新生兔气管软骨细胞的生物学特性

 目的：探讨体外分离培养的新生兔气管软骨细胞的生物学特性。方法：通过酶消化法体外分离培养新生兔气管软骨细胞;倒置显微镜观察软骨细胞形态及生长状况;电镜观察软骨细胞超微结构;运用real-time PCR、免疫细胞化学染色和甲苯胺蓝染色检测气管软骨细胞分泌的细胞外基质成分。结果：体外分离、培养的兔气管软骨细胞呈短小三角形或不规则形贴壁生长。超微结构显示细胞较多突起,孔隙较多,胞质丰富,细胞器发达,细胞内可见大量蛋白分泌物。软骨细胞表达I、II型胶原、蛋白聚糖等,以II型胶原和蛋白聚糖表达为主。免疫细胞化学染色II型胶原和SOX9阳性,I型胶原弱阳性。甲苯胺蓝染色阳性。结论：适宜的酶消化单层培养法获得的新生兔气管软骨细胞具有分泌软骨细胞外基质成分的特性,可初步为体外构建组织工程气管治疗新生兔气管狭窄的实验研究提供种子细胞。     

Transport of putrescine in the isolated rabbit intestine

The transepithelial fluxes of putrescine were studied in sections of the three segments of rabbit intestine mounted in an Ussing chamber. The ileum exhibited the highest mucosal-to-serosal (J _ms) and serosal-to-mucosal (J _sm) unidirectional fluxes of 1 mol/l [³H]putrescine. Putrescine net flux (J _net=J _ms–J _sm) was deduced to be positive through the duodenum (J _net=53.40±14.30 pmol h^–1 cm^–2), not significantly different from zero through the jejunum (J _net=8.90±19.20 pmol h^–1 cm^–2) and negative through the ileum (J _net=–34.30± 13.80 pmol h^–1 cm^–2). Increasing putrescine concentration up to 10 mmol/l led to an increase in J _ms, J _sm and J _net without affecting the transport polarity in the ileum. The tissue retention of putrescine after 100 min was higher by the serosal side than by the mucosal side of the ileum. In parallel experiments, isolated pieces of ileum accumulated putrescine to a five- to sixfold concentration gradient by a ouabain-inhibitable mechanism. In contrast with arginine and in order of decreasing potency, putrescine, cystamine (a transglutaminase inhibitor), spermidine and spermine (1 mmol/l) reduced both unidirectional fluxes of putrescine across the ileum in the Ussing chamber. The latter effectors, except spermine, and N,N-dimethylcasein (1 mg/ml) led to an important, if not complete, suppression of putrescine secretion by the ileum, while the calmodulin antagonist melittin (0.3 g/ml) reversed the polarity of polyamine transport, suggesting the involvement of transglutaminase in putrescine transport. We conclude that the heterogeneous pattern of putrescine transport along the small-intestinal epithelium constitutes an important feature of the regulation of polyamine concentrations in this tissue.     

Effects of theophylline on Na and alanine transport across isolated rabbit ileum.

Addition of theophylline to isolated rabbit ileal mucosa resulted in a persistent increase in short-circuit current (SCC) and a persistent decrease in DC conductance. Under short-circuit condition, net Na flux (JNanet) was reversed by theophylline from absorption to secretion (-0.6 mueq/h cm2) and was due to a decrease in unidirectional mucosal- (M) toserosal (S) flux (JNams). Addition of L-alanine to theophylline-treated m-ucosa increased SCC, JNanet, and JNams. Theophylline reduced JAlanet by 25%. Na flux ratios, determined in the presence of theophylline at +26 and -11 mV (mucosal reference), differed from flux ratios expected for a diffusional process by less than 8%. In contrast, Na flux ratios, determined in the absence of theophylline and presence of glucose at +26 and O mV, differed from diffusional ratios by 35%. The results are consistent with the hypothesis that theophylline inhibits active Na absorption, but the data do not conclusively demonstrate the diffusional nature of unidirectional Na fluxes in the presence of theophylline.     

