Objeetive To explore the value of antisense imaging of 99Tcm-labeled ASON targeting mouse double minute 2(MDM2) mRNA for the diagnosis of human prostate cancer.Methods The ASON targeting MDM2 mRNA and the mismatched oligonucleotide (ASONM) were synthesized and radiolabeled with 99Tcm using the bifunctional chelator HYNIC.The labeling efficiency and radiochemical purity were investigated.Animal models of nude mice bearing human prostate cancer LNCaP were established and divided into 3 groups with 10 mice in each group.99Tcm-HYNIC-ASON,99Tcm-HYNIC-ASONM (study groups) and 99TcmO4-(control group) were injected at the dose of 7.4 MBq through the tail vein,respectively.Tumor imaging was acquired with SPECT and the tumor-to-muscle (T/M) ratio was measured.The data was compared by one-way analysis of variance.Results The labeling efficiencies of ASON and ASONM were (65.15± 2.05) ％ and (64.93±2.18) ％,respectively.Their radiochemical purity was greater than 90％.At 1,4 and 10 h post injection,the T/M ratios of 99Tcm-HYNIC-ASON group were 3.217±0.125,3.749± 0.201 and 4.028±0.186,and those of 99Tcm-HYNIC-ASONM group were 1.579t0.128,1.715±1.140 and 1.683±0.139,and control group 2.146±0.132,1.847±0.124,1.528±0.152,respectively.The T/M ratios in control group and 99Tcm-HYNIC-ASONM group were significantly lower than those in 99Tcm-HYNICASON group at 1,4 and 10 h,respectively (F=213.37-235.41,t=3.527-4.738; all P＜0.01).The T/M ratios of 99Tcm-HYNIC-ASONM group and control group were not significantly different at 1,4 and 10 h (t=2.154,2.287 and 2.236,all P＞0.05).Conclusion The antisense probe of MDM2 can accumulate specifically in prostate cancer tissue in animal models,which might be useful as a non-invasive genetic tool for the early diagnosis of prostate cancer.

Objective To investigate the effect of mouse double minute 2 (MDM2) mRNA ASON and mismatched oligonucleotide (ASONM) radiolabeled with 99Tcm on target gene expression in LNCaP cells.Methods The ASON and ASONM targeted to MDM2 mRNA were synthesized and radiolabeled by 99Tcm with the bifunctional chelator of HYNIC.The labeling efficiency,radiochemical purity,stability and molecular hybridization activity were investigated.The different concentrations of 99Tcm-HYNIC-ASON (0,100,500 nmol/L) and 99Tcm-HYNIC-ASONM (500 nmol/L) coated with lipofectamin 2000 were incubated with prostate cancer cells for 24 h,then RT-PCR and Western blot were carried out to assay the MDM2,p53 mRNA and the corresponding protein level.The variables of RT-PCR and Western blot were analyzed using one-way analysis of variance and q test.Results The labeling efficiency of ASON and ASONM were (65.15± 2.05)％ (n=5) and (64.93±2.18)％ (n=5),respectively.The radiochemical purity were both more than 90％.99Tcm-HYNIC-ASON had a good stability and could hybridize to the sense oligonucleotide (SON).The contents of MDM2 mRNA in 0,100,500 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups were 0.458±0.035,0.250±0.026,0.174±0.032,0.463±0.033,respectively,and there were significant differences between each 2 groups except between 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups (F=33.69,q =24.32-91.45,all P＜0.01).The average density of MDM2 protein in the 4 groups were 90.712±3.042,71.218±2.915,32.775±3.062,88.121±2.710,respectively (F=235.93,q=6.43-19.14,all P＜0.01; except 0 nmol/L99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).The contents of p53 mRNA in the 4 groups were 0.185±0.046,0.203±0.040,0.213±0.027,0.163±0.049,respectively(F =2.18,P＞ 0.05).The average density of p53 protein was 33.865 ± 2.213,70.445±2.180,99.025±3.012,38.351±3.271,respectively (F=53.98,q =3.32-6.74,all P＜0.01 ; except 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).Conclusions The MDM2 antisense probe can accumulate in the prostate cancer cells,and specially hybridize to the MDM2 mRNA and inhibit target gene expression.This novel molecular probe has a promising potential for the diagnosis of prostate cancer at gene level.

