Effect of moxibustion on immune function homeostasis in rats with diarrhea irritable bowel syndrome based on SCF/c-kit signaling pathway / 中国针灸

OBJECTIVE@#To observe the effects of moxibustion on the stem cell factor (SCF)/tyrosine kinase receptor (c-kit) signaling pathway and immune function in rats with diarrhea irritable bowel syndrome (IBS-D), and to explore the mechanism of moxibustion for IBS-D.@*METHODS@#Among 52 young rats born from 6 healthy pregnant SPF rats, 12 rats were randomly selected into the normal group, and the remaining 40 rats were treated with the three-factor combination method of maternal separation, acetic acid enema and chronic restraint stress to establish the IBS-D rat model. Thirty-six rats with successful IBS-D model were randomly divided into a model group, a moxibustion group, and a medication group, 12 rats in each group. The rats in the moxibustion group were treated with suspension moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the medication group were treated with intragastric administration of rifaximin suspension (150 mg/kg). All the treatments were given once a day for 7 consecutive days. The body mass, loose stool rate (LSR), the minimum volume threshold when abdominal withdrawal reflex (AWR) scored 3 were measured before acetic acid enema (35 days old), after modeling (45 days old), and after intervention (53 days old). After intervention (53 days old), HE staining was used to observe the morphology of colon tissue, and spleen and thymus coefficients were measured; ELISA method was used to detect serum inflammatory factors (tumor necrosis factor a [TNF-a], interleukin [IL]-10, IL-8), T-lymphocyte subsets (CD+4, CD+8, CD+45), value of CD+4/CD+8 and immune globulin (IgA, IgG, IgM); real-time PCR method and Western blot method was used to detect the expression of SCF, c-kit mRNA and protein in colon tissue; immunofluorescence staining method were used to detect positive expression of SCF and c-kit.@*RESULTS@#After intervention, compared with the normal group, in the model group, the body mass and the minimum volume threshold when AWR scored 3 were decreased (P<0.01), LSR, spleen and thymus coefficients, serum levels of TNF-α, IL-8, CD+4, CD+45, CD+4/CD+8, IgA, IgG, IgM were increased (P<0.01), serum IL-10 level and protein and mRNA expression of SCF and c-kit in colon tissue were decreased (P<0.01), and the positive expression of SCF and c-kit was decreased (P<0.01). Compared with the model group, in the moxibustion group and the medication group, the body mass and the minimum volume threshold when AWR scored 3 were increased (P<0.01, P<0.05), LSR, spleen and thymus coefficients, serum levels of TNF-α, IL-8, CD+4, CD+8, CD+45, CD+4/CD+8, IgA, IgG, IgM were decreased (P<0.01, P<0.05), serum IL-10 level and protein and mRNA expression of SCF and c-kit in colon tissue were increased (P<0.01), and the positive expression of SCF and c-kit was increased (P<0.01). Compared with the medication group, in the moxibustion group, the level of serum CD+4 was decreased (P<0.05), the value of CD+4/CD+8 was increased (P<0.01), and there was no significant difference in other indexes (P>0.05). The expression of SCF and c-kit mRNA was positively correlated with the minimum volume threshold when AWR scored 3 and IL-10 (P<0.01), and negatively correlated with remaining indexes (P<0.01, P<0.05).@*CONCLUSION@#Moxibustion could reduce visceral hypersensitivity, improve symptoms of abdominal pain and diarrhea in IBS-D rats, and its mechanism may be related to up-regulation of the expression of SCF/c-kit signaling pathway and improvement of IBS-D immune function.

