Objective:To apply chromogenic in situ hybridization(CISH)for detection of HER2 gene amplification in breast cancer tissues and to discuss some modifications of the CISH method.Methods:HER2 gene amplification was detected by CISH in 60 breast cancer specimens with an immunohistochemical score over 2+.The correlation between the results of IHC and CISH was analyzed.Our experience in CISH manipulation was summarized and optimization to CISH was discussed.Results:CISH identified gene amplification in 91%(40/44)specimens with an IHC score of 3+ and in 50%(8/16)specimens with an IHC score of 2+.The total concordance rate between IHC and CISH was 80%(48/60,P

BACKGROUND:Construction of tissue engineering adult cardiac myocytes has been a new research hotspot in cardiovascular fields.
OBJECTIVE:To explore a simple, fast method for the separation of adult rat cardiomyocytes, and preliminarily explore the construction methods of tissue engineering adult cardiac myocytes.
METHODS:Segmented enzymatic digestion method was used to isolate cardiac myocytes from adult rats. Subsequently, cardiac myocytes were transfected with adenovirus and liposome-mediated red fluorescent protein gene. Construction efficiency of tissue engineering cells was qualified using inverted fluorescence microscopy and flow cytometry. Final y, cardiac myocytes were transfected with adenovirus-mediated hypoxia-inducible factor-1a, and the expression of hypoxia-inducible factor-1a was examined by western blot analysis.
RESULTS AND CONCLUSION:A lot of cardiac myocytes were col ected using the segmented enzyme digestion method. Flow cytometric analysis showed that the survival of adult cardiomyocytes was (87.03±0.70)%. Compared with liposomal transfection (transfection efficiency was 0), adenovirus infection efficiency was (70.31± 1.39)%, and the cells expressed red fluorescence under fluorescence microscope. After 4 days of adenovirus infection, transfected cells expressed hypoxia-inducible factor-1a protein. These results showed that segmented enzyme digestion is a fast way to isolate adult cardiac myocytes, and recombinant adenovirus vector is a good vector to transfect cardiac myocytes.

Propionic acid, a major inhibitor to yeast cells, was accumulated during continuous ethanol fermentation from corn meal hydrolysate by the flocculating yeast under stillage backset conditions. Based on its inhibition mechanism in yeast cells, strategies were developed for alleviating this effect. Firstly, high temperature processes such as medium sterilization generated more propionic acid, which should be avoided. Propionic acid was reduced significantly during ethanol fermentation without medium sterilization, and concentrations of biomass and ethanol increased by 59.3% and 7.4%, respectively. Secondly, the running time of stillage backset should be controlled so that propionic acid accumulated would be lower than its half inhibition concentration IC50 (40 mmol/L). Finally, because low pH augmented propionic acid inhibition in yeast cells, a higher pH of 5.5 was validated to be suitable for ethanol fermentation under the stillage backset condition.
Biomass ; Ethanol ; metabolism ; Fermentation ; Flocculation ; Propionates ; chemistry ; Yeasts ; metabolism

