豚鼠膀胱Cajal样细胞超微结构及起搏功能的相关性研究
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摘要
目的：本课题以正常成年豚鼠膀胱Cajal样间质细胞为研究对象,通过对膀胱Cajal样间质细胞(interstitial cells of Cajal, ICCs)超微结构观察及电生理学特性研究,探讨其是否为膀胱的起博细胞,为临床上治疗某些疾病(如detrusor instability, DI)提供新的切入点。
方法:本实验运用采用酶消化法,得到体外培养状态下的ICCs样细胞,透射电镜观察正常成年豚鼠膀胱ICCs样细胞的超微结构；采用激光共聚焦显微镜成像技术记录Fluo4AM负载后的Cajal样间质细胞内钙离子荧光强度的变化,以荧光强度代表细胞内钙离子浓度的变化。；运用膜片钳技术,在全细胞记录模式下观察其是否具有钙电流,以验证其是否为膀胱的起博细胞。
结果：透射电镜观察到CCs样细胞胞浆内线粒体、粗面内质网较丰富,滑面内质网分散分布,中间丝难能见到,未见微管；运用激光共聚焦显微镜成像技术连续扫描,见体外培养的原代膀胱Cajal样间质细胞有2种类型钙震荡：一类振幅大、频率慢而规则的钙震荡；另一类振幅小、频率快且不规则,且形成网络的膀胱Cajal样间质细胞的钙离子震荡的幅度明显高于单个的膀胱Cajal样间质细胞。膀胱逼尿肌细胞钙震荡振幅小不规则,其振幅显著低于Cajal样间质细胞第一类钙震荡振幅；膜片钳试验膀胱Cajal样细胞的钙离子电流不明显。
结论：电镜观察到Cajal样细胞具有独特的超微结构,与胃肠道中ICCs的超微结构有相似；部分膀胱Cajal样细胞具有自发而规律的钙震荡,类似消化道起搏细胞Cajal间质细胞(interstitial cells of Cajal,ICCs)的自发性钙震荡,膀胱逼尿肌无自发性钙震荡。提示：部分膀胱Cajal样细胞是膀胱收缩活动的起搏细胞；另外部分膀胱Cajal样细胞可能起到传导、耦合信息的作用。
Purpose:The purpose of this is to provide proofs to prove that the Cajal-like cells in guinea pig bladder is the pacemaker by observe its utrastructure and electrophysiological, and offer a new starting point linical and treatment of certain diseases (such as the detrusor instability, DI).
Methods:In this experimental we can get the cajal-like interstitial cells by enzyme digestion method. We observe ICCs ultrastructure by transmission electron microscope; Cells were loaded with fluo-4 AM dye. Using laser scanning microscope imaging records the change of calcium fluorescence intensity. Fluorescence intensity was on behalf of changes of the intracellular calcium concentration; by patch clamp technology We observe the calcium current of ICCs in the whole-cell recording mode. It can provide a proof to verify that the ICCs in guinea pig bladder is the pacemaker.
Results:In this experimental the ICCs can be observed to have rich in mitochondria, developed the RER, the sliding surface endoplasmic reticulum, Gorky body. There were 2 different types of spontaneous calcium oscillation in the Cajal-like interstitial cells of bladder:large, slow calcium oscillation and small, irregular calcium oscillation. The first type of calcium oscillation was completely different from those of smooth muscle cells; By the patch clamp technology We can't observe the type calcium current of ICCs in the whole-cell recording mode.
Conclusion:The ICCs in guinea pig bladder have a unique ultrastructure, which similar with the ICCs's in the gastrointestinal tract. We have confirmed that part of the Cajal-like interstitial cells existence of the spontaneous calcium oscillation in the bladder of adult guinea-pig, which like the pacemaker cells of interstitial cells of Cajal in the digestive tract. The spontaneous calcium oscillation was not observed in urinary bladder detrusor. Implying that part of the.Cajal-like interstitial cells may be the pacemaker cells of contraction or activity of bladder. The role of the other part of the Cajal-like interstitial cells in urinary bladder possibly plays the conduction, the coupling information.
