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Objective:To investigate the expression of miRNA-21 in serum of patients with multiple myeloma (MM) and its significance.Methods:The data of 55 MM patients and 20 healthy controls in the Second Hospital of Hebei Medical University from July 2019 to June 2020 were retrospectively analyzed. Among the MM patients, 20 cases were diagnosed without treatment, 15 cases were in complete remission (CR), and 20 cases were clinically relapsed. Blood samples were collected from all subjects, and the relative expression levels of miRNA-21 were detected by real-time quantitative polymerase chain reaction (qRT-PCR), and the correlation of miRNA-21 expression with β 2-macroglobulin (β 2-MG), creatinine, hemoglobin, albumin, t(4;14) mutation, 13q14 mutation and prognosis were analyzed. The MM cell line LP-1 was selected, and normal bone marrow CD138-positive plasma cells were sorted by immunomagnetic beads as control cells. The relative expression levels of miRNA-21 in the two groups of cells were detected by qRT-PCR. Results:The serum relative expression level of miRNA-21 in MM group was higher than that in healthy control group (1.50±0.10 vs. 1.03±0.06, t = 7.04, P = 0.002). The serum expressions of miRNA-21 in newly diagnosed untreated MM patients and relapsed/refractory MM patients were high (1.50±0.10 and 3.13±0.32), and compared with the healthy control group, the differences were statistically significant ( t values were 7.04 and 10.22, both P < 0.05). The relative expression level of miRNA-21 in MM patients with complete remission (CR) was 1.27±0.25, which had no significant difference compared with the healthy control group ( t = 1.76, P = 0.152). The serum expression level of miRNA-21 in MM patients with high β 2-MG, high creatinine, low hemoglobin, low albumin, t(4;14) mutation, 13q14 mutation, non-remission and recurrence was significantly increased (all P < 0.05). The relative expression level of miRNA-21 in LP-1 cell line was higher than that in control cells (1.56±0.05 vs. 1.00±0.06), and the difference was statistically significant ( t = 11.73, P < 0.01). Conclusions:The miRNA-21 may be a molecular marker to assist in the diagnosis and efficacy evaluation of MM.
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Objective To evaluate the effect of continuous infusion of dexmedetomidine on the oxidative stress in patients undergoing percutaneous coronary intervention (PCI).Methods Fifty patients, with acute myocardial infarction who required for emergency PCI, 39 males, 11 females, aged 47-79 years, weighting 45-83 kg, ASA physical status Ⅲ or Ⅳ, were selected and randomly divided into two groups (n=25 each) using a random number table: the control group (group C) and the dexmedetomidine group (group D).In group D, a loading dose of dexmedetomidine 0.5 μg/kg was infused intravenously for 10 min before surgery, and then dexmedetomidine was infused at a rate of 0.2-1.0 μg·kg-1·h-1 during the operation until the end of operation.Patients in group C received the same dose saline in the same way.RASS score was maintained at-2-2 scores in the two groups.Blood samples were collected before the anesthesia induction (T0), at the end of the operation (T1), 6 h after the operation (T2) and 24 h after the operation (T3) to determine the observed changes of PMN, SOD and MDA.The intraoperative adverse reactions including hypotension, bradycardia and hypoxemia were recorded.Results Compared with T0, the number of PMN and the serum concentration of MDA at T1-T3 significantly increased, while effective serum SOD at T1-T3 significantly decreased (P＜0.01 or P＜0.05).The serum concentration of MDA and the number of PMN at T1-T3 in group D were significantly lower than those in group C, while the effective serum SOD was significantly higher than that in group C (P＜0.05).There was no significant difference of intraoperative adverse reactions including hypotension, bradycardia and hypoxemia between the two groups.Conclusion Continuous infusion of dexmedetomidine can decrease oxidative stress thus to alleviate myocardial reperfusion injury.
