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ObjectiveTo observe the effects of modified Shenqiwan on renal function and fibrosis in diabetic nephropathy mice and explore the underlying mechanism based on the glycogen synthase kinase-3β （GSK-3β）/cyclic adenosine monophosphate （cAMP） response element-binding protein （CREB） signaling pathway. MethodFifty male db/db mice and 10 db/m mice were used in this study. The fifty db/db mice were randomly divided into model group， irbesartan group， and low-， medium-， and high-dose modified Shenqiwan groups. The 10 db/m mice were assigned to the normal group. The mice in the low-， medium-， and high-dose modified Shenqiwan groups were administered with modified Shenqiwan in the dosage form of suspension of Chinese medicinal granules by gavage， those in the irbesartan group were given irbesartan suspension by gavage， and those in the normal and model groups were given distilled water of equal volume by gavage. The intervention lasted for 12 weeks. The blood glucose levels， urine albumin-to-creatinine ratio （UACR）， and the protein expression levels of GSK-3β， CREB， transforming growth factor-β1 （TGF-β1）， E-cadherin， Vimentin， fibronectin （FN）， plasminogen activator inhibitor-1 （PAI-1）， and Collagen type Ⅳ （Coll Ⅳ） in the mouse kidneys were recorded before and after treatment. The extent of renal pathological damage was also observed. ResultCompared with the normal group， the model group showed significant increases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， decreased protein expression level of CREB （P<0.05）， and severe renal pathological damage. Compared with the model group， the low-， medium-， and high-dose modified Shenqiwan groups and the irbesartan group showed varying degrees of decreases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， increased expression level of CREB protein （P<0.05）， and improved renal pathological damage. ConclusionModified Shenqiwan can effectively reduce blood glucose levels， improve renal function， and alleviate fibrosis， and the mechanism of action is related to the inhibition of the GSK-3β/CREB signaling pathway.
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ObjectiveTo explore the mechanism of Didangtang （DDT） against the inflammatory cascade triggered by foam cell pyroptosis in high-glucose environment. MethodOxidized low density lipoprotein （ox-LDL， 100 mg·L-1） was used to induce pyroptosis of foam cells. The control group （5.5 mmol·L-1 glucose）， foam cell group （100 mg·L-1 ox-LDL）， high-glucose group （33.3 mmol·L-1 glucose）， DDT group （10% DDT-containing serum）， and NOD-like receptor family pyrin domain-containing 3 （NLRP3） inhibitor group （MCC950， 10 nmmol·L-1） were designed. The cell membrane damage was observed by lactate dehydrogenase （LDH） release assay. The expression of cysteinyl aspartate-specific proteinase-1 （Caspase-1） was detected by immunofluorescence method， and expression of key proteins NLRP3， Caspase-1， gastermin D （GSDMD）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18） in the pyroptosis pathway was determined by Western blot. The release of IL-18 and IL-1β， monocyte chemoattractant protein-1 （MCP-1）， and tumor necrosis factor α （TNF-α） in cell supernatant was measured by enzyme-linked immunosorbent assay （ELISA）. ResultThe expression of NLRP3， Caspase-1， and GSDMD was up-regulated （P<0.01） and the release of IL-1β， IL-18， MCP-1， IL-1α， and TNF-α was increased （P<0.01） in foam cell group compared with those in the control group. The expression of NLRP3， Caspase-1， and GSDMD was higher （P<0.01） and the release of inflammatory factors was more （P<0.01） in the high-glucose group than in the foam cell group. DDT and MCC950 can inhibit expression of NLRP3， Caspase-1， GSDMD and reduce the release of IL-1β， IL-18， MCP-1， IL-1α， and TNF-α. ConclusionDDT can suppress the pyroptosis of foam cells induced by NLRP3/Caspase-1 pathway in high-glucose environment and thereby alleviate the inflammatory cascade.
