目的:建立丙型肝炎病毒(hepatitis C virus,HCV)核心(core,C)蛋白体外表达的非肝细胞模型.方法:核酸酶切法鉴定含有HCV1b基因型C蛋白编码基因的重组质粒pCMH6K的稳定性,将pCMH6K瞬时及稳定转染于中华仓鼠卵巢(China hamster ovary,CHO)...目的:建立丙型肝炎病毒(hepatitis C virus,HCV)核心(core,C)蛋白体外表达的非肝细胞模型.方法:核酸酶切法鉴定含有HCV1b基因型C蛋白编码基因的重组质粒pCMH6K的稳定性,将pCMH6K瞬时及稳定转染于中华仓鼠卵巢(China hamster ovary,CHO)细胞并连续传代110d,免疫荧光法检测转染细胞内HCV C蛋白分布特征,RT-PCR法检测转染细胞内HCVC mRNA.结果:pCMH6K含有与HCV1b基因型C蛋白编码基因(573bp)相一致的特异性片段;在pCMH6K瞬时以及稳定转染的CHO细胞内可见HCV C蛋白主要分布在胞质,少部分在胞膜;在不同时期稳定转染CHO细胞中均可见到与HCV C mRNA相一致的基因特异性片段(267bp).结论:成功建立了能够持续表达HCV C蛋白的CHO细胞株.展开更多
An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr ce...An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr cell by calcium phosphate coprecipitation. Positive clones made up 74% of total clones, which were identified with ELISA. The expression of pSMTPGH was induced by 0.5 μM of Cd￣++. The clone 1-C-3 was found to secrete hGH at the level of 3800 μg/10￣6 cells/24 hrs in media containing 10 μMTX. After 20 generations in culture, the clone was still stable with hGH expression.The molecular weight of secreted protein was the same as that of the natural pGH, 22KD;the identity was further supported by Western blot.展开更多
Objective To evaluate the cytotoxicity of six commonly used copper-bearing intrauterine devices （Cu-IUDs） on Chinese hamster ovary （CHO-K1） cells and to investigate the influence of frame, shape and copper surface...Objective To evaluate the cytotoxicity of six commonly used copper-bearing intrauterine devices （Cu-IUDs） on Chinese hamster ovary （CHO-K1） cells and to investigate the influence of frame, shape and copper surface area of Cu-IUDs on cell toxicity.Methods Cu-IUDs were incubated in 10% FBS-DMEM/F12 culture medium at 37 ℃ for 24 h. The extracts were analyzed by flame atomic absorption spectrometer and were then diluted into different concentrations with culture medium. Finally, cytotoxicity of these original and diluted extracts on CHO-K1 cells was detected by cell counting kit-8 （CCK-8） assay.Results The viabilities of cells treated with the original extracts of six Cu-IUDs （TCu220C bulb, TCu220C, GCu220, GCu300, Yuangong Cu270 and Yuangong Ⅱ- 300） were all below 10% and the cupric ion concentrations in these extracts were 28.22 mg/L, 31.80 mg/L, 92.80 mg/L, 99.74 mg/L, 114.90 mg/L and 119.20 mg/L, respectively. After these original extracts were diluted, significant differences in cytotoxicity were exhibited. IUDs with larger copper surface areas （GCu300 and Yuangong Ⅱ-300） showed more cytotoxicity than those with smaller areas （GCu220 and Yuangong Cu270） respectively; When different shapes of Cu-IUDs were compared, TCu220C bulb showed lower cytotoxicity than TCu220C, and GCu300 exhibited higher toxicity than Yuangong Ⅱ-300; TCu220C displayed significantly lower cytotoxicity than GCu220 due to their differences in frames.Conclusion We presented evidence on the cytotoxic effects of copper ions released from Cu-IUDs on CHO-K1 cells and found that shape, frame together with copper surface area of Cu-IUDs had obvious influence on the cytotoxicity.展开更多
文摘An expressive plasmid pSMTPCH was constructed from porcine growth hormone gene,sheep metallothionein promoter (MT-011)and the vector,pUC19. The linear pSMTPGH and circular pSV2-dhfr were cotransfected into CHO-dhfr cell by calcium phosphate coprecipitation. Positive clones made up 74% of total clones, which were identified with ELISA. The expression of pSMTPGH was induced by 0.5 μM of Cd￣++. The clone 1-C-3 was found to secrete hGH at the level of 3800 μg/10￣6 cells/24 hrs in media containing 10 μMTX. After 20 generations in culture, the clone was still stable with hGH expression.The molecular weight of secreted protein was the same as that of the natural pGH, 22KD;the identity was further supported by Western blot.
基金supported by Public Service Platform of Science and Technology Projects in Data mining of contraceptives monitoring and research of risk assessment model(BM2012062)the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Objective To evaluate the cytotoxicity of six commonly used copper-bearing intrauterine devices （Cu-IUDs） on Chinese hamster ovary （CHO-K1） cells and to investigate the influence of frame, shape and copper surface area of Cu-IUDs on cell toxicity.Methods Cu-IUDs were incubated in 10% FBS-DMEM/F12 culture medium at 37 ℃ for 24 h. The extracts were analyzed by flame atomic absorption spectrometer and were then diluted into different concentrations with culture medium. Finally, cytotoxicity of these original and diluted extracts on CHO-K1 cells was detected by cell counting kit-8 （CCK-8） assay.Results The viabilities of cells treated with the original extracts of six Cu-IUDs （TCu220C bulb, TCu220C, GCu220, GCu300, Yuangong Cu270 and Yuangong Ⅱ- 300） were all below 10% and the cupric ion concentrations in these extracts were 28.22 mg/L, 31.80 mg/L, 92.80 mg/L, 99.74 mg/L, 114.90 mg/L and 119.20 mg/L, respectively. After these original extracts were diluted, significant differences in cytotoxicity were exhibited. IUDs with larger copper surface areas （GCu300 and Yuangong Ⅱ-300） showed more cytotoxicity than those with smaller areas （GCu220 and Yuangong Cu270） respectively; When different shapes of Cu-IUDs were compared, TCu220C bulb showed lower cytotoxicity than TCu220C, and GCu300 exhibited higher toxicity than Yuangong Ⅱ-300; TCu220C displayed significantly lower cytotoxicity than GCu220 due to their differences in frames.Conclusion We presented evidence on the cytotoxic effects of copper ions released from Cu-IUDs on CHO-K1 cells and found that shape, frame together with copper surface area of Cu-IUDs had obvious influence on the cytotoxicity.
