Primary Culture Of Smooth Muscle Cells From Human Esophagogastric Junction And Preliminary Study Of Signal Transduction About Contraction And Relaxation In Circular Muscle Of The Lower Esophagus From Patients With Achalasia

Part one Primary culture and identification of smooth muscle cells from esophagogastric junctionObjective:To explore primary culture of smooth muscle cells?SMCs?of the esophagogastric junction?EGJ?and identify the specific phenotype of SMCs.Methods:Smooth muscles of EGJ were harvested from 24 esophageal cancer patients during the course of esophagostomy from January 2015 to October 2017.Enzymatic dispersion?ED?and explant culture with tissues?EC-T?were carried out for primary culture respectively.In ED,collagenase?and Trypsin/EDTA were selected to explore the best way to get primary cells with different digestive concentrations combined with different temperature and duration such as enzyme injected into tissues?EI?and traditional single enzyme digested in 4?or 37?.The number of visible adherent cells per field of microscope?200×??Cells/200×?and the time required for primary cells growth to the first passage day?FPD?were recorded.In EC-T,traditional explant culture method?T?was compared with trypsin digested-tissue method?TD-T?and residual tissues after collagenase?injected in ED?enzyme injected-tissue method,EI-T?,and the first time to see the cells migrated from the tissue blocks?visibility of cells migration,VCM?,FPD and the proportion of positive tissue blocks that cells migrated from?Positive blocks?%??were recorded.Growth characteristics were observed and proliferation experiments?CCK-8?were tested for ED and EC-T cells that cultured in smooth muscle cell medium?SMCM?and DMEM/F12 containing10%newborn bovine serum?10%-F12?respectively.Smooth muscles and cultured cells were identified by alpha-smooth muscle actin??-SMA?,smooth muscle 22 alpha?SM22??,Vimentin,Desmin,cluster of differentiation 90?CD90?and proliferating cell nuclear antigen?PCNA?with immunohisto-chemistry?IHC?,realtime RT-PCR,immunofluorescence?IF?and incell-western.Results:Both ED and EC-T methods could obtain primary cells.There was no statistical difference in Cells/200×of ED?P=0.864?,but it cost less time in FPD of EI method with low collagenase?concentration?0.5 mg/ml?combined with low temperature?4??and long-term?14-24h?to culture primary cells?median time 8.00d,P<0.05?.In EC-T,EI-T was better than T and TD-T in VCM?median time 6.00d,P<0.05?,FPD?median time 15.50d,P<0.05?and Positive blocks?%??median percentage 86.00%,P<0.05?.In total FPD,ED was less than EC-T?P=0.000?.Primary cells were mainly spindle and long-spindle shaped inhomogeneously.They could grow in a certain direction and show“hills and valleys”morphology.Proliferation curves of cells cultured in SMCM was better than that in 10%-F12,and cells could passage to 6-8 generations in SMCM but only 3-4 generations in10%-F12.They would enlarge gradually with the number of passages and lose the spindle structure in the end.mRNA and protein of?-SMA,SM22?,Vimentin,Desmin,CD90 and PCNA could be detected in tissues and cells,but the expression level of smooth muscle markers was different in culture conditions.Conclusions:Cells obtained from EGJ smooth tissues in ED and EC-T could be identified as SMCs by smooth muscle markers.It was feasible to extract and culture SMCs from EGJ in vitro.The culture cycle of SMCs obtained by ED was short.SMCs cultured with SMCM was beneficial to expand cell population effectively,but cultured with 10%-F12 showed better phenotype of smooth muscle markers.Part two The effects of acetylcholine on intracellular calcium fluorescence in smooth muscle cells of human esophago-gastric junction cultured in vitroObjective:To study effects of acetylcholine?Ach?on the changes of intracellular calcium(Ca²⁺)fluorescence([Ca²⁺]_i)in smooth muscle cells?SMCs?of human esophagogastric junction?EGJ?cultured in vitro in order to determine human EGJ smooth muscle cell model.Methods:Smooth muscles of EGJ were harvested from 9 esophageal cancer patients during the course of esophagostomy from November 2016 to October 2017.Six kinds of primary EGJ SMCs including clasp fiber?Clasp?,sling fiber?Sling?,esophageal circular muscle?EC?,esophageal longitudinal muscle?EL?,gastric circular muscle-sling?GC-S?and gastric circular muscle-clasp?GC-C?,were obtained by enzymatic digestion?ED?and explant culture with enzyme injected-tissue?EI-T?methods.SMCs were cultured with smooth muscle cell medium?SMCM?and DMEM/F-12 medium containing 10%newborn bovine serum?10%-F12?respectively.The third generation in SMCM and second generation in 10%-F12 of SMCs were incubated with Fluo-3/am?5?mol/L?for 30 min.The effect of 10^-6 mM Ach on[Ca²⁺]_i of SMCs in Ca²⁺and Ca²⁺-free buffer was observed under confocal microscopy,and values and corresponding time of fluorescence activity were recorded.Data of cells derived from the same tissue was compared with that of Ca²⁺and Ca²⁺-free buffer before and after Ca²⁺activity.Results:There was different basal[Ca²⁺]_i in EGJ SMCs cultured in