Aim To develop a simple and rapid method to monitor insulin receptor kinase activity and provide a novel cell-based model for screening anti-diabetes drugs.Methods CHO cells were co-transfected by plasmids which respectively contained insulin receptor gene,STAT5b gene and luciferase gene driven by STAT5 response elements.The expression of exogenous gene in transfected cells was examined by RT-PCR.The transfected cells were treated by insulin,and then the concentration and time-dependent response of luciferase expression to insulin induction was examined.Moreover,the specificity was identified by AG1024 treatment and PTP1B gene transfection.Results Expressions of insulin receptor and STAT5b were detected in the transfected CHO cells.The expression of luciferase in transfected cells was induced by insulin in concentration and time-dependent way.The maximal induction fold was 6.25.Moreover,the inducible expression of luciferase by insulin could be specifically blocked by tyrphostin AG1024,an inhibitor of insulin receptor kinase,or co-transfected PTP1B gene.Conclusions The insulin receptor kinase activity can be detected by expression of reporter gene with high sensitivity and specificity in this cell model,and with potential value in high throughput screening for insulin receptor activators and sensitizers.

Objective To approach the neurobiochemical mechanism of chronic central pain (CCP) after spinal cord injury (SCI). Methods 28 SD rats were divided into four groups, the normal group (group A), the pseudosurgery group (group B), and groups with CCP (group C) and without CCP (group D) after L1 spinal cord section injured with WADE method. T13 and L2 segments of rats' spinal cord were took and concentration changes of substance P (SP) in the spinal dorsal horn between two sections were examined by immunofluorescence histochemistry staining combined with confocal laser scanning microscope. Results Concentration of SP in the group D was decreased significantly compared with groups C,A and B (P<0.05-0.01), that of the group C was less than that of group A and B (P<0.05). Conclusion The rat model established by WADE method is proper to study CCP after SCI. SP in dorsal horn of spinal cord may inhibit the CCP after SCI in some degrees.

Objective To investigate the application of time‐resolved fluorescence immunoassay (TRFIA) in the detection of specific antibody of syphilis .Methods Specific antibody of syphilis was detected in serum samples of 240 cases of syphilis and 150 healthy subjects by TRFIA ,Treponemal pallidum particle agglutination(TPPA) and Treponemal pallidum enzyme linked immu‐nosorbent assay(TP‐ELISA) .The sensitivity ,specificity and positivity of these three methods were compared .Results The sensi‐tivity of TRFIA ,TP‐ELISA ,TPPA were 100 .00% ,98 .75% and 97 .92% ,without significantly differences(P>0 .05) ,and the spe‐cificity were 99 .33% ,98 .67% and 100 .00% .The false positive rate of TRFIA was 0 .67% ,and the false negative rate was 0 .00% . The false positive rate of TP‐ELISA was 1 .33% ,and the false negative rate was 1 .25% .False positive rate and false negative rate of TRFIA were lower than TP‐ELISA(P<0 .05) .Conclusion TRFIA could be with high sensitivity and specificity in syphilis spe‐cific antibody test ,and could be used for routine screening of syphilis specific antibody .

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Base Sequence ; Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Genetic Vectors/genetics ; Hep G2 Cells ; K562 Cells ; Molecular Sequence Data ; Receptors, Transferrin/*immunology ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Single-Chain Antibodies/*biosynthesis ; Single-Chain Antibodies/genetics

OBJECTIVETo observe the effects of electroacupuncture (EA) at "Sanyinjiao" (SP 6) on urodynamics indices in rats with overactive bladder (OAB) after cystostomy, and to explore its regulation mechanism on bladder function.

METHODSForty-eight Sprague-Dawley female rats which received cystostomy were randomly divided into a blank group (group A), a blank Sanyinjiao group (group B), a blank non-acupoint group (group C), a model group (group D), a model Sanyinjiao group (group E) and a model non-acupoint group (group F), 8 rats in each one. The model of OAB was established with 1% acetic acid solution perfused into the bladder in the group D, group E and group F. No treatment was given to the group A and group D. Acupuncture was applied at bilateral "Sanyinjiao" (SP 6) in the group B and group E, followed by EA after the arrival of qi. Acupuncture was applied at bilateral non-acupoint in the group C and group F, followed by EA with continuous wave, 2 Hz of frequency for 30 min. The treatment was given for continuous 5 urination cycles. The BL-420 E+ biological function experiment system was used to measure and record the changes of indices of bladder pressure and urodynamics.

RESULTSCompared with the group A, the bladder capacity and urine output in the group B were significantly increased (both P<0.05), and the urination rate was increased in the group C (P<0.05); the differences of each index between group C and group B were not statistically significant (all P>0.05). Compared with the group D, the capacity pressure, bladder capacity, detrusor pressure, urinary output and urination rate in the group E were all increased (all P<0.05). Compared with the group F, the capacity pressure and detrusor pressure in the group E were increased (both P<0.05).

CONCLUSIONThe EA at "Sanyinjiao" (SP 6) could significantly improve urine function in rats with OAB after cystostomy, but its regulation effect on urination is not obvious in rats with non-OAB.

