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BACKGROUND@#Benvitimod cream, a novel synthetic small molecule, was effective in treating mild-to-moderate plaque psoriasis. We conducted a phase III clinical trial to assess the efficacy and safety of benvitimod cream in patients with mild-to-moderate plaque psoriasis.@*METHODS@#We randomly assigned 686 patients (2:1:1) to receive 1% benvitimod cream, 0.005% calcipotriol ointment or placebo twice a day for 12 weeks. The primary efficacy end points were the percentage of patients with a 75% or greater reduction from baseline in the psoriasis area and severity index (PASI 75) score and with a score of 0 or 1 in static physician's global assessment (sPGA) at week 12.@*RESULTS@#The results showed that 50.4% of patients in the benvitimod group achieved PASI 75, which was significantly higher than that in the calcipotriol (38.5%, P < 0.05) and placebo (13.9%, P < 0.05) groups. The proportion of patients achieving an sPGA score 0 or 1 was 66.3% in the benvitimod group and 63.9% in the calcipotriol group, which were both significantly higher than that in the placebo group (34%, P < 0.05). In the long-term follow-up study, 50.8% of patients experienced recurrence. After retreatment with 1% benvitimod, 73.3% of patients achieved an sPGA score of 0 or 1 again at week 52. Adverse events included application site irritation, follicular papules, and contact dermatitis. No systemic adverse reactions were reported.@*CONCLUSION@#During this 12-week study, benvitimod cream was demonstrated with high effectiveness and safety in patients with mild-to-moderate plaque psoriasis.@*TRIAL REGISTRATION@#Chinese Clinical Trial Registry (ChiCTR), ChiCTR-TRC-13003259; http://www.chictr.org.cn/showprojen.aspx?proj=6300.
Subject(s)
Humans , Double-Blind Method , Follow-Up Studies , Ointments , Psoriasis/drug therapy , Resorcinols , Severity of Illness Index , Stilbenes , Treatment OutcomeABSTRACT
Objective:To evaluate preliminarily immunocompetence and immunoprotection of a NMB0315 nucleic acid vaccine,a recombinant protein vaccine and a nucleic acid vaccine plus a recombinant protein vaccine against Neisseria meningitidis serogroup B in mice, and to provide reliable experimental basis for further exploration of the effective immunization methods and pathways of NMB0315 vaccine. Methods:The NMB0315 nucleic acid vaccine [ pcDNA3. 1 (+)/NMB0315 ] and recombinant protein vaccine(pET-30a/NMB0315)were prepared. Female BALB/c mice were inoculated with a NMB0315 DNA vaccine followed by boosting with recombinant protein NMB0315 through intramuscular and intraperitoneal immunization respectively. Next, humoral immunologic response and cellullar immunologic response were detected in female BALB/c mice by ELISA. The survival rate of BALB/c mice was used to evaluate immunoprotection of the vaccines in mice. Results:Specific IgG,IgG1,IgG2a,and sIgA,induced by the NMB0315 DNA vaccine(pNMB0315-CpG),protein NMB0315 vaccine(rNMB0315-FA),NMB0315 DNA vaccine prime-protein boost at week 8, were detected by indirect ELISA,the A450 values were up to(0. 505±0. 042,0. 513±0. 022,0. 342±0. 017,0. 250±0. 015),(0. 823± 0. 061,0. 807±0. 045,0. 596±0. 027,0. 450±0. 028)and(0. 694±0. 053,0. 711±0. 032,0. 455±0. 021,0. 386±0. 024)respectively, which was significantly higher than the PBS control(P<0. 05). The antibody level of protein vaccine was significantly higher than the nucleic acid vaccine group and combined immunization group ( P<0. 05 ) . The stimulation index and IFN-γ level of combined immunization group were significantly higher than the protein vaccine group and nucleic acid vaccine group(P<0. 05). The bactericidal titer of nucleic acid vaccine group, protein vaccine group and combined immunization group reached 1 :64, 1 :128 and 1 :128 respectively,and the protection rates were 70%,95% and 80% respectively. The IgG2a/IgG1 ratios of the nucleic acid vaccine group, the recombinant protein vaccine group and the combined immunization vaccine group were all less than 1 at week 2, 4, 6, 8. Conclusion:The humoral immunity effects( including mucosal immune) induced by the NMB0315 vaccines form high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine;and the cellular immune effects from high to low were as follows:the combined immunization vaccine group, the nucleic acid vaccine group, the recombinant protein vaccine group;The protection effects induced by the NMB0315 vaccines in BALB/c mice within 72 hours from high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine group.