Role of Jiawei Shenqi-wan in Treatment of Cathartic Colon on the Basis of Interstitial Cells of Cajal / 胃肠病学

Background: Cathartic colon belongs to the category of 'constipation' in Traditional Chinese Medicine, and its pathogenesis is related to deficiency of kidney temperament and weak promotion ability, which has become a hot spot of global medical attention. Aims: To investigate the effect and possible mechanism of Jiawei Shenqi-wan (JSQW) on intestinal transmission function and pathological changes of interstitial cells of Cajal (ICCs) in rats with cathartic colon. Methods: Forty rats were randomly divided into blank group, model group, prucalopride group and JSQW group. Fecal moisture content, fecal particle number and intestinal transit rate were detected. The pathological changes of ICCs were observed under transmission electron microscope. RT-qPCR was used to detect the mRNA expressions of water channel proteins (AQP3 and AQP9) and SCF/c-kit pathway. Immunohistochemistry was used to measure the expressions of α-smooth muscle actin (α-SMA), inducible nitric oxide synthase (iNOS) and 5-hydroxytryptamine (5-HT)

Therapeutic effect and mechanism of Shouhui Tongbian Capsules on slow transit constipation model mice / 中国中药杂志

Shouhui Tongbian Capsules was used to explore the therapeutic effect and potential mechanism on slow transit constipation model mice induced by loperamide hydrochloride. In the experiment, loperamide hydrochloride-induced ICR mice were used as the model of slow transit constipation. Fifty ICR mice were divided into the blank group, model group and high, medium and low dose groups of Shouhui Tongbian Capsules extract(100, 200 and 400 mg·kg~(-1)). The model group and the administration groups were then modeled using loperamide hydrochloride intragastrically to obtain slow transit constipation. After successful modeling, high, medium and low doses of drugs were given to each drug group by intragastric administration. After 14 days of administration, the first defecation time, 6 h defecation grain number, 6 h defecation wet weight and dry weight, black feces discharged within 6 h and the fecal water content were measured. Intestinal tissues were taken for c-Kit and SCF immunohistochemical sections to detect the expression of c-Kit and SCF in the blank group, model group and high, medium and low dose groups of the medicinal extract of Shouhui Tongbian Capsules. The tissue changes in the intestinal wall of mice were detected by HE staining. At the same time, partial intestinal tissues were taken to test the activity of ATP synthase and isocitrate dehydrogenase in intestinal tissues of mice. RESULTS:: showed that Shouhui Tongbian Capsules effectively improved the symptoms of slow transit constipation in ICR mice and promoted intestinal movement. Shouhui Tongbian Capsules obviously shortened the time of discharging black stool for the first time, improved the intestinal propulsion rate, increased the water content and amount of feces, and improved the constipation symptoms. Mechanism study revealed that Shouhui Tongbian Capsules increased ATP synthase activity and mitochondrial isocitrate dehydrogenase activity in intestinal tissue, and up-regulated c-Kit/SCF signaling pathway to promote interstitial Cajal cells proliferation, intestinal nerve transmission, intestinal motility and transport capacity.

TNF-α inhibits SCF, ghrelin, and substance P expressions through the NF-κB pathway activation in interstitial cells of Cajal

Ulcerative colitis is a chronic inflammatory disease of the colon where intestinal motility is disturbed. Interstitial cells of Cajal (ICC) are required to maintain normal intestinal motility. In the present study, we assessed the effect of tumor necrosis factor-alpha (TNF-α) on viability and apoptosis of ICC, as well as on the expression of stem cell factor (SCF), ghrelin, and substance P. ICC were derived from the small intestines of Swiss albino mice. Cell viability and apoptosis were measured using CCK-8 assay and flow cytometry, respectively. ELISA was used to measure the concentrations of IL-1β, IL-6, ghrelin, substance P, and endothelin-1. Quantitative RT-PCR was used to measure the expression of SCF. Western blotting was used to measure the expression of apoptosis-related proteins, interleukins, SCF, and NF-κB signaling pathway proteins. TNF-α induced inflammatory injury in ICC by decreasing cell viability and increasing apoptosis and levels of IL-1β and IL-6. TNF-α decreased the levels of SCF, ghrelin, and substance P, but had no effect on endothelin-1. TNF-α down-regulated expressions of SCF, ghrelin, and substance P by activating the NF-κB pathway in ICC. In conclusion, TNF-α down-regulated the expressions of SCF, ghrelin, and substance P via the activation of the NF-κB pathway in ICC.