Acupuncture Points ; Animals ; Cystostomy ; Disease Models, Animal ; Electroacupuncture ; Female ; Humans ; Rats ; Rats, Sprague-Dawley ; Urinary Bladder ; physiopathology ; surgery ; Urinary Bladder, Overactive ; physiopathology ; surgery ; therapy

Objective To measure total hemoglobin concentration (THC) of breast lesion using US-guided diffused optical tomography(DOT) and to investigate the THC optimal threshold value in different size breast lesions.Methods DOT was performed on 500 breast lesions and surgical pathology was as the gold standard.The optimal diagnostic threshold and the efficacy were figured out.Results There were 265 benign and 235 malignant lesions.In malignant lesions,THC of ≥2 cm lesion group was higher than that of ＜2 cm lesion group(P =0.000).In benign lesions,there was no statistical difference between ≥2 cm group and ＜2 cm group (P =0.13).As for ＜2 cm breast lesions,when a THC threshold value of 146.9 μmol/L and 102.2 μmol/L were used,the sensitivity,specificity,positive predictive value,negative predictive value and accuracy were 74.2％,70.0％,71.7％,62.9％,79.9％ and 86.7％,44.4％,61.6％,51.6％,83％,respectively.As for ≥2 cm breast lesions,when a THC threshold value of 210.4 μmol/L were used,the sensitivity,specificity,positive predictive value,negative predictive value and accuracy were 74.0％,86.7％,79.1％,89.2％,69.2％.Conclusions THC of breast cancer increased with the increasing size of lesions.The different diagnostic threshold value should be used according to different size lesions so as to enhance sensitivity,specificity and accuracy.

Objective To investigate the effects and safety of sequential treatments with methotrexate and mifepristone for ectopic pregnancy with fetal cardiac activity.Methods 4 cases of ectopic pregnancy with fetal car- diac activity in our hospital were given by sequential therapy with methotrexate and mifepristone.Serum?-HCG,liv- er function and renal function,blood routine and gastrointestinal response were observed.Results 4 cases of ectopic pregnancy with fetal cardiac activity with 1～4 periods of sequential treatments were cured.Except light gastroin- testinal response,and one had slight rise of serum ALT level and AST level,no one had rnyelosuppression and heavy hepatic injury.Conclusion The sequential therapy with methotrexate and mifepristone is an effective and safe method for the treatment to ectopic pregnancy with fetal cardiac activity.

Objective To study the relationship between pain symptoms and the clinico-pathological features of pelvic endometriosis (EM).Methods One hundred thirty patients with laparoseopic diagnosis of EM were studied retrospectively and the relationship between pain symptoms including dysmenorrhea, chronic pelvic pain(CPP),dyspareunia and dysehezia and the anatomical features of pelvic endometriosis were evaluated.Results One hundred (76.9%)patients with pain symptoms and 30 (23.1%)without were included in this study.The number of patients with mild,moderate and severe dysmenorrhoea was 27 (20.8%),41(31.5%),and 32 (24.6%),respectively.Patients with dyspareunia,CPP and dyschezia were 46(35.4%),45(34.6%) and 67(51.5%),respectively.Compared with patients without dysmenorrhea,the proportion of deep utero-sacral nodules (45.0% vs 13.3%,P=0.00),recto-vaginal nodules (16.0% vs 0,P=0.01),complete obliteration of eul-de sac (41.0% vs 10.0%,P=0.00),and lesions of DIE (51.0% vs 16.7%,P=0.00) was significantly increased in patients with dysmenorrhea. The severity of dysmenorrhea was positively correlated with nodules in uterosacral ligaments (P=0.005,r= 0.302),and invasive depth of uterosacral ligaments (P=0.016,OR=5.085).Among patients with endometrioma,significantly more moderate to severe adhesions were found in patients with dysmenorrhea , compared with those patients without dysmenorrhea(29.1% vs 8.3%,P=0.029).Patients with CPP had more nodules in the utero-sacral ligaments(51.1% vs 30.6%,P=0.018)and DIE lesions(57.8% vs 35.3%,P=0.011),compared with those without.More nodules in the utero-saeral ligaments(46,3% vs 28.6%,P=0.028),recto-vaginal nodules(19.4% vs 4.8%,P=0.01),complete obliteration of cul-de sac(44.8% vs 22.2%,P=0.005)and DIE lesions(53.7% vs 31.7%,P=0.01)were found in patients with dyschezia,compared with those without.Nodules in the recto-vaginal pouch were an independent risk factor of dyspareunia.Conclusion Pain symptoms including dysmenorrhea,dyspareunia, chronic pelvic pain,and dysehezia are remarkedly related to endometriotic nodules at the posterior part of the pelvis or those with deep invasions.

Aged ; Aged, 80 and over ; Humans ; Kidney Diseases ; diagnosis ; etiology ; Male ; Middle Aged ; Nephelometry and Turbidimetry ; Pneumoconiosis ; complications ; Pulmonary Disease, Chronic Obstructive ; complications ; Retinol-Binding Proteins ; urine

Ojective In order to understand the effects of continuous and stable expression of antisense genes on the growth and proliferation of the smooth muscle cells. Method Three combinant retrovial expression vectors, aM1, aM2 and aM3, which was respectively loaded with reverse fragment of c-myc exon 1, 2 and 3, were transferred into rat arterious smooth muscle cells and assayed 1 month after their stable expression. Result aM1 and aM2 could inhibit Myc expression (respectively reduced to 59.7%, 81.3%), and the expression of proliferat cellular nuclear antigen (PCNA) (respectively reduced to 52.4%, 51.9%). aM1 and aM2, especially the latter also inhibited cellular proliferative activity, aM2 slightly hindered cellular cycle and DNA synthesis too, while the effects of aM3 turned out to be contrary to that of aM1 and aM2. In addition, compared with transient expression, the effects of introduced genes were variable in target cells, and the changes brought about by the transfection process would reduce and disappear in a short time with the lowering of cellular compensation. Conclusions Blocking different regions of c-myc can achieve different effects and the blokade of the transcription activity regions usually produces enhanced growth inhibition effects. Moreover, target cells may have some 'correction mechanism ' against the introduced foreign genes.