新西兰雌—雄兔皮肤移植模型供体特异性体内细胞毒性试验方法的建立
发布时间：2018-07-16 16:02
【摘要】：[研究背景] 器官移植后必需应用免疫抑制剂防止急性排斥反应的发生,免疫抑制剂通过抑制T细胞和B细胞等靶细胞的细胞增殖、细胞因子合成而起作用。但是目前仍然无法区分移植抗原引起的免疫排斥反应和病毒细菌感染引起的免疫反应。理想的免疫抑制治疗应该在免疫抑制和保持免疫力间取得平衡,因此对于临床医生来说移植患者管理是一件很困难的事。保存受体免疫力,尽可能减小免疫抑制剂用量或诱导免疫耐受一直是移植免疫学家的主要目标,这方面虽然也取得了一些令人兴奋的成果,但是诱导耐受仍然有大量的工作要做,研究诱导免疫耐受方法,尽可能减小免疫抑制的用量,研究检测免疫耐受及免疫力的免疫学方法将是今后几年器官移植所面临的最重要的挑战。 可靠的免疫状态指标可以为免疫抑制剂的使用提供依据,使抑制剂不仅可以根据排斥反应的程度或耐受情况使用,还可以根据患者的易感性规范化用药,由于长期免疫抑制剂治疗所引起的的高发病率和高死亡率,高度期望规范化用药能够实现。但是,尽管通过血液标本或移植物病理分析检测免疫功能已经可以部分了解宿主抗移植物排斥反应,包括了解浸润细胞的表型及功能,T细胞性质的改变、细胞因子及抗体的合成情况等,但是仍然没有一种可靠的特异的排斥反应检测方法。目前临床常用受体免疫状态的监测的方式仍然是临床症状评估结合移植物活检。但是,活组织病理检查对移植物和宿主均是一种损伤性检查,有其固有的局限性。 现在有学者开始供体特异性体内细胞毒性试验的研究,用带有移植物复合抗原的供体细胞检测受体的免疫杀伤强度,供体细胞在受体内受到的不仅是毒性淋巴细胞的攻击,而且同时要受到抗体、补体、毒性淋巴细胞的多重攻击,因此对移植复合抗原的定量测试不但能反映特异性CTL的活性,而且能全面的反映受体对供体的特异性免疫状态。 本课题设计了一种简单的方法,在新西兰白兔上开展供体特异性体内细胞毒性试验,探索兔体内细胞毒性试验方法建立的技术参数,研究体内细胞毒性试验能否检测出受体的免疫排斥状况。用CFSE标记供体脾细胞,流式细胞仪检测供体细胞在受体内遭受排斥反应过程,同时设置受体脾细胞作为内参照。标记的脾细胞悬液输注给不同的受体,输注后不同时间点采血进行流式细胞分析。我们发现外周血中标记的受体细胞能在8小时内被流式细胞仪检测得到,而不同的主要和次要组织相容性抗原的供体细胞在试验中消失与皮肤移植后排斥动力学相一致。 [目的] 建立新西兰白兔供体特异性体内细胞毒性试验方法,探索细胞毒性试验检测的技术参数、羟基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)荧光染色条件、输注细胞数及确定最佳测试时间点,验证体内细胞毒性实验能否检测出受体的免疫排斥状况,为供体特异性体内细胞毒性试验的临床应用提供新的依据。 [方法] 取4-6月龄清洁级雌、雄新西兰白兔各5只,雌、雄兔各根据体重排序,根据序号雌雄兔配对,雌兔为供体,雄兔为受体,于每只雌兔背部剪下约4cm×2cm的皮肤一块,于雄兔背部移植雌兔皮肤,并用3/0无创伤缝线固定皮缘,移植后2周,移植皮肤完全脱落,建立新西兰白兔雌-雄兔皮肤移植模型,为模型组。另取未作移植的雌、雄新西兰白兔各5只,雌、雄兔各根据体重排序,根据序号雌雄兔配对,为对照组。分别于模型组及对照组配对取雌兔及雄兔脾脏,放入Hank's液中,200目不锈钢网研磨法制备脾细胞悬液,7.5g/L氯化铵裂解红细胞,0.01mol/L PBS稀释制备脾细胞单细胞悬液。雄性脾细胞用0.24μm CFSE染色,雌性脾细胞用高浓度6μm CFSE染色。每对雌、雄兔脾细胞悬液混合,制备1：1混合细胞悬液。细胞混合悬液稀释后,取1滴稀释液和1滴0.5%锥虫蓝溶液混合,静置3min,加至细胞计数板,倒置显微镜下观察,活细胞不着色,死细胞核成蓝色,计数活细胞和死细胞数量,活细胞比例达95%以上(细胞存活率=0.5%锥虫蓝染色阴性细胞/100个细胞/HP×100)。于模型组、对照组雄免输注细胞悬液,注射混合细胞总数为1×109个左右。雄兔输注混合细胞后分别于1,2,4,8h抽取外周血,流式细胞仪检测外周血两种荧光细胞的比例变化,计算供体特异性细胞杀伤率(R)=1-donor cells/recipient cells×100%,比较模型组与对照组的供体特异性细胞杀伤率差异。