Skrtic S, Wallenius K, Gressner A M, Jansson J O
Research Center for Endocrinology and Metabolism, Sahlgrenska University Hospital, Göteborg, Sweden.
Liver. 2000 Apr;20(2):157-64. doi: 10.1034/j.1600-0676.2000.020002157.x.
BACKGROUND/AIMS: Hepatic stellate cells (HSC) are located in close proximity to hepatocytes in Disse's space. Hepatocyte derived factors have earlier been implicated in the paracrine regulation of HSC proliferation. The aim of the present study was to further characterize this mitogenic activity of the parenchymal cell conditioned medium (PCcM).
Primary rat HSC were cultured for 4 days. DNA synthesis was measured by 3H-thymidine incorporation. TGFbeta1 immunoreactivity was quantified by ELISA. PCcM was obtained from hepatocytes cultured in medium without serum or hormones for two days.
Incubation of 4-day-old HSC on plastic surface with PCcM for 2 days increased DNA synthesis, while no effect was seen in HSC cultured on Matrigel. Heat-, acid-, and protease-treatment of PCcM abolished its stimulatory effect. Size fractionations with spin columns indicated that the stimulatory effect was contained in the fractions of a molecular size between 30 and 100 kD. The addition of LY 294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, dose-dependently inhibited the PCcM induced increase in DNA synthesis to about 9% of the control values. The specific MAP kinase (MAPK) inhibitor, PD 98059 only suppressed the PCcM induced DNA synthesis to 35% of control cultures at the highest dose (10 microM). DNA content in the cultures was not affected by either blocker. HSC seemed to produce immunoreactive TGFbeta1. However, addition of latency-associated peptide (LAP), a potent TGFbeta1 blocker, stimulated DNA synthesis to a much less extent than PCcM.
The factor(s) that stimulate DNA synthesis in HSC from hepatocytes are most likely protein(s) with a molecular size between 30-100 kD. These factor(s) rely more on PI3-K than on MAPK for their mitogenic effect and are probably not acting via TGFbeta1 inhibition.
背景/目的：肝星状细胞（HSC）位于狄氏间隙中紧邻肝细胞的位置。肝细胞衍生因子此前已被认为参与肝星状细胞增殖的旁分泌调节。本研究的目的是进一步表征实质细胞条件培养基（PCcM）的这种促有丝分裂活性。
原代大鼠肝星状细胞培养4天。通过3H-胸腺嘧啶核苷掺入法测定DNA合成。通过ELISA定量TGFβ1免疫反应性。PCcM取自无血清或激素培养基中培养两天的肝细胞。
将4日龄肝星状细胞在塑料表面用PCcM孵育2天可增加DNA合成，而在基质胶上培养的肝星状细胞中未观察到影响。PCcM经加热、酸和蛋白酶处理后消除了其刺激作用。用旋转柱进行的大小分级分离表明，刺激作用存在于分子大小在30至100 kD之间的级分中。磷脂酰肌醇3激酶（PI3-K）抑制剂LY 294002的加入剂量依赖性地抑制PCcM诱导的DNA合成增加，使其降至对照值的约9%。特异性丝裂原活化蛋白激酶（MAPK）抑制剂PD 98059在最高剂量（10 microM）时仅将PCcM诱导的DNA合成抑制至对照培养物的35%。两种阻滞剂均未影响培养物中的DNA含量。肝星状细胞似乎产生免疫反应性TGFβ1。然而，加入潜伏相关肽（LAP），一种有效的TGFβ1阻滞剂，刺激DNA合成的程度远低于PCcM。
从肝细胞刺激肝星状细胞DNA合成的因子很可能是分子大小在30 - 100 kD之间的蛋白质。这些因子的促有丝分裂作用更多地依赖PI3-K而非MAPK，并且可能不是通过抑制TGFβ1起作用。