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Objective To observe the expression of microRNA-150 (miR-150) in conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma, so as to investigate its mechanism in proliferation, migration and invasion of conjunctival MALT lymphoma. Methods The expressions of ;m'P-150 and Cbl-b, a possible downstream molecule of miR-150, were measured by qPCR in MALT lymphoma tissues and precancerous tissues collected from 3 patients with conjunctival MALT lymphoma in Changzheng Hospital of Second Military Medical University. Then, we transfected miR-150 inhibitor and negative control into human multiple myeloma cell lines RPM1 8226 by cell transfection. CCK-8 assay and flow cytometry (FCM) method were used to investigate the role of miR-150 in the proliferation and apoptosis of RPM1 8226 cells. Transwell assay was used to analyze the effect of miR-150 on the migration and invasion of RPMI 8226 cells. Western blotting analysis was used to examine the regulation of miR150 on Cbl-b expression in RPMI 8226 cells. Results The expression of miR-150 was significantly increased in the conjunctival MALT lymphoma tissues compared with precancerous tissues (P< 0. 05,P< 0. 01). Compared with negative control group, the proliferation of RPMI 8226 cells was significantly repressed (P< 0. 01), the apoptosis was significantly increased (P< 0. 01), and the migration and invasion were significantly decreased (P< 0. 05,P< 0. 01) after transfection of miR-150 inhibitor. The expression of Cbl-b was significantly up-regulated in MALT lymphoma tissues, and was significantly increased after inhibiting miR-150 expression. Conclusion Up-regulated miR-150 can promote the proliferation, migration and invasion of lymphoma cells and is involved in the generation of conjunctival MALT lymphoma, which may be mediated by inhibiting its downstream target gene Cbl-b.
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Objective:To evaluate preliminarily immunocompetence and immunoprotection of a NMB0315 nucleic acid vaccine,a recombinant protein vaccine and a nucleic acid vaccine plus a recombinant protein vaccine against Neisseria meningitidis serogroup B in mice, and to provide reliable experimental basis for further exploration of the effective immunization methods and pathways of NMB0315 vaccine. Methods:The NMB0315 nucleic acid vaccine [ pcDNA3. 1 (+)/NMB0315 ] and recombinant protein vaccine(pET-30a/NMB0315)were prepared. Female BALB/c mice were inoculated with a NMB0315 DNA vaccine followed by boosting with recombinant protein NMB0315 through intramuscular and intraperitoneal immunization respectively. Next, humoral immunologic response and cellullar immunologic response were detected in female BALB/c mice by ELISA. The survival rate of BALB/c mice was used to evaluate immunoprotection of the vaccines in mice. Results:Specific IgG,IgG1,IgG2a,and sIgA,induced by the NMB0315 DNA vaccine(pNMB0315-CpG),protein NMB0315 vaccine(rNMB0315-FA),NMB0315 DNA vaccine prime-protein boost at week 8, were detected by indirect ELISA,the A450 values were up to(0. 505±0. 042,0. 513±0. 022,0. 342±0. 017,0. 250±0. 015),(0. 823± 0. 061,0. 807±0. 045,0. 596±0. 027,0. 450±0. 028)and(0. 694±0. 053,0. 711±0. 032,0. 455±0. 021,0. 386±0. 024)respectively, which was significantly higher than the PBS control(P<0. 05). The antibody level of protein vaccine was significantly higher than the nucleic acid vaccine group and combined immunization group ( P<0. 05 ) . The stimulation index and IFN-γ level of combined immunization group were significantly higher than the protein vaccine group and nucleic acid vaccine group(P<0. 05). The bactericidal titer of nucleic acid vaccine group, protein vaccine group and combined immunization group reached 1 :64, 1 :128 and 1 :128 respectively,and the protection rates were 70%,95% and 80% respectively. The IgG2a/IgG1 ratios of the nucleic acid vaccine group, the recombinant protein vaccine group and the combined immunization vaccine group were all less than 1 at week 2, 4, 6, 8. Conclusion:The humoral immunity effects( including mucosal immune) induced by the NMB0315 vaccines form high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine;and the cellular immune effects from high to low were as follows:the combined immunization vaccine group, the nucleic acid vaccine group, the recombinant protein vaccine group;The protection effects induced by the NMB0315 vaccines in BALB/c mice within 72 hours from high to low were as follows:the recombinant protein vaccine group, the combined immunization vaccine group, the nucleic acid vaccine group.
