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Objective: To express CTX-M-38 type extended-spectrum-lactamase, and detect its distribution and antibiotic susceptibilities. Methods: Total of 46 strains producing ESBL E.coli was collected from the first affiliated hospital of Zheng zhou University. The CTX-M-38 ESBL gene was selected by PCR using gene recombination technique to construct pET28a-CTX-M-38. The expression of CTX-M-38 in BL21 E.coli and its antibiotic susceptibilities were carried out by liquid dilution test. Testing the enzyme activities of culture supernatant and bacteria sonicate to reflect its distribution. Results: The size of amplified gene product was about 900 bp. The DNA sequence was matched with the information of gene bank. The enzyme activities from bacteria sonicate were stronger than the culture supernatant .The transformant was resistance to penicillins, the first, second and third generations of cephalosporins. It was sensitive to imipenem. The transformant was also sensitive to ceftazidime and aztreonam in vitro, and resistance to antibiotics including beta-lactamase inhibitors except piperacillin/tazobactam. The transformant was also resistance to gentamicin,minocycline, ciprofloxacin and levofloxacin. Conclusion: The CTX-M-38 type ESBL is successfully expressed at designed experimental condition in this study. The product mainly lies inside the bacteria. The transformant shows wide resistance to antibiotics.
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OBJECTIVE To study the genotype distribution of CTX-M type ESBLs in ESBLs-producing Escherichia coli. METHODS All 138 strains of E. coli were collected from the First and Third Affiliated Hospitals,Zhengzhou University from 2006 to 2007. The CTX-M type ESBLs genes were amplified by PCR,the products were sequenced after purification. RESULTS Eighty-two of 138 ESBLs-producing E. coli isolates were classified with CTX-M type-ESBLs genes,and there were 53,27 and 2 strains contained only one type,two types and three types of CTX-M ESBLs,respectively. Among CTX-M type-ESBLs genes,they were identified with CTX-M-1(43 strains),CTX-M-14(56 strains),CTX-M-25(7 strains) and CTX-M-38 (5 strains). CONCLUSIONS The most popular type of CTX-M ESBLs in E. coli is CTX-M14 in Zhengzhou,and two or three kinds of genotypes can exist in 1 strain.
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Objective To study the expression of B7-H1 in gastric mucosa of patients with gastric carcinoma,and to identify its relationship with neoplasm metastasis and prognosis.Methods Expression of B7-H1 in gastric carcinoma cell line SGC-7901 and in freshly-resected gastric mucosa including gastric carcinoma,adjacent tumor tissue and distal normal gastric mucosa were examined by flow cytometric analysis,immunochemical staining,immunofluorescence staining and Western blot.The correlated data was analyzed statistically.And so the correlation among expression level of B7-H1 and the patients,clinicopathological parameters was established.Results B7-H1 expression was detected in SGC-7901 cell line.B7-H1 was found in cell membrane and little cytoplasm.The positive rate of B7-H1 expression in gastric carcinoma was(13/21)62%,and it was(7/21)33% in adjacent tumor tissue,Whereas B7-H1 was absent in distal normal gastric mucosa.Statistical analysis demonstrated a positive correlations of B7-H1 expression in gastric carcinoma with the depth of carcinoma infiltration,lymph node metastasis and pTNM stage(P
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Objective To study the inhibition effects of various gastrin-shNAs on gastrin expression in gastric cancer cell line BGC-823. Methods Four nucleotide sequences of shRNA were designed corresponding to various sites of gastrin gene.Four shRNAs were synthesized by in vitro transcription and transfected into gastric cancer cell line BGC-823 at the final concentration of 10nmol/L,20nmol/L,40nmol/L and 80nmol/L respectively.In situ hybridization and immunohistochemistry techniques were applied to investigate the inhibition of gastrin expression and screen the most effective shRNA.The inhibitory effect on gastrin mRNA of screened shRNA was further identified by RT-PCR.MTT assay was used to determine the inhibitory effect of 4 shRNAs at various final concentrations on the growth of BGC-823 cells. Results The gastrin mRNA and protein exression were suppressed distinctly 24,48,and 72hours after transfection,and exhibited time-and concentration-dependent tendency.The highest suppression efficiency on both mRNA(54.27?0.042)% and protein(41.69?0.038)% level occurred 72 hours later in the cells transfected with shRNAs.The RT-PCR result showed that the inhibitory ratio of shRNA3 on gastrin mRNA of BGC-823 was 48.1%.MTT displayed a proliferative inhibition of the BGC-823 cells after transfection of shRNAs with a concentration-denpendent tendency except the shRNA4 treated cells.Conclusion Four gastrin-shRNAs showed a significant inhibition effect on gastrin expression of gastric cancer cell BGC-823 on mRNA and protein level.shRNAs might be the most effective gastrin-shRNA.Inhibited gastrin expression by shRNAs resulted in a significant decrease of proliferative ability of BGC-823 cells.
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Objective To investigate the reversal effect on mdr1 gene mediated multidrug resistance in gastric cancer SGC7901/VCR cells by small interfering RNA(siRNA).Methods Two siRNAs(mdr1si2631 and mdr1si3071) specifically targeting mdr1 gene were designed and synthesized by transcription in vitro.The siRNA duplexes were used to transfect into the gastric cancer SGC7901/VCR cells.The expression levels of mdr1 mRNA and P-gp were detected by RT-PCR and immunohistochemistry respectively.The accumulation of intracellular adriamycin(ADR)was examined by flow cytometry and the cell sensitivity to ADR was demonstrated by MTT.Results The expression level of mdr1 mRNA treated by siRNAs for 48?hours was decreased in the SGC7901/VCR cells.The mdr1 RT-PCR product in the transfected mdr1si2631 SGC7901/VCR cells could hardly been found,similar to its parental SGC7901 cells,the ratio of mdr1 and ?-actin in the control SGC7901/VCR group was 1.05?0.10,the transfected mdr1si3071 group was 0.16?0.03(P0.05).The RT-PCR results showed that the mdr1 mRNA expression level in the mdr1 si2631 group decline more obviously than that in the mdr1si307l group,near by the level in its parental SGC7901 cells.The P-gp immunoreactivity(IR)in brownish-colored granules was located on the cell membrane.The P-gp IR became weaker in the SGC7901/VCR cells treated by siRNAs for 48 hours and the P-gp expression level in both transfected siRNA groups was decreased.The values of adriamycin-specific fluorescence intensity and the positive rates of intracellular ADR in both transfected siRNA groups were increased.The relative reversal efficiency of the SGC7901/VCR cells to ADR detected by MTT was 79.59%in mdr1 si2631 group and 59.98%in mdr1si3071 group respectively.Conclusion siRNA could reverse mdr1 gene mediated multidrug resistance in gastric cancer SGC790l/VCR cells.