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Objective:To investigate the effects of different doses of Didangtang on myocardial inflammatory lesions in diabetic mice. Method:Sixty C57BL/6J mice were randomly divided into normal group （<italic>n</italic>=10） and model group （<italic>n</italic>=50）. The diabetic mice in the model group were established by intraperitoneal injection of high-fat diet combined with streptozotocin （STZ）. After model reproducing， the mice were fed with high-fat diet. After 8 weeks， the cardiac function of the mice was detected by using an ultrasound imaging platform. If the cardiac function decreased， the diabetic cardiomyopathy mice were modeled successfully. The nonmodel mice were eliminated， and finally 40 model mice were modeled. The rats in the model group were randomly divided into model group， low， medium and high dose of Didangtang group（1.5，3，6 g·kg<sup>-1</sup>） and simvastatin group（0.001 5 g·kg<sup>-1</sup>） according to heart function， with 8 rats in each group. The cardiac function of mice was detected by ultrasound imaging platform， fiber bragg grating（FBG）， triglyceride（TG） and total cholesterol（TC） were detected by automatic biochemical analyzer， hematoxylin-eosin （HE） staining was used to observe the pathological changes of myocardium， and the levels of NOD-like receptor3（NLRP3）， thiomdoxin interaction protein（TXNIP）， cysteinyl aspartate specific proteinase-1（Caspase-1） and Interleukin-1<italic>β</italic>（IL-1<italic>β</italic>） in myocardial tissue， as well as the content of reactive oxygen species（ROS） were detected by Western blot. Result:Compared with the normal control group， the levels of FBG， TC and TG in the model group significantly increased （<italic>P</italic><0.01）； the values of EF and FS significantly decreased （<italic>P</italic><0.01）； the expression of ROS significantly increased （<italic>P</italic><0.05）， and the expressions of NLRP3， TXNIP， Caspase-1 and IL-1<italic>β</italic> in the myocardial tissue significantly increased （<italic>P</italic><0.05）. Compared with the model group， the levels of FBG， TC and TG in the middle and high dose groups of Didangtang and simvastatin groups significantly decreased （<italic>P</italic><0.05）； the EF and FS in each dose group and simvastatin group improved （<italic>P</italic><0.05）， and the change in the middle dose group was more obvious （<italic>P</italic><0.05）. HE staining showed that Didangtang could improve the pathological changes of myocardial tissue in mice， the ROS expression levels of mice in each dose group of Didangtang and simvastatin group significantly reduced， especially in the middle dose group， the expression levels of NLRP3， TXNIP， Caspase-1 and IL-1<italic>β</italic> in each dose group significantly decreased， and the effect of middle dose of Didangtang on reducing expressions of NLRP3， TXNIP and Caspase-1 in myocardial tissue was more obvious， the effect of high dose of Didangtang on reducing the expression of IL-1<italic>β</italic> in myocardial tissue was more obvious. Conclusion:Didangtang can improve myocardial inflammatory lesions in diabetic cardiomyopathy mice by inhibiting the activation of NLRP3 inflammasome.
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Objective:To explore the progression of diabetic macrovascular disease and the effects of Didangtang at different doses on it. Method:Four-week-old male apolipoprotein-E knockout （ApoE<sup>-/-</sup>） mice with diabetic macrovascular disease induced by exposure to high-fat diet combined with streptozotocin （STZ） were randomly divided into the model， simvastatin， as well as high-， medium-， and low-dose Didangtang groups. The age-matched ApoE<sup>-/-</sup> mice of the same batch only fed with a high-fat diet were classified into the ApoE<sup>-/-</sup> （model control） group， and C57BL/6 mice with the same genetic background receiving a regular diet into the normal group. The sampling was conducted at the 8th and 20th weeks of the experiment for observing the pathological characteristics of the aorta and the proportion of plaque area in mice of each group at different time points， followed by the comparison of blood glucose， blood lipid， and oxidized low-density lipoprotein （ox-LDL） levels. The aortic NOD-like receptor protein 3 （NLRP3） and cysteinyl aspartate-specific proteinase-1 （Caspase-1） protein expression was detected by Western blot assay， and the serum interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， interleukin-18 （IL-18）， interleukin-1<italic>α</italic> （IL-1<italic>α</italic>）， and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） levels by enzyme-linked immunosorbent assay （ELISA）. Result:The comparison with the normal group revealed that the proportions of plaque area in the ApoE<sup>-/-</sup> group and the model group were increased （<italic>P</italic><0.01）， while the proportion of plaque area in each administration group was significantly reduced in contrast to that of the model group （<italic>P</italic><0.05）. The aortic NLRP3 and Caspase-1 protein expression levels as well as the serum IL-1<italic>β</italic>， IL-18， IL-1<italic>α</italic>， and TNF-<italic>α </italic>levels in the ApoE<sup>-/-</sup> group and the model group were significantly higher than those in the normal group （<italic>P</italic><0.01）. Compared with the model group， each administration group exhibited a significant reduction in aortic NLRP3 and Caspase-1 protein expression and serum IL-1<italic>β</italic>， IL-18， IL-1<italic>α</italic>， and TNF-<italic>α</italic> levels （<italic>P</italic><0.05）， with the strongest inhibitory effect detected in the medium-dose Didangtang group （<italic>P</italic><0.05）. Conclusion:Didangtang directly alleviates diabetic macrovascular disease possibly by down-regulating NLRP3 and Caspase-1 protein expression and easing the inflammatory cascade.
