Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand
Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand
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DOI：
10.1002/hep.510280229
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发表时间：
1998-08-01
期刊：
影响因子：
13.5
通讯作者：
Gressner, AM
中科院分区：
文献类型：
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作者：
Gong, WR;Pecci, A;Gressner, AM
在组织损伤和炎症中，细胞因子驱动的肝星状细胞（HSC）活化是肝纤维化中的一个重要致病事件，导致产生基质的肌成纤维细胞（MFB）的扩增池，其代表HSC的转化对应物。我们推测，MFB池的扩大也可能是通过调节细胞凋亡来实现的，细胞凋亡在细胞内稳态中与有丝分裂起着相反和互补的作用。我们的特点是在原代培养的HSC和MFB在二次培养的可溶性Fas配体（sFasL）诱导的细胞凋亡的敏感性和相关的Fas（APO-1/CD 95）和一些主要的促凋亡和反凋亡原癌基因的表达水平的影响。MFB显示暴露于sFasL后的剂量依赖性凋亡反应，如核小体DNA片段的强烈增加、细胞DNA的丢失、阳性TUNEL反应和膜联蛋白染色所证明的。只有当蛋白质合成（放线菌酮）或RNA合成（放线菌素D）被逮捕，HSC维持在原代培养的不同时间是完全耐sFasL与放线菌酮的组合，但在后期原代培养（第7天向前）的sFasL介导的细胞凋亡的敏感性增加。通过半定量逆转录聚合酶链反应（RT-PCR）分析和碱性磷酸酶-抗碱性磷酸酶染色，确定Fas受体在HSC和MFB中的表达水平相当。核糖核酸酶保护试验显示，早期HSC中抗凋亡原癌基因bcl-2和bcl-xl的表达明显高于晚期HSC和MFB。半定量RT-PCR和免疫印迹法进一步证实bcl-2的表达。在MSC和MFB中，在RNA水平上发现了相当数量的促凋亡bar，但在蛋白质水平上，MFB显示出bar表达增加。结论：HSC向MFB转化是由于对sFasL介导的凋亡的敏感性增加，这可能与bcl-2和bcl-xl表达的强烈下降有关，导致MFB中促凋亡基因的表达占优势。调控转化型HSC的凋亡敏感性可能是纤维化发病机制中的一个重要补充途径。
Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a ky pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested, HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-antialkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stranger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bar was found in comparable quantities at the RNA level in MSC and MFB but at the protein level MFB showed increased bar expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.