The Regulation Mechanism Of PKCδ In Abnormal Signaling Pathway Of Human Somatotropinoma Cells And Its Corelation With SSTA Theraputic Effect

Part I Expression of PKC8and its Downstream Factors in Primary Cultured Human Somatotropinoma cellsObjective:To investigate the method of primary culture human somatotropinoma cells, and observe the expression and the distribution of PKC8and its downstream factors CREB, ERK1/2in human somatotropinomas.Methods:Tumor tissues of5patients with somatotrophinomas were primary cultured and paraffin embedded simultaneously.The expressions of PKC5,CREB and ERK1/2were detected by immunohistochemistry and Immunofluorescence.Results:Human somatotropinoma cells grew stick to the wall after24hours’s incubation. Cells were closely connected and may grew into a monolayer with a "cobblestone-like" appearance. As those detected by immunohistochemistry and Immunofluorescence techniques,PKC8and ERK1/2were mainly expressed in the cytoplasm,while CREB was mainly expressed in nucleus.Conclusion:Primary culture of human somatotropinoma cells can be successfully achieved. PKCδ and its downstream factors CREB, ERK1/2expressed in human somatotropinomas. Part Ⅱ Effect and Regulation Mechanisms of PKCδ and its Down-stream Factors in Primary Cultured Human Somatotropinoma CellsObjective:To investigate effect and regulation mechanisms of PKCδ and its downstream factors in primary cultured human somatotropinoma cells.Methods:Primary cultured somatotropinoma cells were divided into Control, PMA,Rottlerin and mix groups.After different conditions of invention,GH secretion levels were detected by Elisa kit,proliferation index were detected by cell counting kit8,while phosphorylation levels of PKC8,CREB and ERK1/2were measured by Western blot.Results:PMA increased GH secretion and cell proliferation level,meanwhile the pnosphorylation of PKC8,CREB and ERK1/2were promoted (p<0.05).However, Rottlerin decreased GH secretion and cell proliferation level,meanwhile the pnosphorylation of PKCδ,CREB and ERK1/2were down regulated (p<0.05). Compaired with PMA invention,combination of PMA and Rottlerin treatment decreased GH secretion and cell proliferation level,meanwhile the pnosphorylation of PKCδ,CREB and ERK1/2were down regulated (p<0.05).while compaired with Rottlerin invention,these combination treatment increased GH secretion and cell proliferation level,mean while the pnosphorylation of PKCδ,CREB and ERK1/2were promoted (p<0.05).Conclusion:Activation of PKCδ will lead to an increased GH secretion effect and cell proliferation level.meanwhile the pnosphorylation of PKCδ,CREB and ERK1/2were promoted.As individuals differed from each other,further researchs were required. Part III Effects of Somatostatin Analogs on Growth Hormone Secretion in Human Somatotrophinomas and there Correlation with PKCδ Regulation Pathway&Expression of Gsp OncogeneObjective:To investigate the Effects of Somatostatin Analogs on Growth Hormone Secretion in Human Somatotrophinomas,and there Correlation with PKC8Regulation Pathway&Expression of Gsp Oncogene.Methods:Primary cultured somatotropinoma cells were divided into Control and Somatost atin groups. After different conditions of invention, GH secretion levels were detected by El isa kit, proliferation index were detected by cell counting kit8, while phosphorylation level s of PKCδ, CREB and ERK1/2were measured by Western blot.Meanwhile, Clinical data of20patients of GH-secreting adenomas were analyzed, GH/IGF-1hormone changes before and after SSTA injection were evaluated,then compaired with their in vitro SSTA theraputic effect. DNAs of each sample were used for polymerase chain reaction (PCR), and then used for Gsa gene sequencing.Results:SSTA decreased GH secretion and cell proliferation level,meanwhile the pnosphor ylation of PKCδ,CREB and ERK1/2were all inhibited (p<0.05), Among these20cases, SSTA had different effects on GH secretion,13cases inhibitated,while7cases with no significant effect.Among13inhibitated cases,gsp oncogene observed in6cases,none gsp oncogene was found in another group.Inhibition effect of GH secretion after somatostatin administration showed no significant correlation with gsp oncogene using a chi-square test (p>0.05).Conclusion:SSTA treatment inhibited GH secretion and cell growth of primary cultured human somatotropinoma cells,while individual differences of theraputic effect existed among different samples, meanwhile the pnosphorylation of PKCδ,CREB and ERK1/2were also down regulated.Patients with positive expression of gsp oncogene seems to had a better reaction with SSTA therapy when compared with gsp negative ones.However, no significant variance was found between gsp positive and negative samples according to statistics analysis.As individuals differed from each other,further researchs were required.