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Macroangiopathy is a complication of Type II Diabetes Mellitus (T2DM), which is mainly caused by fibrosis of blood vessels. Using T2DM rat models, we investigated whether the traditional Chinese medicine, Di-Dang Decoction (DDD), exhibited anti-fibrotic actions on great vessels. T2DM rats were randomly divided into non-intervention group, early-, middle-, late-stage DDD intervention groups and control groups, including pioglitazone group and aminoguanidine group. After administration of DDD to T2DM rats at different times, we detected the amount of extracellular matrix (ECM) deposition in the thoracic aorta. The results showed that early-stage intervention with DDD could effectively protect great vessels from ECM deposition. Considering that TGF-β1 is the master regulator of fibrosis, we further validated at the molecular level that, compared to middle- and late-stage intervention with DDD, early-stage intervention with DDD could significantly decrease the expression levels of factors related to the activated TGF-β1/Smad signalling pathway, as well as the expression levels of downstream effectors including CTGF, MMP and TIMP family proteins, which were directly involved in ECM remodelling. Therefore, early-stage intervention with DDD can reduce macrovascular fibrosis and prevent diabetic macroangiopathy.
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Sodium glucose co-transporter 2 (SGLT-2) inhibitors are newly developed but promising medicine for type 2 diabetes. However, patients with a different renal threshold for glucose excretion (RT(G)) may have a different reaction to this medicine. Therefore, the objective of this study was to investigate the characteristics of RT(G) and its impact factors in patients with type 2 diabetes mellitus (T2DM). The clinical and laboratory data of 36 healthy individuals and 168 in-hospital patients with T2DM were collected and analyzed, RTG was calculated using blood glucose (BG) measured by dynamic BG monitoring, urinary glucose excretion (UGE) and estimated glomerular filtration rate (eGFR). The characteristics of RT(G) were investigated. The risk factors for high RT(G) were analyzed using non-conditional logistic regression analysis. Our results found that RT(G) of the T2DM group was higher than that of the healthy individuals (P < 0.05); and 22.22% from the healthy individuals group but 58.33% from the T2DM group had high RT(G). Age, duration of diabetes, body mass index (BMI), and homeostasis model assessment insulin resistance index (HOMA-IR) were independently associated with high RT(G) (P < 0.05). Further stratified analysis revealed that RT(G) in T2DM patients increased with age, duration of diabetes, and BMI. In conclusion, RT(G) is increased in patients with T2DM, especially in those with longer diabetic duration, higher BMI, and those who are older. Therefore, these patients may be more sensitive to SGLT-2 inhibitors.
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The study aimed to investigate the intervening role of Didang decoction (DDD) at different times in macrovascular endothelial defense function, focusing on its effects on the AMP-activated protein kinase (AMPK) signaling pathway. The effects of DDD on mitochondrial energy metabolism were also investigated in rat aortic endothelial cells (RAECs). Type 2 diabetes were induced in rats by streptozotocin (STZ) combined with high fat diet. Rats were randomly divided into non-intervention group, metformin group, simvastatin group, and early-, middle-, late-stage DDD groups. Normal rats were used as control. All the rats received 12 weeks of intervention or control treatment. Western blots were used to detect the expression of AMP-activated protein kinase α1 (AMPKα1) and peroxisome proliferator-activated receptor 1α (PGC-1α). Changes in the intracellular AMP and ATP levels were detected with ELISA. Real-time-PCR was used to detect the mRNA level of caspase-3, endothelial nitric oxide synthase (eNOS), and Bcl-2. Compared to the diabetic non-intervention group, a significant increase in the expression of AMPKα1 and PGC-1α were observed in the early-stage, middle-stage DDD groups and simvastatin group (P < 0.05). The levels of Bcl-2, eNOS, and ATP were significantly increased (P < 0.05), while the level of AMP and caspase-3 were decreased (P < 0.05) in the early-stage DDD group and simvastatin group. Early intervention with DDD enhances mitochondrial energy metabolism by regulating the AMPK signaling pathway and therefore may play a role in strengthening the defense function of large vascular endothelial cells and postpone the development of macrovascular diseases in diabetes.