Objective To develop multiplex polymerase chain reaction (PCR) combined with reversedotblothy bridization (RDB) method for detection of DNA virus in respiratory samples,and provide a surveillance and rapid diagnosis tool of acute viral respiratory infection.Methods We designed multiple PCR primers and the probes referenced to virus nucleic acid sequences in the National Center for Biotechnology Information (NCBI) database,and fixed specific oligonucleotide probes on the nylon membrane.After multiple PCR amplification of virus DNA of human bocavirus (hBOV),karolinska Institutet (KI),adenovirus (AdV),Washington University polyomaviros (WUPyV),and human parvovirus B19 (HPVBI9),the denaturalized amplification products were hybridized with various specific probes,followed by visualization and analysis of the results.The sensitivity and specificity were tested.At the same time,108 cases of clinical specimens of multiple PCR products were analyzed by reverse spot hybridization detection,and compared to the results of culture method.Results The specific probes of multiple PCR-RDB only hybridized with corresponding amplification products without cross-hybridization reaction with other pathogen.The sensitivity of RDB hybridization was 1 colony-forming units (CFU).The positive rate of 34.26％ (37 cases out of 108 cases) with PCR-RDB method was significandy higher than that 27.78％ (30 cases out of 108 cases) with common test method.Conclusions The multiplex PCR combined with RDB might become a rapid and simple method to detect the DNA virus in respiratory samples,which might be a promising tool for clinical application.

Objective To explore the effects of down regulation of osteopontin(OPN)on the bio- logical behavior of MKN28 and SGC7901 cell lines.Methods OPN siRNA was designed according to the relevant literature and was transfected into the two cell lines.Fluorescent labeling was used to test the transfected efficiency.The down-regulation of OPN protein was measured by Western blot.Real- time PCR was used to test the ratio and time difference of down-regulation of OPN mRNA after siRNA transfection.The biological changes before and after OPN siRNA transfected into these two cell lines were tested by flow cytometry(to test cell cycle and apoptosis)and MTT method(to test the prolifera- tion for the consecutive seven days)and the difference between OPN siRNA transfected or non-transfect- ed cells was compared using mixed model.The capability of moving and invasion of cancer cells were tested by Transwell method and analyzed by t-test.Results The transfected efficiency of OPN siRNA were more than 90% in the two cell lines.OPN mRNA down-regulated to 47% at the 72th hour in SGC7901,while 40% at the 48th hour in MKN28.The expression of OPN protein was both down- regulated after siRNA transfection in the two cell lines.The proliferation decreased after transfected with OPN siRNA both in MKN28 andSGC7901(P

OBJECTIVETo study the methodological techniques in measuring the severity of temporomandibulr disorders (TMD) and in evaluating the effectiveness of therapies in clinic.

METHODSBoth Fricton's Craniomandibular Index (CMI) and Helkimo's Clinical Dysfunction Index were calculated from 60 TMD patients. Inter-rater reliability was tested to assess the consistency in use between different examiners. Fricton's CMI was used to assess the clinical improvement after accepting a treatment in 21 TMD patients diagnosed as acute disk displacement without reduction.

RESULTSCorrelation Coefficient for inter-rater reliability in two groups was 0.879 and 0.939 respectively for Fricton's CMI and 0.744 and 0.838 for Helkimo Clinical Dysfunction Index. Fricton's TMJ dysfunction index was decreased from 0.337 to 0.021 (P < 0.001) and Fricton's CMI was decreased from 0.185 to 0.011 (P < 0.001) after the treatment in 21 TMD patients with disk displacement without reduction.

CONCLUSIONSTo avoid using subjective and descriptive report in assessment of the severity of TMD and the effectiveness of therapies, Fricton's CMI is recommended as an objective criteria which is simple in clinical use, and ease in scoring.

Adolescent ; Adult ; Child ; Female ; Humans ; Male ; Middle Aged ; Severity of Illness Index ; Temporomandibular Joint ; pathology ; Temporomandibular Joint Disorders ; pathology ; Temporomandibular Joint Dysfunction Syndrome ; pathology

OBJECTIVETo compare villus cell culture and karyotype analysis with single nucleotide polymorphism (SNP) microarray technology for the detection of chorionic villus chromosome in patients with retention of abortion.

METHODSForty cases were analyzed with the two methods.

RESULTSChorionic villus culturing was successful in 29 cases, among which 10 were found to have an abnormal karyotypes. For the SNP microarray analysis, all 40 cases were successful, among which 16 were shown to have an abnormal molecular karyotype.

CONCLUSIONSNP microarray technology is highly accurate and specific, which is particularly suitable for the detection of chromosomal deletions or duplications, uniparental disomy, low-percentage mosaicism and other chromosomal abnormalities. It has provided an effective supplement to the conventional chorionic villus culture and karyotype analysis.

