急性白血病骨髓间充质干细胞调控免疫的功能及机制

Immunomodulatory effects of mesenchymal stem cells derived from the bone marrow in acuteleukemia patients

目的探讨急性白血病患者骨髓间充质干细胞（MSC）的免疫原性及抑制异体T淋巴细胞增殖的功能和机制。方法采用细胞贴壁法获取急性髓细胞白血病（AML）和急性淋巴细胞白血病（ALL）骨髓MSC，在低血清培养液中培养和扩增。采用流式细胞仪和免疫组化方法检测免疫表型，应用酶联免疫吸附实验检测MSC培养上清液中细胞因子的分泌水平，Transwell检测骨髓MSC抑制T淋巴细胞增殖的能力，混合淋巴细胞反应检测骨髓MSC抑制异体T淋巴细胞增殖的能力。结果ALL骨髓MSC在倒置显微镜下为梭形，CD29、CD44和CD105的表达阳性率分别为98．81％、99．25％和90．52％，而CD31、CD34和CD45均为阴性。AML骨髓MSC表达CD29和CIM4。ALL和AML骨髓MSC不表达人白细胞DR抗原（HLA—DR）和共刺激分子CD80、CD86和CD40。ALL骨髓MSC和AML骨髓MSC均具有分泌TGF-131（567．58±52．64和357．15±33．52）、HGF（647．27±102．54和219．67±62．37）、IL-6（59．67±15．69和54．35--4-12．31）和IL一11（102．58±23．54和78．21±9．67）的功能，但是AML骨髓MSC的TGF—Bl和HGF分泌水平明显低于ALL骨髓MSC（均P〈0．05）。在MSC数量分别为0．5×10。、1×10。、2×10。和5×10。个时，加入AML骨髓MSC的CD3’T淋巴细胞’H-TdT掺入的CPM值分别为（3．58--4-_0．54）×10。、（2．874-0．33）×10。、（1．78-t-O．51）×10。和（1．15±0．15）×10^4，加入ALL骨髓MSC相应的CD3^＋T淋巴细胞^3H-TdT掺人的CPM值分别为（1．96±0．31）×10^4、（1．57±0．28）×10^4、（0．91±0．41）×10^4和（0．22±0．11）×10^4，而未加入MSC的CD3^＋T淋巴细胞^3H-TdT掺人的CPM值为（4．01±0．72）×10^4，AML和ALL骨髓MSC抑制T淋巴细胞的增殖存在明显差异。ALL骨髓MSC抑制T淋巴细胞增殖的能力可以被抗TGF-β1和HGF抗体逆转。结论AIL骨髓MSC具有低免疫原性及体外调节免疫的功能，该免疫调节功能与其分泌细胞因子有关。AML骨髓MSC调控免疫的能力存在病理改变。

Objective To study the immunomodulatory effects and mechanisms of mesenchymal stem cells （MSC） derived from the bone marrow in acute leukemia patients in vitro. Methods Bone marrow mononuclear cells from acute myeloid leukemia （AML） and acute lymphoblastic leukemia （ALL） were obtained and cultured in low serum medium. The immunophenotypes were assessed by FACS and immunol histochemistry. The levels of cytokines were evaluated by enzyme linked immunosorbant assay （ELISA）. T-cell suppression ability was evaluated by Transwell chamber assay. Moreover, the immunoregulatory ability of AML- and ALL-derived MSC was detected by mixed lymphocyte culture assay. Results ALL-derived MSC showed a typical fibroblast-like morphology. They were positive for CD29, CD44 and CD105, the positive rate were 98.81%, 99.25% and 90.52%, respectively, while negative forCD31, CD45 and CD34. Moreover, ALL- and AML-derived MSC didn＇t express HLA-DR and co- stirnulatory molecules （CD40, CD80 and CD86 ）. ALL and AML derived MSC could secret several cytokines, such as TGF-131 （567.58±52.64 and 357.15±33.52）, HGF（647.27 ±102.54 and 219.67±62.37）, IL-6（59.67±15.69 and 54.35±12.31 ） and IL-11 （ 102.58±23.54 and 78.21±9.67 ）, the level of secretion of TGF-131 and HGF were higher in ALL bone marrow derived MSC than that of in AML bone marrow derived MSC. ALL and AML derived MSC significantly suppressed T lymphocyte proliferation in a dose-dependent manner, the counts per minute （CPM） were （ 3.58±0.54） ×10^4, （2.87 ＋ 0.33 ）×^104, （ 1.78±0.51 ）×10^4 and （ 1.15±0.15 ）×10^4 for AML derived MSC, and CPM were （ 1.96±0.31 ）× 10^4, （ 1.57 5±0.28）×10^4, （0.91±0.41 ）× 10^4 and （0.22±0.11 ）×10^4 for ALL derived MSC when MSC were 0.5 ×10^4, 1×10^4, 2×10^4 and 5×10^4. In addition, the CPM was （4.01±0. 72 ）×10^4 in control group. The immunosuppressive ability was different between MSCs derived from AML and ALL. The immunosuppressive effect of ALL derived MSC could be reversed by anti-TGF-β1 and anti-HGF antibody. Conclusion ALL-derived MSC show immunoregulatory effect in vitro and this effect is achieved through eytokines. But MSCs derived from AML display abnormal changes, in T-cell suppression ability.