The effects of theophylline and choleragen on sodium and chloride ion movements within isolated rabbit ileum.

1. Theophylline (10 mM) and choleragen change the direction of net Cl- movements across rabbit ileum, in the short-circuit current condition, from absorption to secretion. The specific activity ratio R of Cl- tracers within the tissue coming from mucosal and serosal solutions respectively is increased, which is consistent with an increase in Cl- exchange flux across the mucosal border. 2. Net Na+ movement is also changed from net absorption to secretion by theophylline and choleragen; the specific activity ratio R of Na+ tracers is raised by theophylline. Because of the large paracellular component to transepithelial Na+ movements, an increase in Na+ exchange flux across the mucosal border is not detected. 3. 2,4,6-Triaminopyrimidine (20 mM) which has been previously shown to block paracellular Na+ movements, blocks both the theophylline and choleragen-dependent reversal of net Na+ movement by preventing the decrease in m-s Na flux. The theophylline-dependent increase in the ratio R of Na+ is still present, and is consistent with an increase in Na+ exchange flux across the mucosal border--unmasked by removal of the paracellular flux components. 4. Ouabain (0.1 mM) abolishes net absorption of Na+ and Cl- in control and net secretion of Na+ and Cl- in theophylline-treated tissue. Ouabain does not affect the theophylline-dependent increase in Cl- exchange across the mucosal border. 5. Replacement of Ringer Cl- with SO24- or Na+ by choline prevents the effects of theophylline and choleragen on Na+ and Cl- fluxes respectively. 6. Ethacrynate (0.1 mM) prevents the theophylline-dependent effects on net Na+ movement. Raising ethacrynate to 0.2 mM abolishes the effects of theophylline on Cl- exchange. An interpretation of these results is that theophylline and choleragen raise the Cl- permeability of the brush border. This increases NaCl leakage from the hypertonic lateral intercellular space into the mucosal solution thereby causing secretion. The selective action of triaminopyrimidine and ethacrynate (0.1 mM) on Na+ flux indicates that Na+ and Cl- move via separate transport pathways across the mucosal border.     

Transport of H plus and other electrolytes across isolated gastric mucosa of the rabbit.

The relationship of transmural and cellular phenomena to the rate of spontaneous luminal HH+ secretion by short-circuited gastric mucosa bathed in HCO3- and CO2-free Ringer solution was studied by the pH-stat technique. The bulk of luminal acidification can be accounted for by the appearance of H+ and not by organic acids. Acid secretion requires the presence of O2 and exogenous substrate but is not limited by endogenous CO2 production. The rate of H+ secretion is matched by the serosal appearance of alkali. The greater appearance of CO2 on the serosal side of the mucosa is consistent with hydroxylation of CO2 and greater permeability of the serosal border to HCO3-. Luminal changes in H+ concentrations affect H+ secretion but serosal changes do not, suggesting that the gradient produced by H+ secretion is at the luminal border. Steady-state isotopic Na+ and Cl- fluxes indicate net secretion of both ions, but net K+ transport is negligible.     

Hypothermia enhances acetylcholine-induced contraction of isolated rat ileum

The amplitude of acetylcholine (ACh)-induced contraction of isolated rat ileum was enhanced at medium temperatures lower than normal body temperature. Maximum enhancement was achieved between 30 and 25 degrees C. Changes in medium pH and activities of the enteric nervous system due to temperature changes were not essential for this enhancement.     

Methylprednisolone increases absorptive capacity of rabbit ileum in vitro

The effect of glucocorticoids on intestinal ion transport was studied in ileum in vitro from control and methylprednisolone (MP)-treated (40 mg im for 2 days) rabbits under the following conditions: a) basal rates of Na and Cl transport, b) the response to an individual absorptive stimulus (alanine, glucose, or epinephrine), and c) the response to a combination of the three absorptive stimuli. The results indicate that MP 1) increases basal absorption of Na and Cl and secretion of bicarbonate (as measured by residual ion flux), 2) does not alter the specific transport pathways stimulated by maximal doses of alanine, glucose, or epinephrine, but 3) significantly increases the absorptive capacity of ileum. After addition of combined alanine, glucose, and epinephrine, MP-treated ileum absorbed 15.8 mueq X cm-2 X h-1 Na (vs. 6.6 in controls, P less than 0.001) and 9.5 mueq X cm-2 X h-1 Cl (vs. 4.1 in controls, P less than 0.005). Additionally MP did not alter the Na dependence of either the short-circuit current or Cl absorption found in controls, although there appears to be a portion of residual ion flux insensitive to epinephrine inhibition. These data suggest that the MP-induced increase in absorptive capacity is due to an increase in a postapical transport step, most probably the Na pump.     