方法:本实验运用采用酶消化法,得到体外培养状态下的ICCs样细胞,透射电镜观察正常成年豚鼠膀胱ICCs样细胞的超微结构；采用激光共聚焦显微镜成像技术记录Fluo4AM负载后的Cajal样间质细胞内钙离子荧光强度的变化,以荧光强度代表细胞内钙离子浓度的变化。；运用膜片钳技术,在全细胞记录模式下观察其是否具有钙电流,以验证其是否为膀胱的起博细胞。
结果：透射电镜观察到CCs样细胞胞浆内线粒体、粗面内质网较丰富,滑面内质网分散分布,中间丝难能见到,未见微管；运用激光共聚焦显微镜成像技术连续扫描,见体外培养的原代膀胱Cajal样间质细胞有2种类型钙震荡：一类振幅大、频率慢而规则的钙震荡；另一类振幅小、频率快且不规则,且形成网络的膀胱Cajal样间质细胞的钙离子震荡的幅度明显高于单个的膀胱Cajal样间质细胞。膀胱逼尿肌细胞钙震荡振幅小不规则,其振幅显著低于Cajal样间质细胞第一类钙震荡振幅；膜片钳试验膀胱Cajal样细胞的钙离子电流不明显。
结论：电镜观察到Cajal样细胞具有独特的超微结构,与胃肠道中ICCs的超微结构有相似；部分膀胱Cajal样细胞具有自发而规律的钙震荡,类似消化道起搏细胞Cajal间质细胞(interstitial cells of Cajal,ICCs)的自发性钙震荡,膀胱逼尿肌无自发性钙震荡。提示：部分膀胱Cajal样细胞是膀胱收缩活动的起搏细胞；另外部分膀胱Cajal样细胞可能起到传导、耦合信息的作用。
Purpose:The purpose of this is to provide proofs to prove that the Cajal-like cells in guinea pig bladder is the pacemaker by observe its utrastructure and electrophysiological, and offer a new starting point linical and treatment of certain diseases (such as the detrusor instability, DI).
Methods:In this experimental we can get the cajal-like interstitial cells by enzyme digestion method. We observe ICCs ultrastructure by transmission electron microscope; Cells were loaded with fluo-4 AM dye. Using laser scanning microscope imaging records the change of calcium fluorescence intensity. Fluorescence intensity was on behalf of changes of the intracellular calcium concentration; by patch clamp technology We observe the calcium current of ICCs in the whole-cell recording mode. It can provide a proof to verify that the ICCs in guinea pig bladder is the pacemaker.
Results:In this experimental the ICCs can be observed to have rich in mitochondria, developed the RER, the sliding surface endoplasmic reticulum, Gorky body. There were 2 different types of spontaneous calcium oscillation in the Cajal-like interstitial cells of bladder:large, slow calcium oscillation and small, irregular calcium oscillation. The first type of calcium oscillation was completely different from those of smooth muscle cells; By the patch clamp technology We can't observe the type calcium current of ICCs in the whole-cell recording mode.
Conclusion:The ICCs in guinea pig bladder have a unique ultrastructure, which similar with the ICCs's in the gastrointestinal tract. We have confirmed that part of the Cajal-like interstitial cells existence of the spontaneous calcium oscillation in the bladder of adult guinea-pig, which like the pacemaker cells of interstitial cells of Cajal in the digestive tract. The spontaneous calcium oscillation was not observed in urinary bladder detrusor. Implying that part of the.Cajal-like interstitial cells may be the pacemaker cells of contraction or activity of bladder. The role of the other part of the Cajal-like interstitial cells in urinary bladder possibly plays the conduction, the coupling information.
引文
1 Andersson K E, Amer A. Urinary bladder contraction and relaxation:physiology and pathophysiology. Physiol Rev,2004,84:935-986.
2. ICCs.S.RSur les ganglions et plexus nerveux de I'intestin. Compt Rend Soc Biol,1893,45:217-233.
3 lino S, Horiguchi K. Interstitial cells of cajal are involved in neurotransmission in the gastrointestinal tract. Acta Histochem Cytochem 2006;39:145-153
4 Ordog T, Ward SM, Sanders KM. Interstitial cells of cajal generate electrical slow waves in the murine stomach. J Physiol,1999,518:257-269.