Effects of different methods of acupuncture and moxibustion on functional constipation in rats:a comparative study / 中国针灸

<p><b>OBJECTIVE</b>To compare the effects of acupuncture, electroacupuncture (EA) and moxibustion on functional constipation in rats.</p><p><b>METHODS</b>Sixty male Sprague-Dawley rats were divided into a control group (=8), a model group (=11), a medication group (=8), an acupuncture group (=11), an EA group (=11) and a moxibustion group (=11) by random number table. The rats in the model group, medication group, acupuncture group, EA group and moxibustion group were treated with intragastric administration of loperamide hydrochloride for 6 days continuously to establish the functional constipation models, while equal volume of drinking water was administrated to rats in the control group at the same time. The rats in the acupuncture group, EA group and moxibustion group were respectively treated with acupuncture, EA and moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) one hour after intragastric administration; rats in the medication group were treated with intragastric administration of cisapride suspension. All the treatment was given once a day for 6 days. At the last day of intervention, the 24-hour food intake, stool quantity and its water content were measured in each group; the pushing rate of intestine was measured; the structure of colon tissue and acidic mucus in its mucous layer were observed by hematoxylin-eosin dyeing and alcian blue dyeing; the expression of stem cell factor (SCF) and c-kit mRNA was detected by real-time PCR.</p><p><b>RESULTS</b>Compared with the control group, the 24-hour food intake and stool quantity were reduced in the model group (both<0.01), and the water content of stool and pushing rate of intestine were reduced (both<0.01); compared with the model group, the stool quantity and its water content were increased in the medication group, acupuncture group, EA group (<0.05,<0.01), which were not significantly different from those in the moxibustion group (both>0.05). The pushing rate of intestine in each intervention group was increased (all<0.01). The 24-hour food intake and stool quantity in the medication group were not significantly different from those in the acupuncture group, EA group and moxibustion group (all>0.05), and the water content of stool was only reduced in the moxibustion group (<0.01). The pushing rate of intestine in the acupuncture group and moxibustion group was lower than that in the medication group (both<0.01), while that in the EA group was not significantly different from that in the medication group (>0.05). The water content of stool in the moxibustion group was lower than that in the acupuncture group and EA group (both<0.01). The pushing rate of intestine in the acupuncture group and moxibustion group was lower than that in the EA group (both<0.01). The HE staining result indicated the structure of colon tissue was normal, complete and similar in each group; the alcian blue staining indicated the acidic mucosubstance in the model group was lower than that in the control group; compared with the model group, the acidic mucosubstance in the medication group, acupuncture group, EA group and moxibustion group was all increased. Compared with the control group, the expression of SCF and c-kit mRNA was reduced in the model group (both<0.05); compared with the model group, the expression of SCF and c-kit mRNA was increased in the medication group, acupuncture group, EA group and moxibustion group (all<0.05); compared with the moxibustion group, the expression of c-kit mRNA was reduced in the acupuncture group and EA group (both<0.05).</p><p><b>CONCLUSIONS</b>Acupuncture, EA and moxibustion all can play a positive regulative role on functional constipation in rats, in which EA has the best efficacy, followed by acupuncture.</p>

Effects of Electroacupuncture on Ultrastructure of Interstitial Cells of Cajal and Stem Cell Factor-kit Signal Pathway of Gastric Antrum in Diabetic Gastroparesis Rats / 针刺研究

