Isolation and characterization of radiation-resistant lung cancer D6-R cell line / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To isolate an isogenic radioresistant cancer cell line after fractioned X-ray radiation and characterize the resistant cells.</p><p><b>METHODS</b>D6 cells were exposed to repeated X-ray irradiation, and after a total dose of 5200 cGy in 8 fractions, a radioresistant monoclone D6-R was obtained. The radiosensitivity and drug sensitivity of the novel radioresistant D6-R cells, together with their parent D6 cells, were measured using clonogenic assay and MTT assay respectively. Cell cycle distribution was analyzed by flow cytometry. Fluorescence microscopy and flow cytometry were applied for apoptosis detection. Comet assay was used for the detection of DNA damage and repair.</p><p><b>RESULTS</b>D6-R cells showed higher and broader initial shoulder (D0=2.08 Gy, Dq=1.64 Gy, N=2.20) than the parent D6 cells (D0=1.84 Gy, Dq=0.34 Gy, N=1.20). They were 1.65-fold more radioresistant than D6 cells in terms of SF2 (63% vs 38%) and were more resistant to ADM (3.15-fold) and 5-FU (3.86-fold) as compared with the latter. It was found that D6-R cells had higher fractions of cells in S phase (53.4% vs 37.8%) and lower fractions of cells in G1 (44.1% vs 57.2%) and G2-M phase (2.5% vs 5%). There was no difference in radiation-induced apoptosis between D6-R and D6 cells. D6-R cells showed less initial DNA damage and increased capacity in DNA repair after irradiation, as compared with the parent cells.</p><p><b>CONCLUSIONS</b>D6-R cells have been isolated by exposing the parental D6 cells to repeated irradiation. The difference in cell cycle pattern together with the induction and repair of DNA damage might, at least partially, explain the mechanism of the radioresistance.</p>

Screening for metronidazole-resistance associated gene fragments of Helicobacter pylori by suppression subtractive hybridization / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To screen metronidazole (MTZ) -resistance associated gene fragments of Helicobacter pylori (H.pylori) by suppression subtractive hybridization(SSH).</p><p><b>METHODS</b>The suppression subtractive hybridization (SSH) was used to screen the different DNA fragments between MTZ-resistant and -susceptible clinical strains of H.pylori. The resistant strains specific gene fragments were obtained by SSH and identified by dot blotting.</p><p><b>RESULT</b>Among the 120 subtractive colonies which were randomly chosen, 37 DNA fragments were different (>or=2 times) in DNA-copy numbers between resistant and susceptible strains and 17 of them were significantly different (>or=3 times). These 17 DNA fragments were sequenced subsequently. Ten of them were new sequences and the other 7 were duplicated sequences. These sequences represented respectively: depeptide ABC transporter periplasmic dipeptide-binding protein (dppA), permease protein (dppB), et al.</p><p><b>CONCLUSION</b>Gene fragments specific to MTZ-resistant H. pylori strains were obtained by SSH and these genes may associated with MTZ-resistance of H.pylori.</p>

Relation between mutant p53 and multidrug resistance in gastric cancer / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore the relationship between mutant p53 and multidrug resistance in gastric cancer.</p><p><b>METHODS</b>Mutant p53 (mp53) and mp53+sv40Tag were transferred to gastric cancer cell line SGC-7901. The MDR-1 mRNA was examined using RT-PCR, and the difference in chemotherapeutic sensitivity of SGC-7901 cells with mutant p53 was compared with those with mp53+sv40Tag and controls by MTT method.</p><p><b>RESULTS</b>SGC-7901 cells with mutant p53 showed higher MDR-1 mRNA than that of other two groups. SGC-7901 cells with mutant p53 showed higher chemotherapeutic sensitivity to 5-Fu than that with mp53+sv40Tag and control (P<0.05), but no difference between those with mp53+sv40Tag and control (P>0.05). SGC-7901 cells with mutant p53 and those with mp53+sv40Tag showed higher chemotherapeutic sensitivity to ADM than control (P<0.05), but no difference between those with mp53 and with mp53+sv40Tag (P>0.05). There was no difference in chemotherapeutic sensitivity of SGC-7901 cells with mutant p53 compared with those with mp53+sv40Tag and control to CDDP (P>0.05).</p><p><b>CONCLUSION</b>Mutante p53 genes relates to multidrug resistance of gastric cancer.</p>

