The Effects And Mechanisms Of N Pkcs In The Proliferation Of Pulmonary Artery Smooth Muscle Cells Induced By Hypoxia

Part ?: Isolation and Culture of Mouse Pulmonary Artery Smooth Muscle Cells by Magnetic SeparationObjective: Isolation of mouse primary pulmonary artery smooth muscle cells by magnetic separation method to improve the success rate of cell separation and to cultivate primary pulmonary artery smooth muscle cells in vitro and construct hypoxic model to lay a good experimental foundation.At the same time,a new idea was proposed for the isolation of mouse primary pulmonary artery smooth muscle cells.Methods: 1.Healthy adult C57 BL / 6J mice weighing about 20 g were selected and anesthetized by intraperitoneal injection of 4% chloral hydrate(0.01 ml / g)under aseptic conditions.2.After anesthesia,the mice were planed to expose the kidneys and the renal arteries were severed.After the thorax was exposed,3-5 ml of sterilized PBS solution was slowly infused from the right ventricle to the pulmonary artery until the lungs turned white.Then,using a blunt separation device to separate the trachea.3.Slowly inject 3-5 ml PA agarose into the right lung until the lungs turn gray.2-3 ml of Lung agarose is injected into the trachea until the gel fills the lungs.Remove heart and lungs and place in ice-cold PBS for about 5 minutes to allow the gel to coagulate.4.The lung tissue is shredded and then transferred to a 50 ml sterile centrifuge tube.Themagnet is attached and the ferrous tissue moves to the wall of the centrifuge tube near the magnet.The PBS solution is then aspirated and the lung tissue is washed 3 times with 5 ml sterile PBS solution.5.6 ml of collagenase solution was added to the centrifuge tube,poured into a petri dish and digested for 1 hour at 37?.One hour later,lung tissue and collagenase were repeatedly aspirated with a 20 ml syringe and transferred to a sterilized 50 ml centrifuge tube.The magnet was connected and the supernatant was aspirated and washed three times with 5 ml of complete medium to inactivate the collagenase.Finally,add complete medium and then transfer the cell suspension into Petri dish and incubate overnight(5% CO2,37?).6.On the second day,the tissue pieces in the culture dish were poured into a 50 ml centrifuge tube.Connect the magnet,use a complete culture medium to clean the centrifuge tube 3 times,and then into a new sterile Petri dish,placed in 5% CO2,37 ? incubator to continue culturing.3-5 days to replace the medium.During the culturing process,magnetic separation techniques may again be used to separate the tissue fragments in the culture vessel according to cell growth.7.Pulmonary artery smooth muscle cells became predominant cells in the collected cells by adjusting the medium.8.In the first pass,the cell suspension is placed close to the magnet column to remove the remaining iron particles in the cells to obtain purer cells.The cells are then stably cultured.9.The isolated cells were identified by ordinary light microscopy and laser confocal microscopy.Results:1.After the first isolation of pulmonary artery smooth muscle cells,small pieces of microvascular tissue that were black and contained iron particles were visible by ordinary light microscopy.It was shown that iron powder and gel were successfully injected into the right lung and the iron powder bound to pulmonary artery tissue through the gel.After 3 days,cells were seen to crawl out of the small iron-containing vessels.The morphology of the cells showed long fusiform radial.After passage,vascular smooth muscle cells showed "peak-valley" growth after 7-10 days.Thus,initially identified from the morphology of pulmonary artery smooth muscle cells.2.Immunofluorescence staining of mouse pulmonary artery smooth muscle cells was observed under a laser scanning confocal microscope.Blue was the nucleus,green fluorescence was smooth muscle protein expression,and red fluorescence was smooth muscle myosin heavy chain positive.By immunofluorescence in the two smooth muscle