自体树突状细胞活化的骨髓细胞对慢性粒细胞白血病骨髓的净化

Bone Marrow Cells Activated by Autologous Dendritic Cells Purges Bone Marrow from Patients with Chronic Myelogenous Leukemia

为考察慢性粒细胞白血病 (CML)患者自体树突状细胞 (DCs)活化骨髓细胞、净化骨髓的作用 ,采用免疫磁珠从 2例血液学缓解的CML骨髓单个核细胞 (BMMNCs)分离DCs,另取BMMNCs置T 2 5塑料培养瓶建立Dexter培养体系 ,分为 3份 ,第 1份为对照 ,第 2份加rhIL 2 ,第3份加自体DCs。每周取半量非贴壁细胞、计数、行CFU GM检测 ,然后加等量新鲜培养基继续培养。当非贴壁细胞总数 <2× 10 5时终止培养 ,收集贴壁细胞做CFU GM集落形成试验并检测CD34和P2 10表达。取贴壁细胞形成的CFU GM集落 ,抽提RNA ,用反转录聚合酶链反应 (RT PCR)检测BCR ABL的表达。结果发现 ,在DCs加入CML骨髓Dexter培养体系培养后 1- 2周 ,非贴壁细胞的CFU GM产量明显下降 ,与rhIL 2的体系基本平行 ,同时体系中的P2 10阳性细胞显著减少。不过 ,培养 3周后含DCs的体系CFU GM产量下降减缓 ,而含rhIL 2的体系CFU GM产量持续下降。在含自体DCs的Dexter体系 ,贴壁细胞中CD34+ 细胞和P2 10 + 细胞比例最低 ,只是贴壁细胞产生的CFU GM集落中 ,BCR ABL(+ )的集落比例与不含DCs的体系比较无明显差别。这些结果说明自体DCs加入CML骨髓Dexter培养体系能减少白血病的祖细胞 ,包括成熟的祖细胞和原始的祖细胞 ,自体DCs有可能用于CML骨髓的体外净化。

In order to investigate the effect of autologous dendritic cells(DCs) activating bone marrow cells and purging bone marrow from chronic myelogenous leukemia(CML) patients, DCs were separated by negative selection system of human cells from bone marrow mononuclear cells(BMMNCs) of 2 CML patients in hematological remission and harvested after 3 days of culture in IMDM containing autologous plasma, rhGM CSF and rhTNFα at 37℃, 5% CO 2 humidified atmosphere. BMMNCs from the patients were also used to set up long term culture(LTC) system in T 25 plastic flasks. The LTCs included three groups, i.e., control, addition of rhIL 2, and co culture with autologous DCs. Half of non adherent cells were collected, counted and assayed for CFU GM weekly. Then, equivalent volume of fresh medium was replaced to maintain the culture. The culture was discontinued if the non adherent cells count was less than 2×10 5. Adherent cells were collected for CFU GM assay and flow cytometry for CD34 and P210. The colonies originating from the adherent cells were picked up under the inverted microscope. RNA was extracted, and BCR/ABL measured by nested reverse transcription polymerase chain reaction(RT PCR). The results showed that the CFU GM yields of non adherent cells declined after 1 to 2 weeks co cultured with autologous DCs, and it paralleled with group with rhIL 2. P210 + cell percentage was also decreased. From the third week on, however, the decrease of CFU GM yields slowed down, while CFU GM in the system with rhIL 2 continued to fall. In system co cultured with autologous DCs, the adherent cells contained the least percentagcs of CD34 + cells and P210 + cells percentage. However, the expression of BCR/ABL in CFU GM colonies derieved from the adherent cells of DCs co cultured had no significant difference with those from the culture without DCs. Our results suggest that co culture of marrow cells with autologous DCs could significantly diminish the leukemic progenitors cells including both mature and primitive progenitor cells. Autologous dendritic cells might be used for ex vivo purging of CML marrow.