Immortalization of human umbilical vein endothelial cells by transfected with hTERT and SV40LT / 中华心血管病杂志

<p><b>OBJECTIVE</b>To immortalize human umbilical vein endothelial cells (HUVECs) by ectopic expression of the telomerase reverse transcriptase enzyme (hTERT), and by Simian Virus 40 Large T (SV40LT) antigen without malignant transformation.</p><p><b>METHODS</b>Two different retroviruses that contained hTERT/SV40LT cDNA fragment and drug resistance gene were constructed, and were used to transfect normal primary HUVECs. The transfected cells were screened with 500 microg/ml G418 and 4 microg/ml puromycin. Drug resistance cell clones were selected 3 days after transfection and cultured for further studies. An under inverted microscope and a scanning electron microscope were used to observe the morphology and growth of the cells. The expression of VIII factor and transfected DNA fragments were detected for identification of the endothelial origin and successful transfection. And the expression of E-selectin and endothelial lipase with or without the stimulus of TNF-alpha were also assayed to analyze the biological activity of the transfected cells.</p><p><b>RESULTS</b>The cells were homogenous, closely apposed, large, flat, and polygonal, displayed a characteristic ovoid nucleus with one or two nucleoli and formed monolayer with polygonal shape without overlapping. Immunocytochemical staining showed the existence of VIII factor. SV40LT/hTERT antigen expressed by the transfected cells was detected, while the contrasts had non-expression. Telomerase activity of the cell was detected in the transfected cells, which was 0.36 at 12 th passage and 0.38 at 50 th passage. However, the activity in the normal HUVECs was 1.12 at the first passage and 0.06 at the third passage assayed by PCR-ELISA. Both E-selectin and endothelial lipase were all specific in endothelial cells. The expressions of these two were also detected. And the expression of E-selectin can be up-regulated with the stimulus of TNF-alpha, while the expression of endothelial lipase was not unregulated significantly.</p><p><b>CONCLUSION</b>Ectopic expression of hTERT and SV40LT can effectively immortalize HUVECs without tumorigenesis.</p>

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen.</p><p><b>METHODS</b>Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.</p><p><b>RESULTS</b>The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-alpha (tumor necrosis factor-alpha), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found.</p><p><b>CONCLUSION</b>Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.</p>

Conditionally immortalized human colorectal crypt cell line / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish human colorectal crypt cell line.</p><p><b>METHODS</b>Colorectal crypt cells were separated from human fetal gut by dispase I digestion, AKP-negative cells from fetal colorectal crypt were collected and cultured on Matrigel matrix. Subsequently the primary cultured cells were transfected with recombinant retrovirus containing human telomerase reverse transcriptase (hTERT) and simian virus 40 large T antigen (SV40 LT) in 48 h. The characterization of immortalized cells was identified after the transfection and cells were screened with antibiotics for 12 approximately 16 weeks and expanded.</p><p><b>RESULTS</b>Mucin, cytokeratin-pan, 8, 19 were presented in immortalized cells by immunohistochemical staining; ectopic expressions of both hTERT and SV40 LT were also found in immortalized cells by Western blotting. Agarose electrophoresis showed that the cells expressed Musashi-1 mRNA. No evidence of carcinogenesis was found in nude mouse experiment and soft-agarose cloning test.</p><p><b>CONCLUSION</b>The immortalized human colorectal crypt cells were characterized and the established cell line may be an ideal target for carcinogenesis study in vitro.</p>

