Analysis of passenger cell composition of human fetal pancreas: implications for transplantation.
Analysis of passenger cell composition of human fetal pancreas: implications for transplantation.
复制标题
人胎儿胰腺过客细胞组成的分析：对移植的影响。
DOI：
10.1016/s0041-1345(98)01601-7
复制
发表时间：
1999
影响因子：
0.9
通讯作者：
Hullett,DA
中科院分区：
文献类型：
--
作者：
MacKenzie,DA;Sollinger,HW;Hullett,DA
人胎儿胰腺（HFP）是治疗胰岛素依赖型糖尿病（IDDM）的潜在胰岛来源。在链脲佐菌素（STZ）诱导的糖尿病裸鼠的肾包膜下移植HFP外植体或HFP proislets（通过有限的胶原酶消化产生的内分泌富集细胞），可在移植后3个月内恢复正常。先前已报道HFP proislets比未消化的HFP组织具有更低的免疫原性。本研究的目的是了解乘客白细胞的组成，并确定一种制备的HFP与降低免疫原性适用于临床移植。对1-2 mm 3 HFP外植体进行3分钟胶原酶消化后，获得外分泌富集细胞的单细胞悬浮液。收集剩余的内分泌富集的原胰岛簇，并进一步用分散酶消化成单细胞。通过流式细胞术，使用CD 14决定簇分析外分泌或内分泌富集细胞的单细胞悬浮液中是否存在单核细胞/巨噬细胞，使用CD 3、CD 4或CD 8决定簇分析T细胞。外分泌细胞群含有大量的CD 14+细胞（占总细胞的4.9±2%，n= 9），而内分泌富集群中的CD 14+细胞显著较少（占总细胞的0.8±0.4%，n= 9）。如使用人PBMC测定的，CD 14决定簇不受分散酶消化的影响。观察到CD 3 + T细胞（CD 4和CD 8表型）的数量变化范围为总细胞群的0.2%至2.5%，并且存在于外分泌和内分泌富集的细胞群中。使用经辐照的外分泌富集或内分泌富集的细胞作为刺激物和成人PBMC作为应答物，以5：1的应答物与刺激物比率建立MLR培养物。与用内分泌富集的HFP细胞刺激的MLR培养物相比，用外分泌富集的HFP细胞刺激的MLR培养物中IFNγ和IL 10的产生显著更大（IFNγ：第4天p= 0.0003，第7天p= 0.02; IL 10：第4天p= 0.02，第7天p= 0.007，n= 5种不同的HFP/PBMC组合）。比较CTL活性正在评估中。现在认识到，直接和间接的同种异体识别途径都可能导致移植物丢失。直接T细胞识别是宿主T细胞与供体APC上的同种异体MHC分子直接相互作用的结果，而在间接途径中，宿主T细胞识别由自身APC加工和呈递的同种异体肽。CD 14+细胞数量的减少可能部分地解释了内分泌富集的HFP细胞群的免疫原性的降低，其通过直接途径刺激的能力降低。使用简单消化方案从移植群体中选择性地消耗过客细胞的能力可以显著增强成功移植HFP胰岛的能力。
Human fetal pancreas (HFP) is a potential source of transplantable islets for the treatment of insulin dependent diabetes mellitus (IDDM). Transplantation of either HFP explants or HFP proislets (endocrine enriched cells generated by limited collagenase digestion) beneath the kidney capsule of streptozotocin (STZ)-induced diabetic nude mice restores euglycemia within 3 months post-transplant. HFP proislets have previously been reported to be less immunogenic than non-digested HFP tissue. The aim of this study is to understand the passenger leukocyte composition of HFP and to identify a preparation of HFP with decreased immunogenicity suitable for clinical transplantation. Single cell suspensions of exocrine enriched cells were obtained following a 3 minute collagenase digestion of 1-2 mm 3 HFP explants. The remaining endocrine enriched proislets clusters were collected and further digested with dispase to single cells. The single cell suspensions of either exocrine or endocrine enriched cells were analyzed for the presence of monocytes/macrophages using the CD14 determinant and T cells using the CD3, CD4 or CD8 determinates by flow cytometry. The exocrine cell population contained a significant number of CD14+ cells (4.9±2% of total cells, n= 9), whereas there were significantly fewer CD14+ cells in the endocrine enriched population (0.8±0.4% of total cells, n= 9). The CD14 determinant was unaffected by the dispase digestion as determined using human PBMC. CD3+ T cells (both CD4 and CD8 phenotypes) were observed in varying amounts ranging from 0.2% to 2.5% of the total population of cells, and were present in both the exocrine-and endocrine-enriched cell populations. MLR cultures were established at a responder to stimulator ratio of 5: 1 using either irradiated exocrine-enriched or endocrine-enriched cells as stimulators and adult human PBMC as responders. Both IFNγ and IL10 production was significantly greater in MLR cultures stimulated with exocrine enriched HFP cells compared to MLR cultures stimulated with endocrine enriched HFP cells (IFNγ: p= 0.0003 on day 4, p= 0.02 on day 7; IL10: p= 0.02 on day 4 and p= 0.007 on day 7, n= 5 different HFP/PBMC combinations). Comparative CTL activity is being evaluated. It is now recognized that both direct and indirect pathways of allorecognition can contribute to graft loss. Direct T cell recognition is the result of host T cells interacting directly with allo-MHC molecules on donor APC's whereas in the indirect pathway host T cells recognize allopeptides processed and presented by self APC's. The decreased numbers of CD14+ cells may account in part for the decreased immunogenicity of the endocrine enriched HFP cell population via a decreased ability to stimulate via the direct pathway. The ability to selectively deplete passenger cells from the transplanted population using simple digestion protocols may significantly enhance the ability to successfully transplant HFP islets.