Screening for specific biomarkers in serum of patients with rheumatoid arthritis using proteomic fingerprint techniques

AIM: To detect the serum protein biomarkers and establish a diagnostic model for rheumatoid arthritis (RA) adopting matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with weak cationic exchange (WCX) magnetic beads, and to study the clinical significance of the model in early diagnosis of RA. METHODS: The serum samples from 60 patients with RA, 35 patients with osteoarthritis (OA) and 36 healthy controls were prepared with WCX magnetic beads, and then analyzed on PBSⅡ-C mass spectrometer reader. The protein spectra of the serum samples were normalized by Ciphergen ProteinChip software. The peak labeling was performed by Biomarker Wizard software. The specific protein biomarkers were screened by Biomarker Pattern software to construct a diagnostic model for RA. RESULTS: Totally 33 discriminative mass-to-charge (m/z) ratio were identified to be related with RA (P<0.05). Among these, the m/z peaks at 15 715.5D, 7 771.4D, 8 959.4D, 8 469.8D and 8 710.8D were used to construct a diagnostic model in a training set. In a test set, the sensitivity and specificity of the model were 86.7%and 90.0%, respectively. CONCLUSION: The potential protein biomarkers for RA are discovered in serum by MALDI-TOF-MS combined with WCX magnetic beads. The model of multiple biomarkers provides a powerful and reliable diagnostic method for RA diagnosis with high sensitivity and specificity.     

Analysis of serum biomarkers in human knee osteoarthritis

AIM: To analyze the proteomic components of the sera from knee osteoarthritis patients and normal people, and to search proteins that might serve as serum biomarkers for osteoarthritis diagnosis, treatment or pathogenesis. METHODS: Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was applied to analyze the sera obtained from the patients with knee osteoarthritis (n=4) and normal controls (n=4). The differentially expressed proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western blotting analysis was applied to confirm the results. RESULTS: Comparative proteomic data of serum from the patients with osteoarthritis was successfully obtained. Eight differentially expressed protein spots were observed. Five up-regulated and 3 down-regulated proteins were identified. Western blot analysis confirmed that α₂-macroglobulin was increased. CONCLUSION: There are significant differences between serum proteins obtained from the patients with knee osteoarthritis and normal controls. α₂-Macroglobulin might be utilized as potential biomarkers for the diagnosis and treatment of osteoarthritis.     

Diagnostic and prognostic application of proteomic patterns in breast cancer

AIM: To detect the serum proteomic patterns in patients of breast cancer by the method of SELDI-TOF-MS and CM10 ProteinChip, and to screen the biomarker candidates, build and validate the diagnostic models, and evaluate its clinical value in surveillance and follow-up after operation. METHODS: The SELDI-TOF-MS technology and CM10 ProteinChip were used to detect the proteomic patterns of serum from 63 breast cancer patients and 40 healthy women. The biomarker candidates were screened and the diagnostic models were constructed by ZJU-PDAS software. Meanwhile, the model was blind-validated in another 23 patients and 20 healthy women. At the same time, 16 serum samples were detected to evaluate its value in surveillance and follow-up after operation. RESULTS: The best model was composed by two protein peaks (BC1/3.9 kD and BC2/5.6 kD) with its sensitivity and specificity of 87.30% (55/63) and 95.00% (38/40), respectively. The sensitivity and specificity in the blind-validation of new cases were 95.65% (22/23) and 85.00% (17/20), respectively. The diagnostic efficacies were the same to the patients of different stages (P>0.05). The expression of BC1 increased while BC2 decreased after operation. The expression of BC2 in the patients with recurrence or metastasis was higher than that in the tumor-free survivors (P<0.05). CONCLUSION: This method shows its potential in detection, surveillance and follow-up after operation. The method is also useful for screening the novel and better biomarkers in breast cancer.     