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<p><b>BACKGROUND</b>Atopic dermatitis (AD) is an inflammatory skin disease characterized by chronic recurrent dermatitis with profound itching. Most patients have personal and/or family history of atopic diseases. Several criteria have been proposed for the diagnosis of AD. Although the clinical features of childhood AD have been widely studied, there has been less large-scale study on adult/adolescent AD. The aim of this study was to investigate the clinical features of adult/adolescent patients with chronic symmetrical eczema/AD and to propose Chinese diagnostic criteria for adult/adolescent AD.</p><p><b>METHODS</b>A hospital-based study was performed. Forty-two dermatological centers participated in this study. Adult and adolescent patients (12 years and over) with chronic symmetrical eczema or AD were included in this study. Questionnaires were completed by both patients and investigators. The valid questionnaires were analyzed using EpiData 3.1 and SPSS 17.0 software.</p><p><b>RESULTS</b>A total of 2662 valid questionnaires were collected (1369 male and 1293 female). Of all 2662 patients, 2062 (77.5%) patients had the disease after 12 years old, while only 600 (22.5%) patients had the disease before 12 years old, suggesting late-onset eczema/AD is common. Two thousand one hundred and thirty-nine (80.4%) patients had the disease for more than 6 months. One thousand one hundred and forty-four (43.0%) patients had a personal and/or family history of atopic diseases. One thousand five hundred and forty-eight (58.2%) patients had an elevated total serum IgE and/or eosinophilia and/or positive allergen-specific IgE. Based on these clinical and laboratory features, we proposed Chinese criteria for adult/adolescent AD. Of all 2662 patients, 60.3% were satisfied with our criteria, while only 48.2% satisfied with Hanifin Rajka criteria and 32.7% satisfied with Williams criteria, suggesting a good sensitivity of our criteria in adult/adolescent AD patients.</p><p><b>CONCLUSION</b>Late-onset of eczema or AD is common. The clinical manifestations of AD are heterogeneous. We have proposed Chinese diagnostic criteria for adolescent and adult AD, which are simple and sensitive for diagnosis of adult/adolescent AD.</p>
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Dermatitis, Atopic , Diagnosis , Allergy and Immunology , Eczema , Diagnosis , Immunoglobulin E , Blood , Retrospective Studies , Surveys and QuestionnairesABSTRACT
<p><b>OBJECTIVE</b>To study the molecular mechanism of curcumin in human esophageal carcinoma cell line (EC109).</p><p><b>METHODS</b>EC109 cells were cultivated in vitro. When 80%-90% confluence was reached, they were treated with curcumin in different concentrations (15-120 µmol/L). The effects on cell proliferation were examined by CCK-8 colorimetry. The ultrastructure of EC109 cells were detected with transmission electron microscope(TEM). The cells apoptosis was observed with laser confocal microscope(LCM) by AnnexinV-FITC/PI double staining. The proteins level of PTEN, AKT, GSK3β and Caspase 3 were tested by flow cytometry(FCM) .</p><p><b>RESULTS</b>CCK-8 test showed that curcumin could inhibit the proliferation of EC109 cells in a time- and concentration-dependent manner. TEM and LCM examinations indicated that curcumin could make EC109 cells apoptosis. The data of FCM showed that curcumin could increase the expression of PTEN, GSK3β and Caspase 3, decreased the expression of AKT.</p><p><b>CONCLUSION</b>The effects of curcumin on inhibiting proliferation and promoting apoptosis of EC109 cells were related with increased expression of PTEN and inhibition of PI3K/AKT signaling pathway.</p>
Subject(s)
Humans , Apoptosis , Caspase 3 , Metabolism , Cell Line, Tumor , Cell Proliferation , Curcumin , Pharmacology , Esophageal Neoplasms , Metabolism , Glycogen Synthase Kinase 3 , Metabolism , Glycogen Synthase Kinase 3 beta , PTEN Phosphohydrolase , Metabolism , Phosphatidylinositol 3-Kinases , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To investigate the inhibition effect of curcumin on the proliferation of the human esophageal carcinoma cell line Ec109 and its impact on PEN/PI3K/Akt signaling pathway.</p><p><b>METHODS</b>Esophageal carcinoma Ec109 cells were cultured in vitro conventionally and were treated with curcumin at different concentrations. The cell proliferation level was examined by MIT colorimetry, the ultrastructure of curcumin-treated Ec109 cells were detected with transmission electron microscope (TEM) and cell apoptosis was observed by FCM with AnnexinV-FITC/PI double staining. The protein levels of PTEN, Akt, GSK3P and Caspase 3 of curcumin-treated Ec109 cells were detected by Western blot.