INVITRO FORMATION AND EXPANSION OF CYSTS DERIVED FROM HUMAN RENAL-CORTEX EPITHELIAL-CELLS
INVITRO FORMATION AND EXPANSION OF CYSTS DERIVED FROM HUMAN RENAL-CORTEX EPITHELIAL-CELLS
复制标题
DOI：
10.1038/ki.1992.184
复制
发表时间：
1992-05-01
影响因子：
19.6
通讯作者：
GRANTHAM, JJ
中科院分区：
文献类型：
--
作者：
NEUFELD, TK;DOUGLASS, D;GRANTHAM, JJ
成人获得性肾囊肿起源于终末分化的小管上皮，这是上皮细胞增殖、液体积聚和细胞外基质重塑增加的结果。为了更好地了解人类上皮囊肿是如何形成并逐渐扩大的，我们将正常成人肾皮质原代培养的细胞分散在聚合的I型胶原中。透明基质允许在光镜下反复观察单个肾细胞形成的囊肿。囊肿细胞与远端肾细胞组织化学标记物（细胞角蛋白抗体AE1/AE3、上皮膜抗原和花生凝集素）反应强烈，但与近端肾小管标记物（紫斑四角龙凝集素和寻常Phaseolus vulgaris红凝集素）反应不一致或根本没有反应。在标准显微镜视野下形成的球形、充满液体的上皮囊肿的数量量化了囊肿的形成。囊肿的进展是由囊肿直径（表面积）的增加决定的，代表增生事件。在无血清的培养基中，需要EGF或TGF（α）才能使分散在基质中的单个上皮细胞形成囊肿；需要胰岛素作为辅助因素。EGF的EC50约为0.1 ng/ml，胰岛素的EC50约为1 mu-g/ml。早期培养的正常皮层在胶原基质中分散形成的囊肿比在凝胶中悬浮前传代几次的细胞更有效。腺苷酸环化酶（PGE1、AVP、VIP、PTH、forskolin、霍乱毒素）、甲基异丁基黄嘌呤和8-Br-cAMP的激动剂，虽然在确定的培养基中不能单独引起囊肿形成，但在EGF和胰岛素的存在下，促进了囊肿的发生和进展。血管紧张素II、肿瘤坏死因子（α）、β -雌二醇和百日咳毒素在EGF和胰岛素不存在或不存在的情况下没有作用。百日咳毒素抑制EGF和福斯克林引起的囊肿的发生和扩大，而增强EGF和PGE1引起的囊肿的发生和扩大。TGF（β -1）和2-氯腺苷抑制囊肿的形成和扩张。在可透膜上生长的人肾皮质细胞的极化单层被用来独立量化激动剂对细胞从基底外侧到根尖表面的溶质和水的净分泌的影响。PGE1、forskolin和8-Br-cAMP刺激净分泌液持续数天；EGF增强福斯克林刺激的液体分泌。我们得出结论，孤立的人皮质细胞体外囊肿的形成和扩张取决于细胞增殖和液体分泌之间的协调相互作用。孤立上皮细胞的囊肿形成主要由EGF或TGF（α）决定（环AMP起次要作用），而液体积聚的速率由环AMP控制（EGF或TGF（α）起次要作用）。这些实验支持了肾囊肿的形成和生长可能受内分泌、自分泌和旁分泌因素的调控。
Acquired renal cysts derive from terminally differentiated tubular epithelium in adults as a consequence of increased epithelial cell proliferation, fluid accumulation and extracellular matrix remodelling. To understand better how human epithelial cysts may be initiated and progressively expand, cells from primary cultures of normal human adult renal cortex were dispersed in polymerized type I collagen. The transparent matrix permitted repeated observation by light microscopy of cyst formation from individual renal cells. The cyst cells reacted strongly with distal nephron histochemical markers (cytokeratin antibodies AE1/AE3, epithelial membrane antigen, and Arachis hypogaea lectin) but inconsistently or not at all to markers of proximal tubules (Tetragonolobus purpureas lectin and Phaseolus vulgaris erythroagglutinin lectin). The number of spherical, fluid-filled epithelial cysts that developed in a standardized microscope field quantified cyst initiation. Cyst progression was determined from the increase in the diameter (surface area) of cysts and represents a hyperplastic event. EGF or TGF(alpha), were required in serum-free defined medium to cause cysts to develop from individual epithelial cells dispersed in the matrix; insulin was required as a co-factor. The EC50 for EGF was approximately 0.1 ng/ml, and for insulin 1-mu-g/ml. Early cultures of normal cortex formed cysts more efficiently when dispersed in collagen matrix than cells passaged several times before suspension in the gel. Agonists of adenylate cyclase (PGE1, AVP, VIP, PTH, forskolin, cholera toxin), methylisobutylxanthine, and 8-Br-cAMP, though incapable of causing cyst formation alone in defined medium, enhanced cyst initiation and progression in the presence of EGF and insulin. Angiotensin II, TNF(alpha), beta-estradiol, and pertussis toxin had no effect in the absence or presence of EGF and insulin. Pertussis toxin inhibited cyst initiation and expansion caused by EGF and forskolin but potentiated cyst initiation and expansion caused by EGF and PGE1. Cyst formation and expansion were inhibited by TGF(beta-1) and 2-chloroadenosine. Polarized monolayers of human renal cortical cells grown on permeable membranes were used to independently quantify the effects of agonists on the net secretion of solute and water from the basolateral to the apical surface of the cells. PGE1, forskolin, and 8-Br-cAMP stimulated net fluid secretion that was sustained for several days; EGF enhanced forskolin-stimulated fluid secretion. We conclude that the formation and expansion of in vitro cysts derived from solitary human cortex cells depends on the coordinated interplay between cellular proliferation and fluid secretion. The initiation of cyst formation from solitary epithelial cells is determined primarily by EGF or TGF(alpha) (cyclic AMP has a secondary role), whereas the rate of fluid accumulation is controlled by cyclic AMP (EGF or TGF(alpha) have secondary roles). These experiments support the view that renal cyst formation and growth may be regulated by endocrine, autocrine and paracrine factors.