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Objective@#To investigate the efficacy and safety of IA regimen which contains idarubicin (IDA) 8 mg/m2, 10 mg/m2 or 12 mg/m2 as induction chemotherapy for adult patients with de-novo acute myeloid leukemia (AML) .@*Methods@#A total of 1 215 newly diagnosed adult AML patients, ranging from May 2011 to March 2015 in the First Affiliated Hospital of Soochow University and other 36 clinical blood centers in China were enrolled in the multicenter, single-blind, non-randomized, clinical controlled study. To compare the response rate of complete remission (CR) , adverse events between different dose idarubicin combined with cytarabine (100 mg/m2) as induction chemotherapy in newly diagnosed patients of adult AML.@*Results@#Of 1 207 evaluable AML patients were assigned to this analysis of CR rate. The CR rates of IDA 8 mg/m2 group, IDA 10 mg/m2 group and IDA 12 mg/m2 group were 73.6% (215/292) , 84.1% (662/787) and 86.7% (111/128) , respectively (P<0.001) . After adjusted for age, blast ratio of bone marrow, FAB classification and risk stratification, the odds ratios (95% CI) of IDA 10 mg/m2 group and IDA 12 mg/m2 group were 0.49 (0.34-0.70) and 0.36 (0.18-0.71) , as compared with the IDA 8 mg/m2 group (P<0.001, P=0.003) . In the intermediate and favorable groups, CR rates was 76.5% (163/213) , 86.9% (506/582) and 86.1% (68/79) in different doses of IDA (P=0.007) . Interestingly, IA regimen with IDA 10 mg/m2 was the only beneficial factor affecting CR in this group after adjusted for age, blast ratio of bone marrow and FAB classification[OR=0.47 (95% CI 0.31-0.71) , P<0.001]. CR rates in adverse group was 50.0% (18/36) , 60.6% (43/71) and 81.8% (18/22) respectively (P=0.089) . However, the odds ratios (95% CI) of IDA 12 mg/m2 when compared with the IDA 8 mg/m2 was 0.22 (0.06-0.80) , after adjusted for age, blast ratio of bone marrow and FAB classification. The median time (days) of neutrophil count less than 0.5×109/L in IDA 8 mg/m2 group, IDA 10 mg/m2 group and IDA 12 mg/m2 group were 14 (11-18) , 15 (11-20) and 18 (14-22) , respectively (P=0.012) and of platelet count lower than 20×109/L were 14 (7-17) , 15 (11-20) and 17 (15-21) , respectively (P=0.001) . The incidences of lung infection in the three groups were 9.8%, 13.5% and 25.2%, respectively (P<0.001) .@*Conclusions@#For young adult patients (aged 18-60 years) with AML in China, intensifying induction therapy with idarubicin 10 mg/m2 is clinically superior to IDA 8 mg/m2 and IDA 12 mg/m2 in favorable intermediate AML subgroup. However, idarubicin 12 mg/m2 is more suitable to adverse AML subgroup.
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Objective To explore the clinical effect of limited internal fixation combined with miniature double side external fixing support in the treatment of midfoot fracture dislocation.Methods From March 2010 to March 2015,200 patients of midfoot fracture dislocation in our hospital were randomly divided into the control group and the observation group according to random figure table,100 cases in each group.The patients of the observation group were given limited internal fixation combined with miniature double side external fixing support treatment,and the patients of the control group were treated with conventional therapy.The clinical effects of the two groups were observed and compared.Results The operation time and wound healing time of the observation group[(67.46 ± 4.53)min,(837 ±3.43)weeks]were significantly shorter than those of the control group[(113.25 ±5.63)min, (16.44 ±5.63)weeks](t =8.37,12.24,all P <0.05).After different surgical treatment,the incidence rate of lim-ited activity of the observation group (0.00%)was significantly lower than 30.00% of the control group.The disease recovery excellent rate of the observation group (100.00%)was significantly higher than that of the control group (70.00%),and the difference was statistically significant (χ2 =35.29,P <0.05).Conclusion For midfoot frac-ture dislocation patients,limited internal fixation combined with miniature double side external fixing support treatment is very good to patients,which can correct fracture and dislocation,fast recovery,decrease infection opportunity,greatly reduce the patients'pain,it is worthy of widely used in clinic.
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Objective To analyze the effect of Yaotongning capsule in the treatment of lumbar osteoarthritis patients.Methods From March 2013 to March 2015 in our hospital, 80 cases of lumbar osteoarthritis patients according to the digital table method were divided into two groups: the control group and the experimental group.The patients of the control group were given Bitongning, and the patients of the experimental group were given Yaotongning capsule, and the therapeutic effect, serum indexes and Japanese Orthopaedic Association ( JOA ) of two groups were compared. Results The curative effect of the experimental group was higher than control group(95.00%vs.67.50%) , the JOA score of the experimental group was higher than control group [(27.46 ±1.07)points vs.(21.06 ±1.89)points] (P<0.05).After treatment, the MMP-3(65.28 ±4.37) ng/L and IL-1β(12.43 ±1.01) ng/L of the experimental group were significantly lower than MMP-3(79.56 ±5.36) ng/L, IL-1β(16.44 ±1.03) ng/L of the control group, and the difference was significant (P<0.05).Conclusion The lumbar osteoarthritis patients with Yaotongning capsule has obvious effect, and the function of lumbar intervertebral joint improves obviously, and the serum index is normal, safe and reliable.
