c mice successfully.
3. Compared with those in cont
引文
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