博士 / 國立陽明大學 / 臨床醫學研究所 / 87 / 英 文 摘 要
E6/E7 genes of human papilloma virus type 16 were used to immortalize a primary culture of marginal cells (MC) from gerbils. One of the cloned lines was selected which demonstrated preservation of the main characteristics of the MC, both morphologically and physiologically. Electromicroscopic examination showed well-developed junctional complexes and apical microvilli which suggested its epithelial origin. Polymerase chain reaction (PCR) demonstrated the incorporation of E6/E7 genes with the genome. Reverse transcription PCR revealed the existence of mRNA of IsK channel, a unique marker of MC among the inner ear cells, in this clone. Analyzed by flow cytometry, the DNA content of this cell line was diploid. The formation of well-organized domes could be demonstrated after confluence of the monolayer cells. Electrophysiologic studies displayed evidence of apical K+ and Na+ channels which were blocked by Ba2+ (2 mM) and amiloride (10-5 M), respectively. Existence of basolateral Na,K-ATPase and Na+/Cl-/K+ cotransporter was shown by blockage by ouabain (10-3 M) and bumetanide (50 mM), individually. Injection of the cell line to nude mice failed to induce growth of tumors. This cell line was serum-, density- and anchorage-dependent when cultured in plastic dishes.
Because cytoplasmic cAMP has been reported to be the secondary messenger mediating K+ transport in MC of freshly isolated stria vascularis, the possible role of cAMP in ion transport processes of this immortalized marginal cell line (MCPV-8) showing evidence of K+ and Na+ reabsorption was evaluated in this study. Confluent MCPV-8 monolayers were mounted into Ussing chambers and perfused on both sides with perilymph-like Ringer''s solution. Transepithelial short-circuit current (ISC), resistance (RT) and open-circuit voltage (VT) were measured using voltage clamp technique. The following results were obtained: 1). Addition of forskolin (10-4 M) to the basolateral perfusate increased ISC to 311 ± 42 %; no significant change in RT was observed. Addition of BaCl2 (2 mM) to the apical perfusate at the maximal response of forskolin blocked 50-60 % of ISC and subsequent addition of amiloride (10-5 M) to the apical perfusate further blocked ISC to a value close to 0. 2). To evaluate the effect of cellular cAMP on Ba2+-sensitive K+ current, amiloride-sensitive Na+ current was blocked first by addition of amiloride (10-5 M) to the apical perfusate; subsequent addition of 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or N6,2''-O-dibutyryladenosine 3'',5''-cyclic monophosphate (dbcAMP, 1 mM) to the basolateral perfusate increased ISC to 175 ± 13 % and 411 ± 32 %, respectively. The stimulated ISC was blocked to close to 0 by addition of BaCl2 (2 mM) to the apical perfusate. N2,2''-O-dibutyrylguanosine 3'',5''-cyclic monophosphate (dbcGMP, 1 mM) had no effect on ISC. 3). To access the effect of cellular cAMP on amiloride-sensitive Na+ current, Ba2+-sensitive K+ current was blocked in advance by addition of BaCl2 to the apical perfusate; subsequent addition of IBMX or dbcAMP to the basolateral perfusate increased ISC to 219 ± 21 % and 388 ± 39 %, respectively. The stimulated ISC was blocked to close to 0 by addition of amiloride to the apical perfusate. dbcGMP had no effect on ISC. Hence, these results suggest that cellular cAMP is the secondary messenger that mediates the transepithelial transport of both K+ and Na+ in MCPV-8 monolayers. In conclusion, this cell line shows characteristics of well-differentiated MC maintaining the major ionic transport processes, and provides us a good model to study the possible mechanisms and regulating factors of endolymph production.
| Identifer | oai:union.ndltd.org:TW/087YM000521005 |
| Date | January 1998 |
| Creators | Tzong-Yang Tu, 杜宗陽 |
| Contributors | Jen-Hwey Chiu, Chiang-Feng Lien, 邱仁輝, 連江豐 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 102 |
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