Abortion, Missed ; genetics ; Adult ; Chorionic Villi ; chemistry ; Chromosome Aberrations ; Female ; Humans ; Karyotyping ; Male ; Oligonucleotide Array Sequence Analysis ; methods ; Polymorphism, Single Nucleotide ; Pregnancy ; Pregnancy Trimester, First ; genetics

Objective:To explore the level of tibial growth plate chondrocyte mitophagy in young rats with chronic renal failure (CRF) and its effect on chondrocyte apoptosis.Methods:Male 4-week-old Sprague-Dawley rats were randomly divided into two groups according to random number table method: normal control group ( n=20, intragastric administration with distilled water) and CRF group ( n=20, given adenine suspension 150 mg·kg -1·d -1). All the young rats were sacrificed after continuous gavage for 6 weeks. The length of tibia was measured on X ray film, the width of tibia growth plate was measured and compared on histological section, and the apoptosis rate of chondrocytes in growth plate was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The growth plate chondrocytes of two groups were isolated and cultured to the third generation in vitro, and the apoptosis rate of chondrocytes was detected by TUNEL assay. The co-localization of mitochondria and autophagy lysosomes in chondrocytes was observed by double fluorescence staining. Western blotting was used to detect the level of mitochondrial marker protein translocate of the outer mitochondrial membrane-20 (Tom-20) and autophagy marker light chain-3 protein (LC-3). The mitophagy of growth plate chondrocytes was observed by transmission electron microscope. Results:Compared with the normal control group, the tibia length of CRF group was shorter [(27.32±5.81) mm vs (35.43±3.61) mm, t=5.226, P<0.001], and the relative width of growth plate in histological section was narrower (0.56±0.19 vs 1.00±0.21, t=6.744, P<0.001). The apoptosis rate of chondrocytes in growth plate in CRF group was higher than that in the normal control group (17.2%±4.8% vs 5.1%±3.4%, t=6.505, P<0.001). The apoptosis rate of chondrocytes cultured in vitro in CRF group was higher than that in the normal control group (11.8%±6.2% vs 3.1%±1.2%, t=4.357, P<0.001). The result of double influorescence staining showed that there was co-localization between mitochondria and autophagy lysosomes in CRF group. Western blotting results showed that the levels of LC-3 protein ( t=8.944, P<0.001) and Tom-20 protein ( t=6.708, P<0.001) in CRF group were lower than those in the normal control group. Conclusion:The level of tibial growth plate chondrocyte mitophagy in young rats with CRF increases, which will lead to a decrease in the number of mitochondria, an increase in the apoptosis and a decrease in the number of chondrocytes, and eventually lead to dysplasia of tibia.

Objective To investigate the role of proteasome inhibitor bortezomib (BTZ) in inflammatory response in abdominal aortic aneurysm (AAA) formation induced by angiotensin Ⅱ (Ang Ⅱ). Methods Ang Ⅱ-induced ApoEmice AAA models were established. Forty male ApoEmice (8-10-week-old) were randomly and equally divided into four groups:Sham group,BTZ group,Ang Ⅱ group,and Ang Ⅱ+BTZ group.HE staining,immunohistochemical staining,and flow cytometry were used to analyze the inflammatory response. Real-time quantitative polymerase chain reaction (qPCR) was used to analyze the mRNA expression of intercellular cell adhesion molecule-1 (ICAM-1). Western blotting was used to analyze the activation of nuclear factor κB signaling (NF-κB). Results The mean maximum suprarenal aortic diameter (Dmax) of Sham group,BTZ group,Ang Ⅱ group,and Ang Ⅱ+BTZ group were (1.00±0.01),(0.99±0.01),(1.50±0.13),and (1.20±0.04)mm,respectively (F=8.959,P=0.000). The Dmax of Ang Ⅱ group was significantly larger than those of Sham group (P=0.000) and Ang Ⅱ+BTZ group (P=0.015). The incidence of AAA in Ang Ⅱ group,Ang Ⅱ+BTZ group,and Sham group were 60%,17%,and 0,respectively. HE staining revealed that the abdominal aortic wall thickening was more severe in Ang Ⅱ group than in Sham group and Ang Ⅱ+BTZ group,similar with the infiltration of inflammatory cells. Immunohistochemical staining demonstrated that the CD3T lymphocyte count was significantly higher in Ang Ⅱ group than in Sham group (107.9±15.9 vs. 0,P=0.000) and Ang Ⅱ+BTZ group (107.9±15.9 vs. 0.8±0.5,P=0.000). Flow cytometry also demonstrated that the proportion of the CD3T lymphocytes of the Ang Ⅱ group [(13.50±0.69)%] was significantly higher than that in the Ang Ⅱ+BTZ group [(10.40±0.78)%] at week 1 (t=3.009,P=0.040),and the proportion of the CD3T lymphocytes of the Ang Ⅱ group [(22.70±0.93)%] was significantly higher than that in the Ang Ⅱ+BTZ group [(15.10±0.97)%] at week 4 (t=5.654,P=0.005). The qPCR analysis showed that the mRNA expression of ICAM-1 was significantly up-regulated in Ang Ⅱ group than in Sham group (1.93±0.54 vs. 1.00±0.15,P=0.011) and Ang Ⅱ+BTZ group (1.93±0.54 vs. 0.83±0.08,P=0.009). Western blot analysis showed a lower phosphorylation level of inhibitor of NF-κB in the Ang Ⅱ group compared with the Sham group or Ang Ⅱ+BTZ group,accompanied with an increased phosphorylation level of p65. Conclusion Proteasome inhibitor BTZ can attenuate AAA formation partially by regulating T lymphocytes infiltration through regulating the mRNA expression of ICAM-1 regulated by the activation of NF-κB signaling pathway.
Angiotensin II ; adverse effects ; Animals ; Aortic Aneurysm, Abdominal ; chemically induced ; drug therapy ; Apolipoproteins E ; genetics ; Bortezomib ; pharmacology ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NF-kappa B ; metabolism ; Phosphorylation ; Proteasome Inhibitors ; pharmacology ; Random Allocation ; Signal Transduction ; T-Lymphocytes ; cytology

