CD4^＋CD25^＋CD127^dim/-调节性T淋巴细胞对肝星状细胞增殖及功能的影响

CD4^＋CD25^＋CD127^dim/- regulatory T lymphocytes promote the proliferation and functions of hepatic stellate cells

目的了解CD4^＋CD25^＋CD127^dim/-调节性T淋巴细胞在体外对肝星状细胞（HSC）增殖以及功能的影响，初步探讨调节性T淋巴细胞促肝纤维化的机制。方法传代培养HSC LX-2，将免疫磁珠细胞分选（MASC）法分离所得慢性乙型肝炎患者调节性T淋巴细胞与HSC LX-2按不同方式共培养5d，以单独培养的HSC作为对照，细胞计数试剂盒-8（CCK-8）法检测共培养HSC增殖情况，ELISA法检测上清液中转化生长因子（TGF）β1含量，放射免疫法检测HSC分泌HA、PCⅢ水平。统计学处理采用LSD-t检验。结果5例调节性T淋巴细胞与HSC比例为1．5：1时HSC增殖最为明显，10例直接接触共培养与使用Transwell系统共培养调节性T淋巴细胞与HSC吸光度值分别为（0．713±0．032）、（0．735±0．028）cpm，均较对照组的（0．677±0．029）cpm增殖明显（t=5．4003，8．7878；均P〈0．01）。10例直接接触共培养与Transwell组细胞上清液中TGFCβ1浓度分别为（781．59±76．45）、（813．53±60．62）pg／mL，显著高于对照组的（722．51±59．66）pg／mL（t=4．0014，6．1653；均P〈0．01）；HA浓度分别为（433．57±27．90）、（445．40±23．73）ng／mL，显著高于对照组的（415．83±19．44）ng／mL（t=3．3124，5．5231；均P〈0．01）；PCⅢ浓度分别为（21．93±1．71）、（23．12±1．87）ng／mL，显著高于对照组的（20．10±1．49）ng／mL（t=4．8082，7．9436；均P〈0．01）。且Transwell组各项结果均显著高于直接接触组（t=3．3875，2．1639，2．2107，3．1354；均P〈0．05）。结论CD4^＋CD25^＋CD127^dim/-调节性T淋巴细胞可促进共培养的HSC增殖及其HA、pCⅢ的分泌。体外实验证明，CD4^＋CD25^＋CD127^dim/-调节性T淋巴细胞具有促进肝纤维化的重要作用。

Objective To investigate whether CD4^＋CD25^＋CD127^dim/- regulatory T lymphocytes （Treg） can induce the proliferation of hepatic stellate cells （HSC） and expression of fibrosis related factors on HSC in vitro and further to explore the mechanism of Treg inducing fibrogenesis. Methods HSC LX 2 cells were subeultured. CD4^＋CD25^＋CD127^dim/- cells were purified using magnetic cell separation. The HSC were co cultured with Treg by direct contact or by Transwell system in vitro. The HSC cultured alone was used as control. Cell proliferation was measured by CCK 8 assay. The expression of transforming growth factor （TGF）-β1 was detected by enzyme-linked inmunosorbent assay （ELISA）, and the expressions of hyaluronic acid （HA） and precollagen Ⅲ （PCⅢ） were detected by radio immunoassay （RIA）. The data were analyzed by LSD-t test. Results HSC proliferation was strongest when Treg：HSC=1.5：1. The absorbance in direct contact co culture group and Transwell system co-culture group was （0.713±0.032） cpm and （0.735±0.028） cpm, respectively, both of which were higher than that in control group [（0. 677±0. 029） cpm] （t=5. 4003 and 8. 7878, respectively; both P〈0. 01）. The concentrations of TGF-131 in the supernatant were （781. 59 ± 76.45） pg/mL and （813. 53 ± 60. 62） pg/mL, respectively in direct contact co-culture group and Transwell system co-culture group, which were significantly higher than that in control group [（722. 51± 59. 66 ） pg/mL] （ t = 4. 0014 and 6. 1653, respectively; both P 〈 0.01 ）. The concentrations of HA were （433.57±27. 90） ng/mL and （445.40±23.73） ng/mL, respectively in direct contact co-culture group and Transwell system co-culture group, which were higher compared to that in control group [（415. 83＋19. 44） ng/mL] （t =3. 3124 and 5. 5231, respectively; both P〈 0.01）. Likewise, the concentrations of PCⅢ were （21.93±1. 71） and （23. 12±1. 87） ng/mL in direct contact group and Transwell group, respectively compared to （20.10±1.49） ng/mL in control group （t = 4. 8082 and 7. 9436, respectively; both P〈 0.01）. Furthermore, the absorbance, concentrations of TGF-β1, HA and PC Ⅲ in Transwell co culture group were all higher compared to direct contact group （t =3. 3875, 2. 1639, 2. 2107 and 3. 1354, respectively; all P〈0. 05）. Conclusions The cell proliferation and the expressions of fibrosis-related factors in HSC increase greatly after co-cultured with CD4^＋CD25^＋CD127^dim/- Treg. Therefore, Treg may play an important role in inducing liver fibrogenesis.