Objective To observe the influence of insulin resistance(IR) and isosorbide mononitrate(ISMN) on myocardial cellular apoptosis in spontaneously hypertensive rats (SHR) .Methods Forty male 14‐week old Wistar(W) rats and SHR(S) each were respectively or jointly fed with normal diet (ND) ,high fat and high glucose(HFHG) diet ,normal saline(NS)and ISMN by ga‐vage .Then they were randomly divided into the normal and NS group (normal W and normal S ) ,HFHG and NS group(HFHG W and HFHG S) ,normal and ISMN group(ISMN W and ISMN S) ,HFHG and ISMN group(HI W and HI S) ,with 10 rats in each group .After 12‐week feeding ,carotid arterial blood was collected for detecting blood glucose concentration and insulin level and cal‐culating insulin resistance index (HOMA‐IR);4 myocardial tissue samples were taken for respectively observing the morphology under microscope ,and detecting the NO level ,myocardial Bcl‐2 ,Bax gene and their protein expression levels in myocardial tissue . Results Myocardial NO level ,Bax gene mRNA and related protein levels in the HFHG and ISMN intervention groups were higher than those in the normal group ,while the bcl‐2 gene mRNA and related protein expression were on the contrary ;myocardial tissue NO level ,Bax gene mRNA and related protein expression in the S groups were increased compared with the corresponding W groups ,while the bcl‐2 gene mRNA and related protein expression were on the contrary ;in the HFHG W group ,the myocardial tis‐sue NO level had significantly positive correlation with HOMA‐IR ,and in the ISMN W group ,HOMA‐IR was positively correlated with the NO level in the myocardial tissue .Conclusion Myocardial cellular apoptosis of SHR is increased compared with Wistar rats ;both IR and ISMN can aggravate the apoptosis of SHR myocardial cells ,moreover IR has a mutual induction and reciprocal causation with ISMN .

Objective:To construct the recombinant eukaryotic expression vector plasmids with mutant C-kit cDNA and to study the effect of the mutant C-kit gene on cell proliferation and cell cycle in gastrointestinal stromal tumor (GIST). Methods: Wild-type C-kit cDNA was cloned from human embryonic brain tissue by RT-PCR technique.Site-directed mutagenesis of the wild type C-kit cDNA was performed according to the C-kit mutations we cloned. Recombinant plasmids were stably transfected into human embryonic kidney cell line and the cells expressing mutant C-kit were selected by special cell culture medium containing G418.Expressions of C-kit protein of the transfectants were detected by Western blot.Cell proliferation and cell cycle of the transfectants were detected by MTT clolorimetic assay and flow cytometry,respectively.Whether HEK cell with mutated C-kit cDNA could grow autonomously in nude mice or not was also detected. pcDNA3 vector transfected and recombinant plasmids with wild-type C-kit cDNA transfected HEK cell were used as the control groups. Results: The mutant C-kit cDNA was obtained by site-directed mutagenesis of the wild type C-kit cDNA. Compared with the 2 control groups ,the growth rate and proliferative activity of the HEK cells with mutant C-kit cDNA were increased significantly.The analysis of cell cycle showed that more HEK cells with mutanted C-kit cDNA remained in proliferation phase (S+G 2-M)than the groups without mutated C-kit cDNA.HEK cells with the mutated C-kit also grew autonomously in nude mice.Conclusion: Mutation of C-kit gene can increase proliferation of human cells,causing malignant transformation of human normal cells,which may play an important role in the malignant transformation of GIST.

Objective To design and implement a portable electrocardiogram (ECG) monitor for multiple users. Methods Multilevel menu on liquid crystal displayer(LCD) for switching among users based on digital signal processor(DSP) was developed. Record header in ECG records for different users was appended, and communication protocol between monitor and hospital center was made. Results This monitor could record three users’s ECG signal and transmit the ECG records to remote hospital center via digital communication method. The hospital center could receive and file the ECG records of different users. Conclusion This monitor can be used for three users and is more efficient in the application of ECG monitoring.

Objective:To analyze the consistency of HER-2 expression among endoscopic biopsy and radical operation specimens of gastric adenocarcinoma and to investigate the clinical application value of HER-2 detection in trastuzumab treated patients. Methods:From March 2013 to February 2014, 167 patients from Shanghai Changhai Hospital were diagnosed with gastric adenocarcinoma using endoscopic biopsy specimens. The corresponding surgical specimens were collected for pathological analysis. The relevant clinical and pathological data were collected. HER-2 protein expression of endoscopic biopsy specimens was detected by immunohistochemistry (IHC). HER-2 protein expression and gene amplification status of the corresponding tumor resection specimens were detected by IHC and fluorescence in situ hybridization (FISH). Results were analyzed for clinicopathological characteristics. Results:Among the 167 cas-es, 18 cases (10.8%) were HER-2 positive, including 10 cases showing IHC3+and 8 cases showing IHC2+with FISH positive. The consis-tency rate result among endoscopic biopsy and surgical operation specimens was 82%. Excluding the cases showing IHC2+, the true positive rate and the true negative rate were 73.3%and 97.0%, respectively. Conclusion:HER-2 detection of endoscopic biopsy speci-men by IHC shows great predictive value. The main reason for the difference of surgery and biopsy specimens is the heterogeneity of tumor expression. Increasing the number of specimens and combined testing with FISH are important methods to reduce misjudge-ment.