OBJECTIVE: To observe the effects of electroacupuncture (EA) on gastrointestinal motility and the ultrastructure of interstitial cells of Cajal (ICC) and the expressions of c-kit receptor protein and stem cell factor (SCF) mRNA in diabetic gastroparesis (DGP) rats, so as to explore its mechanism. METHODS: Fifty SD rats were randomly divided into normal, model, acupoint, non-acupoint and metoclopramide groups (n＝10 rats/group). DGP model was established by intraperitoneal injection of streptozotocin (STZ, 2%), and raised with high-sugar high-fat diet irregularly. EA (sparse-dense, 10 Hz/50 Hz, 2 mA, 20 min) was applied at "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sanyinjiao" (SP 6), and the corresponding non-acupoints of the 3 acupoints, daily for 15 days. The rats in metoclopramide group received intragastric administration of metoclopramide (1.7%, 1 mL/100 g) for 15 days, once a day. Blood sugar was determined with One Touch blood glucose test paper. The gastric emptying rate (GER) and the intestinal propulsion rate (IPR) were measured by intragastric phenol red. The ultrastructure of ICC was detected by transmission electron microscopy. The expression levels of c-kit receptor protein and SCF mRNA of gastric antrum were examined respectively by Western blot and RT-PCR. RESULTS: Compared with the normal group, the blood glucose significantly increased in the model group (P<0.01), while the GER, IRP and the expression level of SCF mRNA in the gastric antrum significantly decreased (P<0.01), and the ultrastructure of ICC appeared apoptosis-like changes. The blood glucose of the EA group was obviously decreased compared with that of the model group (P<0.05); the GER and IRP significantly increased(P<0.05, P<0.01); the expression level of SCF mRNA increased (P<0.01), the number of ICC increased and its ultrastructure was repaired. There was some relief on ICC ultrastructure in the acupoint group compared with that in the non-acupoint group; and SCF mRNA increased (P<0.05). There was no significant difference on c-kit receptor expression among all the modeling groups (P＞0.05). CONCLUSIONS: EA at ST 36, etc. can regulate the blood glucose and improve gastrointestinal emptying in DGP rats. The mechanism may be related to up-regulating SCF mRNA, repairing ICC ultrastructure, restoring the pacing function, and improving gastrointestinal motility.

AFT024-SCF construction and functional identification / 中国病理生理杂志

AIM:To construct AFT024-SCF cell line and HPC-Lhx2 cell line for confirming the biological function of AFT024-SCF.METHODS:The HPC-Lhx2 cell line, AFT024-SCF cell line and AFT024-GFP cell line were constructed by retro-viral infection.The expression of stem cell factor(SCF) in AFT024-SCF cells was detected by real-time PCR and Western blot.SCF in the supernatant of AFT024-SCF was detected with ELISA.The supernatant of AFT024-SCF and AFT024-GFP were collected and then diluted (1:10) with basic IMDM medium.So we made 4 culture medium:AFT024-SCF medium was used for experiment group, AFT024-GFP medium was used for endogenous negative control, IM-DM basic medium was used for exogenous negative control, and IMDM basic medium with SCF was used for positive con-trol.SCF-dependent HPC-Lhx2 cell line was cultured in these 4 different medium for 72 h.According to MTT method and colony forming experiment, the biological function of AFT024-SCF was confirmed by the proliferation ability of SCF-depend-ent HPC-Lhx2 cell line.RESULTS:SCF was highly expressed in AFT024-SCF cells.After cultured for 72 h, neither IM-DM basic medium nor GFP-AFT024 medium support HPC-Lhx2 cell line proliferation.However, AFT024-SCF medium supported HPC-Lhx2 cell line expansion as well as the positive control medium.CONCLUSION:AFT024-SCF cells ex-press SCF successfully and recombinant SCF can be replaced by the supernatant of AFT024-SCF culture medium for expan-ding HPC-Lhx2 cell line in vitro.

Relação da expressão de fatores de crescimento celular (IGF-1) e (SCF) com fatores prognósticos e o alvo da rapamicina em mamíferos (m-TOR) em mastocitomas cutâneos caninos / IGF-1 and SCF protein expression in cutaneous mast cell tumors in dogs and relation to prognostic factors and mammalian target of rapamycin (m-TOR)

