IDO基因转染组织工程化软骨细胞诱导T细胞免疫耐受的研究

发布时间：2018-05-20 08:48

本文选题：组织工程 + 软骨细胞　；参考：《第四军医大学》2008年硕士论文

【摘要】： 软骨缺损的修复是临床一大难题,由于自体软骨移植来源有限,组织工程化软骨移植将是解决损伤软骨修复的重要途径,然而,组织工程化软骨具有免疫原性。本课题拟通过转染吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase, IDO)基因以达到改变软骨细胞移植后局部T细胞免疫应答负调的目的,诱导移植受体的T细胞对同种异体组织工程化软骨移植物的免疫耐受,为发展转基因方法预防临床同种异体移植后自身免疫排斥的技术奠定基础,实现组织工程化的种子细胞能用于不同免疫遗传背景的患者,克服MHC(major histocompatibility complex,主要组织相容性复合物)的多态性和多基因性所形成的同种异体间的免疫屏障,达到组织工程化种子细胞的“通用化”,开辟了从移植物局部解决免疫排斥的新途径。主要内容及结果如下: (一)小鼠软骨细胞的分离和培养 1.C57小鼠关节软骨的获取:体外经0.2%Ⅱ型胶原酶37℃消化3～4 h后离心,收集可以获取供实验用的大量软骨细胞,台盼蓝染色消化法获取的软骨细胞活力在95%以上。 2.原代培养的软骨细胞,MTT法检测软骨细胞生长曲线,发现软骨细胞贴壁后于第2～5 d进入指数生长期,7 d左右可以铺满瓶底,但是随着细胞传代次数的增加,细胞形成一代的时间明显延长。 3.软骨细胞分泌的细胞外基质,对甲苯胺蓝染色呈明显蓝色异染反应,本实验获取的软骨细胞经甲苯胺蓝染色后,发现细胞纯度在85%以上,但是随着传代次数增多软骨细胞分泌细胞外基质的能力下降,因此,选择第2、3代软骨细胞是用于实验的最佳时期。 (二)软骨细胞pEGFP-N1-IDO质粒转染及基因表达 1.在最优化的脂质体和质粒比例下(2:1),质粒pEGFP-N1-IDO被脂质体转染至原代培养的软骨细胞,转染后的软骨细胞仍能贴壁生长。 2.荧光显微镜和激光共聚焦显微镜对转染后的软骨细胞中EGFP表达的监测表明,绿色荧光均匀地分布于整个细胞内,于转染后12 h开始并在48 h时有较强表达。EGFP在胞浆和胞核中均有表达,但胞浆中表达明显高于胞核表达。 3.于转染后不同时间点收集软骨细胞,多聚甲醛固定后,流式细胞术检测软骨细胞中EGFP的表达,转染后随着时间的延长,转染效率呈先上升,后下降的趋势。于转染后24 h达到最高峰,48 h后转染效率可高达36%。 4.软骨细胞基因转染48 h后,RT-PCR在RNA水平可检测到mIDO表达,与β-actin的光密度值相比,软骨细胞中基因的相对表达量稳定。 (三) mIDO转染后软骨细胞体外对T淋巴细胞增殖的影响 1.利用高压液相色谱(HPLC)分析mIDO转染软骨细胞培养液中色氨酸的含量,24 h后单纯软骨细胞组色氨酸含量为4.93～5.71 mg/L,平均为5.3±0.39 mg/L,而基因转染软骨细胞组未检测到色氨酸。在细胞培养液中色氨酸的降解,同时也伴随着犬尿氨酸代谢产物的增加,对细胞上清液中色氨酸消除动力学研究表明,转基因软骨细胞上清液中色氨酸的降解明显快于单纯软骨细胞组。 2.转基因软骨细胞体外混合淋巴细胞反应,以2×105个经3 000 radγ射线照射的脾淋巴细胞作为抗原递呈细胞和3×105个淋巴结淋巴细胞作为免疫应答细胞,于混合反应后第5 d、6 d、7 d、8 d通过MTT法检测各组淋巴细胞增殖情况。结果显示,较单纯淋巴细胞脾细胞组,转基因和未转基因组淋巴细胞均出现了增殖(P0.02),且转IDO基因组淋巴细胞增殖较未转基因组明显减慢(P0.05)。 3.混合淋巴细胞反应中以CFSE标记免疫应答细胞,共同培养96 h后经流式细胞术检测应答T细胞的增殖,转基因组和未转基因组应答细胞均出现了增殖(P0.01),共形成9代细胞。IDO基因转染组96 h后总体增殖指数为1.122±0.017,单纯淋巴细胞阴性对照组为1.026±0.016,阳性单纯软骨细胞组为1.201±0.026,较阳性对照组,IDO基因组同种异体T淋巴细胞增殖得到明显抑制。结论:消化法可获取供实验用的大量原代培养的软骨细胞,且第2、3代软骨细胞是用于实验的最佳时期;脂质体有望成为原代培养软骨细胞良好的基因转导载体,mIDO被导入原代培养的软骨细胞后,对EGFP监测表明,IDO在细胞中得到了稳定表达,且主要表达于胞浆;转基因细胞体外培养中明显降解培养液中色氨酸浓度,为IDO通过降解局部微环境中色氨酸浓度而抑制同种异体T细胞增殖提供科学依据;体外混合淋巴细胞反应中,表达mIDO转染软骨细胞明显抑制同种异体T细胞增殖,为基因转染后软骨细胞通过降解局部微环境中色氨酸的浓度抑制T细胞的增殖从而延长移植物的存活时间提供了思路。
