目的：探讨雌激素影响人精子顶体反应的可能机制。方法：应用异硫氰酸荧光素标记豌豆凝集素荧光染色法（FITC-PSA）分析精子顶体反应（AR）、以分光光度比色法测定顶体酶（ACE）活性。结果：17β-雌二醇（17β-E2）可促进精子发生AR，并增强精子顶体酶的活性；去除培养液中的Ca2+后，17β-E2不能诱导精子发生AR；PKC抑制剂能明显降低17β-E2所诱导的AR；E2-BSA亦能够促进精子发生AR，其作用与17β-E2无显著差异。结论：雌激素对人精子顶体反应有一定的促进作用，增强精子顶体酶活性可能是其作用途径之一，此过程涉及了胞外Ca2+、PKC及精子膜上的ER或雌激素结合位点的参与。
Objective To study the effect of substance P ( SP) on int racellular f ree calcium concent ration in human poorly-differentiated gast ric cancer cell in vitro. Methods Human gast ric cancer cell line MKN45 was cultured in RPMI 1640. Then the cells were loaded with specific calcium fluorescent probe Furu23/ AM. ASN21377642 (NK21 receptor antagonist) , Nicardipine (calcium channel blocker) and different concent rations of SP were used to treat gast ric cancer cells. The concent ration changes of int racellular free calcium were detected by laser scanning confocal microscope. Results It was found that 10 , 50 and 100 nmol/ L SP could significantly increase the int racellular free calcium concent ration of gast ric cancer cells in Hanks solutions , which contain ext racellular calcium ( P lt;0. 05) , and the change was in a dose-dependent manner ( P lt; 0. 05) . When there was ext racellular calcium existed ,the increasing amplitude of intracellular f ree calcium concent ration was significantly higher than that when there was no extracellular calcium ( Plt; 0. 05) . And when Hanks solutions were pretreated with ASN21377642 and Nicardipine , the effects of 100 nmol/ L SP were partly inhibited , and the concent rations of int racellular f ree calcium were significantly lower than those in group s without pret reatment s ( P lt; 0. 05) . Conclusion SP can significantly increase free calcium concent ration in the gastric cancer cells. Releasing of stored calcium in the cells and influx of extracelluar calcium may contribute to the elevation of int racellular free calcium concentration.
From this experiment we found that acetylcholine (Ach) was the agonist of the colonic smooth muscles, and noradrenaline (NA) the internal anal sphincter(IAS). The contractions of colonic smooth muscles were significantly inhibited under the calcium-free solution (Ｐ＜0.01), but the IAS was not affected. Ryanodine, which can exhaust the intracellular calcium, remarkably depressed the contractions of IAS (Ｐ＜0.01),but had no effect on the colonic smooth muscles. It can be concluded that the contractions of colonic smooth muscles are mainly related to the influx of extracellular calcium; the release of intracellular calcium plays an important role in the contractions of IAS.
Objective To study the influence of three different ways of myogenic induction on Ca2+ regulation of mesenchymal stem cells (MSCs) derived from umbilical cord blood. Methods From January 2007 to April 2010, three different ways of myogenic induction including the adoptions of 5azacytidine, extraction of myocardium, and myocardial differentiation medium were used to induce MSCs derived from the umbilical cord blood of dogs in Xinhua Hospital of Shanghai Jiaotong University. Confocal laser scanning microscope was used to detect cells induced by the three abovementioned methods, cardiomyocytes and Ca2+ combined with Fluo3/AM inside the MSCs. For each group of cells, 2 to 5 visual fields were chosen, and 30 visual fields were recorded for each kind of cells. The mean fluorescence intensity of ten images shot in one minute was used to reflect the concentration of free intracellular Ca2+. Furthermore, the change of the concentration was continuously monitored by optical density(OD) value. Results After induction, the Ca2+ concentration inside the MSCs was significantly higher than that inside the cardiomyocytes (F=59.400, P=0.000). There was a statistical difference among the intracellular Ca2+ concentration induced respectively by 5azacytidine, extraction of myocardium, and myocardial differentiation medium (F=18.988, P=0.000). No significant difference existed between the intracellular Ca2+ concentration induced by 5-azacytidine and extraction of myocardium (OD value: 1 076.88±44.65 vs. 1 040.90±37.48, P=0.186), while the intracellular Ca2+ concentration induced by 5azacytidine was significantly higher than that induced by myocardial differentiation medium (OD value: 1 076.88±44.65 vs. 973.91±46.49, P=0.001), and the intracellular Ca2+ concentration induced by extraction of myocardium was significantly higher than that induced by myocardial differentiation medium (OD value: 1 040.90±37.48 vs. 973.91±46.49, P=0.001). The concentration of intracellular Ca2+ induced by all the three different methods fluctuated spontaneously, which was quite similar with the cardiomyocytes, but the frequency and the scope of the fluctuation were quite different. Ca2+ was released instantly by KCl stimulation in the two groups of MSCs pretreated by 5-aza and extraction of myocardium. Though MSCs pretreated by myocardial differentiation medium had response to KCl stimulation, Ca2+ could not be released in this group. On the contrary, the duration of Ca2+ release was prolonged. Conclusion Ca2+ regulation system of MSCs derived from umbilical cord blood can be influenced by these myogenic inductions. However, the reason and effect of the differences need to be elucidated by further investigation.