cells related to the specific expression of the protein,indicating that the magnetic separation of cells isolated from pulmonary artery smooth muscle cells.Conclusion: The method of magnetic separation of pulmonary artery smooth muscle cells in mice is feasible,and the isolated pulmonary artery smooth muscle cells are in good condition for the follow-up experiments.Part ?: Effects of hypoxia on pulmonary artery smooth muscle cell proliferation and n PKCs expressionObjective: 1.To investigate the effects of hypoxia on pulmonary artery smooth muscle cell proliferation.2.To investigate the effects of hypoxia on the expression of n PKCs in pulmonary artery smooth muscle cells.Methods: 1.Primary pulmonary artery smooth muscle cells of healthy adult C57 BL / 6J mice were isolated by magnetic separation and passaged to cells for stable growth in vitro.2.Select the growth of mouse pulmonary artery smooth muscle cells.According to the different hypoxia treatment time,the cells were divided into 24 h group,48 h group and 72 h group,and each group was set as normoxia control group.The CCK-8 and Brdu method was used to detect the cell proliferation rate in each group.3.Select the growth of mouse pulmonary artery smooth muscle cells.According to the different hypoxia treatment time,the cells were divided into 0h group,24 h group,48 h group and 72 h group Flow cytometry and Western blot was used to detect the expression of PKC?,PKC?,PKC? and PKC? under different hypoxic induction time.4.The experimental results of each group data sorting and statistical analysis.Results: 1.Hypoxia can promote pulmonary artery smooth muscle cell proliferation.The results of CCK-8 showed that there was no significant difference in cell viability between Hypoxia 24 h group and Normoxia 24 h group(P> 0.05),and there was no significant difference between Hypoxia 48 h group and Normoxia 48 h group(P > 0.05).However,the cell viability of 72 h Hypoxia group was significantly higher than that of Normoxia 72 h group(P <0.05).The results of cell proliferation detected by Brdu showed that there was no significant difference between Hypoxia 24 h group and Normoxia 24 h group(P> 0.05).The survival rate of 48 h Hypoxia group was higher than that of Normoxia 48 h group(P <0.05).The survival rate of 72 h Hypoxia group was significantly higher thanthat of Normoxia 72 h group(P <0.05).The results of CCK-8 and Brdu were consistent,showing that hypoxia can promote the proliferation of pulmonary artery smooth muscle cells and hypoxia-induced proliferation of pulmonary artery smooth muscle cells significantly increased,so choose hypoxia-induced 72 h as a follow-up experimental conditions.2.Hypoxia can induce the upregulation of PKC? and PKC? in pulmonary artery smooth muscle cells.The results of flow cytometry showed that the expressions of PKC? and PKC? in hypoxia 72 h were significantly increased(P <0.05)compared with those in normoxia group,while PKC? and PKC? were not significantly different at 72 h P> 0.05).Western blot also showed that the expression of PKC? and PKC? increased significantly at 72h(P <0.05),while PKC? and PKC? had no significant difference at 72h(P> 0.05).From the above two results we can see that in normal pulmonary artery smooth muscle cells,hypoxia can induce the upregulation of PKC?,PKC? expression in pulmonary artery smooth muscle cells and PKC?,PKC? expression no significant change.Conclusion: 1.Hypoxic conditions can induce the proliferation of mouse pulmonary artery smooth muscle cells and the proliferation of hypoxic treatment at 72 h the most significant effect.2.Hypoxia can upregulate the expression of PKC? and PKC? in mouse pulmonary artery smooth muscle cells.Part ?: Hypoxia pro motes t he proliferation of pul monary artery s mooth muscle cells by upregulating PKC? and PKC?Objective: 1.To investigate the relationship between hypoxia-induced up-regulation of n PKCs and the proliferation of pulmonary artery smooth muscle cells.2.To explore the mechanism of hypoxia-induced up-regulation