O mastocitoma cutâneo (MTC) é a neoplasia maligna mais comum na pele dos cães e seu comportamento biológico é muito variável. Dentre os fatores prognósticos estudados nos MTCs, a classificação histopatológica, o índice proliferativo e o padrão de expressão doc-KIT são os que apresentam uma associação mais relevante com o provável prognóstico deste tumor. O objetivo deste trabalho foi avaliar a expressão proteica de fator de crescimento semelhante à insulina tipo 1 (IGF-1), fator de célula tronco (SCF) e sua relação com o receptor tirosina quinase (c-KIT), alvo da rapamicina em mamíferos (m-TOR), grau histológico, índice proliferativo pelo KI-67e o número de figuras de mitose (IM) com dados clínicos de cães com MTCs . Foram utilizadas 133 amostras de MTCs, provenientes de 133 cães, dispostas em lâminas de microarranjo de tecidos (TMA). A técnica de imuno-histoquímica foi utilizada para a avaliação destas proteínas. Observou-se associação entre SCF e, a graduação histopatológica proposta em 2011, índice mitótico, proliferação celular (KI-67), escore de IGF-1, local da lesão, idade dos animais e padrão imuno-histoquímico do receptor c-KIT. A relação de dependência também foi observada entre IGF-1 e o porte dos animais, IM, m-TOR e c-KIT. A expressão de SCF teve relacção com a agressividade dos MTCs caninos, uma vez que foi mais freqüente em MTCs com c-KIT citoplasmático. A relação entre a expressão de IGF-1, SCF, c-KIT e m-TOR pode estar associada à integralização de suas vias de ação. A expressão de IGF-1 está associada à MTCs em cães de porte grande.

Cutaneous mast cell tumor (MCT) is one of the most common neoplasms in the skin of dogs and express variable biological behavior. Among the MTC aspects studied, histological classification, proliferative index and protein expression of c-KIT show the most defined connection with the tumor prognostic. The aim of this study was to evaluate the protein expression of insulin-like growth factor type 1 (IGF-1), steam cell factor (SCF) and theit relationship with tyrosine kinase receptor (c-KIT), mammalian target of rapamycin (m-TOR), histological classification (KI-67), proliferative and mitotic index and epidemiological data in MTCs. In this study 133 MTC samples from 133 animals were used, arranged in tissue microarray (TMA) slides. The TMA was used for evaluation the proteins. An association was observed between SCF and histological grade proposed in 2011, mitotic index, cell proliferation, IGF-1, lesion site, age of the animals, and immunohistochemical pattern c-KIT receptor. The dependence relationship was also observed between IGF-1 and animal size, mitotic index, m-TOR and c-KIT. The SCF protein expression was related to canine MTCs aggressiveness, since it is more frequent in MCTs with c-KIT cytoplasmic. The relationship between the expression of IGF-1, SCF, c-KIT e m-TOR can be associated with the integration of its actions ways. The IGF-1 expression is associated with large dog breeds MTCs.

La vía ubiquitina-proteasoma ¿destruir o construir? ese es el dilema / Ubiquitin-proteasome pathway to built or to destroy? that is the question

Introducción: la construcción de nuevas células es un proceso complejo y para lograr que sea unidireccional e irreversible la célula utiliza el mecanismo de destruir proteínas que se oponen al paso de una etapa a la siguiente. Objetivo: demostrar que la destrucción de proteínas contribuye a la reproducción celular. Método: se analizaron más de 500 artículos de los últimos 5 años publicados en revistas nacionales y de circulación internacional, disponibles en las bases de datos HINARI, PubMed y Perii y localizados mediante el sitio infomed. Desarrollo: primero, se hizo una exposición sobre la vía ubiquitina-proteasoma. Después, se analizó la participación en el ciclo celular de los dos grandes complejos con actividad de ubiquitina-ligasa que son los encargados de marcar las proteínas que deben ser destruidas. Estos complejos actúan consecutiva y coordinadamente, y sus acciones determinan el progreso en un solo sentido del ciclo de vida de la célula. Conclusiones: la destrucción selectiva de proteínas mediante la vía ubiquitina proteasoma permite la formación de nuevas células y con ello la perpetuación de la vida.

Introduction: the generation of new cells is an extremely complex process. To become this process unidirectional and irreversible cells destroy proteins which actions are oppose to the transition from one phase to the next one. Objective: to show that protein destruction is essential for cell reproduction. Material and Methods: more than 500 papers published during the last five years in national and international journals were analyzed. These articles are available in HINARI, PubMed, and Perii databases and were localized through infomed. Development: first, the ubiquitin-proteasome pathway is presented. Next, the contribution of the complexes SCF an anaphase promoting complex to the progression of the cell cycle is analysed. These complexes act consecutively and coordinately and their actions determine de progression of the cell´s life. Conclusions: the selective destroy of specific proteins by mean of ubiquitin-proteasome pathway, allow new cells formation, and ensure the continuity of life.