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Objective To investigate the clinical effect of external fixation combined with limited internal fixation in the treatment of C type Tile pelvic fractures.Methods 60 patients with C type Tile pelvic fractures treated in our hospital were selected,and the patients treated with single external fixation were selected as group A,the patients treated with limited internal fixation were selected as group B,the patients treated with external fixation com-bined with limited internal fixation were selected as group C.The postoperative fracture reduction,functional recovery and complications of the occurrence were compared.Results The excellent and good rate of fracture reduction (95%)and excellent rate of fracture function recovery(90%)of group C had statistically significant differences com-pared with those of group A(65%,75%)and group B(75%,70%)(χ2 =4.91,4.14,3.97,3.92,all P <0.05). The incidence rate of postoperative complications of group C(5%)was significantly lower than group A(30%)and group B(25%),there were statistically significant differences(χ2 =4.33,3.92,all P <0.05).Conclusion C Tile type of pelvic fractures with external fixation combined with external fixation has more advantages in clinical applica-tion,can further improve the functional recovery of patients,reduce the incidence of complications.
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Objective To investigate the role of microRNA-155 (miR-155) on post-transcription regulation of SH2 domain-containing inositol 5'-phosphatase 1 (SHIP1) gene expression in the pathogenesis of acute myeloid leukemia (AML).Methods Quantitative real-time polymerase chain reaction (RT-PCR) was performed to detect the expression of miR-155 and SHIP1 mRNA in the AML patients and controls.miR-155 mimics was transfected into U937cells (U937m) by using X-treme GENE siRNA transfection reagent.Cells without transfection (U937c) and cells with negative transfection (U937mc) were used as controls.RT-PCR was performed to detect the expression of miR-155 and SHIP1 mRNA in these cells.The expression of SHIP1,TAKT and pAKT were detected by Western blot in U937 cells.Apoptosis was studied by flow cytometry (FCM).Results The average level of SHIP1 protein content in 15 samples of patients with AML-M4 or AML-M5 from 30 AML patients was significantly lower compared with that of patients with the other types of AML,and the levels of miR-155 were significantly higher in the same group of patients (P ＜ 0.05).Significantly decreased levels of SHIP1 protein were found in U937m cells compared that of U937c and U937mc (P ＜ 0.05).Significantly decreased rate of apoptosis was observed in U937m cells compared with that of U937mc and U937c.U937m cells also exhibited no alteration in total AKT content,while increased p-AKT levels were found (P＜ 0.05).Conclusion SHIP1 was also a primary target of miR-155 in AML.Overexpression of miR-155 could activate PI3K-AKT pathway to depressing SHIP1 and decrease the apoptosis rate of AML cells.
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Parabiosis is an experimental animal model in medical studies.This kind of animal models can realize the exchange of blood and body fluid between the two united animals very well, and makes it possible to observe all influences of these exchange in different aspects, ranging from blood to immunity.Over the past few years, based on parabiosis studies, heterochronic parabiosis animals are preferred to be used as conjoint models and to provide experimental basis for researches in cancer, endocrine, transplantation, neurology and stomatology and so on.