Abstract: Objective To observe the combined protective effects of U50 488H and hypothermia preservation on isolated rabbit hearts preconditioned. Methods Forty rabbits were randomly divided into five groups, 8 rabbits in each group. The perfusion model of isolated rabbit hearts was established by the Langendorff device. In the control group: the isolated rabbit hearts were preserved with the University of Wissconsin solution (UW ) for six hours; groupI : the isolated rabbit hearts were preconditioned with St. ThomasII cardioplegic solution containing U50 488H (1. 6mmo l/L ) and then preserved with hypothermic preservation for four hours; groupII ; the precondition was the same as group II , hypothermic preservat ion fo r six hours; group III : the precondit ion was the same as group I , hypothermic preservation for eight hours; group IV : the precondit on was the same as group I , hypothermic preservation for ten hours. The cardiac function, myocardial sarcoplasmic reticulum calcium ion adenosine triphosphatase (SRCa2+ -ATPase) act ivity and calcium ion concentrations in mitochondria were determined at thirty minutes after reperfusion. Results As the hypothermic preservation time increased from four to ten hours, the recovery rate of each index of cardiac function, coronary artery flow (Cf) and SRCa2+ -ATPase activity also decreased, but the calcium ion concentrations in the mitochondria increased. Cardiac function index recovery rates in group I and group II w ere higher than those in group III and groupIV respectively (P lt; 0. 05, 0. 01) ,meanwhile recovery rates of cardiac function index in group III were higher than that in group IV (P lt; 0. 05). Recovery rate of Cf in groupII ( 84. 56%±10. 38%)were higher than those in group III (79. 45%±9. 67% ) and group IV (68. 31%±6. 84% , P lt;0.01) , meanwhile the recovery rate of Cf in group III was higher than that in group IV (P lt; 0. 05). SRCa2+ -A Tpase activity in group II (4. 43±0. 41μmo l/m g?h)were higher than those in control group (3. 04±0. 22Lmo l/mg?h ) , group III (3. 26±0. 29Lmo l/m g?h) and group IV (2. 57±0. 63Lmo l/m g?h, P lt; 0. 05) , SRCa2+ -ATPase activity in group III was higher than that in group IV (P lt; 0. 01). The calcium ion concentrations in mitochondria in group II (38176±4. 30μmo l/g ?dw ) and in the control group (40. 23±3. 75μmol/g ?dw )were less than those in group III (43125±5116μmol/g?dw ) and groupIV (45. 78±3. 26μmol/g?dw , P lt; 0. 05, 0. 01) respect ively. Conclusion The hypothermic preservation time for isolated dono r’s hearts p re-treated with St. Thomas II cardioplegic solution containing U 50 488H should the kep tunder 8h. The myocardial protection effects of both UW solution and U50 488H- containing St. Thomas II cardioplegic solution on isolated dono r’s hearts appear to be the same at 6 hours.
Myocardial stunning is the main pathological basis of heart dysfunction after open heart operation, its exact pathogenesis hasn’t been clarified until today.In recent years,the molecular and cellular studies have revealed possibly crucial pathogenesis of myocardial stunning that delayed recovery of myocardial glucose oxidation causes intracellular H + accumulation which augments H + Na + exchange thus leading to [Na +] i overload.[Na +] i overload increases Na + Ca 2+ exchange resulting in t...