Expression and significance of c-kit messenger ribonucleic acid and c-kit protein in diabetic cystopathy in guinea pig / 中国病理生理杂志

AIM: To determine the expression of c-kit mRNA and protein in the bladders in guinea pigs with diabetic cystopathy (DCP) and to explore the correlation and mechanisms between c-kit expression and DCP. METHODS: Sixty guinea pigs were divided randomly into control group (n=20) and experimental group (n=40). The guinea pigs in experimental group were injected with streptozotocin (STZ) to induce diabetes mellitus. After fed for 10 weeks, the animals in both groups were tested with urodynamics, and the guinea pigs in experimental group were divided into the subgroups of DCP and the diabetic no-cystopathy (NDCP) group according to the results of urodynamics. mRNA expression of c-kit was detected by reverse transcription polymerase chain reaction (RT-PCR) and protein expression of c-kit was tested and analyzed by laser scanning confocal microscope. RESULTS: Decreased expression of c-kit mRNA was observed in DCP group compared to control and the NDCP group. The ratio of c-kit mRNA and GAPDH was 5.66±0.54 in controls (P<0.05), 5.54±1.28 in NDCP group (P<0.05) and 4.65 ±0.47 in DCP group. c-kit protein expression significantly declined in DCP group. The mean value of fluorescence intensity was 856.52± 53.03 in control group, 844.67± 59.24 in NDCP group and 548.69± 48.51 in DCP (P<0.01).CONCLUSION: The declined expression of c-kit) gene at transcription and translation levels destroys the SCF/c-kit signal pathway, leading to the dysfunction of Cajal-like) cells in DCP guinea pig, so the abnormal expression of c-kit gene is involved in the pathogenesis of DCP.

SCF protects the cortical neuron from apoptotic cell death in diabetic mice / 中国神经精神疾病杂志

Objective To investigate the role of SCF on neuronal apoptosis induced by diabetes and its possible mechanism.Methods Twenty-seven male C57 mice were randomly divided into control group,diabetes group,and diabe-tes plus stem cell factor(SCF)group.The diabetic mice were induced by streptozotocin.TUNEL staining was used to assess neuronal apoptosis and western blot were used to detect the protein level of BCL-2,BAX,CASPASE 3 and P-ERK/ERK.Results Compared with the controls,the number of apoptotic neuron death and the protein levels of active CASPASE 3 were significantly increased in the cortex of diabetic mice.Treatment with SCF significantly reduced apoptotic neuron death and attenuated the increased in protein levels of active CASPASE 3 in the cortex of diabetic mice.The levels of BCL-2 and BAX were significantly increased in the diabetic animals compared to the controls.Treatment with SCF could significantly attenuated the increase in the expression of BAX but could not affect the level of BCL-2 in the cortex of diabetic mice.P-ERK was significantly decreased in the diabete group but not in dibete plus SCF group.Conclusions SCF can protect a-gainst diabete-induced apoptotic neuron death through increasing the phosphorylation of ERK and influencing the expression of BCL-2/BAX.

Effects of tacrolimus on cell proliferation of and secretion of stem cell factors by cultured human keratinocytes / 中华皮肤科杂志

Objective To study the effects oftacrolimus on the cell proliferation of and secretion of stem cell factors (SCF) by cultured human keratinocytes. Methods Human keratinocyte cell line HaCaT was cultured and treated with various concentrations (0, 10, 102, 103, 104 nmol/L) of tacrolimus. After 48 hours of treatment, cell proliferation was measured with MTT assay, the levels of stem cell factor in the culture supernatant of HaCaT cells were detected with ELISA. Results Inhibited proliferation was observed in HaCaT cells treated with tacrolimus of 103-104 nmol/L. The secretion of SCF by HaCaT cells was enhanced in the presence of 10-102 nmol/L of tacrolimus, was inhibited in the presence of tacrolimus of 104 nmol/L, and remained unchanged with 103 nmol/L. Conclusion Certain concentrations of tacrolimus could enhance the expression Of SCF in HaCaT cells, which may be associated with the efficacy of tacrolimus in the treatment of vitiligo.

