| BackgroundIn the late 19th century,Professor Erdheim first identified a class of epithelial neoplasms associated with the eustachian tube during embryonic development in the Sella region.Subsequently,professor Cushing,an expert in neurosurgery,named it craniopharyngioma in 1932 and it is still used today.According to the world health organization(WHO)of the central nervous system tumor pathology classification in 2007,craniopharyngioma can be fall into adamantinomatous craniopharyngioma(ACP)and papillary craniopharyngioma,and ACP accounts for more than 90%.ACP originates from the survivable epithelial cells which located in the Rathke cyst in the saddle area during embryonic development.Although it is a benign tumor,ACP is often a biological manifestation of malignancy.The prevalence of hypopituitarism in ACP is higher than other tumors in saddle area,especially the GH deficiency(GHD).Hypopituitarism refers to the presence of endocrine function defects along one or more endocrine axes accompanied by related pituitary pathological manifestations.Adult GHD syndrome has been well defined and the clinical manifestation is changes in body composition,reduced exercise capacity and quality of life(QoL),and a series of adverse changes in cardiovascular function.Studies have insisted that tumor compression of pituitary gland and related blood supply vessels is the main cause of GHD.However,some scholars later found that endocrine level and tumor size is not absolutely related.Therefore,the mechanism of GHD in ACP remains to be further studied.Interleukin-1?(IL-1?)was first discovered by Gery in 1972 as a classic pro-inflammatory factor,divided into precursor and mature forms,both of which can promote inflammatory response through its receptor IL-1R1 and play a core role in regulating immune response.Studies have demonstrated that in chornic obstructive pulmonary disease,activation of lung fibroblasts and secretion of extracellular matrix are directly effected by pro-inflammatory factors,and blocking IL-1? expression can completely reverse pulmonary fibrosis.Here,We discovered the maximum extensive correlation between the incidence of GHD and IL-1? secreted by means of ACP through clinical data evaluation.We hypothesized that IL-1? secreted by ACP induces pituitary fibrosis and GHD by promoting the activation of pituitary pericytes.Therefore,it is of great significance to elucidate the pathogenesis of GHD in ACP.In addition,we can use anti-fibrosis drugs and anti-inflammatory drugs to improve GHD.Content and methods1.Collection of clinical dataFrom September 2017 to January 2018,15 surgical specimens of craniopharyngioma were collected from the Department of Neurosurgery,Nanfang Hospital,Southern Medical University.All specimens were diagnosed as ACP by the Department of Pathology,Nanfang Hospital.GHD specimens accounted for 10 cases,NO-GHD specimens accounted for 5 cases,male specimens accounted for 8 cases,female specimens accounted for 7 cases.An overall of 15 specimens were collected,some of which underwent routine fixed dehydration and paraffin embedding,and some fresh tumor tissues were preserved in liquid nitrogen or used in the culture of primary ACP cells.Clinical data of all patients,including endocrine examination results and imaging results,were recorded.2.Improved method of culturing somatotropic cells from rat adenohypophysis200gą10g male Sprague-Dawley(SD)rats had been offered from The Experimental Animal Center of Nanfang Hospital,Southern Medical University.The rats had been anesthetized with the aid of isoflurane inhalation and the pituitary tissues had been randomly divided into agencies.An improved method become used in group A(digesting adenohypophysis with 0.25%trypsin-EDTA,observed by using getting rid of pericytes with the aid of the usage of double filtration and using serum-unfastened medium for culturing somatotropic cells).The conventional technique became used in group B(digesting adenohypophysis with 0.35%collagenase,using serum medium for culturing somatotropic cells,and casting off pericytes via converting the culture dish).The variety of somatotropic cells in group A and B changed into counted from 1 to 6 days.The secretion of GH was compared by ELISA.The activation level of pericyts in the two media was tested by western blot,and cell scratch,CCK-8,cell migration and other functional experiments were used to detect the viability of pericyts.3.Exploring the potential mechanism of the influence of IL-1? secreted by ACP on pituitary GH secretion.To prognosis of GHD,the Body Mass Index(BMI),Insulin Like Growth Factor-1(IGF-1)and height GH values after insulin stimulation test of 15 ACP patients have been recorded.Histological staining was executed on the ACP samples.Levels of 9 proinflammatory cytokines in tumor tissue and cell supernatant were checked by way of Millipore bead arrays.The impact of IL-1? on GH secretion modified into evaluated.Western blot,qRT-PCR and cell functional assays have been used to find out the capability mechanism through which IL-1? acts on GH secretion.The stereotactic ALZET osmotic pump method changed into used to simulate the process of ACP secretinginflammatory cytokines.Recombinant IL-1?(rrIL-1?)and conditioned media(CM)organized from the supernatant of ACP cells become infused at once into the intra-sellar at a charge of 1 ?l/h over 28 days,and then the consequences of IL-1?remedy on pathological changes of pituitary gland and GH secretion had been measured.To similarly verify whether or not IL-1? influences GH secretion through IL-1R1,an IL-1R1 blocker(IL-1R1a,10 mg/kg body weight,once day by day)modified into administered subcutaneously from the number one day until day 28.4.Statistical methodSPSS 19.0 software become used for statistical evaluation of the information.Comparisons among groups had been finished using impartial sample T check or one-way evaluation of variance(ANOVA).Spearman correlation evaluation became used for the correlation test of the two indexes.P<0.05 changed into considered statistically enormous.Results1.Improved method of culturing somatotropic cells from rat adenohypophysisWith the passage of time,the survival charge and GH secretion level of number one somatotropic cells cultured through the modified approach have been substantially better than those cultured by way of the conventional approach.In addition,pericytes commenced to multiply unexpectedly after 3 days via the conventional methods,and their activation index ?-SMA and collagen expression ranges had been substantially elevated.2.Exploring the potential mechanism of the influence of IL-1? secreted by ACP on pituitary GH secretion.There turned into a enormous high best correlation among pituitary fibrosis and GHD(rS=0.756,P=0.001).A variety of cytokines,specifically IL-1?,interleukin-8(IL-8),and monocyte chemoattractant protein-1(MCP-1),have been improved in tumor tissue and mobile supernatant.Only IL-1? showed a giant distinction among the GHD group and the No-GHD institution(P<0.001,F=6.251 in tumor tissue;P=0.003,F=1,529 in mobile supernatant).IL-1? extensively decreased GH secretion in coculture of GH3 and pericytes.The activation of pericytes due to the use of IL-1?changed into mediated through the IL-1R1 signaling pathway.In vivo,IL-1? induces pituitary fibrosis,similarly primary to a reduced diploma of GH.IL-1R1a can antagonize this pathological change.ConclusionThe stepped forward approach of culturing somatotropic cells from rat adenohypophysis solves the hassle of pericyte infection,and extends the way of life time of primary somatotropic cells in vitro.The mobile morphology and hobby of number one somatotropic cells are extensively advanced in comparison with the strategies said in the literature at domestic and overseas.This method can be used for reference via special primary cultures infected through no-goal cells.Also,we located that the move speak among ACP cells and stroma cells in the pituitary,i.E.Pericytes,is vital detail on the Underlying mechanisms of GHD,and we suggest that neutralization of IL-1? signaling might be a capability therapy for GHD in ACP. |
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