妊娠对小鼠皮肤移植的影响及可能机制的初步探索

发布时间：2018-02-14 11:12

本文关键词： 皮肤移植母胎免疫 HO-1 小鼠　出处：《浙江大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的高效非特异性免疫抑制剂的迅速发展和临床广泛应用使同种器官移植取得了长足的进步,急性排斥率明显降低,器官短期存活率显著提高。但是数十年来,由于慢性排斥和免疫抑制剂的毒副作用,移植器官的长期存活率仍无明显改善。因此诱导特异性移植耐受,预防急慢性排斥反应,减少药物的毒副作用,避免长期服用免疫抑制剂就成为解决移植器官长期存活的关键问题。 Medawar在1953年提出胎儿是类似于母体的同种异体移植物这个概念来阐释母体与胎儿之间的免疫关系。在妊娠过程中母体免疫系统必须对持续表达父源性抗原的同种异基因胎儿保持耐受而不影响母体抗感染的免疫反应。所以认为妊娠过程是处于免疫耐受状态。在人类发现许多自身免疫性疾病如类风湿关节炎、多发性硬化等患者在妊娠时期症状有所缓解而在分娩后复发,并且在相关动物实验中也有类似发现。因此,我们希望通过观察妊娠过程中进行外周器官移植是否可以延长移植物的存活时间来评价妊娠过程是否对于外周器官移植具有保护作用。本研究旨在通过提取妊娠小鼠与非妊娠小鼠脾中的单个核细胞与父系雄鼠的脾细胞进行混合淋巴细胞反应来评价妊娠时期母体外周细胞免疫反应的变化;通过在妊娠初期的小鼠背部移植父系雄鼠的皮肤来观察妊娠是否能够对外周移植器官起保护作用;并且通过比较妊娠前后脾与子宫HO-1 mRNA表达的差异来初步探讨妊娠过程中保护性分子HO-1在外周器官脾以及与胎儿直接接触的子宫的变化差异。实验方法采用清洁级雌性健康小鼠C57BL/6(H2~b)54只,雄性小鼠BALB/c(H2~d)24只,体重为18——23g。先通过6只C57BL/6小鼠互相行同系皮肤移植来建立皮肤移植模型。将48只C57BL/6雌鼠随机平均分成细胞反应组与皮肤移植组,其中每小组又分为妊娠组(Pre)与空白对照组(N-Pre),每组12只。妊娠组小鼠与BALB/c雄性小鼠同笼使之怀孕。细胞反应组小鼠于孕14天被处死,提取脾脏PBMC与BALB/c雄鼠脾细胞进行单向混合淋巴细胞反应,通过计算刺激指数(Stimulating Index,SI)来评价其细胞免疫反应的强度;同时取脾、子宫组织约100 mg,无菌PBS清洗,-80℃保存,通过RT-PCR检测评价妊娠与非妊娠小鼠外周(脾)与母胎接触面(子宫)HO-1mRNA表达的差异,通过计算Ct值的差异来衡量;皮肤移植组小鼠于孕6天接受来自于BALB/c雄鼠的皮肤移植,用平均存活时间(mean survival time,MST)来进行比较。统计分析用SPSS13.0统计软件完成。结果 BALB/c雄鼠的脾细胞分别与妊娠组与非妊娠对照组C57BL/6雌鼠的外周单个核细胞与进行混合培养,刺激效应细胞增殖程度的差异无统计学意义(P＞0.05)。同系C57BL/6小鼠皮肤移植皮片平均存活时间＞30天,同种异基因皮肤移植中对照组(未妊娠组)小鼠移植皮片平均存活时间为(7.08±1.08)d,妊娠组的皮片平均存活时间为(7.67±0.89)d,移植皮片存活时间没有出现明显延长,差别不具有统计学意义。用实时荧光定量PCR的方法对妊娠组与非妊娠组脾与子宫HO-1 mRNA表达差异进行定量检测,以管家基因GAPDH作为对照进行比较。结果显示妊娠组脾脏HO-1 mRNA表达水平较非妊娠组增加了(1.56±0.05)倍,而妊娠组的子宫HO-1 mRNA的表达水平则增加了(2.21±0.03)倍。妊娠组子宫中HO-1 mRNA表达的升高程度较脾中的升高更为显著,差异具有统计学意义(P＜0.05)。结论 1、小鼠妊娠组母体外周细胞免疫反应并未比非妊娠组有所减弱 2、在小鼠妊娠早期进行的皮肤移植与未妊娠小鼠相比移植物的存活时间没有出现明显改变 3、妊娠组小鼠外周器官脾脏与母胎直接接触的器官子宫的保护性因子HO-1 mRNA表达水平与非妊娠组相比明显升高,并且妊娠组子宫HO-1mRNA表达升高的程度明显高于脾脏。因此小鼠妊娠对来自父系的同种异基因皮肤移植不具有保护作用。
[Abstract]:objective
The rapid development of non specific immune inhibitors and clinical application to renal transplantation has made considerable progress, the acute rejection rate decreased significantly, the survival rate of organ short-term increased significantly. But decades, due to chronic rejection and immunosuppressive, organ transplant long-term survival has not been improved accordingly. Tolerance induction, prevention of acute and chronic rejection, reduce the side effects of drugs, avoid long-term use of immunosuppressive agents becomes the key problem of long-term survival of transplanted organs.
Medawar in 1953 is similar to the maternal fetal allograft to explain this concept between the maternal and fetal immune relationship. During pregnancy on the maternal immune system must be sustained expression of paternal antigen of allogeneic fetal maintain tolerance without affecting the maternal immune responses against infections. So it is in the process of pregnancy immune tolerance. It is found that many autoimmune diseases such as rheumatoid arthritis in humans, patients with multiple sclerosis during pregnancy symptoms eased and relapse after childbirth, and there are similar to those found in related animal experiment. Therefore, we hope that the peripheral organ transplantation can prolong the survival time of the graft. The evaluation process of pregnancy has protective effect for peripheral organ transplantation by observing the process of pregnancy.
The purpose of this study is to change through the extraction of pregnant mice and non mouse spleen mononuclear cells of male rats and paternal pregnancy spleen cells mixed lymphocyte reaction to evaluate the pregnancy period of maternal cellular immunity; in early pregnancy mice back skin transplantation paternal male rats to observe the protective effect of pregnancy to peripheral transplantation organ; and by comparing the differences before and after pregnancy and uterine HO-1 spleen mRNA expression to investigate molecular HO-1 in peripheral organs spleen and changes in direct contact with the fetal uterus during pregnancy. The difference of protection