5于彬,梅峰,童卫东等。慢传输型便秘患者结肠壁内ICCs细胞的形态学研究。消化外科,2004,3：185-189
6 Rolle U, Piotrowska AP, Nemeth L, et al. Altered distribution of interstitial cells of ICCs in Hirschsprung disease. Arch Pathol Lab Med,2002,126(8):928-33.
7 Hanani M, Freund HR. Interstitial cells of ICCs-their role in pacing and signal transmission in the digestive system. Acta Physiol Scand,2000; 170(3):177-190
8 Vannucchi MG. Receptors in interstitial cells of ICCs:identification and possible physiological roles. Microsc Res Tech,1999,47:325-335.
9 Van Der AA F, Roskams T, Blyweert W, et al. Identification of kit positive cells in the human urinary tract. J Urol,2004,171:2492-2496.
10 Metzger R, Schuster T, Till H, et al. ICCs-like cells in the upper urinary tract:comparative study in various species. Pediatr Surg Int,2005,21:169-74.
11 McCloskey KD, Alison GM. Kit positive cells in the guinea pig bladder. J Urol,2002,168:832-836.
12 Sergeant G P, Hollywood M A, McCloskey K D, et al. Specialised pacemaking cells in the rabbit urethra. J Physiol,2000,526:359-366.
13 Biers SM, Reynard JM, Doore T, Brading AF. The functional effects of a c-kit tyrosine inhibitor on guinea-pig and human detrusor BJU Int.2006,97(3):612-616
14 Piaseczna Piotrowska A, Rolle U, Solari V, et al. Interstitial cells of ICCs in the human normal urinary bladder and in the bladder of patients with megacystis-microcolonintestinal hypoperistalsis syndrome. BJU Int,2004,94:143-146.
15 Campbell J, Chempbell G, Ross R. The smooth cell in culture. Physiol Rev,1979,59:1.
16 Ashman LK. The biology of stem cell factor and its receptor C-kit. Int J Biochem CellBiol,1999, 31(10):1037-51.
17. Lecoin L, Gabella G, Le Douarin N. Origin of the c-kit-positive interstitial cells in theavian Dlwel.
18 Ward SM, Burns AJ, Torihashi S, Harney SC, Sanders KM. Impaired development of interstitial cells and intestinal electrical rhythmicity in steel mutants. Am J Physiol,1995,269:C1577-C1585 Development, 1996,122:725-733.
19 Lecoin L, Gabella G, Le Douarin N. Origin of the c-kit-positive interstitial cells in theavian DIwel. Development,1996,122:725-733.
20 Torihashi S, Nishi K, Tokutomi Y, et al. Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype. Gastroenterology,1999,117:140-148.
21于彬,周德山,何扬涛等。胚胎小肠ICCs细胞的发育研究。中国组织化学与细胞化学杂志,2003,12：263-267。
22 Nemeth L, Maddur S, Puri P. Immunoloealizafon of the gap junction protein Connexin43 in the intersfifial ceils of C al in dIe normal an dHirschsprung's disease bowel [J]. J Ped/atr Surg,2000,35(6): 823-828.
23 Davidson RA, McCluskey KD. Morphology an d localization interstifialcells in the gllille8 pig bladder:Structural relationships with smooth mnsoleand neurous[J]. J Urol,2005,173(4):1385-1390.
24 Christensen J. A commentary on the morphological identification of interstitial cells of Cajal in the gut [J]. J Auton Nerv Syst,1992,37:75.
25 Piotrowska PA, Rolie U, Solari V, d. Interstitial cells of ICCs in the human normal urinary bladderan dinthebladder of ellt8with tong a. cystis-micrecolon-intestinal hypoper-istalsis syndrome[J]. BJU Int, 2004,94(1):143.146.
26唐尚杰郭涛杜云惠.食管内心脏电生理检查[M]。云南科技出版社,2005： 3-4
27 Nakayama s, Ohya s, Liu HN, et al. Sulphonylurea receptors differently modulate ICC pacemaker Ca2+ activity and smooth muscle contractility.J Cell Sci.2005 Sep 15;118(Pt 18):4163-4173
28 Anne Epperson, William J. Hatton, Brid Callaghan,etc. Molecular markers expressed incultured and freshly isolated interstitial cells of ICCs.Am J Physiol Cell Physiol,2000,279:C529-C539.