实验数据应用SPSS13.0软件进行统计学分析,所有数据示为x±s,组间杀伤率比较用t检验,P0.05认为差异有显著性意义。 [结果] 1.实验动物数量分析模型组及对照组新西兰白兔各10只,每组分别5对雌、雄兔。全部进入结果分析,无脱失。 2.皮肤移植模型观察结果皮肤移植后15d,移植皮肤完全脱落,雌-雄兔皮肤移植排斥模型建立,皮肤病理证实。 3. CFSE染色混合脾细胞悬液集中在淋巴细胞所在的小细胞区,单核细胞区也有一部分阳性细胞,但是染色不均一,选用小淋巴细胞区作为检测门。CFSE染色时需要较大浓度差才能完全把两群脾细胞完全分离开,在浓度相差25倍时流式图才显示为两组独立的点图,即CFSE低浓度用0.24μM,高浓度用6μM。 4.流式细胞学分析结果CFSE染色后的混合细胞在输注前供体、受体细胞各为49.79%及49.85%。移植模型组供体雌兔脾细胞在输注后1h剩4.73%,在2-4h后剩0.30%,8h后基本清除,而同基因的雄兔脾细胞同对照组一样缓慢下降。模型组供体特异性细胞杀伤率(R)在混合细胞输注后1h为48.26%+2.31%,2h为64.18%±6.23%,4h为73.41%±3.87%,8h为89.66%±6.96%。而对照组杀伤率在混合细胞输注后1h为5.49%±2.51%,2h为13.23%±2.23%,4h为21.53%±2.23%,8h为31.51%±3.21%。在混合细胞输注后的1h、2h、4h、8h,各时间点模型组供体特异性细胞杀伤率(R)明显高于对照组,差异有统计学意义(t=25.284,13.624,24.215,15.146,所有P0.01)。 [结论] 新西兰雌-雄兔皮肤移植模型体内抗原特异性细胞毒性试验流式检测设定在淋巴细胞门,两种细胞CFSE的染色浓度差控制在25倍左右,输注细胞1,2h模型组杀伤率高达48.26%±2.31%,64.18%±6.23%,选择这一时间点可以准确判定两组白兔所处的免疫状态,且能满足临床要求的快速检测的目的。供体特异性体内细胞毒性试验的特点有： 1、供体特异性体内细胞毒性试验是体内试验,不用模拟体内复杂的免疫环境,本身就是移植物所处的免疫环境。 2、检测方法准确简单,应用荧光标记流式细胞技术检测,可准确到单个细胞水平。 3、检测靶细胞为供体来源细胞(脾细胞),抗原性与移植物相同,为复合抗原,容易获得,能准确反映移植物所受免疫攻击情况。 4、内参照细胞为受体来源同基因细胞(脾细胞),控制了试验偶然误差和系统误差；内参照细胞在体外处理及体内免疫反应的全过程始终与试验靶细胞处于同样的反应体系中,消除了由于操作及体内非特异性反应所致的误差。
[Abstract]:BACKGROUND OF THE STUDY
Immunosuppressive therapy should be used to prevent acute rejection after organ transplantation , and the immune inhibitor can play a role in inhibiting cell proliferation and cytokine synthesis of target cells such as T cells and B cells . However , it is difficult to differentiate between immune rejection caused by transplantation antigen and immune response caused by virus bacterial infection .
The reliable immune status indicator can provide a basis for the use of an immunosuppressive agent , so that the inhibitor can be used not only according to the degree or tolerance of the rejection reaction , but also can be realized according to the susceptibility of the patient .