</p><p><b>RESULTS</b>MTT test showed that curcumin could inhibit the proliferation of Ec109 cells in a time and concentration-dependent manner. TEM examination indicated that curcumin could induce Ec109 cell apoptosis. FCM detection showed that Ec109 cell apoptotic rate increased significantly with the increase of drug concentration. On the other hand, curcumin could promote the expression of PTEN, GSK3beta and Caspase 3 yet reduce the expression of Akt.</p><p><b>CONCLUSION</b>Curcumin could obviously up-regulate the expression of PTEN, GSK3beta and Caspase 3, surpress PI3K/Akt signaling pathway and hence inhibit the proliferation of Ec109 cells.</p>
Subject(s)
Humans , Apoptosis , Caspase 3 , Metabolism , Cell Line, Tumor , Cell Proliferation , Curcumin , Pharmacology , Glycogen Synthase Kinase 3 , Metabolism , Glycogen Synthase Kinase 3 beta , Oncogene Protein v-akt , Metabolism , PTEN Phosphohydrolase , Metabolism , Phosphatidylinositol 3-Kinases , Metabolism , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To test the hypothesis that pharmacological postconditioning with lactic acid and low dose edaravone could mimic the upper trigger of mechanical postconditioning and relieve reperfusion injury through mitochondrial pathway.</p><p><b>METHODS</b>Rats were randomly divided into 6 groups (n = 18 each): sham, reperfusion/injury(I/R), postconditioning (IP), lactic acid (Lac, 60 µl), low dose edaravone (Eda, 3 µg/kg), and Lac+Eda. After 45 min myocardial ischemia, different drugs or saline were administrated around the infarct border according to different groups using micro syringe at the time of reperfusion. After 10 min reperfusion, right atrial plasma pH value was determined in all rats. Then the rats were sacrificed at 1, 6 and 24 h (n = 6 each), apoptotic index was measured by TUNEL, infarct area and ischemic area were measured through Evans blue-TTC double staining, mitochondrial absorbance, the contents of MDA and SOD in ischemic myocardium were detected by spectrophotometry, and the expression of apoptotic pathway molecules, such as Bcl-2, Bax and Cytochrome c (Cyt-c) , were detected by Western blot.</p><p><b>RESULTS</b>Right atrial plasma pH value was significantly lower, the content of MDA was significantly lower, and the content of SOD was significantly higher in IP and Lac+Eda groups than in I/R group (all P < 0.05). The mitochondrial absorbance in Lac+Eda group at all time points were all significantly higher than those in I/R group (all P < 0.05). The level of Bcl-2 in ischemic myocardium in Lac+Eda group was significantly higher than in I/R group (1.02 ± 0.19 vs.0.02 ± 0.01, P < 0.05), the level of Bax (0.38 ± 0.07 vs.2.40 ± 0.45, P < 0.05) and Cyt-c(0.78 ± 0.05 vs.6.54 ± 1.86, P < 0.05) were all lower than those in I/R group. The content of CK[(849 ± 228) vs.(1249 ± 211) U/L, P < 0.05] and CK-MB[(470 ± 266) vs. (966 ± 263) U/L, P < 0.05] in Lac+Eda group were all significantly lower than in I/R group, apoptotic index [(10.51 ± 1.52)% vs. (15.00 ± 1.90) %, P < 0.05] and infarct area [(27.12 ± 5.55)% vs. (45.66 ± 10.81)%, P < 0.05] in Lac+Eda group were all significantly lower than those in I/R group.</p><p><b>CONCLUSION</b>Pharmacological postconditioning with lactic acid and low dose edaravone could mimic the upper triggers of mechanical postconditioning and attenuate myocardial reperfusion injury through mitochondrial pathway.</p>
Subject(s)
Animals , Male , Rats , Antipyrine , Pharmacology , Apoptosis , Cytochromes c , Metabolism , Disease Models, Animal , Ischemic Postconditioning , Lactic Acid , Pharmacology , Mitochondria , Metabolism , Myocardial Reperfusion Injury , Metabolism , Pathology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To investigate the work-related musculoskeletal disorders among automobile assembly workers, to discusses the related risk factors and their relationship.</p><p><b>METHOD</b>The selected 1508 automobile assembly workers from a north car manufacturing company were regarded as the study object. The hazard zone jobs checklist, Nordic musculoskeletal symptom questionnaire (NMQ) and pain questionnaire were used to perform the epidemiological cross-sectional and retrospective survey and study for the General status, awkward ergonomics factors and related influencing factors, and musculoskeletal disorders of workers.