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<p><b>OBJECTIVE</b>To summarize a case of acute myeloid leukemia（AML） with severe infection and a rare translocation of t（10;11）（q22;q23）who got spontaneous remission.</p><p><b>METHODS</b>The laboratorial examination results and clinical data in this case were summarized in couple with the light of published literatures.</p><p><b>RESULTS</b>Like most of the spontaneous remission cases, severe infection happened to this case of AML patient, but the different point was that a rare translocation of t（10;11）（q22;q23）was disclosed in this patient. There were only 6 cases of this kind of translocation reported by the literatures up to now. This patient got spontaneous remission after the controlled infection without any chemotherapy. The rare translocation of t（10;11）（q22;q23）disappeared after he got remission.</p><p><b>CONCLUSION</b>Spontaneous remission of acute leukemia was a rare phenomenon, the underlying mechanism was unclear, maybe due to the inflammatory factors triggered by infection, or the activated immune system by the infection, or even the role of gene mutation factors. Accumulating data might shed insight into this rare kind of disease.</p>
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Humanos , Masculino , Enfermedad Aguda , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 11 , Leucemia Mieloide Aguda , Remisión Espontánea , Translocación GenéticaRESUMEN
<p><b>OBJECTIVE</b>To investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia (CML).</p><p><b>METHODS</b>The expression level of SHP-1 mRNA and protein in bone marrow or peripheral blood mononuclear cells from CML patients were detected by Western blot and SYBR Green-based qRT-PCR. The methylation status of SHP-1 were assessed by methylation-specific polymerase chain reaction (MSP) assay. K562 cells were infected with the lentiviral plasmids pEX-SHP-1-puro-Lv105 (K562-SHP-1) or pEX-EGFP-puro-Lv105 (K562-EGFP). The levels of proteins and phosphorylated proteins were detected by Western blot. qRT-PCR assay was used to test the level of BCR-ABL mRNA.</p><p><b>RESULTS</b>The relative levels of SHP-1 mRNA were sharply decreased in advanced stages CML compared to chronic phase (CP)-CML (0.79±0.37 vs 1.18±0.64, P= 0.009). The level of SHP-1 protein was lower in advanced stages CML compared to CP-CML (0.57±0.02 vs 1.02±0.04, P=0.039). The frequency of SHP-1 gene promoter methylation at selected loci in CP-CML was 23.8% (10/42), and the methylated regions were detected in all advanced CML samples (P<0.01). SHP-1 was stably transfected into K562 cells and selected with puromycin. Overexpression of SHP-1 inhibited the proliferation and induced the apoptosis of K562 cells, meanwhile leaded to G0/G1 phase arrest. After transfection, the level of BCR-ABL mRNA was not affected in K562-SHP-1 cells (1.32±0.34) compared to K562-EGFP cells (1.18±0.20, P=0.644), but overexpression of SHP-1 caused a slight decrease in BCR-ABL protein in K562-SHP-1 cells compared to K562 -EGFP cells (0.78±0.15 vs 1.27±0.24, P=0.040). Overexpression of SHP-1 resulted in a remarkable decrease in MYC protein, phosphorylated forms of JAK2, STAT5, Akt and MAPK. However, the un-phosphorylated forms of these molecules were not significantly affected.</p><p><b>CONCLUSION</b>Decreased expression of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulation of BCR-ABL, Akt, MAPK, MYC, JAK2 and STAT5 signaling.</p>
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Humanos , Apoptosis , Metilación de ADN , Progresión de la Enfermedad , Proteínas de Fusión bcr-abl , Janus Quinasa 2 , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva , Leucocitos Mononucleares , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , ARN MensajeroRESUMEN
ObjectiveTo investigate the modification method of Cervical Spine Injury Severity Score System and discuss diagnosis and treatment strategy of lower cervical spine injuries.Methods Treatments of lower cervical spine injuries were selected according to the injury severity graded by the modified Moore' s classification system.Conservative therapy could be adopted for the patients with stability quantification rating ＜ 3 points or for the those with stability quantification rating =3 points but without spinal cord or nerve root compression.Surgical treatment was recommendable for the patients with stability quantification rating =3 points and with spinal cord or nerve root compression.Surgical therapy could be required for the patients with stability quantification rating ≥4 points and with risk of lower cervical instability.The higher the stability quantification score implied the stronger the surgical indications.Lower cervical spine injury combined with spine cord or nerve root compression had absolute surgical indications.At the same time,therapies were selected based on patients' other factors.ResultsBased on basic principles of the modified Moore' s classification system together with opinions of the patients and their relatives,14 patients were managed with surgical treatment and 16 with conservative treatment.Among the patients with complete spinal cord injury (Grade A),two patients treated surgically showed no obvious signs of spinal function recovery,but their nerve root irritation symptoms disappeared; the other one patient who needed surgery but received conservative treatment had no change of the spinal cord function and nerve root irritation.The patients with incomplete spinal cord injury (Grades B,C and D) treated surgically obtained certain degree of spine cord function recovery,with their American Spinal Injury Association (ASIA) score raised by 0.5 grade on average.However,the patients who needed surgery but received conservative treatment gained average increase of ASIA score for 0.5 grade.Imaging examination showed that patients without combined spinal injuries obtained interbody fusion after surgery,with normal alignment and height of the cervical vertebra but without presence of vertebral shift or instability. ConclusionsThe modified Moore' s classification system takes patients' spiaal injury condition and other factors into consideration in selection of conservative or surgical treatment,which improves the Cervical Spine Injury Severity Score System to some extent and has prospect of clinical application.