29 Kito Y, Ward SM, Sanders KM. Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine. Am J Physiol Cell Physiol 2005;288:C710-720
1 Smet PJ, Jonavicius J, Marshall VR. Distribution of nitric oxide Synthase-immunoreactive nerves and identification of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cGMP immunohistochemistry [J]. Neuroscience,1996,71(2):337-348.
2 Sanders KM, Koh SD,Ward SM. Interstitial cells of cajal as pacemakers in the gastrointestinal tract[J]. Annu Rev Physiol,2006,68:307-343.
3 Hashitani H, Yanai Y, Suzuki H. Role of interstitial cells and gap junctions in the transmission of spontaneous Ca2+ signals in detrusor smooth muscles of the guinea2p ig urinary bladder[J]. J Physiol, 2004,559 (Pt 2):567-581.
4 Davidson RA,McCloskey KD. Morphology and localization interstitial cells in the guinea-pig bladder:Structural relationship swith smooth muscle and neurous [J]. J Urol,2005,173 (4):1385-1390.
5 Sui GP, Rothery S,Dupont E, et al. Gap junctions and connexin expression in human suburothelial interstitial cells [J]. BJU Int,2002,90 (1):118-129.
6 Hashitani H, Fukuta H, Takano H, et al. Origin and p ropagation of spontaneous excitation in smooth muscle of the guinea2p ig urinary bladder[J]. J Physiol,2001,530 (2):273-286.
7 Shafik A, E12Sibai O, Shafik AA, et al. Identification of interstitial cells of Caial in human urinary bladder:concep t of vesical pacemaker[J]. Urology,2004,64 (4):809-813.
8 Piaseczna Piotrowska A, Rolie U, SolariV, et al. Interstitial cells of Cajal in the human normal urinary bladder and in the bladder of patients with megacystis microcolon intestinal hypoperistalsis syndrome. [J]. BJU Int,2004,94 (1):143-146.
9 Hescheler J, HalbachM, Egert U, et al. Determination of electrical properties of ES cell2derived cardiomyocytes using MEAs [J]. J Electrocardiol,2004,37 (Supp 1):110-116.
10 Koh SD, Jun JY, Kim TW, et al. A Ca (2+) inhibited nonselective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal[J]. J Physiol,2002,540 (Pt 3):803-814.
11 Sergeant GP, Thornbury KD,McHale NG, et al. Interstitial cells of cajal in the urethra[J]. J CellMolMed,2006,10 (2):280-291.
12 Lang RJ, ZoltkowskiBZ, Hammer JM, et al. Electrica I characterization of interstitia I cells of Cajal21ike ce 11s and smo th muscle cells isolated from the mouse ureteropelvic iunction [J]. J Uro I, 2007,177(4):1573-1580.
13 McHale NG, Hollywood MA, Sergeanl GP, et al. Organization and function of ICC in the urinary tract [J]. J Physiol,2006,576 (Pt3):689-694.
14 Skin KS, Rothbery BS, Yellen G. Blocker state dependence and trapping in hyterpolarization2activated cation channels:edidence for an intracellular activation gate[J]. J Gen Physiol, 2001,117 (2):91-101.
15 McCloskey KD, Gurney AM. Kit positive cells in the guinea pig bladder[J]. J Urol,2002,168 (2):832-836.
16 Biers SM, Reynard JM, Doore T. The functional effects of a c-kit tyrosine inhibitor on guinea-pig and human detrusor. [J]. BJU Int,2006,97 (3):612-616.
17 Kubota Y, Kajioka S,Biers SM. Investigation of the effect of the c-kit inhibitor Glivec on isolated guinea2p ig detrusor p reparations[J]. Auton Neurosci,2004,115(1/2):64-73.
