Zhang Xin, Yu Wei-Ping, Gao Lei, Wei Kai-Bin, Ju Jiu-Long, Xu Jia-Zhang
Department of Pathology and Pathophysiology, Basic Medical School, Southeast University, Nanjing 210009, Jiangsu Province, China.
World J Gastroenterol. 2004 Feb 15;10(4):610-3. doi: 10.3748/wjg.v10.i4.610.
To study the effects of Kupffer cell-conditioned medium (KCCM) derived from lipopolysaccharide (LPS) treatment on proliferation of rat hepatic stellate cells (HSC).
HSC and Kupffer cells were isolated from the liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz and cultured. KCCM was prepared and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect HSC proliferation. The content of type IV collagen and laminin secreted by HSC in the HSC-conditioned medium was determined by radioimmunoassay. TGF-beta(1) production in the KCCM was detected by enzyme-linked immunosorbent assay (ELISA).
HSC and Kupffer cells isolated had high purity. One microgram per mililiter LPS-activated KCCM and unstimulated KCCM could significantly promote HSC proliferation [0.132+/-0.005 and 0.123+/-0.008 vs control group (0.100+/-0.003), P<0.01], and there was a difference between them (P<0.05). Ten microgram per mililiter LPS-activated KCCM (0.106+/-0.010) was unable to promote HSC proliferation (P>0.05). Adding anti-TGF-beta(1) antibodies could suppress the proliferation promoted by unstimulated KCCM and LPS (1 microg/ml)-activated KCCM (0.109+/-0.009 vs 0.123+/-0.008, 0.115+/-0.008 vs 0.132+/-0.005, P<0.01). LPS (1 microg/ml or 10 microg/ml) could not promote HSC proliferation immediately (0.096+/-0.003 and 0.101+/-0.004 vs 0.100+/-0.003, P>0.05). There was a parallel behavior between HSC proliferation and increased ECM level. One microgram per mililiter LPS-activated KCCM contained a larger amount of TGF-beta(1) than unstimulated KCCM.
The technique for isolation of HSC and Kupffer cells described here is simple and reliable. KCCM stimulated by LPS may promote HSC proliferation and collagen accumulation, which are associated with hepatic fibrogenesis.
研究脂多糖（LPS）处理后的库普弗细胞条件培养基（KCCM）对大鼠肝星状细胞（HSC）增殖的影响。
通过用链霉蛋白酶和胶原酶原位灌注及用Nycodenz进行密度梯度离心从Wistar大鼠肝脏中分离HSC和库普弗细胞并进行培养。制备KCCM，采用噻唑蓝（MTT）比色法检测HSC增殖。采用放射免疫分析法测定HSC条件培养基中HSC分泌的IV型胶原和层粘连蛋白含量。采用酶联免疫吸附测定法（ELISA）检测KCCM中转化生长因子-β1（TGF-β1）的产生。
分离的HSC和库普弗细胞纯度高。每毫升1微克LPS激活的KCCM和未刺激的KCCM均可显著促进HSC增殖[分别为0.132±0.005和0.123±0.008，与对照组（0.100±0.003）相比，P<0.01]，且二者之间存在差异（P<0.05）。每毫升10微克LPS激活的KCCM（0.106±0.010）不能促进HSC增殖（P>0.05）。加入抗TGF-β1抗体可抑制未刺激的KCCM和LPS（1微克/毫升）激活的KCCM所促进的增殖（分别为0.109±0.009与0.123±0.008，0.115±0.008与0.132±0.005，P<0.01）。LPS（1微克/毫升或10微克/毫升）不能立即促进HSC增殖（分别为0.096±0.003和0.101±0.004与0.100±0.003，P>0.05）。HSC增殖与细胞外基质水平升高呈平行关系。每毫升1微克LPS激活的KCCM中TGF-β1含量高于未刺激的KCCM。
本文所述的HSC和库普弗细胞分离技术简单可靠。LPS刺激的KCCM可能促进HSC增殖和胶原积累，这与肝纤维化形成有关。