Research on Fbx4 protein / 国际外科学杂志

Protein degradation mediated by ubiquitin-proteasome pathway is involved in many biological processes.SCF complex is a sort of important E3 ubiquitin-ligase enzyme.Fbx4 protein is one of F-box protein family members,an important component of substrate-specific ScF Fbx4/αβ-crystallin ligase,and a subunit of SCF complex.Inactivation of Fbx4 protein or alteration of its expression level will influence activity of SCF Fbx4/αβ-crystallin ligase,thus will influence expression level of Cyclin D1.And Fbx4 protein is also an important regulatory factor for telomere maintenance.

Synergistic activation of p70S6 kinase associated with stem cell factor in MO7e cells

Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.

Serum Level of G-CSF and GM-CSF and CFU-GM Response to Hematopoietic Growth Factors in a Patient with Cyclic Neutropenia

PURPOSE: Serum levels of G-CSF and GM-CSF were measured and CFU-GM assay using G- CSF, GM-CSF and SCF was conducted to evaluate the influence of hematopoietic growth factor on the precursor cells of cyclic neutropenia. METHODS: A 7-year-old male with cyclic neutropenia was studied. Marrow mononuclear cells were isolated at neutrophil nadir and recovery and cultured in methylcellulose media with or without G-CSF, GM- CSF and SCF. CD34 positive cells were evaluated using flow cytometry. Serum levels of G-CSF and GM-CSF were measured by ELISA. RESULTS: The Numbers of CFU-GM without growth factors were 50 at neutrophil nadir and 33 at the recovery phase in the patient and show increased colony forming capacity. CD34 positive cells were 9.32% at nadir and 14.17% at recovery. Increasement of CFU-GM with G-CSF at nadir and recovery were 46% and 118% and those with GM-CSF were 70% and 78% respectively, compared with 54.4% and 78.2% in control groups. In contrast, the presence of SCF did not enhance CFU-GM number in the patient, but in the control group, increasement with SCF was 28.9 %. There an was inverse relationship between serum G-CSF levels and peripheral neutrophil count whereas those of GM-CSF were constant. CONCLUSION: Serum G-CSF level showed inverse relationship with neutrophil counts. The response of progenitor cells to G-CSF and GM-CSF was not impaired. The presence of SCF did not enhance CFU-GM number in the patient. This result suggests that the abnormality in hematopoiesis in cyclic neutropenia may involve more immature progenitor cells responsive to SCF.

c-kit and Ligand Expression in the Mouse Hippocampus after Kainate-induced Seizure / 대한해부학회지

In the present study, we have etamined the role of c-kit and KL ligand in the mouse brain after kainate-induced seizure. To investigate whether c-kit receptor and KL ligand might involved in kainate-induced apoptosis, the expression patterns of c-kit and KL mRNA and localization of immunoreactivity for c-Kit, SCF and Bcl-2 protein were examined by in situ hybridization technique and immunohistochemical method, respectively, in the mouse hippocampus after kainate treatment. This report is the first demonstration for the role of c-kit receptor and KL ligand in the kainate-induced apoptosis. Our conclusion is based on : 1] c-kit and KL mRNA expressions were increased in CA3 region of the hippocampus in 1h after kainate treatment, 2] immunoreactivities for c-Kit protein and SCF were detected higher level in the CA1 and CA3 sectors in 24h after kainate treatment, 3] expression level for Bcl-2 protein was increased in the CA3 region of the hippocampus 24h after kainate treatment. These results suggest that bcl-2 could promote cell survival of injured neurons in CA3 after kainate-induced seizure. And increased translations of c-kit receptor and KL ligand after kainate injection in this area susgest that c-kit receptor and KL ligand could have a role in the kainate-induced apoptosis.