[Abstract]:The repair of cartilage defects is a major problem in clinical practice. Due to the limited source of autologous cartilage transplantation, tissue engineered cartilage transplantation will be an important way to solve the repair of damaged cartilage. However, tissue engineered cartilage has immunogenicity. This topic is to be transfected through the transfection of indolamine 2,3- dioxygenase (indoleamine 2,3-dioxygenase, IDO) gene to achieve the goal. The aim of changing the negative modulation of the local T cell immune response after the transplantation of chondrocytes is to induce the immune tolerance of the transplant recipient's T cells to the allograft tissue engineered cartilage graft, which lays the foundation for the development of the transgenic method to prevent the autoimmune rejection after the allograft transplantation, and the tissue engineered seed cells can be used for the application. Patients with different immune genetic backgrounds have overcome the polymorphism of the MHC (major histocompatibility complex, the major histocompatibility complex) and the immune barrier formed by multiple genotypes to achieve the "generality" of the tissue engineered seed cells, and open up a new way to solve the immune rejection locally from the graft. The contents and results are as follows:
(1) isolation and culture of murine chondrocytes
The acquisition of articular cartilage in 1.C57 mice: centrifugation after digestion of 0.2% type of collagenase at 37 C for 3~4 h in vitro, a large number of chondrocytes for experimental use were collected. The vitality of chondrocytes obtained by trypan blue staining method was more than 95%.
2. primary cultured chondrocytes, MTT method was used to detect the growth curve of chondrocytes. It was found that the chondrocytes entered the exponential growth period from second to 5 d after the chondrocytes adhered to the wall, and it could be filled with the bottom of the bottle at about 7 d, but the time of cell formation was obviously prolonged with the increase of the number of cells.
3. the extracellular matrix secreted by chondrocytes showed a clear blue staining reaction to toluidine blue. The chondrocytes obtained by this experiment were stained with toluidine blue, and the purity of the cells was above 85%. However, with the increase of the number of cells, the capacity of the cartilage cells to secrete the extracellular matrix was decreased. Therefore, the selection of the chondrocytes of the 2,3 generation was used as a real one. The best time for testing.
