Objective To develop an ideal cultural method to amplify embryonic hepatic stem cells and inhibit their differentiation in vitro. Methods Suspension of ED 14 Fischer (F) 344 rat em-bryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly. The cells in group 1 were seeded into type I collagen-coated plates by adherent culture while those in group 2 were seeded into soft agar medium by suspen-sion culture. After culture for 2 weeks, the morphology and ultrastructure of cells in both groups were observed and compared by inverted microscope and transmission electron microscope, respectivley.The expression of CD90. 1 and CD49F, the two specific stem cell surface markers, was tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase stai-ning was used to detect stem cell differentiation. Result Embryonic hepatic stem cells in group 2 were characterized by higher nucleus-cytoplasm ratio and less cell organelles, higher expression of CD90. 1 and CD49F, and stronger positive reaction for alkaline phosphatase staining compared with those in group 1. Moreover, the cells in group 1 showed significant differentiation features. Conclusion Em-bryonic hepatic stem cells cultured suspendedly in soft agar medium experience less differentiation than those adherently cultured in serum-added culture medium, and can proliferate and form clone ball with a specific stem cell feature.

OBJECTIVETo evaluate the effect of beta irradiation on intimal proliferation and apoptosis in vein grafts.

METHODSAutogenous vein graft model was established in 80 rats by transplanting the internal branch of the jugular vein to the carotid artery by end to end anastomosis. The veins were irradiated by (32)P solution before anastomosis. Two dose schedules were studied: control group (graft, nonirradiated) and radiation group (20 Gy). The grafted veins were harvested at 3, 1, 2 and 4 week respectively after the operation. intimal hyperplasia (IH), smooth muscle cell (SMC), proliferation, p53, bcl-2 and bax were observed pathologically and immunohistochemically. They were analyzed by a computerized system. The presence of apoptotic VSMC was demonstrated by TUNEL method.

RESULTThere was a significant decrease in the average intimal thickness at 7, 14 and 28 days (t = 15.694, P < 0.05) in the radiation group. Immunohistochemical analysis of PCNA indicated decreased positive cells in the radiation group compared with the controls at 1 and 2 weeks (t = 60.157, P < 0.01). Apoptosis of VSMC was higher in the radiation group than in the control group at 2 weeks (t = 56.176, P < 0.01). There was no significant difference in expression of P(53) between the two groups, and there was a significant increase in bax/bcl-2 in the radiation group at 2 weeks (t = 9.783, P < 0.05).

CONCLUSIONThese preliminary results demonstrated that low dose of beta irradiation in the vein graft inhibits SMC proliferation and induces the apoptosis of VSMC in rats.

Animals ; Apoptosis ; Beta Particles ; Brachytherapy ; Disease Models, Animal ; Graft Occlusion, Vascular ; metabolism ; pathology ; In Situ Nick-End Labeling ; Male ; Muscle, Smooth, Vascular ; pathology ; radiation effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Rats ; Tumor Suppressor Protein p53 ; metabolism ; Tunica Intima ; pathology ; radiation effects

Objective To study phylogenies, epidemiology and genetic environment of CTX-M type of ESBLs produced by Escherichia coli and Klebsiella pneumoniae isolated from nine hospitals in Guangzhou. Methods The phylogenies of CTX-M type of ESBLs were analyzed by PCR Genetic environment of CTX-M-15 encoding gene (bla_(CTX-M-15)) were investigated by conjugation test and plasmid analysis. The clonal relationship of strains producing CTX-M-15 was determined by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Results A total of 361 ESBLs-producing isolates of Escherichia coli and Klebsiella pneumoniae were collected. 67.3% of ESBLs strains were detected to produce CTX-M-type ESBLs, and the commonest genotypes in Escherichia coli and Klebsiella pneumoniae were CTX-M-14 (35.4% and 28.3%), CTX-M-15(21.5% and 26.1%) EBIC-PCR products of all CTX-M-15-producing strains show 39 strains of Escherichia coli were classified into 27 genotypes while 43 strains of Klebsiella pneumoniae were divided into 30 genotypes. Furthermore, the genotypes of CTX-M-55, CTX-M-19, CTX-M-27, with ceftazidime-hydrelyzing activity, were detected in this study. The great majority of bla_(CTX-M-15) genes were found to locate on a 65 000 bp-conjugative plasmid, and there was no blaTEM-1, bla_(OXA-1), blaDSA-1 or aac (6')-Ib-cr gene coexisted on the plasmid, ISEcp1-like insertion sequences, relative to mobilization of bla_(CTX-M-15) gene, were detected in all bla_(CTX-M-15) positive strains, and the distances between the end of ISEcp1-like insertion sequences and the start cedon of bla_(CTX-M-15) were equal, with 48 base pairs. Conclusion CTX-M-14 is still the most common genotype of ESBLs in Guangzhou, but high prevalence of CTX-M-15 ESBLs hydrolyzing ceftazidime already appears in south China.