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Objective: To investigate the effects of Qingyi Huaji (QYHJ) decoction, a compound traditional Chinese herbal medicine, on tumor inhibition rate and serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) in nude mice with transplanted tumors of human pancreatic cancer. Methods: The tumor-bearing mice model was established by subcutaneously inoculating with xenografts of pancreatic cancer into the right armpit of 40 BALB/c nude mice. After successful modeling, the mice were randomly divided into untreated group (Arabic gum), capecitabine group, low-dose QYHJ decoction group (36 g/kg) and high-dose QYHJ decoction group (72 g/kg), with 10 mice in each group. Citrate buffer solution (containing 5% Arabic gum), capecitabine suspension and QYHJ decoction were administered to four groups by gavage respectively. After 5-week treatment, the concentrations of serum IL-6, IL-8 and TNF-alpha were examined by enzyme-linked immunosorbent assay (ELISA) using blood sample from eye socket. Then the mice were euthanized by cervical dislocation. Tumor weight and the tumor inhibition rate were calculated. Results: Tumor weight in the low-dose QYHJ decoction group decreased significantly as compared with the untreated group (P<0.05). Serum levels of IL-6 and TNF-alpha in low- and high-dose QYHJ groups were extremely significantly lower than those in the untreated group (P<0.01). Serum level of IL-8 in the low-dose QYHJ group was significantly lower than that in the untreated group (P<0.05). Correlation analysis showed that transplanted tumor weight of the mice was linearly positively correlated with serum levels of IL-6, IL-8 or TNF-alpha (P<0.01). Conclusion: Conventional dose of QYHJ decoction is effective in suppressing pancreatic carcinoma in nude mice. The mechanism may be related to down-regulation of serum cytokines such as IL-6, IL-8 and TNF-alpha.
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Objective To investigate the expression of SHP-1 and JAK1 mRNA in acute leukemia patients and their impact on disease development,and outcome of the primary chemotherapy.Methods Semi-quantitative reverse transcription polymerase chain reaction was used to measure the expression of SHP-1 and Janus kinase 1(JAK1)mRNA in 93 patients with acute leukemia(AL)and 20 healthy adults as normal 、controls(NC).Results The expression of SHP-1 mRNA in de novo AL patients was significantly lower than that in NC group(P=0.000),which was elevated when complete remission(CR)was achieved(P=0.032)and decreased after the disease relapsed (P=0.015).The expression of JAK1 mRNA in NC group was a lower than that in de novo AL group, but with no statistical significance(P=o.051).While there was statistical significance between NC group and relapsed AL group(P=0.047).The complete remission(CR)rate of the primary chemotherapy in SHP-1 positive group Was 88.9%,but 60.38%in negative group,and there was a statistical significance between them(P=0.018).There Was a negative correlation between the expression level of SHP-1 and JAKI mRNA (P=0.048).Conclusion The expression of SHP-1 mRNA Was significantly decreased or absent in the specimens of acute leukemia patients,and the positive expression of SHP-1 mRNA may be proposed as a factor of preferable therapeutic efficacy in de novo AL and a marker for the progress of the disease.The abundance of JAK1 mRNA was possibly elevated in patients with acute leukemia.