Objective: To explore the sensitivity of Kit or PDGFRA mutants related to gastrointestinal stromal tumor (GIST) to Gleevec.Methods: The recombinant plasmids of KIT Del559-560, KIT Ins IPYD579, PDGFRA D842V and PDG-FRA L839P gene mutants were transiently transformed into the CHO cells by liposome methods.Western blot was used to detect the expression of the related protein and their phosphorylated forms after the cells were incubated with Gleevec for 90 min.At 72 hours after incubation with Gleevec, MTT was used to detect cell proliferation.Results: Western blot results showed that Gleevec at 0.1 μM can notably reduce phosphorylation of KIT Del559-560.Gleevec at 1μM completely blocked phosphorylation of KIT Ins IPYD579 and PDGFRA L839P, but did not affect PDGFRA D842V phosphorylation.MTT analy-sis indicated that growth of CHOPDGFRA L839P was inhibited by Gleevec at 1μM, however, CHOPDGFRA D842V was re-sistant to Gleevec at 5 μM.Conclusion: Gleevec can decrease the expression of phosphorylated protein CHOPDGFRA L839P and CHOKIT Ins IPYD579, and can remarkably inhibit the proliferation of cells containing PDGFRA L839P mutant.

Objective To study the expression of human giant larvae-1 (Hugl-1) in ovarian carcinoma and its clinical significance.Methods Hugl-1 mRNA expression in 31 ovarian cancer and the corresponding adjacent tissues was examined by real time fluorescence quantitative RT-PCR and in situ hybridization.Moreover,analysis was done while taking into consideration of the clinicopathologic parameters of ovarian cancer.Results The expression of Hugl-1 mRNA in ovarian cancer tissue was significantly higher than that in the corresponding adjacent tissues ([0.051 ? 0.029]vs [0.026 ? 0.043],P

OBJECTIVETo investigate the clinicopathological and prognostic significance of the c-kit protein in gastrointestinal stromal tumor (GIST).

METHODSParaffin embedded materials from 53 benign GISTs, 13 potentially malignant and 55 malignant cases were analysed for c-kit expression by immunohistochemical method, while using leiomyomas and schwannomas as controls. Positive signals were shown in cytoplasma and cell membrane.

RESULTSOf 122 GISTs, 118 (97%) were positive for c-kit. Localization of positive signals was accurate. The rate of c-kit protein in benign, potentially malignant and malignant cases was 98% (53/54), 93% (12/13), 96% (53/55) respectively. Compared with benign GIST, the positivity of c-kit in metastasis or recurrent cases decreased, but c-kit protein expression rate was not significantly different between the three patterns of GIST (chi(2) = 1.167, P > 0.05). Leiomyomas and schwannomas were typically c-kit negative.

CONCLUSIONAs a sensitive and specific marker of GIST, c-kit seems to be a useful antibody in the diagnosis and differential diagnosis of GIST, but it may not be used as a prognostic index.

Biomarkers, Tumor ; biosynthesis ; genetics ; Diagnosis, Differential ; Gastrointestinal Neoplasms ; diagnosis ; metabolism ; Humans ; Immunohistochemistry ; Mutation ; Prognosis ; Proto-Oncogene Proteins c-kit ; biosynthesis ; genetics