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Animales , Proteínas Quinasas Activadas por AMP , Metabolismo , Adenosina Trifosfato , Metabolismo , Aorta , Metabolismo , Enfermedades Cardiovasculares , Metabolismo , Caspasa 3 , Metabolismo , Diabetes Mellitus Experimental , Quimioterapia , Metabolismo , Diabetes Mellitus Tipo 2 , Quimioterapia , Metabolismo , Dípteros , Medicamentos Herbarios Chinos , Farmacología , Usos Terapéuticos , Células Endoteliales , Metabolismo , Endotelio Vascular , Metabolismo , Metabolismo Energético , Sanguijuelas , Mitocondrias , Metabolismo , Óxido Nítrico Sintasa de Tipo III , Metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Metabolismo , Fitoterapia , Proteínas Proto-Oncogénicas c-bcl-2 , Metabolismo , Prunus persica , Ratas Sprague-Dawley , Rheum , Transducción de SeñalRESUMEN
[Summary] A total of 128 individuals with type 2 diabetes underwent continuous glucose monitoring for 3 consecutive days.The dawn phenomenon was defined by three different parameters according to the previous research:(1)the absolute increase of glucose level from nocturnal nadir to prebreakfast value(?G) above 20 mg/dl;(2)?G above 10 mg/dl;( 3 ) insulin requirement increased at least 20%.The participants were secondarily separated by presence/absence of a dawn phenomenon based on the definitions above.The impact on blood glucose fluctuation of different groups was assessed according to the standard deviation of blood glucose( SDBG) , the area under curve above 10 mmol/L ( AUC ) , and the mean amplitude of glycemic excursions ( MAGE ) , etc.The frequencies of dawn phenomenon were 64.8%(?G≥20mg/dl), 85.2%(?G≥10 mg/dl), and 59.4%(rise in insulin requirement≥20%)respectively.The impacts on SDBG, AUC, MAGE, and MODD were without statistical difference(P>0.05) between the presence and absence of the dawn phenomenon patients when?G≥10 mg/dl.However, the differences reached statistical significance(P<0.05) when ?G≥20 mg/dl and the increase in insulin requirement≥20%. Besides, the incidence of dawn phenomenon was positively correlated with HOMA-IR, HbA1C , and free C-peptide.Dawn phenomenon is a very frequent event in type 2 diabetes and not only impacts the overall glycemic control but also exaggerates glucose fluctuation.To be clinically relevant, ?G≥20mg/dl should be taken as the quantitative criterion of the dawn phenomenon.
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Objective To observe the effects of the intervention of Didang Decoction at different times on changes of AMPK signaling pathway related factors in macrovascular endotheliocytes of diabetic rats; To discuss the mechanism of mitochondria energy metabolism regulating the AMPK signaling pathway for macrovascular endothelial defense function. Methods Injection of STZ into the caudal vein and administration of high fat diet wer used to generate diabetic rat model. All rats were randomly divided into the following 7 groups: control, model, metformin, simvastatin, early-, middle-, and late-stage Didang Decoction group. Western blot was used to detect the expressions of APMKα1 and PGC-1α in rat aortic endothelial cells. Changes in the intracellular AMP and ATP levels were detected by ELISA. Real time fluorescence quantitative PCR was used to detected mRNA expressions of Caspase-3, eNOS, and Bcl-2 in tissue of thoracic aorta. Results Compared with the model group, the expressions of AMPKα1 and PGC-1α in the early-stage and middle-stage Didang Decoction group and simvastatin group increased (P<0.05); the gene expressions of Bcl-2, and eNOS significantly increased in the early-stage Didang Decoction group and simvastatin group (P<0.05), while the expressions of Caspase-3 decreased significantly (P<0.05). The expression of ATP increased significantly and the expression of AMP decreased significantly in the early-stage Didang Decoction group and simvastatin group (P<0.05), and the best effects were shown in the early-stage Didang Decoction group. Conclusion Early intervention of Didang Decoction can enhance energy metabolism in the mitochondria of macrovascular endothelial cells by regulating the AMPK signaling pathway, and then plays a role in strengthening the defense function of macrovascular endothelial cells.
