未成熟树突状细胞诱导小鼠同种异体牙移植免疫耐受的实验研究

发布时间：2018-06-24 01:13

本文选题：同种异体牙移植 + 树突状细胞　；参考：《重庆医科大学》2009年硕士论文

【摘要】： 本研究旨在运用未成熟树突状细胞(immature dendritic cells, imDC)诱导特异性免疫耐受的特性,研究其对小鼠同种异体牙移植后排斥反应的抑制作用。在该研究中建立了小鼠同种异体牙及自体牙异位移植模型,分别通过尾静脉和移植局部注射imDC的方式,将供体小鼠骨髓来源的imDC回输入受体小鼠体内,并与自体牙移植组和未经处理的同种异体牙移植组比较,HE染色和免疫组化法观察不同回输方式的imDC对小鼠同种异体牙移植后排斥反应的抑制效果。研究共分为两个部分,内容如下: 第一部分:不同浓度粒巨噬细胞集落刺激因子在未成熟树突状细胞培养中的效果研究目的:探讨不同浓度细胞因子在培养小鼠骨髓来源未imDC中的作用,寻找培养所需最佳细胞因子浓度。方法:分别用低、中、高剂量粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)联合或不联合白细胞介素4(Interleukin-4, IL-4)培养小鼠骨髓源性imDC,对其进行形态学观察和细胞表型检测。结果:培养第7 d,低剂量组DC数量最少、突起细小,CD80、CD86及MHCII(major histocompatibility complex)类分子表达低,联合IL-4培养可增加DC数量,CD80、CD86及MHCII类分子的表达亦相应升高;中等剂量组与高剂量组DC数量增多,突起较低剂量组更明显,CD80、CD86及MHCII类分子表达也较前者提高,但二组无明显差异,在中等剂量组和高剂量组中,不论是否联合IL-4培养,所诱导出的DC在数量和成熟度上的差异亦不显著。结论:GM-CSF低剂量时,可诱导出纯度较高的imDC,但DC的数量较少,GM-CSF中等剂量和高剂量时,可使DC数量增多,但所诱导出的DC为成熟与未成熟DC的混合物。IL-4仅在GM-CSF低剂量时表现出一定程度增加DC产量和促进DC成熟的作用,当GM-CSF中等剂量和高剂量时,无论是否联合诱导IL-4培养,诱导imDC的产量及纯度无明显差异。第二部分:未成熟树突状细胞作用于小鼠同种异体牙移植排斥反应的效果研究目的:研究采用不同输注方式的供体小鼠骨髓来源imDC对受体小鼠同种异体牙异位移植后排斥反应的抑制效果。方法:建立小鼠自体牙及同种异体牙异位移植模型,运用50U/mlGM-CSF诱导出小鼠骨髓来源imDC,分别于同种异体牙移植术前7d以尾静脉注射方式或同种异体牙移植术后第0、3d以局部注射的方式2*106/只回输入受者体内,并与自体牙移植组和未经处理的同种异体牙移植组比较,组织病理切片和免疫组化观察imDC对小鼠同种异体牙移植后排斥反应的抑制效果。结果:自体牙移植组未见明显排斥反应;未经处理的同种异体牙移植组出现相对明显的排斥反应,表现为移植部位皮肤呈紫红色,耳廓肿胀,皮温较高,组织包裹移植牙松弛,镜下可见大量淋巴细胞浸润,移植牙周围组织血管扩张充血出血,可见大量红细胞;尾静脉回输imDC组排斥反应程度较轻,表现为移植部位充血、可见红色结痂及少量稀薄渗出物,皮温较对侧稍高,组织包裹移植牙不甚紧密,镜下观中性粒细胞及部分淋巴细胞浸润,移植牙周围组织血管可见扩张、充血,出现小范围细胞变性和单核细胞浸润;局部注射imDC组表现与未经处理的同种异体牙移植组类似,出现较明显的排斥反应。对免疫组化结果进行半定量分析表明,自体牙移植组CD4+T细胞和CD8+T细胞的阳性率在所有实验组中为最低;经过尾静脉回输imDC处理的实验组,CD4+T细胞和CD8+T细胞阳性率较局部注射imDC组和异体牙移植组明显降低;但局部注射imDC组和异体牙移植组之间CD4+T细胞和CD8+T细胞阳性率无显著差异。结论:供体骨髓来源的imDC于术前7d经尾静脉回输入受者体内后可减轻受者同种异体牙移植后的排斥反应,但不能完全消除排斥反应,而局部注射供者骨髓来源的imDC在本研究中发现对受体同种异体牙移植后的排斥反应没有明显抑制作用。
[Abstract]:The purpose of this study was to use the characteristics of immature dendritic cells (imDC) to induce specific immune tolerance, and to study the inhibition effect on rejection after allograft tooth transplantation in mice.
In this study, a model of heterotopic transplantation of allogenic teeth and autologous teeth in mice was established. By the way of the tail vein and the local injection of imDC, the imDC of the bone marrow from the donor mice was returned to the recipient mice. Compared with the autologous tooth transplantation group and the untreated allograft group, the HE staining and immunohistochemistry were observed. The inhibitory effect of imDC with different reinfusion methods on rejection after allograft tooth transplantation in mice is divided into two parts.
Part I: effect of granulocyte macrophage colony-stimulating factor on immature dendritic cells cultured in vitro
Objective: To explore the effect of different concentrations of cytokines on the culture of mouse bone marrow, and to find the best cytokine concentration needed for culture. Methods: granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with or not combined with or without interleukin 4 (Interle) 4 (Interle). Ukin-4, IL-4) cultured mouse bone marrow derived imDC. Morphological observation and cell phenotype detection. Results: Seventh D, low dose group DC, CD80, CD86 and MHCII (major histocompatibility complex) molecules are low. The increase in the number of DC in the medium and high dose group, the lower dose group is more obvious, the expression of CD80, CD86 and MHCII is also higher than the former, but there is no significant difference between the two groups. In the medium dose group and the high dose group, no matter whether