调节性T细胞对肝星状细胞增殖及透明质酸分泌的影响

The influence of regulatory T cells in hepatic stellate cell proliferation and HA secretion

目的研究CD4+CD25+调节性T细胞(Treg)对肝星状细胞(HSC)增殖及透明质酸(HA)分泌的影响。方法 MASC法分离慢性乙型肝炎患者血中CD4+CD25+Treg细胞;传代培养人HSC LX-2(空白组),不同比例CD4+CD25+Treg细胞与HSC直接共培养(共培养组),不同剂量的重组人转化生长因子(TGF)β1和HSC共培养(对照组);使用CCK-8法检测HSC增殖、放射免疫法检测HA分泌情况。组间比较采用单因素方差分析。结果共培养组与空白对照组(0.6623±0.02702)相比,在比例为2.5∶1时HSC增殖最为明显(0.6836±0.00560),比例为1∶1、5∶1及7.5∶1时HSC增殖均明显减少,且随着比例升高其增殖水平逐渐降低,各组与空白对照组相比有统计学差异(F=11.421,5.272,84.514,490.639;均P<0.05)。不同浓度TGFβ1对照组的HSC均比空白对照组(0.3883±0.00189)细胞增殖明显(0.6720±0.00753,0.6773±0.00585,0.5373±0.00387),并于8 ng/μl时增殖达到高峰,各亚组与空白组相比有统计学差异(F=196.175,799.624,236.461;P<0.01)。共培养组与对照组中细胞上清液中HA浓度与HSC增殖呈正相关(r=0.661、0.871,均P<0.01)。结论 CD4+CD25+Treg细胞促HSC增殖并促进其分泌HA,并在体外证实CD4+CD25+Treg细胞在慢性乙型肝炎进展过程中发挥重要作用。

Objective To investigate the effect of CD4+CD25+ regulatory T cells(Treg)in the proliferation of hepatic stellate cells(HSC) and the secretion of Hyaluronic Acid(HA).Methods By using magnetic activated cell sorter(MRCS),Treg cells were isolated from peripheral blood of chronic hepatitis B patients The hepatic stellate cells(HSC)cultured alone were used as blank group.The LX-2 were co-cultured with Treg cells in vitro as co-culture group;adding TGFβ1 to HSC co-cultured as control group.CCK-8 method for the detection of the proliferation of HSC,ELISA analysis for the detection of HA secretion was performed.One-way analysis of variance was used for the comparison.Results Compared with the blank group(0.6623±0.02702) the proliferation of HSC of the co-culture group was significantly higherand the most prominent effect was 2.5∶ 1(0.6836±0.00560),while the proliferation of HSC decreased at 1∶ 1、5∶ 1 and 7.5∶ 1,and the discrepancy between co-culture subgroups and blank group had statistical significance(F=11.421,5.272,84.514,490.639;P<0.05).Compared with blank group,the proliferation of HSCs of different concentrations of TGFβ1 control subgroup increased significantly(0.6720±0.00753,0.6773±0.00585,0.5373±0.00387) and reached a peak at 8 ng/μl,and the discrepancy between control subgroups and blank group had statistical significance(F=196.175,799.624,236.461;P<0.01).Linear correlation analysis showed concentrations of HA and the proliferation of HSC in co-culture group was positively correlated with control group(r=0.661,0.871;P<0.01).Conclusion CD4+CD25+ Treg cells could accelerate the cultivation of HSCs proliferation and HA secretion.And,in vitro,we confirmed CD4+CD25+ Treg cells play an important role in the process of chronic hepatitis B.