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Objective To investigate the effects of heat-killed Staphylococcus aureus (HKSA) on the apoptosis and expression of iNOS and IL-1β in THP-1 monocytes in the presence of high concentration of glucose.Methods THP-1 cells were cultured in medium containing 25.0 mmol/L(HG) or 5.5 mmol/L (LG,control) D-glucose for 12 h-8 d.The THP-1 cells cultured for 6 d were extracted on the 0-48 h with or without HKSA,then apoptosis and expression of iNOS and IL-1β were examined.Apoptosis was analyzed by flow cytometry and expressions of IL-1β and iNOS were quantitated by real-time PCR.Results The expression of iNOS and IL-1β in THP-1 monocytes was increased significantly in the presence of high concentration of glucose for 12-48 h(P＜0.05),reaching the highest level at 24 h and returned to baseline after 4 d.The expression was significantly lower than that of control after 4-6 d.Apoptosis rate was also increased significantly after 48 h to 4 days.HKSA infection enhanced apoptosis,but inhibited the expression of iNOS and IL-1 β in the presence of high concentration of glucose.The expression of iNOS and IL-1β increased significantly at 6 h(P＜0.01),reaching the highest level at 12 h,but the levels were significantly lower than those in control groups (P＜0.05).Conclusion These data suggest that high concentration of glucose can interfere with the anti-bacterial function of monocytes by reducing their expression of iNOS and IL-1β and enhancing their apoptosis.
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According to the analysis of 9 237 hospitalized type 2 diabetic patients, the prevalence of diabetic retinopathy ( DR )was 32.9％ , with the prevalence of mild, moderate, and serious non-proliferative DR and proliferative DR being 10. 1％, 18. 3％, 3.2％, and 1.3％ respectively. The prevalence of diabetic macular edema ( DME ) was 3.56％ in type 2 diabetics and i 0. 8％ in patients with DR. Diabetes duration and proteinuria were the common risk factors of DR and DME.
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This study was aimed to detect the telomere length and the telomerase expression activity in patients with chronic lymphocytic leukemia (CLL), and investigate their relation to prognosis of CLL. The telomere length and the telomerase expression activity of peripheral blood and / or bone marrow mononuclear cells were examined by Tel-FISH, a semi-quantitative method and by TRAP-ELISA respectively; the expressions of ZAP70 and CD38 were detected by flow cytometry. The results showed that comparing the telomere length in different stages, there was a tendency that the telomere became prolonged when the stage raised up. There was statistical significant difference between Rai stages III-IV and stage 0, Rai stages III-IV and stages I-II, Binet stage C and stage A, Binet stage C and stage B; while no statistical significant difference existed between Rai stage 0 and stages I-II, Binet stage A and stage B. The telomere length in ZAP70 negative group was found similar as in ZAP70 positive group. The telomere length in CD38 positive group was shorter than that in CD38 negative group, but there was no statistical difference between them. Comparing the telomerase expression activity between different stages, there was a tendency that it increased when the stages went up; comparing the telomerase expression activity at different Rai stages, it increased at the higher stages. One case of CLL demonstrated that telomerase expression did not show at remission stage, but was found at relapse stage, which suggested that telomerase expression may relate to prognosis of disease. It is concluded that the telomerase length is in relation to Rai and Binet stage, which was shorter at higher stage than that at lower stage and intermediate stage. It seemed that the telomerase expression activity increased at higher stages. The expression of telomerase in mononuclear cells is stable and not influenced by treatment.
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Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Leucemia Linfocítica Crónica de Células B , Metabolismo , Patología , Telomerasa , Metabolismo , Telómero , GenéticaRESUMEN
The purpose of this study was to determine the changes of pathogens in hematological ward and susceptibility of patients received chemotherapy to antibiotics. The pathogens were taken from blood, urine and sputum of patients who accepted chemotherapy from years 2001 to 2005, then were isolated and identified. The susceptibility test was performed by disk diffusion method. The results showed that the total of 418 strains were detected. Gram-negative bacteria were the most common of nosocomial infection. Pseudomonas aeruginosa, Enterobacter cloacae, E. coli account for the most of Gram negative- bacteria infection and most resistant to broad-spectrum penicillin, Acinetobacter baumannii showed a trend of increase. The ratios of gram positive bacteria and fungi were increased slowly, mainly as Enterococcus and Candida. Enterococcus is the most common cause of Gram-positive bacterial infection. Vancomycin resistance did not occur. It is concluded that Gram-negative bacteria are main cause of nosocomial infection in patients with hematological malignancies. Gram positive bacteria and fungi had been more frequent. Strains resistant to antimicrobial agents increase.