2. ICCs.S.RSur les ganglions et plexus nerveux de I'intestin. Compt Rend Soc Biol,1893,45:217-233.
3 lino S, Horiguchi K. Interstitial cells of cajal are involved in neurotransmission in the gastrointestinal tract. Acta Histochem Cytochem 2006;39:145-153
4 Ordog T, Ward SM, Sanders KM. Interstitial cells of cajal generate electrical slow waves in the murine stomach. J Physiol,1999,518:257-269.
5于彬,梅峰,童卫东等。慢传输型便秘患者结肠壁内ICCs细胞的形态学研究。消化外科,2004,3：185-189
6 Rolle U, Piotrowska AP, Nemeth L, et al. Altered distribution of interstitial cells of ICCs in Hirschsprung disease. Arch Pathol Lab Med,2002,126(8):928-33.
7 Hanani M, Freund HR. Interstitial cells of ICCs-their role in pacing and signal transmission in the digestive system. Acta Physiol Scand,2000; 170(3):177-190
8 Vannucchi MG. Receptors in interstitial cells of ICCs:identification and possible physiological roles. Microsc Res Tech,1999,47:325-335.
9 Van Der AA F, Roskams T, Blyweert W, et al. Identification of kit positive cells in the human urinary tract. J Urol,2004,171:2492-2496.
10 Metzger R, Schuster T, Till H, et al. ICCs-like cells in the upper urinary tract:comparative study in various species. Pediatr Surg Int,2005,21:169-74.
11 McCloskey KD, Alison GM. Kit positive cells in the guinea pig bladder. J Urol,2002,168:832-836.
12 Sergeant G P, Hollywood M A, McCloskey K D, et al. Specialised pacemaking cells in the rabbit urethra. J Physiol,2000,526:359-366.
13 Biers SM, Reynard JM, Doore T, Brading AF. The functional effects of a c-kit tyrosine inhibitor on guinea-pig and human detrusor BJU Int.2006,97(3):612-616
14 Piaseczna Piotrowska A, Rolle U, Solari V, et al. Interstitial cells of ICCs in the human normal urinary bladder and in the bladder of patients with megacystis-microcolonintestinal hypoperistalsis syndrome. BJU Int,2004,94:143-146.
15 Campbell J, Chempbell G, Ross R. The smooth cell in culture. Physiol Rev,1979,59:1.
16 Ashman LK. The biology of stem cell factor and its receptor C-kit. Int J Biochem CellBiol,1999, 31(10):1037-51.
17. Lecoin L, Gabella G, Le Douarin N. Origin of the c-kit-positive interstitial cells in theavian Dlwel.
18 Ward SM, Burns AJ, Torihashi S, Harney SC, Sanders KM. Impaired development of interstitial cells and intestinal electrical rhythmicity in steel mutants. Am J Physiol,1995,269:C1577-C1585 Development, 1996,122:725-733.
19 Lecoin L, Gabella G, Le Douarin N. Origin of the c-kit-positive interstitial cells in theavian DIwel. Development,1996,122:725-733.
20 Torihashi S, Nishi K, Tokutomi Y, et al. Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype. Gastroenterology,1999,117:140-148.
21于彬,周德山,何扬涛等。胚胎小肠ICCs细胞的发育研究。中国组织化学与细胞化学杂志,2003,12：263-267。
22 Nemeth L, Maddur S, Puri P. Immunoloealizafon of the gap junction protein Connexin43 in the intersfifial ceils of C al in dIe normal an dHirschsprung's disease bowel [J]. J Ped/atr Surg,2000,35(6): 823-828.
23 Davidson RA, McCluskey KD. Morphology an d localization interstifialcells in the gllille8 pig bladder:Structural relationships with smooth mnsoleand neurous[J]. J Urol,2005,173(4):1385-1390.
24 Christensen J. A commentary on the morphological identification of interstitial cells of Cajal in the gut [J]. J Auton Nerv Syst,1992,37:75.
25 Piotrowska PA, Rolie U, Solari V, d. Interstitial cells of ICCs in the human normal urinary bladderan dinthebladder of ellt8with tong a. cystis-micrecolon-intestinal hypoper-istalsis syndrome[J]. BJU Int, 2004,94(1):143.146.