At present , some scholars begin to study the cytotoxicity test of donor - specific in vivo , and use donor cells with graft composite antigen to detect the immune killing intensity of the receptor . The donor cells are not only attacked by toxic lymphocytes in the body , but also can be subjected to multiple attacks of antibodies , complement and toxic lymphocytes , so that the quantitative test of the grafted composite antigen can not only reflect the activity of the specific CTL , but also can comprehensively reflect the specific immune status of the receptor to the donor .
In this study , a simple method was designed to study the cytotoxicity of donor - specific in vivo in New Zealand white rabbits . The technique parameters established in vivo cytotoxicity test were explored . The donor spleen cells were labeled with CFSE . The donor spleen cells were detected by flow cytometry .
Purpose of the project
New Zealand white rabbit donor specific in vivo cytotoxicity test method was established to explore the technical parameters of cytotoxicity test , the fluorescent staining conditions of hydroxyfluorescein acetoacetic acid succinimide ester ( CFSE ) , the number of infusion cells and the determination of the optimal test time point , to verify whether the in vivo cytotoxicity test can detect the immune rejection condition of the receptor , and provide a new basis for clinical application of the donor specific in vivo cytotoxicity test .
Methodology
Male and female rabbits were divided into 4 - 6 months old female and male New Zealand white rabbits according to their weight . The male and female rabbits were divided into two groups according to the weight of the male and female rabbits .
The result is not valid .
1 . There were 10 rabbits in the model group and control group , 5 pairs of female and male rabbits in each group . All the results were analyzed without loss of loss .
2 . After skin transplantation , 15 days after skin transplantation , the skin was completely peeled off , and the female - male rabbit skin graft rejection model was established , and the skin pathology was confirmed .
3 . CFSE staining mixed spleen cell suspension concentrated in the small cell area in which the lymphocytes were located , but the mononuclear cell area also had some positive cells , but the staining was not uniform , and the small lymphocyte region was used as the detection gate . When CFSE staining , the two groups of spleen cells were completely separated , and the flow graph was shown as two groups of independent point graphs at 25 times the concentration of CFSE , namely , the concentration of CFSE was 0.24 . m u.M and the high concentration was 6 . m
4 . The results of flow cytometry analysis showed that the donor and recipient cells were 49.79 % and 49.85 % after the infusion of the mixed cells . The total cell killing rate ( R ) of the donor - specific cells in the model group was 48.26 % + 2.31 % , 2h was 64.18 % 卤 6.23 % , 2h was 13.23 % 卤 3.87 % , 8h was 89.66 % 卤 6.96 % . The difference was statistically significant ( t = 25.284 , 13.624 , 24.215 , 15.146 , all P0.01 ) .
Conclusion
in New Zealand female - male rabbit skin transplantation model in vivo antigen - specific cytotoxicity assay was set up in lymphocyte door , and that difference of staining concentration of two cell CFSE was controlled at about 25 times , and the killing rate of the two groups of cells was up to 48.26 % 卤 2.31 % , 64.18 % 卤 6.23 % .
1 . The cytotoxicity test of donor - specific in - vivo cytotoxicity test is in vivo test without simulating the complex immune environment in vivo , which is the immune environment at which the graft is located .
and 2 , the detection method is accurate and simple , and the detection of the fluorescence labeled flow cytometry can be applied to the single cell level accurately .
3 . The detection target cells are donor - derived cells ( spleen cells ) , the antigenicity of which is the same as that of the graft , is a composite antigen , is easy to obtain , and can accurately reflect the immune attack condition of the graft .
4 . The internal reference cell is the receptor - derived co - gene cell ( spleen cell ) , which controls the experimental error and systematic error .
The internal reference cell is always in the same reaction system as the test target cell in the whole process of in vitro treatment and in vivo immune response , and the error caused by the operation and the nonspecific reaction in vivo is eliminated .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2126898
[Abstract]:BACKGROUND OF THE STUDY
Immunosuppressive therapy should be used to prevent acute rejection after organ transplantation , and the immune inhibitor can play a role in inhibiting cell proliferation and cytokine synthesis of target cells such as T cells and B cells . However , it is difficult to differentiate between immune rejection caused by transplantation antigen and immune response caused by virus bacterial infection .
The reliable immune status indicator can provide a basis for the use of an immunosuppressive agent , so that the inhibitor can be used not only according to the degree or tolerance of the rejection reaction , but also can be realized according to the susceptibility of the patient .