Serum comparative proteomic analysis of radiation-induced hepatic injury in cirrhotic rats

AIM: To investigate the differential expression of serum proteins in cirrhotic SD rats for exploring the pathogenesis and identifying the potential biomarkers of radiation-induced hepatic injury. METHODS: Liver cirrhosis was induced in 8 healthy SD rats with subcutaneous injection of carbon tetrachloride (CCl₄) for 6 weeks and then the animals were randomly divided into 2 groups (4 rats in each group): control group (CCl₄ alone) and experimental group (CCl₄ plus radiation). The latter received hemi-liver radiation with single dose of 15 Gy while the former did not receive radiation. Total serum proteins of the 2 groups were extracted 6 h after radiation. Two-dimensional gel electrophoresis (2-DE) were performed to look for differentially expressed proteins. These proteins were then analyzed and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western bloltting was used to validate the expression of heparanase and hepatocyte growth factor receptor in all independent series of serum samples. RESULTS: Two-dimensional gel images were acquired with good resolution and repetition. Thirty-three differentially expressed proteins were selected and 12 proteins were successfully identified by MS, in which 5 were up-regulated while 7 were down-regulated. The increased level of heparanase and decreased level of hepatocyte growth factor receptor were further confirmed by Western blotting. CONCLUSION: Twelve proteins associated with radiation-induced hepatic injury are successfully characterized by serum comparative proteomics. Heparanase and hepatocyte growth factor receptor might be useful for detecting and monitoring radiation-induced hepatic injury.     

Identification of specific serum biomarkers in gastric adenocarcinoma patients

AIM: To screen the possible serum biomarkers for the diagnosis of gastric adenocarcinoma. METHODS: The surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to screen the serum samples from 109 cases of gastric adenocarcinoma and 106 control subjects (60 healthy subjects, 30 patients with chronic superficial gastritis and 16 cases of chronic atrophic gastritis). The differentially-expressed protein peaks were selected and isolated with high-performance liquid chromatography (HPLC), and processed with enzyme before liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS) analysis. The data mining was performed with software Xcalibur program component Bioworks 3.2. RESULTS: Three differentially-expressed protein peaks were selected as potential serum biomarkers of gastric adenocarcinoma patients.The m/z peak at 5 906.5 showed the increase (8.53±4.33 in cancer group, and 0.88±0.31 in control group). The m/z peaks at 6 635.7 and 8 716.3 showed the decrease (6.54±2.44 and 0.93 ± 0.29, respectively, in cancer group and 17.56±4.43 and 2.16±0.98, respectively, in control group, P<0.01). The 3 m/z peaks were identified as fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI,respectively. The combined use of the 3 biomarkers distinguished the samples in the cancer patients out of the controls at a sensitivity of 93.85% (61/65) and a specificity of 94.34% (50/53). CONCLUSION: The fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI identified as potential markers for gastric adenocarcinoma show diagnostic values in clinical application.     

Expression of ERK5 in colorectal cancer tissues and its correlation with distant metastasis in colorectal cancer patients

AIM: To investigate the expression of extracellular signal-regulated kinase 5 (ERK5) in primary colorectal cancer (CRC) and adjacent normal mucosa, and to analyze the relationship between ERK5 expression and clinicopathological parameters for exploring the functions of ERK5 in the occurrence and development of CRC. METHODS: The expression of ERK5 in carcinoma tissues and normal mucosa was examined by a set of tissue microarrays and the method of immunohistochemistry. The potential relationship between ERK5 expression and clinicopathological features was also analyzed. RESULTS: ERK5 expression was significantly higher in CRC tissues (134/338, 39.6%) than that in normal tissues (21/80, 26.2%; P<0.05). Overexpression of ERK5 in CRC tissues was significantly correlated with distant metastasis (P<0.05). However, no correlation between ERK5 expression and age at surgery, sex, tumor location, the depth of invasion, lymph node metastasis, TNM staging or differentiation grade was found (P>0.05). According to the Kaplan-Meier analysis, there is no significant difference in 5-year overall survival between the patients with ERK5 expression at high level and at low level. CONCLUSION: ERK5 protein is highly expressed in CRC with distant metastasis. This may be a promotive factor in the process of distant metastasis.     