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[Objective] To investigate the effect of the secretory proteins of the ventral prostate glands on the sperm membrane proteins in golden hamsters. [Methods] The sperm was collected from female hamsters uteri after mated with the males with or without ventral prostate gland. Male golden hamsters were divided into four experimental groups: (i) all accessory sex glands (ASG) removed; (ii) ventral prostate gland removed; (iii) all ASG removed except ventral prostate gland and (iv) sham-operated controls. Each group contained 6 hamsters. The sperm membrane proteins were extracted from uterine sperm and analyzed by SDS-PAGE and two dimension electrophoresis. [Results] The SDS-PAGE results of sperm membrane showed that the ventral prostate glands added up some proteins or increased the quantity of some proteins, which contained molecular mass (MM)15 k, 29 k, 38 k, 55 k, and 91 k proteins, to the sperm membrane. 2D-electrophoresis of sperm membrane showed that some extra protein spots were appeared in with ventral prostate gland groups, their MM and isoelectric point (IP) corresponding to 16 k/8.60, 16.6 k/9.20, 28 k/5.88, 28 k/6.10, 29 k/5.98, 32 k/6.35, 32 k/6.50, 32 k/7.20, 61 k/5.90,and 83 k/6.40, respectively. The quantity of some protein spots increased in sperm membrane of ventral prostate glands group, their MM/IP coresponding to 17 k/5.95, 17.5 k/6.50, 24 k/7.20, 26 k/5.40, 26 k/5.60, 27 k/7.20, 27 k/7.50, 28 k/5.70, 29 k/8.50, 42 k/6.50, and 42 k/7.00, respectively. [Conclusion] The ventral prostate gland secretion plays a role in regulating the sperm membrane proteins and the latter may in turn affect the male fertility or embryo development.
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Objective To investigate the expression of suppressor of cytokine signaling genes (SOCSs) and JAKs mRNA in the acute myloid leukemia(AML) patients. Methods The expression of SOCSs and JAKs mRNA as well as TYK2 in AML patients and healthy adults as normal contrals (NC) was measured with RT-PCR. Results The expression of SOCS 1,4,5 and 7 in AML patients was lower than those in normal control and AML with remis-sion (P<0.01),but the expression of SOCS 3 and 6 was higher than those in normal control and remission AML(P<0.01),however there was no significant difference in SOC2 between groups. The expressions of JAK2 ,JAK3 and TYK2 in AML were significantly higher than those in patients with remission and normal control(P<0.05). The ex-pression of JAK1 mRNA in relapsed AML was higher than that in normal control group(P<0.05),but the latter has no statistical significance between beginning treatment and normal group(P>0.05). Conclusion The deletion and degradion of SOCS 1,4,5 and 7 present in AML patients and JAKs expression is significantly increased, suggesting that both of them may co-participate in the pathogenesis of AML.
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Background and purpose:For the past few years, HPV DNA has been detected in the peripheral blood in the patients with cervical cancer and believed to be a promising tumor marker, but because the concentration in the patients' serum was extremely low, it made it difficult to detect the circulating HPV DNA. In order to find a way to improve the sensitivity of the assay, this study compared and evaluated three methods that are currently being used to measure HPV DNA in the extraction from serum of cervical cancer patients. Methods:51 patients were pathologically proved to be cervical cancer and enrolled into the study. DNA from their sera was extracted by three methods:(1) Phenol-chloroform extraction, (2) UNIQ-10 collumn virus DNA kit(SANGON,Shanghai), (3) QIAamp MinElute Virus spin kit(QIAGEN,Germany). Then the HPV DNA was quantitated by PCR using GP5+/GP6+ primers.Results:Either fresh or short-term storage sera has been used The positive rates were 14.3%, 5.7%, and 57.1% with three assays, respectively. But when long-term storage samples were used to quantitate, the positive rates were low regardless of the methods being used.Conclusions:QIAamp MinElute Virus spin kit was the most sensitive for the content of serum HPV DNA, it could improve the positive rate for the assay if the serum was not stored for long time.