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Humanos , Infección Hospitalaria , Epidemiología , Microbiología , Farmacorresistencia Bacteriana , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas , Epidemiología , Microbiología , Enfermedades Hematológicas , Microbiología , Neoplasias Hematológicas , Microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
The purpose of this study was to construct the IgHV and IL-2 coexpressed vector. The IgHV gene fragments were obtained from the peripheral blood of patients with lymphoma, and were cloned into eukaryotic expression vector. Meanwhile, the gene fragments of IgHV linked with gene of IL-2 were inserted into pcDNA3.0 to form a fusion gene of IgHV-IL-2. Then fusion genes were transfected into COS cells by Lipofectin and the expression of IL-2 was detected by ELISA. The results showed that the IgHV/pcDNA3.0 expression vector was successfully constructed. The 3' end of IgHV was linked to IL-2 gene, and IL-2 could be correctly expressed. In conclusion, the expression vector of IgHV-IL-2 can express IL-2 correctly in COS cells.
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Humanos , Vacunas contra el Cáncer , Alergia e Inmunología , Células Eucariotas , Metabolismo , Genes de Inmunoglobulinas , Vectores Genéticos , Cadenas Pesadas de Inmunoglobulina , Genética , Región Variable de Inmunoglobulina , Genética , Interleucina-2 , Genética , Linfoma , Alergia e Inmunología , Proteínas Recombinantes de Fusión , Genética , Alergia e Inmunología , Vacunas de ADN , Genética , Alergia e InmunologíaRESUMEN
Pi-dan is a signifi cant conception,which comes from Inner Canon of Huangdi.From the vivid description of Pi-dan in Inner Canon of Huangdi,we consider that the formation and process of Pi-dan is equivalent to metabolic syndrome.They are have the same etiological factor:obesity,the same pathogenesis:abdominal fullness and interior heat,and they can result in diabetes,hypertension,hyperlipidemia,fat liver,gout and a series of severity vascular complication.The theory of Pi-dan exactly points out that obesity the original cause of metabolic syndrome,and suggests that early prevention and treatment for obesity is an important method to prevention metabolic syndrome and its complications.The main pathogenesis of Pi-dan is abdominal fullness and interior heat,and the main therapeutic methods are Xiaogaojiangzhuo,Kaiyuqingre,and Huoxuetongluo.
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<p><b>OBJECTIVE</b>To explore the feasibility of regulated expansion and committed differentiation potential of JAK2 gene modified hematopoietic stem/progenitor cells in vitro.</p><p><b>METHODS</b>A murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein containing two copies of modified FK506 binding protein (F36v) linked tyrosine kinase JAK2 was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GpE + 86 packaging cell was transfected with this vector. Bone marrow cells from C57BL/6 mice were transduced by co-cultured with irradiated (1500 cGy) GpE + 86 producer clone for 48 h. Transduced marrow cells were expanded in X-VIVO 15 and divided into four groups as follows: (1) control group; (2) AP20187 alone group; (3) SCF alone group and (4) AP20187 + SCF group. The phenotypes of the expanded cells were analyzed by directly phycoerythrin-labeled anti-Sca1, c-kit, CD(34), Gr1, CD(11b), TER119, CD(41), B220 and CD(3) monoclonal antibodies for flow cytometry. Committed differentiation, progenitor colony assay and spleen colony forming units (CFU-S) were further evaluated.</p><p><b>RESULTS</b>A significant sustained outgrowth of transduced marrow cells was obtained only in the AP20187 + SCF group. Cells expanded up to 10(14)-fold after 80 days culture. The doubling time was about 30 hs. The phenotypes of the expanded cells were homogeneously strong positive for CD(34), c-kit and Sca1, while were almost negative for Gr1, CD(11b), TER119, CD(41), B220 and CD(3). Functional assay demonstrated that the expanded cells had multipotential to differentiate into granulocyte, macrophage, erythrocyte, or B-cells under different cytokines combinations. A prominent megakaryocytic differentiation was observed when cultured with SCF/Tpo/IL-11 combination. The expanded cells were also capable of forming BFU-E, CFU-GM and CFU-Mix in methylcellulose colony assay. The expanded cells over three months could still form CFU-S.</p><p><b>CONCLUSIONS</b>AP20187 combined SCF mediated activation of JAK2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed to differentiate into multilineage cells. This system may provide important insights into stem cell biology and may be promising for gene and cell therapy.</p>