Analysis of proteomic spectra in serum from patients with colorectal cancer by SELDI-TOF mass spectrometry

AIM: To study the changes of proteomic spectra in serum from patients with colorectal cancer in order to detect the specific protein markers. METHODS: Proteomic spectra of sixty-four serum samples from patients with colorectal cancer (preoperation and postoperation) and forty from normal individuals were generated by IMAC-Cu proteinchip array and SELDI-TOF mass spectroscopy (surface enhanced laser desorption/ionization-time of flight). The discriminatory profiling between cancer and normal samples was analyzed by Biomarker Wizard software and Biomarker Pattern softwar. RESULTS: Nineteen differentially expressed proteins in serum were screened by analysis of protemic spectra of preoperative patients and normal individuals. Five proteins (5972.67 D, 5927.21 D, 6113.48 D, 5908.55 D and 4292.51 D) were obtained for making up marker patterns that was able to class the patients-team and normal-team. Corresponding correct ratio were 97.5% (56/64) and 80% (32/40). The proteins that overexprssed in preoperation were obviously down-regulated when postoperation. CONCLUSION: The preliminary results suggest that classification system will provide a highly accurate and innovative approach for the early diagnosis of colorectal cancer and judgment of prognosis. SELDI-TOF mass spectrometry is a useful tool for the detection and identification of new protein markers in serum.     

Changes of serum autoantibodies against β1 and M2 receptors in the patients of obstructive sleep apnea syndrome with chronic obstructive pulmonary disease

AIM: To study the changes of serum autoantibodies against β₁-adrenergic and M₂-muscarinic receptors in obstructive sleep apnea syndrome (OSAS) patients with chronic obstructive pulmonary disease (COPD), that is, overlap syndrome (OS). METHOD: Serum autoantibodies against β₁ and M₂ receptors in 26 cases with OS, 32 with OSAS, 30 with COPD and 28 normal subjects were determined by using enzyme-linked immunosorbent assay(ELISA). RESULTS: The positive rates and titer of β₁ and M₂ receptor autoantibodies are significantly increased in OS group (92.2％,57.7% and 1:98, 1:67), compared to OSAS (71.9%, 40.6% and 1:83, 1:30) or COPD group (70.0%, 36.7% and 1:79, 1:28) (P＜0.05), and they are higher in these groups than in the control (25.0%, 14.3% and 1:20, 1:20) (P＜0.01). CONCLUSION: Serum β₁-and M₂ receptor autoantibodies are significantly increased in the patients with COPD, OSAS or OS, compared to the control, and the highest is in OS.     

Association between ERK5 -322G/T polymorphism and genetic susceptibility to sporadic colorectal cancer

AIM: To investigate the correlation between extracellular signal-regulated kinase 5 ( ERK5 ) -322G/T polymorphism (rs3866958) and the susceptibility to sporadic colorectal cancer (CRC) in southern Chinese population. METHODS: ERK5 -322G/T genotypes were determined by Taqman-MGB probes in 835 CRC cases and 908 healthy controls. RESULTS: No significance of ERK5 -322G/T genotype distribution between CRC patients and controls was observed, but -322G/T decreased the susceptibility to CRC in fat people whose BMI was ≥ 24 kg/m². Compared to GG genotypes, the carriers with GT and TT genotypes had a significant decrease in the risk of CRC(OR=0.576,95%CI 0.413-0.804, P<0.01). CONCLUSION: ERK5 -322G/T polymorphism (rs3866958) has no significant relevance with sporadic CRC susceptibility, but decrease, the risk of CRC in people with fatness. The T variant genotype is an independent protective factor against sporadic CRC of overweight patients in southern Chinese population.     