Experimental method
The clean grade healthy female mice C57BL/6 (H2~b) 54, BALB/c male mice (H2~d) 24, the weight of 18 23g. by 6 C57BL/6 mice for each other was made skin transplantation model. 48 female C57BL/6 rats were randomly divided into cell reaction group and skin transplantation group, each group divided into pregnancy group (Pre) and control group (N-Pre), 12 rats in each group. Pregnant group were mated with BALB/c male mice. The mice were sacrificed on the cell response group pregnant 14 days, PBMC from spleen and spleen cells of BALB/c mice by mixed lymphocyte reaction, by calculating the stimulation index (Stimulating Index, SI) to evaluate the cellular immune response of spleen strength; at the same time, the uterus is about 100 mg, aseptic PBS cleaning, -80 C preservation, by RT-PCR detection and evaluation of pregnant women and non pregnant mouse peripheral (spleen) contact with the maternal (uterine) HO-1mRN The difference of A expression was calculated by calculating the difference of Ct value. Skin transplantation group received skin transplantation from BALB/c male rats on the 6 day of pregnancy, and the average survival time (mean survival time, MST) was compared. Statistical analysis was done by SPSS13.0 statistical software.
Result
BALB/c male rats with spleen cells respectively in pregnancy group and non pregnancy control group C57BL/6 female rats of peripheral mononuclear cells and the mixed culture, stimulate the effect degree of cell proliferation was not statistically significant (P > 0.05). Male C57BL/6 mice skin graft, the average survival time of 30 days, the allogeneic skin transplantation in the control group (non pregnancy group) mice skin graft average survival time was (7.08 + 1.08) d, skin pregnancy group the average survival time was (7.67 + 0.89) d, skin graft survival time did not appear significantly prolonged, the difference has statistical significance. Using real-time quantitative PCR to pregnant group and non pregnant group spleen and uterine HO-1 mRNA expression were detected, the housekeeping gene GAPDH as control for comparison. The results showed that the expression level of spleen HO-1 mRNA pregnancy group than non pregnancy group increased (1.56 + 0.05) times, and pregnancy The expression level of HO-1 mRNA in the uterus increased by (2.21 + 0.03) times. The increase of HO-1 mRNA expression in the uterus of pregnant group was more significant than that in the spleen, and the difference was statistically significant (P < 0.05).
conclusion
1, the immune response of the maternal peripheral cell in the pregnant group was not less than that in the non pregnant group.
2, there was no significant change in the survival time of the graft compared with the non pregnant mice during the early stage of the skin transplantation in mice.
3, the expression level of mRNA protection factor HO-1 pregnant mice peripheral organs spleen and fetal organs in direct contact with the uterus and non pregnancy group increased significantly, and the expression of HO-1mRNA increased the uterine pregnancy group was obviously higher than that of the spleen. It does not have protective effects on the pregnant mice from parent allogeneic skin transplantation.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R714;R392

【共引文献】