or not the IL-4 culture is combined, the differences in the quantity and maturity of the induced DC are not significant. Conclusion: GM-C At low doses of SF, a higher purity of imDC can be induced, but the number of DC is less, the number of DC can be increased when the GM-CSF is medium and high, but the induced DC is a mixture of mature and immature DC,.IL-4 only increases DC production and promotes DC maturation at a low dose of GM-CSF, when GM-CSF medium and high doses are used. No significant difference was found in the yield and purity of imDC induced by IL-4 culture.
The second part: the effect of immature dendritic cells on allograft rejection in mice.
Objective: To study the inhibitory effect of bone marrow derived imDC from donor mice with different infusion methods on rejection of allogenic tooth allograft in recipient mice. Methods: to establish autologous teeth and allograft model of allogenic teeth in mice, and to induce imDC in mouse bone marrow by 50U/mlGM-CSF, and 7d before allograft. 2*106/ only returned to the recipient after the tail vein injection or the local injection of 0,3d, and compared with the autologous tooth transplantation group and the untreated allograft group. Histopathology and immunohistochemistry were used to observe the inhibitory effect of imDC on the rejection after allograft tooth transplantation. There was no obvious rejection in the autologous tooth transplantation group, and the untreated allograft group had a relatively obvious rejection reaction, which showed that the skin of the transplanted site was purple red, the auricle swelled, the skin temperature was high, the tissue parcel was relaxed, a large number of lymphocytes were soaked in the microscope, and the blood vessels around the transplanted teeth were dilated and bleed. A large number of red blood cells; the rejection in the imDC group of the tail vein transfusion was light, showing the hyperemia of the transplant site, the red scab and a small amount of exudate, the skin temperature was slightly higher than the opposite side, the tissue wrapped in the transplanted teeth was not very close, the neutrophils and some lymphocytes infiltrated under the microscope, and the tissue vessels around the transplanted teeth were dilated, congested and small. The local injection of imDC was similar to the untreated group of allograft teeth, and there was a significant rejection. Semi quantitative analysis of the immunohistochemical results showed that the positive rate of CD4+T and CD8+T cells in the autologous tooth transplantation group was the lowest in all the experimental groups; through the caudal vein. The positive rate of CD4+T cells and CD8+T cells in the experimental group treated with imDC was significantly lower than that in the local imDC group and the allograft group, but there was no significant difference in the positive rate of CD4+T and CD8+T cells between the local injection of imDC and the allograft teeth. Conclusion: the imDC of the donor bone marrow derived from the tail vein of the donor bone marrow was entered into the recipient's body before the operation. The rejection reaction after the allograft tooth transplantation was alleviated, but the rejection reaction was not completely eliminated, and the local injection of donor bone marrow imDC in this study found no significant inhibitory effect on rejection after the allograft tooth allograft.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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