Measurement of serum lipopolysaccharide binding protein for diagnosis and prognosis prediction in septic patients

AIM: To investigate the role of lipopolysaccharide binding protein (LBP) for diagnosis and prognosis prediction in the septic patients. METHODS: A total number of 80 ICU patients were enrolled. The patients were divided into systemic inflammatory response syndrome (SIRS) group and sepsis group, the patients in sepsis group were divided into non-survivor sub-group and survivor sub-group. We collected the serum samples and analyzed acute physiology and chronic health evaluation (APACHE) II score on the first day of the patients hospitalized in ICU. In addition, we also selected 10 healthy volunteers and collected their serum samples. The serum concentrations of LBP, C-reactive protein (CRP) and procalcitonin (PCT) were measured by ELISA. ROC analysis of LBP, CRP, PCT and APACHE II score was conducted to discriminate among critically ill patients with sepsis and predict the prognosis of the patients with sepsis. RESULTS: The levels of the 4 indicators in the septic patients were higher than those in the patients of SIRS (P<0.05). In addition, serum LBP and APACHE II score in the non-survivor sub-group were higher than those in the survivor sub-group (P<0.05), whereas no difference of the PCT and CRP levels between survivors and non-survivors with sepsis was observed. LBP levels greater than 26.84 mg/L had 97.1% sensitivity and 95.9% specificity to discriminate between SIRS and sepsis. LBP levels greater than 54.16 mg/L had 85.2% sensitivity and 80.0% specificity for prognosis of unfavorable outcome.CONCLUSION: LBP level was more accurately correlated with diagnosis or prognosis prediction than CRP or PCT in patients with sepsis.     

Diagnostic and prognostic value of serum AQP4 antibody on neuromyelitis optica

AIM: To investigate the diagnostic and prognostic value of the serum AQP4 antibody on neuromyelitis optica (NMO). METHODS: Sera of the patients with NMO,transverse myelitis （TM）, optic neuritis (ON), multiple sclerosis (MS) and other neurological disease (OND) were all collected and detected for the presence of AQP4 antibody by CBA. The positive rates and the titers of AQP4 antibody were compared among all the groups. The characteristics of NMO patients with AQP4 antibody positive and those AQP4 antibody negative were also compared. The sensitivity and specificity of serum AQP4 antibody for diagnosis of NMO were evaluated with receiver operating characteristic(ROC) curve. The Kaplan-Meier curve was used to examine the accumulated rate of AQP4 antibody for the progress to NMO.RESULTS: The AQP4 antibody positive rate in NMO group was the highest (87.50%). Orderly, rLETM group was 85.71%，RION group was 80.00%, LETM group was 30.70%, BON group was 8.00%, and MS group was 3.85%. The AQP4 antibody was negative in OND group. There was significant difference between AQP4 antibody positive and AQP4 antibody negative NMO patients on gender, the number of patients with poor visual outcome, time of NMO diagnose confirmed(P<0.05). The sensitivity and specificity were 87.5% and 85.4% for discriminating NMO from other diseases. The Kaplan-Meier curve showed that AQP4-Ab-positive patients were more likely to become NMO compared to the AQP4-Ab-negative patients.CONCLUSION: The serum AQP4 antibody is extensively important in the early diagnosis and prognosis of the NMO with high sensitivity and high specificity.     

Changes of serum T-PSA,F-PSA and F/T ratio in patients with prostate cancer (PCa) and its clinical significance

AIM: To evaluate the changes of serum total prostate specific antigen (T-PSA), free prostate specific antigen (F-PSA) and the ratio of F-PSA to T-PSA (F/T) in patients with prostate cancer and its clinical significance. METHODS: The concentrations of T-PSA and F-PSA in serum were measured by micropartical enzyme immunoassay (MEIA) using AxSYM System, and the F/T ratio was calculated. RESULTS: Before operation, the concentrations of T-PSA and F-PSA in patients with PCa were much higher and F/T ratio was significantly lower than that in patients with benign prostate hyperplasia (BPH). T-PSA and F-PSA levels decreased, but F/T ratio increased after operation in PCa and BPH. F/T ratio in 83.5% PCa and 6.5% BPH was less than 0.16. To diagnosis PCa, the sensitivity of F/T ratio was 83.5%, and the specificity was 86.7%. CONCLUSION: Serum T-PSA, F-PSA and F/T ratio are important parameters for the early diagnosis of prostate cancer.     