Migrating action-potential complexes in vitro in cholera-exposed rabbit ileum

The objective of this study was to determine whether cholera-exposed rabbit ileum exhibits altered myoelectric activity in vitro, without central nervous system connections. Whole-cell lysate of Vibrio cholerae, 100 mg in 1 ml saline, was injected into the jejunum of New Zealand White rabbits. Segments of ileum were removed at 12 and 24 h after inoculation and studied in vitro using myoelectric recording techniques. Propagating ring contractions were visualized and corresponded to intense action-potential activity that propagated over consecutive electrode sites. This altered myoelectric activity was similar to the previously described migrating action-potential complex (MAPC) in vivo after infection of rabbit ileum with live V. cholerae, its wholecell lysate, or the purified enterotoxin choleragen, with one exception. All MAPC activity propagated aborally in the in vivo-infected loops; in contrast 26% of the MAPCs propagated retrograde in the in vitro loops. Control segments were injected with saline, and no in vitro MAPCs were observed. Thus, the MAPC stimulated by cholera toxin may be maintained by the enteric nervous system of the gut wall. Although a role for extrinsic nerves is not excluded, our observations suggest that the small intestine may work autonomously, independent of the central nervous system.     

Effects of scorpion venom on electrolyte transport by rabbit ileum

Scorpion venom, which depolarizes nerves, was used to obtain further evidence that intramural nerves affect ion transport by the rabbit ileum. Ileal epithelium, stripped of muscularis propria, was mounted in a flux chamber modified to permit electrical field stimulation (EFS) of the tissue. Response of the short-circuit current (Isc) to venom was most rapid on the serosal surface, and the response was eliminated by tetrodotoxin. Isc response was influenced by venom batch number and by factors within the tissue. Venom (10 micrograms/ml) and EFS each caused chloride secretion by reducing mucosal-to-serosal movement and by increasing serosal-to-mucosal movement. Sodium transport and residual ion fluxes did not change. In the presence of venom, EFS caused no further changes in ion transport, but tissues still responded to glucose and to aminophylline. The early peak of Isc was reduced about 40% by atropine, implying that acetylcholine, released by venom, stimulates muscarinic receptors. The blockade of the Isc response to venom with tetrodotoxin is further evidence that venom depolarizes intramural nerves and liberates transmitters that cause chloride secretion. The identity of the other transmitters is not known.     

Short-circuit current and total conductance measurements on rabbit ileum.

1. Short-curcuit current (SCC) and total conductance (Gt) measurements were made in in vitro preparations of rabbit terminal ileum. 2. Successive additions of D-glucose increased the SCC and Gt in a stepwise way and both effects were seen to correlate linearly. 3. Measurements of SCC ant Gt were also made after replacing Na in the Krebs solution by K or sucrose. The SCC decreased to nearly zero in both cases but the decrease in Gt was larger when sucrose was used. 4. SCC and Gt were monitored after the addition of ouabain. SCC decreased to nearly zero but Gt remained constant. 5. On successive additions of sucrose after the preparation was mounted in 1/4 Na Krebs without osmolal compensation, SCC decreased and Gt increased, a linear relationship being found between both changes. 6. Successive additions of 2-deoxy-td-glucose caused a stepwise decrease of SCC and an increase in Gt. These effects seemed independent of the presence of glucose. 7. A model in which a resistance in series is added to the basic model by Ussing & Windhager (1964), modified by Rose & tschultz (1971), is proposed to account for these findings.