(two) transfection and gene expression of pEGFP-N1-IDO plasmid in chondrocytes
1. under the optimal proportion of liposomes and plasmids (2:1), plasmid pEGFP-N1-IDO was transfected into the primary cultured chondrocytes by liposomes, and the chondrocytes after transfection could still grow on the wall.
The monitoring of the expression of EGFP in the transfected chondrocytes by 2. fluorescence microscopy and confocal microscopy showed that the green fluorescence was evenly distributed throughout the whole cell. The expression of.EGFP in the cytoplasm and nucleus was expressed at 12 h after transfection and at 48 h, but the expression in the cytoplasm was obviously higher than that of the nucleus.
3. after transfection, the chondrocytes were collected at different time points. After the polyformaldehyde was fixed, the expression of EGFP in cartilage cells was detected by flow cytometry. After the transfection, the transfection efficiency increased first and then decreased. After transfection, the highest peak was reached by 24 h, and the transfection efficiency could reach 36%. after 48 h transfection.
After transfection of 4. chondrocyte gene to 48 h, the expression of mIDO was detected at the level of RNA, and the relative expression of gene in cartilage cells was stable compared with the light density value of beta -actin.
(three) effects of chondrocytes transfected with mIDO on proliferation of T lymphocytes in vitro
1. the content of tryptophan in chondrocyte culture fluid transfected by mIDO was analyzed by high pressure liquid chromatography (HPLC). After 24 h, the content of tryptophan in pure chondrocyte group was 4.93 ~ 5.71 mg/L, with an average of 5.3 0.39 mg/L, while the gene transfected chondrocyte group was not detected by tryptophan. The study on the removal of tryptophan in cell supernatant by the increase of acid metabolites showed that the degradation of tryptophan in the supernatant of the transgenic chondrocytes was significantly faster than that in the simple chondrocyte group.
2. transgenic chondrocytes were mixed lymphocyte reaction in vitro, with 2 x 105 splenic lymphocytes irradiated by 3000 rad gamma rays as antigen presenting cells and 3 x 105 lymph node lymphocytes as immune response cells. After the mixed reaction, fifth D, 6 D, 7 d, 8 d were detected by MTT method. The results showed that the lymphocyte proliferation was more simple. In lymphocyte splenocytes group, proliferation (P0.02) was found in both transgenic and non transgenomic lymphocytes, and the proliferation of IDO genome was significantly slower than that of the non transgenome genome (P0.05).
3. the immune response cells were labeled with CFSE in the mixed lymphocyte reaction, and the proliferation of T cells was detected by flow cytometry after 96 h co culture. The proliferation of T cells was detected in both the transgenic and non transgenomic response cells (P0.01), and the total body proliferation index of the 9 generation cell.IDO gene transfected group was 1.122 + 0.017 after 96 h, and the simple lymphocyte negative was negative. The control group was 1.026 + 0.016, and the positive chondrocyte group was 1.201 + 0.026. Compared with the positive control group, the proliferation of IDO T cells was significantly inhibited.
Conclusion: the digestion method can obtain a large number of primary cultured chondrocytes for experimental use, and the 2,3 generation chondrocytes are the best time for the experiment. Liposome is expected to be a good carrier of gene transduction in the primary cultured chondrocytes. MIDO is introduced into the primary cultured chondrocytes. The monitoring of EGFP shows that IDO has obtained a stable table in the cell. The concentration of tryptophan in the culture medium in vitro culture of transgenic cells provides a scientific basis for IDO to inhibit the proliferation of allogenic T cells by degrading the concentration of tryptophan in the local microenvironment. In the mixed lymphocyte reaction in vitro, the expression of mIDO transfected to chondrocytes inhibits the increase of allogenic T cells. Colonization, after gene transfection, chondrocytes can inhibit the proliferation of T cells by degrading the concentration of tryptophan in local microenvironment and thus prolonging the survival time of the grafts.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R318.0;R392.4

【参考文献】