Effects of drynaria total flavonoids on serum levels of leptin,interleukin 6, prostaglandin E2 and expression of bone β2-adrenergic receptor in ovariectomized osteoporosis rats

AIM: To investigate the effects of drynaria total flavonoids on serum levels of leptin (LEP), interleukin 6(IL-6), prostaglandin E₂(PGE₂) and the expression of bone β₂-adrenergic receptor (ADRB2) in a rat model of ovariectomized osteoporosis(OP). METHODS: The osteoporosis model was established by ovariectomy. Twelve weeks after modeling,bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry to verify successful modeling.Enzyme-linked immunosorbent assay was applied to detect the concentrations of LEP, IL-6 and PGE₂ in serum. The expression of ADRB2 was determined by immunohistochemical technique. RESULTS: Compared with sham group,BMD of the rats in model group significantly decreased in multiple regions 12 weeks after modeling(P<0.01). The serum levels of LEP, IL-6 and PGE₂ in model group were significantly higher than those in sham group(P<0.05). The levels of LEP, IL-6 and PGE₂ in drynaria total flavonoids group were significantly lower than those in model group(P<0.01). No significant difference of PGE₂ between these 2 groups was observed. The ADRB2 expression in sham group and treatment group was significantly different from that in model group, and no significant difference between sham group and treatment group was found. CONCLUSION: The serum levels of LEP, IL-6 and PGE₂ and the expression of bone ADRB2 increased in OP rats.Drynaria total flavonoids reduce the production of LEP, IL-6 and the expression of ADRB2, and suppress the bone absorption, which may be one of the mechanisms in treating OP.     

Effects of hypoxia inhalation therapy on the number of eosinophil and CD4+T-lymphocyte in remission stage in asthmatic guinea pigs

AIM:To explore the mechanism underlying the therapeutic effects of hypoxia inhalation on asthma. METHODS:Guinea pigs were randomized into the normal group(NG), asthmatic group(AG) and the hypoxia inhalation-treated group(HITG). The model of asthma was established in the latter two groups through sensitization and induction with 10% ovalbumin(OA) and 1% OA, respectively. The animals in HITG were treated with hypoxia inhalation (13.0%±0.5% O₂/N₂ mixed gas). The content of serum cortisol, the number of eosinophils(EOS) and percentage of hypodense eosinophils(HEOS) in bronchoalveolar lavage fluid(BALF),the number of CD₄⁺T-lymphocyte in peripheral blood(PB) and the tension of airway muscle were determined. RESULTS:(1)The content of serum cortisol was significantly higher in NG and HITG than in AG(P＜0.01); (2)The number of EOS and percentage of HEOS in BALF was significantly lower in HITG than in AG(P＜0.01); (3) The number of CD₄⁺T-lymphocyte in PB was significantly higher in AG than in HITG(P＜0.01).CONCLUSION:After treatment with hypoxia inhalation, the content of serum cortisol in asthmatic guinea pigs was significantly increased to result in marked decreased of the number of EOS, the percentage of HEOS in BALF, and the number of CD₄⁺T-lymphocyte in PB, thus result in the tension of airway muscle and alleviation of the airway hyperresponsiveness. All these may be beneficial to preventing the relapse of asthma.     