Biological activity of the virulence factor cagA of Helicobacter pylori / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>China is one of the countries with the highest incidence of H. pylori and more than 9090 isolates possessed the cagA gene. This study was to evaluate the biological activity of the H. pylori virulence factor cagA isolated from Chinese patients.</p><p><b>METHODS</b>cagA DNA fragments were amplified from the genomic DNA and subsequently cloned into the mammalian expression vector for cell transfection and DNA sequencing. cagA protein, phosphorylated-tyrosine cagA and the complex of cagA precipitated with SHP-2 were identified respectively by western blot in the crude cell lysate from conditionally immortalized gastric epithelial cells at 48 hours after transfection with cagA DNA. In addition, the ability of induction of scattering phenotype was examined after transient expression of cagA in AGS cells.</p><p><b>RESULTS</b>The C-terminal half of cagA contained only one repeated sequence and three tandem five-amino-acid motifs glutamic acid-proline-isoleucine-tyrosine-alanine (EPIYA). Moreover, the amino acid sequence of D2 region in repeated sequence was aspartic acid-phenylanaline-aspartic acid (D-F-D) which was significantly distinguished from the three repeated sequences and aspartic acid-aspartic adid-leucine (D-D-L) in the western standard strain NCTC11637. Western blot revealed that cagA became phosphorylated in tyrosine site and bound with SHP-2 after transient expression of cagA DNA in gastric epithelial cells. Transient expression of cagA in AGS cells showed that cagA was able to induce the elongation phenotype although to a lesser extent than western strains.</p><p><b>CONCLUSIONS</b>cagA perturbs cell signaling pathways by binding with SHP-2. However, significant difference exists in amino acid sequence and biological function of cagA in Chinese compared with those of western countries.</p>

Preparation of monoclonal antibodies against oh(8)dG and their biological characteristics / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies against oh(8)dG and to evaluate the relationship between Hp infection and oxidative DNA damage by detecting oh8dG in gastric mucosa.</p><p><b>METHODS</b>BALB/C mice were immunized with BSA-oh(8)dG conjugate, monoclonal antibodies were prepared by hybridoma technique, the biological characteristics of antibodies were analysed by competitive ELISA, Western blot and immunohistochemistry.</p><p><b>RESULTS</b>Two strains of hybridoma cell were obtained. ELISA and Western blot indicated that the antibodies were fairly specific for oh(8)dG. In immunohistochemistry,the positive rate of oh(8)dG expression in Hp positive tissues and Hp negative tissues was 55% and 5%, respectively(P<0.01).</p><p><b>CONCLUSION</b>The prepared antibodies can specially recognize oh(8)dG and immunohistochemistry with the monoclonal antibodies showed Hp infection can increase oh(8)dG level in gastric mucosa.</p>

Correlation of autoantibodies against oxidized low density lipoprotein and its immune complex with coronary artery disease / 浙江大学学报·医学版

OBJECTIVE: To evaluate the relationship between autoantibodies against oxidized low density lipoprotein(ox- LDL), immune complex and coronary artery disease(CAD). METHODS: The levels of autoantibodies against ox-LDL and immune complex were assayed by ELISA, the content of ox-LDL and lipid levels were also measured. The serum samples were taken from 61 patients with CAD, 116 patients with essential hypertension(EH) and 123 healthy individuals as control. RESULTS: The patients with CAD had significantly higher levels of anti-ox-LDL IgG[21.48(17.58 approximate, equals 29.01)U/L], anti-ox-LDL IgM4.71[3.88 approximate, equals 7.06)U/L] and ox-LDL[0.87(0.44 approximate, equals 1.08) mg/L] than EH group[15.93(11.12 approximate, equals 22.26) U/L 2.54(1.17 approximate, equals 5.05) U/L 0.32(0.16 approximate, equals 0.61) mg/L]and healthy control group[11.12(4.70 approximate, equals 16.57)U/L 1.61(0.60 approximate, equals 3.03)U/L 0.23(0.12 approximate, equals 0.36)mg/L], P<0.001. However, the serum ox-LDL immune complex[2.63(1.69 approximate, equals 5.90)U/L] was significantly lower in CAD than that in EH group[15.71(6.25 approximate, equals 28.74)U/L] and that in healthy control group[12.54(8.28 approximate, equals 23.90)U/L], P<0.001. There were discrepancies in the association between ox-LDL and autoantibodies against ox-LDL among different groups. CONCLUSION: The changes of autoantibodies against ox-LDL and immune complex in patients with CAD may be related to the role of ox-LDL and autoantibodies against ox-LDL in the process of coronary atherosclerosis.