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<p><b>OBJECTIVE</b>To investigate the synergistic antitumor effects of combined use of p14ARF gene and 5-fluorouracil (5-Fu) in pancreatic cancer.</p><p><b>METHODS</b>A human pancreatic cancer cell line PC-3 was transfected with lipofectin-mediated recombinant p14ARF gene, and was then administered with 5-Fu. Cell growth, morphological changes, cell cycle, apoptosis, and molecular changes were measured using the MTT assay, flow cytometry, RT-PCR, Western blotting, and immunocytochemical assays.</p><p><b>RESULTS</b>After transfection of p14ARF, cell growth was obviously inhibited, resulting in an accumulation of cells in the G(1) phase. The proportion of cells in the G(1) phase was significantly increased from 58.51% to 75.92%, and in the S and G(2)/M phases decreased significantly from 20.05% to 12.60%, and from 21.44% to 11.48%, respectively, as compared with those of the control groups. PC-3/p14ARF cells that underwent 5-Fu treatment had significantly greater G(2)/M phase accumulation, from 11.48% to 53.47%. The apoptopic index was increased in PC-3/p14ARF cells from 3.64% to 19.62%. The MTT assay showed p14ARF-expressing cells were significantly more sensitive to 5-Fu (0.01 - 10 mg/L) than those devoid of p14ARF expression (P < 0.01). Western blotting showed p14ARF upregulates p53 expression.</p><p><b>CONCLUSION</b>Combined use of p14ARF gene and 5-Fu acts synergistically to inhibit pancreatic cancer cell proliferation, suggesting a new anticancer strategy.</p>
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Humanos , Fluorouracilo , Farmacología , Neoplasias Pancreáticas , Genética , Terapéutica , Transfección , Células Tumorales Cultivadas , Proteína p14ARF Supresora de Tumor , Genética , Proteína p53 Supresora de Tumor , Genética , Regulación hacia Arriba , FisiologíaRESUMEN
Objective To investigate the role of P21 protein and survivin on the cell apoptosis of non-small cell lung cancer (NSCLC) induced by mevastatin. Methods The inhibitory effect of mevastatin on A549 and NCI-H520 cell line was evaluated by MTT assay. The cell cycle distribution and apoptosis induction were determined with flow cytometer and transmission electron microscope. The expression of p21 protein was assessed with flow cytometer. The mRNA expression of p21 and survivin was assessed with RT-PCR. Results Flow cytometry showed that mevastatin induced G_0/G_1 cell arrest in NSCLC cell lines. The results indicated that mevastatin caused apoptosis in concentration-dependent manner. Mevastatin produced no change in expression of P21 mRNA and total P21 protein. Concomitantly, P21 protein localized on cellular membrane was decreased. It was also found that mevastatin suppressed the expression of survivin mRNA in NSCLC cell lines. All these effects were reversed by mevalonate. Conclusions Mevastatin inhibited the proliferation of NSCLC cell lines. Mevastatin exerts growth inhibitory effect and induces apoptosis effect by inhibiting mevalonate synthesis. Its mechanisms might involve blockade of the isoprenylation of p21 protein and down-regulation of the expression of survivin mRNA.
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AIM: To investigate the role of mitochondrial ceramidase in mitochondrial functions, especially in the regulation of apoptosis. METHODS: pCDNA3.1/His-MtCDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transfected into K562 cells by liposome, and G418 was used to screen the positive clones. A stable transfected K562 cell line was established and defined as ‘K562TC’. The differences between K562 and K562TC cells in serum withdrawal resistance and Bcl-2 protein expression were evaluated by annexin V/PI test, flow cytometry and Western blotting, respectively. RESULTS: K562TC cells with elevated Bcl-2 protein expression level identified by FCM or Western blotting showed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N'-dimethylsphingosine (DMS), a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate (SPP) production, also abrogated Bcl-2 protein expression in K562TC cells, while exogenous sphingosine-1-phosphate up-regulated Bcl-2 protein level in K562 cells. CONCLUSION: Mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form, indicating that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.
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Objective To explore the expression changes in SHIP1 gene in patients with chronic granulocytic leukemia (CGL), healthy volunteers and the K562 cells (one of human CGL cell lines), and to explore the effects of BCR/ABL expression in K562 cells on the SHIP1 gene by blocking the BCR/ABL expression in K562 cells with a specific siRNA. Methods The expression levels of SHIP1 mRNA in leukocytes of the patients with CGL, healthy controls and K562 cells were assessed with RQ-PCR. In addition, K562 cells were divided into three groups: control group (untreated), non-specific interference group (treated with non-specific siRNA), and specific interference group (treated with specific siRNA to block BCR/ABL gene). RQ-PCR and Western blotting were employed to examine the expression level of mRNA and protein (P210 and P145) of BCR/ABL and SHIP1 genes. Results The mRNA expression of SHIP1 in CGL patients and K562 cells was significantly lower than that of the healthy controls. The expression of BCR/ABL gene was down-regulated, while SHIP1 gene up-regulated, in the specific interference group. No statistical significant difference was found between the non-specific interference group and the control group. Conclusions The mRNA expression of SHIP1 is lower in patients with CGL than in the healthy control. The specific siRNA can block the expression of BCR-ABL and further suppress the expression of SHIP1.