Limb ischemic preconditioning protects human umbilical vein endothelial cells from oxidative stress induced by H2O2

AIM: To investigate the effects of the sera from the rats after limb ischemic preconditioning (LIPC) on human umbilical vein endothelial cells (HUVECs) injured by hydrogen peroxide (H₂O₂). METHODS: The HUVECs were divided into 5 groups: the cells in control group were cultured without any intervention; the cells in model group (M) were damaged by 1 mmol/L H₂O₂ for 2 h; the cells in early preconditioning serum (EPS) group, delayed preconditioning serum (DPS) group or sham limb ischemic preconditioning serum (SPS) group were treated with the corresponding serum at 5% for 12 h, respectively, and then treaed with H₂O₂ for 2 h. The viability of the HUVECs was analyzed by flow cytometry. The lactate dehydrogenase (LDH) in the culture media was detected. The cell adhesion molecules in the HUVECs were detected by real-time PCR. The mRNA and protein expression of heme oxygenase-1 (HO-1) was also determined. RESULTS: The viability of HUVECs incubated with 1 mmol/L H₂O₂ for 2 h significantly decreased compared with the control cells, which was accompanied with the augmentations of LDH in the medium and the cell adhesion molecules in cells, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Preincubation with EPS and DPS derived from the rats subjected LIPC attenuated these injuries. Furthermore, pretreatment with EPS and DPS increased the expression of HO-1 at mRNA and protein levels. CONCLUSION: LIPC protects the HUVECs from H₂O₂-induced injury.     

Effects of Sini decoction on the expression of TGF-β1 in balloon injured iliac artery of rabbits

AIM:To investigate the effect of Sini decoction (SND) on vascular stenosis and the expression of transfoming growth factor-β1 (TGF-β₁) in iliac artery balloon injured rabbits. METHODS:24 male New Zealand albino rabbits were divided into three groups:control group, model group and SND treatment group. The iliac arteries were injured by balloon in model and SND groups. Four weeks later, serum TGF-β₁ level was assayed by ELISA. Endothelial hyperplasia, TGF-β₁ protein and mRNA expression were observed in injured iliac artery. RESULTS:Light microscope showed that the vascular lumina were narrower, intima was thicker in model group control and SND treatment group. The serum TGF-β₁ level was lower in control than model group and SND treatment group, and the serum TGF-β₁ level in SND treatment group was lower than that in model group. Immunohistochemistry and RT-PCR results showed that TGF-β₁ protein and mRNA expression was lower in rabbit iliac artery of control group than that in model group and SND treatment group, and the expression of TGF-β₁ protein and mRNA decreased significantly in SND treatment group compared with model group. CONCLUSION:SND could lessen intimal hyperplasia and vascular stenosis in balloon injured iliac artery, which might be related to decrease in TGF-β₁ protein and gene expression in iliac artery.      

Expression of IL-1α and TNF-α modified by Toll-like receptor 4 genetic polymorphism is associated with colorectal carcinoma

AIM: To investigate the association of D299G, T399I and A896G polymorphisms of Toll-like receptor 4 (TLR4) and colorectal carcinoma (CRC). METHODS:
The genotypes of these 3 loci among 268 patients with CRC and 268 healthy controls were determined by polymerase chain reaction-restriction fragment lengthy polymorphism (PCR-RFLP). The protein levels of IL-1α, IL-8, TGF-β and TNF-α in the homogenate of CRC biopsies were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: No significant difference of the genotype frequencies of TLR4 A896G and D299G between the cases and the controls was observed. CT combined TT genotype of T399I was significantly associated with increased CRC risk. The individuals with the T allele of T399I showed a 1.843-fold increase in CRC risk as compared with the C allele. The concentrations of IL-1α and TNF-α in CRC biopsies were significantly elevated in the individuals with the genotype of T399I CT combined with TT as compared with the genotype of CC. CONCLUSION: TLR4 T399I promotes the development of CRC by modifying the expression of IL-1α and TNF-α in CRC tissues.     