At present , some scholars begin to study the cytotoxicity test of donor - specific in vivo , and use donor cells with graft composite antigen to detect the immune killing intensity of the receptor . The donor cells are not only attacked by toxic lymphocytes in the body , but also can be subjected to multiple attacks of antibodies , complement and toxic lymphocytes , so that the quantitative test of the grafted composite antigen can not only reflect the activity of the specific CTL , but also can comprehensively reflect the specific immune status of the receptor to the donor .
In this study , a simple method was designed to study the cytotoxicity of donor - specific in vivo in New Zealand white rabbits . The technique parameters established in vivo cytotoxicity test were explored . The donor spleen cells were labeled with CFSE . The donor spleen cells were detected by flow cytometry .
Purpose of the project
New Zealand white rabbit donor specific in vivo cytotoxicity test method was established to explore the technical parameters of cytotoxicity test , the fluorescent staining conditions of hydroxyfluorescein acetoacetic acid succinimide ester ( CFSE ) , the number of infusion cells and the determination of the optimal test time point , to verify whether the in vivo cytotoxicity test can detect the immune rejection condition of the receptor , and provide a new basis for clinical application of the donor specific in vivo cytotoxicity test .
Methodology
Male and female rabbits were divided into 4 - 6 months old female and male New Zealand white rabbits according to their weight . The male and female rabbits were divided into two groups according to the weight of the male and female rabbits .
The result is not valid .
1 . There were 10 rabbits in the model group and control group , 5 pairs of female and male rabbits in each group . All the results were analyzed without loss of loss .
2 . After skin transplantation , 15 days after skin transplantation , the skin was completely peeled off , and the female - male rabbit skin graft rejection model was established , and the skin pathology was confirmed .
3 . CFSE staining mixed spleen cell suspension concentrated in the small cell area in which the lymphocytes were located , but the mononuclear cell area also had some positive cells , but the staining was not uniform , and the small lymphocyte region was used as the detection gate . When CFSE staining , the two groups of spleen cells were completely separated , and the flow graph was shown as two groups of independent point graphs at 25 times the concentration of CFSE , namely , the concentration of CFSE was 0.24 . m u.M and the high concentration was 6 . m
4 . The results of flow cytometry analysis showed that the donor and recipient cells were 49.79 % and 49.85 % after the infusion of the mixed cells . The total cell killing rate ( R ) of the donor - specific cells in the model group was 48.26 % + 2.31 % , 2h was 64.18 % 卤 6.23 % , 2h was 13.23 % 卤 3.87 % , 8h was 89.66 % 卤 6.96 % . The difference was statistically significant ( t = 25.284 , 13.624 , 24.215 , 15.146 , all P0.01 ) .
Conclusion
in New Zealand female - male rabbit skin transplantation model in vivo antigen - specific cytotoxicity assay was set up in lymphocyte door , and that difference of staining concentration of two cell CFSE was controlled at about 25 times , and the killing rate of the two groups of cells was up to 48.26 % 卤 2.31 % , 64.18 % 卤 6.23 % .
1 . The cytotoxicity test of donor - specific in - vivo cytotoxicity test is in vivo test without simulating the complex immune environment in vivo , which is the immune environment at which the graft is located .
and 2 , the detection method is accurate and simple , and the detection of the fluorescence labeled flow cytometry can be applied to the single cell level accurately .
3 . The detection target cells are donor - derived cells ( spleen cells ) , the antigenicity of which is the same as that of the graft , is a composite antigen , is easy to obtain , and can accurately reflect the immune attack condition of the graft .
4 . The internal reference cell is the receptor - derived co - gene cell ( spleen cell ) , which controls the experimental error and systematic error .
The internal reference cell is always in the same reaction system as the test target cell in the whole process of in vitro treatment and in vivo immune response , and the error caused by the operation and the nonspecific reaction in vivo is eliminated .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前3条
1 张明明;周佩军;邵琨;王祥慧;徐达;;转移性体内迟发型超敏反应监测法的建立及临床应用[J];上海交通大学学报(医学版);2010年09期
2 蒋泽生;高毅;;荧光染料CFSE差异浓度标记活细胞体内示踪方法[J];中国组织工程研究与临床康复;2010年15期
3 蒋泽生;高毅;;供体特异性体内免疫状态的定量检测[J];中国组织工程研究与临床康复;2010年31期
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