26唐尚杰郭涛杜云惠.食管内心脏电生理检查[M]。云南科技出版社,2005： 3-4
27 Nakayama s, Ohya s, Liu HN, et al. Sulphonylurea receptors differently modulate ICC pacemaker Ca2+ activity and smooth muscle contractility.J Cell Sci.2005 Sep 15;118(Pt 18):4163-4173
28 Anne Epperson, William J. Hatton, Brid Callaghan,etc. Molecular markers expressed incultured and freshly isolated interstitial cells of ICCs.Am J Physiol Cell Physiol,2000,279:C529-C539.
29 Kito Y, Ward SM, Sanders KM. Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine. Am J Physiol Cell Physiol 2005;288:C710-720
1 Smet PJ, Jonavicius J, Marshall VR. Distribution of nitric oxide Synthase-immunoreactive nerves and identification of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cGMP immunohistochemistry [J]. Neuroscience,1996,71(2):337-348.
2 Sanders KM, Koh SD,Ward SM. Interstitial cells of cajal as pacemakers in the gastrointestinal tract[J]. Annu Rev Physiol,2006,68:307-343.
3 Hashitani H, Yanai Y, Suzuki H. Role of interstitial cells and gap junctions in the transmission of spontaneous Ca2+ signals in detrusor smooth muscles of the guinea2p ig urinary bladder[J]. J Physiol, 2004,559 (Pt 2):567-581.
4 Davidson RA,McCloskey KD. Morphology and localization interstitial cells in the guinea-pig bladder:Structural relationship swith smooth muscle and neurous [J]. J Urol,2005,173 (4):1385-1390.
5 Sui GP, Rothery S,Dupont E, et al. Gap junctions and connexin expression in human suburothelial interstitial cells [J]. BJU Int,2002,90 (1):118-129.
6 Hashitani H, Fukuta H, Takano H, et al. Origin and p ropagation of spontaneous excitation in smooth muscle of the guinea2p ig urinary bladder[J]. J Physiol,2001,530 (2):273-286.
7 Shafik A, E12Sibai O, Shafik AA, et al. Identification of interstitial cells of Caial in human urinary bladder:concep t of vesical pacemaker[J]. Urology,2004,64 (4):809-813.
8 Piaseczna Piotrowska A, Rolie U, SolariV, et al. Interstitial cells of Cajal in the human normal urinary bladder and in the bladder of patients with megacystis microcolon intestinal hypoperistalsis syndrome. [J]. BJU Int,2004,94 (1):143-146.
9 Hescheler J, HalbachM, Egert U, et al. Determination of electrical properties of ES cell2derived cardiomyocytes using MEAs [J]. J Electrocardiol,2004,37 (Supp 1):110-116.
10 Koh SD, Jun JY, Kim TW, et al. A Ca (2+) inhibited nonselective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal[J]. J Physiol,2002,540 (Pt 3):803-814.
11 Sergeant GP, Thornbury KD,McHale NG, et al. Interstitial cells of cajal in the urethra[J]. J CellMolMed,2006,10 (2):280-291.
12 Lang RJ, ZoltkowskiBZ, Hammer JM, et al. Electrica I characterization of interstitia I cells of Cajal21ike ce 11s and smo th muscle cells isolated from the mouse ureteropelvic iunction [J]. J Uro I, 2007,177(4):1573-1580.
13 McHale NG, Hollywood MA, Sergeanl GP, et al. Organization and function of ICC in the urinary tract [J]. J Physiol,2006,576 (Pt3):689-694.
14 Skin KS, Rothbery BS, Yellen G. Blocker state dependence and trapping in hyterpolarization2activated cation channels:edidence for an intracellular activation gate[J]. J Gen Physiol, 2001,117 (2):91-101.
15 McCloskey KD, Gurney AM. Kit positive cells in the guinea pig bladder[J]. J Urol,2002,168 (2):832-836.
16 Biers SM, Reynard JM, Doore T. The functional effects of a c-kit tyrosine inhibitor on guinea-pig and human detrusor. [J]. BJU Int,2006,97 (3):612-616.
17 Kubota Y, Kajioka S,Biers SM. Investigation of the effect of the c-kit inhibitor Glivec on isolated guinea2p ig detrusor p reparations[J]. Auton Neurosci,2004,115(1/2):64-73.
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