</p><p><b>RESULTS</b>The predominant body sites of occurring WMSDs among automobile assembly workers were mainly low back, wrist, neck and shoulders, the predominant workshop section of occurring WMSDs were mostly concentrated in engine compartment, interior ornament, door cover, chassis and debugging section. The predominant body site of WMSDs among engine compartment and chassis section workers was low back, interior ornament workers were low back and wrist, door cover workers was wrist, chassis workers was low back, debugging workers were neck and low back. Neck musculoskeletal disorders had the trend with the increase of a body height; Smoking may increase the occurrence of musculoskeletal disorders.</p><p><b>CONCLUSION</b>The WMSDs appears to be a serious ergonomic proble assem among automobile assembly workers, predominant occurring site of WMSDs is with different workshop section, its characteristics is quite obvious, probably related to its existing awkward work position or activities. The worker height and smoking habits may be important factors which affect musculoskeletal disorders happen.</p>
Subject(s)
Adult , Humans , Male , Young Adult , Cross-Sectional Studies , Cumulative Trauma Disorders , Epidemiology , Ergonomics , Musculoskeletal Diseases , Epidemiology , Occupational Diseases , Epidemiology , Prevalence , Retrospective Studies , Risk Factors , Surveys and QuestionnairesABSTRACT
The aim of this study was to investigate the population pharmacokinetic characteristics of high-dose methotrexate (HDMTX) administered in children with acute lymphoblastic leukemia (ALL) for individualizing methotrexate dose regimen with routine plasma concentration data. The data of 329 children with acute lymphoblastic leukemia administering HDMTX in hospital were collected from 2003 to 2006. Population pharmacokinetics model, parameters, inter-and intra-individual variation were estimated by nonlinear mixed effect modes (NONMEM) software. The effects of the covariates including sex, age, body weight, alkalization and hydration volumes as well as biochemical parameters on the clearance and apparent volume of distribution of methotrexate were explored and the final model was established. The results showed that according to one compartment open pharmacokinetic model with first-order absorption and first-order elimination used in the modeling procedure, the factor that significantly influenced the clearance and apparent volume of distribution of methotrexate was weight (p<0.05). The final model in this study was: CL=7.0 x [1 + 0.0218 x (WT-31.1)] x EXP (eta(CL)) (L/h); V=32.8 x [1 + 0.0288 x (WT-31.1)] x EXP (eta(V)) (L), where 31.1 (kg) was the average body weight of the population. The inter individual variation (CV) of CL and V were 19.4% and 31.0% respectively. It is concluded that the population pharmacokinetic model of HDMTX in children with ALL is established. The obtained final model and population pharmacokinetics parameters of methotrexate may be useful for individualization treatment.
Subject(s)
Child , Child, Preschool , Female , Humans , Male , Antimetabolites, Antineoplastic , Pharmacokinetics , Antineoplastic Combined Chemotherapy Protocols , Therapeutic Uses , Dose-Response Relationship, Drug , Methotrexate , Pharmacokinetics , Models, Biological , Nonlinear Dynamics , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Drug Therapy , MetabolismABSTRACT
Exposure of endothelial cells (ECs) to hypoxia leads to a decrease in EC proliferation. However, the mechanism by which hypoxia inhibits EC proliferation is unclear. Perlecan has been reported to play an important role in regulating EC proliferation. We hypothesized that perlecan was involved in the hypoxia-induced inhibition of EC proliferation. To test this hypothesis, rat cardiac microvascular ECs were cultured under normoxic or hypoxic conditions for 12 h and harvested for determination of perlecan mRNA expression using real-time reverse transcription-polymerase chain reaction (RT-PCR). The results showed that exposure of ECs to hypoxia for 12 h induced a decrease in perlecan mRNA expression (61.72%, P<0.05). Concomitantly, the down-regulation of endogenous perlecan induced by hypoxia or the neutralization of endogenous perlecan with anti-perlecan antibody significantly inhibited EC proliferation and responsiveness to basic fibroblast growth factor (bFGF), and decreased focal adhesion kinase (FAK) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. These data indicate that down-regulation of perlecan expression contributes to hypoxia-induced inhibition of rat cardiac microvascular EC proliferation by suppressing FAK-mediated and ERK1/2-dependent growth signals.