different media.The[Ca²⁺]_i of cells in each group was changed after stimulated by Ach about 2s.The Ca²⁺fluorescence activity of EL,GC-S and GC-C was relatively gentle compared with that of Clasp,Sling and EC.In Ca²⁺buffer,the basic[Ca²⁺]_i of cells was stronger than that in Ca²⁺-free buffer with long-term Ca²⁺activity,but the fluorescence peak was not sharp.In Ca²⁺-free buffer,the[Ca²⁺]_i of cells obtained by ED showed typical intracellular Ca²⁺release with or without long-term Ca²⁺activity.SMCs of Clasp,Sling,EC and GC-C were mainly characterized by release of intracellular Ca²⁺stores,but for EL and GC-S were influx of extracellular Ca²⁺.The statistical difference in the peak fluorescence of Ca²⁺activity of Clasp,Sling,EC and EL cultured in 10%-F12 by ED method was found between Ca²⁺and Ca²⁺-free buffers.In SMCs obtained by EI-T method,there was a statistical difference in the peak fluorescence of Sling cultured in 10%-F12and GC-S in both mediums,and the activity of Ca²⁺in each group was not typical as that of ED method.Conclusions:During the changes of[Ca²⁺]_i in the EGJ SMCs induced by Ach in vitro,Clasp,Sling,EC,EL,GC-S and GC-C did not simply depend on one kind of Ca²⁺activity,release of intracellular Ca²⁺stores or influx of extracellular Ca²⁺,but to combine them to maintain a long-term Ca²⁺activity.The model of human EGJ SMCs in vitro could be created by SMCs obtained with ED method and cultured in DMEM/F-12 medium with 10%newborn bovine serum.Part three Preliminary study of calcium-related proteins and nitric oxide synthase expression in circular muscle of the lower esophagus from patients with achalasiaObjective:To investigate the expression of Ca²⁺-related signal molecules and nitric oxide synthase?NOS?in esophageal achalasia.Methods:Circular muscles from the lower esophagus were harvested from achalasia patients and esophageal cancer patients?controls?.Subtypes of L-type calcium channel?LTCC?,inositol 1,4,5-trisphosphate receptors?IP₃Rs?,ryanodine receptors?RyRs?and nitric oxide synthases?NOSs?were detected by real-time RT-PCR and immunohistochemistry?IHC?,combined with base sequencing of RT-PCR products.mRNA expression of these proteins in achalasia was analyzed by linear correlation analysis with integrated relaxation pressure?IRP?of esophageal manometry.Results:There were ten patients the achalasia group and seventeen patients in the control group.i NOS mRNA was positive but IHC staining was negative in both groups.mRNA and protein expression of n NOS,Cav1.2,Cav1.3,IP₃R1,IP₃R2,IP₃R3 and RyR2,could be detected in both groups,but eNOS,Cav1.1,RyR1 and RyR3 were not.In achalasia,iNOS,n NOS,Cav1.3,IP₃R1 and IP₃R2 increased at mRNA level,but only IP₃R2 increased significantly at protein level as mRNA.iNOS and Cav1.3 mRNA were positively correlated with IRP,and there were no linear relations in others.Conclusions:In achalasia pathogenesis,NOSs was still involved in the regulation of the pathological status of esophageal smooth muscle,and i NOS mRNA increased significantly accompanying with reduced of the expression of n NOS in smooth muscle cells.Calcium related proteins might play an important role in the pathophysiology of achalasia as an myogenic factor.Conclusions:1.Cells obtained from esophagogastric junction?EGJ?smooth tissues in enzymatic digestion?ED?and explant culture with tissues?EC-T?could be identified as smooth muscle cells?SMCs?by smooth muscle markers.In ED,method with enzyme?collagenase??injected into tissues?EI?in low concentration?0.5mg/ml?combined with low temperature?4??and long-term?14-24h?to culture primary cells was efficient,and its culture cycle of SMCs was short.The residual tissues could be used directly in explant culture method to obtain SMCs with the best efficiency.It was feasible to extract and culture SMCs from EGJ in vitro.SMCs cultured with smooth muscle cell medium?SMCM?was beneficial to expand cell population effectively,but cultured with DMEM/F-12 medium with 10%newborn bovine serum?10%-F12?showed better phenotype of smooth muscle markers.2.Basing on the changes of calcium fluorescence induced by Ach of SMCs cultured in vitro,intracellular calcium release was the main feature of Clasp fiber,Sling fiber,esophageal circular muscle?EC?and gastric circular muscles-calsp?GC-C?,but for esophageal longitudinal muscle?EL?and gastric circular muscle-sling?GC-S?was extracellular calcium influx.These cells did not simply depend on one kind of Ca²⁺activity as release of intracellular Ca²⁺stores or influx of extracellular Ca²⁺,but to combine them to maintain a long-term Ca²⁺activity.The model of human EGJ SMCs in vitro could be created by SMCs obtained with ED method and cultured in 10%-F12.3.In achalasia pathogenesis,nitric oxide synthases?NOSs?were still involved in the regulation of the pathological status of esophageal smooth muscle as inducible NOS?iNOS?mRNA increased significantly accompanying with reduced of the expression of neuronal NOS?n NOS?in SMCs.Calcium related proteins might play an important role in the pathophysiology of achalasia as an myogenic factor.