Objective To explore the effect of hydrostatic pressure on intracellular free calcium concentration ([Ca2+]i) and the gene expression of transient receptor potential vanilloid (TRPV) in cultured human bladder smooth muscle cells (hb-SMCs), and to prel iminarily probe into the possible molecular mechanism of hb-SMCs prol iferation stimulated by hydrostatic pressure. Methods The passage 6-7 hb-SMCs were loaded with Ca2+ indicator Fluo-3/AM. When the hb-SMCs were under 0 cm H2O (1 cm H2O=0.098 kPa) (group A) or 200 cm H2O hydrostatic pressure for 30 minutes (group B) and then removing the 200 cm H2O hydrostatic pressure (group C), the [Ca2+]i was measured respectively by inverted laser anningconfocal microscope. When the hb-SMCs were given the 200 cm H2O hydrostatic pressure for 0 hour, 2 hours, 6 hours, 2 hours, and 24 hours, the mRNA expressions of TRPV1, TRPV2, and TRPV4 were detected by RT-PCR technique. Results The [Ca2+]i of group A, group B, and group C were (100.808 ± 1.724), (122.008 ± 1.575), and (99.918 ± 0.887) U, respectively; group B was significantly higher than groups A and C (P lt; 0.001). The [Ca2+]i of group C decreased to the base l ine level of group A after removing the pressure (t=0.919, P=0.394). The TRPV1, TRPV2, and TRPV4 genes expressed in hb-SMCs under 200 cm H2O hydrostatic pressure at 0 hour, 2 hours, 6 hours, 12 hours, and 24 hours, but the expressions had no obvious changes with time. There was no significant difference in the expressions of TRPV1, TRPV2, and TRPV4 among 3 groups (P gt; 0.05). Conclusion The [Ca2+]i of hb-SMCs increases significantly under high hydrostatic pressure. As possible genes in stretch-activated cation channel, the TRPV1, TRPV2, and TRPV4 express in hb-SMCs under 200 cm H2O hydrostatic pressure. It is possible that the mechanical pressure regulates the [Ca2+]i of hb-SMCs by opening the stretch-activated cation channel rather than up-regulating its expression.
Objective To study the effect and mechanism of the apoptosis of hypertrophic scar fibroblasts (HSF) induced by artesunate(Art). Methods HSFs were isolated and cultured from human earlobe scars by the tissue adherence method. The 3th to 5th generation cells were harvested and divided into two groups. HSF was cultured with normal medium in control group and with medium containing60, 120 and 240 mg/L (5 ml)Art in experimental group. Apoptosis and cell cycle were identified by light microscopy, electronmicroscopy and flow cytometry. Then, HSF was cultured with normal medium in control group and with medium containing 30, 60 and 120 mg/L Art in experimental group. The changes of intracellular calcium concentration were observed. Results The primary HSF was fusiform in shape and adherent. The vimentin positive expression was analyzed by immunocytochemistry. Art could induce apoptosis of HSF in the range of 60-240 mg/L under inverted microscope. The effect was dose and timedependent. Clumping of nuclear chromatin showed margination in the experimentalgroup. And the disaggregation of the nucleolus were observed under electronmicroscopy. There were significant differences in the proportion of HSF apoptosis and HSF at G0-G1,S, G2-M stages between the two groups(P＜0.05). Apoptotic peak was shown in experimental group by flow cytometry. The peak became more evident asArt concentration increased. The intracellular calcium concentration elevated markedly in HSF with 30-120 mg/L Art treatment for 24 hours, showing significant differences between the two groups (P＜0.05). Conclusion The Art facilitates HSF cells apoptosis in vitro by the change of cell cycle. It is suggested that intracellular calcium variation may be one of the mechanisms of HSF apoptosis induced by Art.
Free calcium ions, as a kind of message-transport substance, is important in cellular activity such as cell movement, cell differentiation and cell proliferation. In order to investigate the relationship between free calcium ions and scar contracture, the fibroblasts which originated from hypertrophic scar, keloid and normal skin were used as the experimental target. The fibroblasts from 4th-6th generations of different sources were used; Then the intracellular free calcium ions concentrations were measured respectively by the fluorescent Ca2+ indicator Fura-2/AM and Image analysis system. The results showed that the level of Ca2+ in fibroblasts of hypertrophic scar was higher than that in keloid and normal skin (P lt; 0.01). There was no significant difference between the level of Ca2+ in keloid and in normal skin. The conclusion was that the concentration of intracellular free calcium ions played an important role in the scar contract, but the exact mechanism was still unclear and required further study.
We correct some misunderstandings of hypertension therapy and update the knowledge of hypertensive drugs by reviewing the progress of evidence-based research of hypertension in 2004.