1.Change in Expression of Keratin and Proto-oncogene Induced by Beta-propiolactone in HaCaT Cell.
Yin LIU ; Eon Gi SUNG ; In Hwan SONG ; Dongyi DU ; Dae Kwang KIM ; Jeong Hyun PARK ; Hoon Ki SUNG ; Yungchang LEE ; Joo Young KIM
Korean Journal of Anatomy 2001;34(4):389-404
To investigate the relationship between the morphologic changes and the expression of keratin and proto-oncogene induced by Beta-propiolactone (BPL), we assessed on the expression of keratins (K8, K10, K13) and proto-oncogenes (c-fos, c-jun, c-myc) in human HaCaT cell line. The cells were treated with 0, 0.1, 1 microgram/ml BPL for 2 or 6 hours. Morphologic studies revealed that BPL changed the cells into fibrocyte-shaped, caused highly lobulated nuclei and reduced desmosomes in their number. Findings of immunofluorescence and Northern blotting indicated that BPL consistently decrease expression of K10 representing a normal differentiation marker of keratinocytes, while increasing expression of K8 and K13 associated with a pathologic differentiation. This reagent also up-regulated expression of c-fos and c-jun, and down-regulated expression of c-myc. Together with staining for each keratin or proto-oncogene and DNA content in flow cytometry, BPL increased K8 expression dramatically at S-G2-M phase. The induction of c-Fos at S-G2-M phase appeared within 2 hours, and c-Jun gradually occurred. However, c-Myc was inhibited regardless of phases of cell cycle. In conclusion, these changes caused by BPL demonstrate a close relationship between the morphologic evidence and the altered expression of each keratin and proto-oncogene.
Blotting, Northern
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Cell Cycle
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Cell Line
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Desmosomes
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DNA
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Flow Cytometry
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Fluorescent Antibody Technique
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Humans
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Keratinocytes
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Propiolactone*
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Proto-Oncogenes*
2.Conditions for SARS-CoV cultivation and inactivation.
Song-le ZHANG ; Li-juan MA ; Guang TIAN ; Liang-yan ZHANG ; Xue-ying ZHANG ; Xi-liang WANG
Chinese Journal of Experimental and Clinical Virology 2005;19(2):135-137
OBJECTIVETo study the method for cultivation and inactivation of SARS-CoV.
METHODSIn order to choose the sensitive cell strain and the best infection dose of the virus, Vero, Vero-E6 and 2BS cell lines were infected with SARS-CoV. The cultivation temperature was selected among 25 degrees C, 33 degrees C and 37 degrees C. The best inactivating time and effect were observed with beta-propiolactone whose concentration ranged from 1:2000 to 1:20,000 at room temperature.
RESULTSVero and Vero-E6 cell lines were sensitive to SARS-CoV. The cytopathic changes of the cells were 75% at 37 degrees C in 5 percent CO2 incubator after infection. SARS-CoV was inactivated completely in beta-propiolactone (1:4000). The toxicity of beta-propiolactone was hydrolyzed completely when the inactivated virus was cultured for 16 hours at 2 degrees C, 8 degrees C and in water bath for 2 hours at 37 degrees C.
CONCLUSIONThe titer of SARS-CoV was the highest when it was cultured in Vero or Vero-E6 cells for 72 hours at 37 degrees C in 5 percent CO2 incubator. SARS-CoV was inactivated completely in beta-propiolactone when its concentration was 1:4000 and the interaction time was 1 hour.
Animals ; Cercopithecus aethiops ; Dose-Response Relationship, Drug ; Propiolactone ; pharmacology ; SARS Virus ; drug effects ; growth & development ; Temperature ; Time Factors ; Vero Cells ; Virus Inactivation ; drug effects
3.The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector.
Hong-Lan ZHAO ; Fong QIOU ; Qing-Ling MENG ; Yao Y I ; Rui-Guang TIAN ; Jian LU ; Jing-Yuan CAO ; Sheng-Li BI
Chinese Journal of Experimental and Clinical Virology 2011;25(6):450-452
OBJECTIVETo find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods.
RESULTSThe expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition.
CONCLUSIONSThe application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.
Animals ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Formaldehyde ; pharmacology ; Genetic Vectors ; Hepatitis A Antigens ; genetics ; immunology ; Hepatitis A Vaccines ; immunology ; Humans ; Propiolactone ; pharmacology ; Recombinant Proteins ; immunology ; Vaccines, Synthetic ; immunology ; Vaccinia virus ; genetics