Subject(s)
Animals , Male , Rats , Capillaries , Cell Biology , Cell Hypoxia , Cell Proliferation , Cells, Cultured , Coronary Circulation , Down-Regulation , Endothelial Cells , Cell Biology , Metabolism , Focal Adhesion Kinase 1 , Metabolism , Heparan Sulfate Proteoglycans , Genetics , Metabolism , MAP Kinase Signaling System , Oxygen , Metabolism , RNA, Messenger , Genetics , Metabolism , Rats, Sprague-Dawley , Real-Time Polymerase Chain ReactionABSTRACT
<p><b>UNLABELLED</b>From large-scale sequence of human fetal liver cDNA library, we have obtained a full-length cDNA from an EST after further sequencing. It has been demonstrated by the alignment comparison with data base available that it is a novel member of Ubc family and got the number from GeneBank: UBF-F1 AF 294842.</p><p><b>AIM AND METHODS</b>To demonstrate its authenticity, UBF was amplified from the total RNA of human fetal liver and HL-60 cell line using RT-PCR, and the PCR products were further sequenced and compared with the original UBF sequence. To evaluate the expression level and subcellular location of UBF in human multiple tissues, in situ hybridization was carried out on the frozen section of human fetal multiple tissues and HL-60 cell line with DIG-labeled UBF cDNA probes.</p><p><b>RESULTS</b>The experimental results of RT-PCR and sequencing showed that the sequence of RT-PCR products were the same as the original UBF. The experimental results of in situ hybridization showed that UBF was expressed widely by human multiple fetal tissues and the expression level were very high in HL-60 cells.</p><p><b>CONCLUSION</b>It is suggested that the special structure of UBF is authentic, and the expression profiling research of UBF shows that UBF is expressed widely by human multiple fetal tissues and the expression level is very high in HL-60 cells, implying that UBF plays the important function in the developing tissues and leukemia cells. It is also suggested that UBF may be functionally related with the nucleic-involving cellular activities based on the results of sub-cellular localizations.</p>
Subject(s)
Humans , Amino Acid Sequence , Cloning, Molecular , Gene Expression Profiling , HL-60 Cells , Molecular Sequence Data , Pol1 Transcription Initiation Complex Proteins , Genetics , Reading Frames , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Ubiquitin-Conjugating Enzymes , Classification , UbiquitinationABSTRACT
Hematopoietic stromal cells, being the essential ingredient of the hematopoietic microenvironment, play very important roles in the control and regulation of self-renewal, proliferation and differentiation of hematopoietic stem cells (HSC) via complex interactions of cell-cell, cell-humoral and cell-extracellular matrix. Evidence from in vivo experiment has proved that HSC derived from normal mice could reconstitute hematopoiesis of mice with HSC defects but failed to reconstitute hematopoiesis of those mice with microenvironment defects, showing the importance of hematopoietic microenvironment in the maintenance of hematopoiesis in vivo. A well-known long-term culture (LTC) system established by Dexter demonstrated in another way that stromal cell layer in the system could support ex vivo hematopoiesis for several months, even more than one year under the optimal conditions. It, however, has not been demonstrated that what is the key elements and in which way the ex vivo hematopoiesis could be maintained for so long time. As the inventions for the large-scale screening methodologies the suppression subtractive hybridization (SSH) was chosen for the screening differentially expressed genes expressed by LTC cultured stromal cells but not by the uncultured bone marrow cells (BMC). mRNA extracted from both cultured adherent cells (tester) and BMC (driver) were hybridized according to the protocol provided by CLONTECH. Total of 130 clones differentially expressed by cultured cells were randomly picked up and 106 ESTs were obtained after sequencing. They represent 26 identical or similar genes and 7 novel genes after the bioinformatics analysis. 5 of the novel genes with the entire open reading frame, without functional clues, have been cloned into the mammalian expression vectors and the functions of them in the control of proliferation and differentiation of HSC will be further exploring. The most interesting discovery is that 3 novel genes have signal peptides, implying the potential discovery of novel growth factors as 80% known growth factors have signal peptides. Our experimental results suggest that: (a) based on the results of subtractive efficiency, the SSH could be a reliable method to screen differentially expressed genes; (b) gene expression may be regulated by multiple factors, even conditioning-dependent, in this experiment the genes expressed by bone marrow stromal cells are LTC-cultivation inducible; (c) it is possible to find interesting genes or special gene after relatively large-scale screen.