本文關(guān)鍵詞： 皮膚移植 母胎免疫 HO-1 小鼠　出處：《浙江大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 高效非特異性免疫抑制劑的迅速發(fā)展和臨床廣泛應(yīng)用使同種器官移植取得了長(zhǎng)足的進(jìn)步,急性排斥率明顯降低,器官短期存活率顯著提高。但是數(shù)十年來,由于慢性排斥和免疫抑制劑的毒副作用,移植器官的長(zhǎng)期存活率仍無明顯改善。因此誘導(dǎo)特異性移植耐受,預(yù)防急慢性排斥反應(yīng),減少藥物的毒副作用,避免長(zhǎng)期服用免疫抑制劑就成為解決移植器官長(zhǎng)期存活的關(guān)鍵問題。 Medawar在1953年提出胎兒是類似于母體的同種異體移植物這個(gè)概念來闡釋母體與胎兒之間的免疫關(guān)系。在妊娠過程中母體免疫系統(tǒng)必須對(duì)持續(xù)表達(dá)父源性抗原的同種異基因胎兒保持耐受而不影響母體抗感染的免疫反應(yīng)。所以認(rèn)為妊娠過程是處于免疫耐受狀態(tài)。在人類發(fā)現(xiàn)許多自身免疫性疾病如類風(fēng)濕關(guān)節(jié)炎、多發(fā)性硬化等患者在妊娠時(shí)期癥狀有所緩解而在分娩后復(fù)發(fā),并且在相關(guān)動(dòng)物實(shí)驗(yàn)中也有類似發(fā)現(xiàn)。因此,我們希望通過觀察妊娠過程中進(jìn)行外周器官移植是否可以延長(zhǎng)移植物的存活時(shí)間來評(píng)價(jià)妊娠過程是否對(duì)于外周器官移植具有保護(hù)作用。 本研究旨在通過提取妊娠小鼠與非妊娠小鼠脾中的單個(gè)核細(xì)胞與父系雄鼠的脾細(xì)胞進(jìn)行混合淋巴細(xì)胞反應(yīng)來評(píng)價(jià)妊娠時(shí)期母體外周細(xì)胞免疫反應(yīng)的變化;通過在妊娠初期的小鼠背部移植父系雄鼠的皮膚來觀察妊娠是否能夠?qū)ν庵芤浦财鞴倨鸨Ｗo(hù)作用;并且通過比較妊娠前后脾與子宮HO-1 mRNA表達(dá)的差異來初步探討妊娠過程中保護(hù)性分子HO-1在外周器官脾以及與胎兒直接接觸的子宮的變化差異。 實(shí)驗(yàn)方法 采用清潔級(jí)雌性健康小鼠C57BL/6(H2~b)54只,雄性小鼠BALB/c(H2~d)24只,體重為18——23g。先通過6只C57BL/6小鼠互相行同系皮膚移植來建立皮膚移植模型。將48只C57BL/6雌鼠隨機(jī)平均分成細(xì)胞反應(yīng)組與皮膚移植組,其中每小組又分為妊娠組(Pre)與空白對(duì)照組(N-Pre),每組12只。妊娠組小鼠與BALB/c雄性小鼠同籠使之懷孕。細(xì)胞反應(yīng)組小鼠于孕14天被處死,提取脾臟PBMC與BALB/c雄鼠脾細(xì)胞進(jìn)行單向混合淋巴細(xì)胞反應(yīng),通過計(jì)算刺激指數(shù)(Stimulating Index,SI)來評(píng)價(jià)其細(xì)胞免疫反應(yīng)的強(qiáng)度;同時(shí)取脾、子宮組織約100 mg,無菌PBS清洗,-80℃保存,通過RT-PCR檢測(cè)評(píng)價(jià)妊娠與非妊娠小鼠外周(脾)與母胎接觸面(子宮)HO-1mRNA表達(dá)的差異,通過計(jì)算Ct值的差異來衡量;皮膚移植組小鼠于孕6天接受來自于BALB/c雄鼠的皮膚移植,用平均存活時(shí)間(mean survival time,MST)來進(jìn)行比較。統(tǒng)計(jì)分析用SPSS13.0統(tǒng)計(jì)軟件完成。 結(jié)果 BALB/c雄鼠的脾細(xì)胞分別與妊娠組與非妊娠對(duì)照組C57BL/6雌鼠的外周單個(gè)核細(xì)胞與進(jìn)行混合培養(yǎng),刺激效應(yīng)細(xì)胞增殖程度的差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。同系C57BL/6小鼠皮膚移植皮片平均存活時(shí)間＞30天,同種異基因皮膚移植中對(duì)照組(未妊娠組)小鼠移植皮片平均存活時(shí)間為(7.08±1.08)d,妊娠組的皮片平均存活時(shí)間為(7.67±0.89)d,移植皮片存活時(shí)間沒有出現(xiàn)明顯延長(zhǎng),差別不具有統(tǒng)計(jì)學(xué)意義。用實(shí)時(shí)熒光定量PCR的方法對(duì)妊娠組與非妊娠組脾與子宮HO-1 mRNA表達(dá)差異進(jìn)行定量檢測(cè),以管家基因GAPDH作為對(duì)照進(jìn)行比較。結(jié)果顯示妊娠組脾臟HO-1 mRNA表達(dá)水平較非妊娠組增加了(1.56±0.05)倍,而妊娠組的子宮HO-1 mRNA的表達(dá)水平則增加了(2.21±0.03)倍。妊娠組子宮中HO-1 mRNA表達(dá)的升高程度較脾中的升高更為顯著,差異具有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 結(jié)論 1、小鼠妊娠組母體外周細(xì)胞免疫反應(yīng)并未比非妊娠組有所減弱 2、在小鼠妊娠早期進(jìn)行的皮膚移植與未妊娠小鼠相比移植物的存活時(shí)間沒有出現(xiàn)明顯改變 3、妊娠組小鼠外周器官脾臟與母胎直接接觸的器官子宮的保護(hù)性因子HO-1 mRNA表達(dá)水平與非妊娠組相比明顯升高,并且妊娠組子宮HO-1mRNA表達(dá)升高的程度明顯高于脾臟。因此小鼠妊娠對(duì)來自父系的同種異基因皮膚移植不具有保護(hù)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R714;R392

【共引文獻(xiàn)】

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相關(guān)碩士學(xué)位論文 前3條

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