Study on the binding capacity of IgA1 from patients with IgA nephropathy to human mesangial cells

AIM: To examine and compare the ability of serum IgA 1, from both the patients with IgA nephropathy(IgAN) and the healthy control, to bind to human mesangial cells(HMC). METHODS: Serum IgA was isolated with jacalin column, heated to aggregated form(IgA₁) and labeled with [¹²⁵I]. Binding capacity of IgA₁ to primary HMC was evaluated by radioligant binding assay, specificity of binding was determined by competitive inhibition, and relative affinities was compared by cross competitive inhibition. RESULTS: Both IgA₁ from normal control and patients with IgAN bound to MC in a dose-dependent, saturatable manner, but the binding of IgA₁ from patients was saturated at approximately 200 pmol while that from healthy was at 400 pmol. The Scatchard analysis revealed a Kd of(8.9±2.1)×10^-8 mol/L for patient' s IgA₁ versus(4.3±1.2)×10^-7mol/L for normal IgA₁(P<0.05). Competition inhibition showed that both serum albumin and IgG could not block the binding of IgA₁ to HMC while mIgA 1 could partially block that. Cross competition inhibition demonstrated that patient' s IgA₁ blocked normal IgA₁ binding significantly(P<0.01), however, normal IgA₁ was unable to inhibit the binding of patient' s IgA₁. CONCLUSIONS: ①There are IgA binding proteins or receptors on mesangial cells. ②IgA₁ from patients with IgAN has a higher binding capacity than that from healthy.     

Roles of maspin in biological behaviors of PC-3 prostate cancer cells

AIM: To investigate the roles of maspin in the biological behaviors of prostate cancer cells. METHODS: Specific shRNA targeting maspin gene was designed. The plasmid targeting maspin gene was constructed and lentiviral expression system was used for transfection. qRT-PCR and Western blotting were performed to identify the stable maspin-shRNA-transfected PC-3 cells. The expression of apoptosis-related genes was analyzed by qRT-PCR. Dynamic observation of cell growth and doubling time were conducted by an xCELLigence system. The cell death upon proteasome inhibitor treatment was determined by flow cytometry analysis. The expression levels of RelA and RelB were detected by Western blotting. RESULTS: The recombinant plasmid containing maspin-shRNA was successfully constructed. Limited dilution was performed to obtain monoclonal PC-3-siMaspin cells. The doubling time of PC-3-siMaspin cells was 26.83 h while that of PC-3-control cells was 37.95 h. The mRNA expression of bcl-2 and A20 in PC-3-siMaspin cells was increased, while that of bax and bim was down-regulated. The cell death rates of PC-3-control cells and PC-3-siMaspin cells after treated with MG-132 were 27.1% ?5.6% and 7.5% ?2.3% at 8 h, 24.2% ?3.7% and 8.2% ?2.5% at 24 h, and 28.7% ?3.7% and 7.6%?2.5% at 36 h after treatment,respectively. RelA expression was decreased in PC-3-control cells treated with MG-132 while that in PC-3-siMaspin cells stayed unchanged. CONCLUSION: Maspin expression is increased in androgen-independent prostate cancer PC-3 cells. Maspin silencing significantly reduces the doubling time and accelerates the cell growth. Maspin silencing markedly reduces the sensitivity of PC-3 cells to proteasome inhibitor, which may be linked to the abolishment of RelA degradation.     

利用免疫磁性分离–PCR检测安祖花细菌性枯萎病病原菌

 根据地毯草黄单胞菌花叶万年青致病变种（Xanthomonas axonopodis pv. dieffenbachiae）的RAPD序列设计特异性引物,并对羧基化磁珠的结合性能进行检验,从而建立和优化了免疫磁性分离–PCR体系,对安祖花细菌性枯萎病进行早期检测。结果表明：1 mg磁珠对多克隆抗体最大吸附值为0.268 mg,进行免疫磁性捕获时免疫磁珠的最佳浓度是0.566 ~ 0.741 mg ?mL^-1。免疫磁性分离–PCR可以减少PCR反应中的抑制物质,对X. axonopodis pv. dieffenbachiae的检测灵敏度可以达到10 ~ 100 cfu ?mL^-1,比常规PCR检测灵敏至少100倍。