Advanced gastric cancer (AGC) has a high recurrence rate (especially peritoneal relapse) and a poor prognosis. Systematic chemotherapy or targeted therapy have not been able to significantly reduce the major cause of an unfavorable prognosis, namely the high peritoneal AGC recurrence rate post-surgery. Further studies concerning the application of hyperthermic intraperitoneal chemotherapy (HIPEC) post curative surgery for AGC patients, namely the prophylactic HIPEC (P-HIPEC), have involved a prophylactic approach to prevent peritoneal relapse following curative gastrectomy in high-risk patients. Theoretically, breaking the "plasma-peritoneal barrier" increases cytotoxic chemotherapy activity via a synergistic hyperthermic effect; therefore, HIPEC can eradicate free cancer cells and micro-metastasis within the peritoneal cavity intraoperatively or soon after curative gastrectomy to reduce peritoneal recurrence. Many clinical trials have shown that P-HIPEC can reduce peritoneal recurrence and improve prognosis of AGC patients. However, some studies applying HIPEC at an early stage have revealed a high rate of complications that limited generalizability. This procedure has been increasingly adopted, given the complication rate has now been reduced and safety has been proven. Recently, for assessing the important role of HIPEC, many high-quality prospective randomized controlled clinical trials have been conducted to further investigate the best guidance for P-HIPEC and to demonstrate its effectiveness and safety with a higher grade of evidence. With theory development, the technique, equipment, and management of HIPEC and the role of P-HIPEC for AGC continues to evolve. This study summarizes the progress of P-HIPEC for high-risk AGC patients.
Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Chemotherapy, Cancer, Regional Perfusion ; Combined Modality Therapy ; Humans ; Hyperthermia, Induced ; Neoplasm Recurrence, Local ; Peritoneal Neoplasms ; drug therapy ; Randomized Controlled Trials as Topic ; Stomach Neoplasms ; drug therapy

Objective To investigated the changes of angiopoietin-like protein 2(Angptl2) in patients with arteriosclerotic occlusion (ASO). Methods A total of 140 subjects including 75 ASO patients (ASO group) and 65 healthy subjects (control group) were enrolled in this study. Angptl2 and adiponectin were evaluated by using enzyme-linked immunosorbent assay. Biochemical data and high sensitive C reactive protein were measured and recorded as well. Results Compared to the control group,the ASO group presented with significantly higher level of plasma Angptl2 [(13.55±9.17) μg/L vs. (9.04±4.79) μg/L,P=0.010]. Plasma Angptl2 level of critical limb ischemia subjects was significantly higher than that of intermittent claudication subjects [(17.01±10.20)μg/L vs. (10.53±6.97) μg/L,P=0.003]. The best diagnostic cutoff value of Angptl2 was 13.67 μg/L,with a sensitivity of 60.34% and a specificity of 81.25%. In addition,type 2 diabetes mellitus patients with ASO exhibited significantly higher serum Angptl2 levels [(18.67±9.84)μg/L] than those without ASO [(13.01±3.47) μg/L] (P=0.021). In ASO group,serum Angptl2 levels were negatively correlated with ankle brachial index (r=-0.244,P=0.035). Conclusion The plasma level of Angptl2 increases in ASO patients. Its level is remarkably increased when the disease progressions to critical limb ischemia. Angptl2 can be a potential biological marker of disease progression.