of n PKCs and which leads to proliferation of pulmonary artery smooth muscle cells.Methods: 1.Primary pulmonary artery smooth muscle cells of healthy adult C57 BL / 6J mice were isolated by magnetic separation and passaged to cells for stable growth in vitro.2.Pulmonary artery smooth muscle cells with good growth status were selected and divided into the following groups according to different interventions.PKC?: Normoxia group,Normoxia+PMA group,Hypoxia group,Hypoxia+Rottlerin group.PKC?: Normoxia group,Normoxia+PMA group,Hypoxia group,Hypoxia+ PKC? inhibitor pepitide group.3.A lentiviral vector containing sh RNA fragments for inhibiting PKC? and PKC?,respectively,was constructed and the control group was set up with no-load lentiviral vector.Group by different interventions: Normal group,Scramble group,PKC? knockdown group,PKC? knockdown group.4.Western blot was used to detect the expression of PKC?,PKC?,P-AKT,AKT,P-ERK and ERK in each group.5.Brdu detection of cell proliferation in each group.6.The experimental results of each group data sorting and statistical analysis.Results:1.Hypoxia upregulates PKC? and PKC? leading to pulmonary artery smooth muscle cell proliferation.Brdu test results show that,compared with Hypoxia group,Normoxia+PKC? group and Normoxia+PKC? group of cell proliferation was no significant difference(P> 0.05);compared with Normoxia group,there was no significant difference in cell proliferation between Hypoxia+Rottlerin group and Hypoxia+PKC? inhibitor pepitide group(P> 0.05),these tesults indicates that the inhibitory and agonistic effects of inhibitors and agonists of PKC? and PKC? were in accordance with the experimental requirements.Compared with Normoxia group,the proliferation of Normoxia+PMA group were significantly increased(P <0.05),indicating that simply up-regulating PKC? and PKC? can induce pulmonary artery smooth muscle cell proliferation.Compared with Hypoxia group,the cell proliferation of Hypoxia+Rottlerin group and Hypoxia+PKC? inhibitor pepitide group was significantly reduced.Inhibition of protein kinase C in hypoxia caused pulmonary artery smooth muscle cell proliferation was inhibited.2.Hypoxia induces AKT and ERK phosphorylation by upregulating PKC? and PKC?.Western blot results showed that compared with Normoxia group,the expressions of PKC? and PKC? in Normoxia+PMA group were significantly increased(P <0.05).Compared with hypoxia group,The expressions of PKC? and PKC? in Hypoxia+Rottlerin group and Hypoxia+PKC? inhibitor pepitide group were significantly decreased(P <0.05),which indicated that the agonistic and inhibitory effects of PKC? and PKC? were in line with experimental requirements.Compared with Normoxia group,phosphorylation of AKT and ERK in Normoxia+PMA group was increased significantly(P <0.05).Compared with the Hypoxia group,the phosphorylation of AKT and ERK in Hypoxia+Rottlerin group and Hypoxia+PKC? inhibitor pepitide group was significantly inhibited(P <0.05).Hypoxia can increase the phosphorylation of AKT and ERK by inducing the upregulation of PKC? and PKC?.3.Hypoxia induces proliferation of pulmonary artery smooth muscle cells by inducing anincrease in phosphorylation of AKT and ERK by upregulating PKC? and PKC?.After lentiviral transfection,hypoxia culture 72 h.The result of Western blot showed that the knockout of PKC? and PKC? was successful.Compared with the Normal hypoxia group,the phosphorylation of AKT and ERK in the knockout group of PKC? and PKC? was significantly inhibited(P <0.05),Compared with the Normal hypoxia group,PKC? and PKC? knockout group cell proliferation was significantly inhibited.It shows that protein kinase C is knocked down,resulting in the decrease of phosphorylation of AKT and ERK,which leads to the inhibition of the proliferation of pulmonary artery smooth muscle cells.Conclusion: In pulmonary artery smooth muscle cells,hypoxia can induce the increase of phosphorylation of AKT and ERK by inducing the upregulation of PKC? and PKC?,thereby promoting cell proliferation.