[转移帖]HepG2.2.15细胞中能检测到cccDNA吗

cao1976

原帖由yaoming发表于 2009-7-22 16:32 ：

能否在HepG2.2.15细胞细胞中检测到cccDNA现在仍然存在争议，

我们最近用taqman realtime检测不到，请问有检测到的战友吗？

cao1976

bigben发表于 2009-7-22 19:57 ：

查了一下，有一篇文章是做这个的，就是用PCR检测2.2.15的cccDNA
Liu, M.C., et al. , Dynamic analysis of hepatitis B virus DNA and its antigens in 2.2.15 cells. Journal of Viral Hepatitis, 2004. 11(2): p. 124--129.

SUMMARY. The 2.2.15 cells-derived from HepG2 cells
transfected with a plasmid containing hepatitis B virus
(HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids
and virions (Proc Natl Acad Sci U S A 1987; 84:
1005–1009). The latter elicit acute hepatitis in chimpanzees
(Proc Natl Acad Sci U S A 1987; 84: 4641–4644). We studied
the presence of intracellular and extracellular HBV covalently
closed circular (ccc) DNA in this culture system by
polymerase chain reaction (PCR), kinetically analysed
HBsAg and hepatitis B e antigen (HBeAg) released in the
culture media by quantitative enzyme-linked immunosorbent
assay and quantitated by real-time PCR but HBV DNA
from intracellular and extracellular HBV-DNA. HBV cccDNA
was found both intracellularly and extracellularly. A significant
correlation was seen between the extracellular HBV
DNA levels and virus antigens (r ¼ 0.833; P ¼ 0.01 and
r &frac14; 0.939; P < 0.01 for HBsAg and HBeAg, respectively),
whereas there was no statistical correlation between intracellular
HBV DNA levels and virus antigen levels (r &frac14; 0.024;
P &frac14; 0.955 and r &frac14; 0.177; P &frac14; 0.625 for HBsAg and
HBeAg, respectively). These data would be valuable in
studies of the HBV life cycle and of potential anti-viral
agents.

cao1976

bigen发表于 2009-7-22 20:24：


这篇文章里面说cccDNA的提取方法：
To enrich for ccc forms, DNA was isolated from cytoplasmic and nuclear fractions of
2.2.15 cells by two extractions with phenol, followed by two extractions with phenol and chloroform (1:1) without prior treatment with proteinase K.

Sells, M.A., et al. , Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. Journal of Virology, 1988. 62(8): p. 2836--2844.

cao1976

gong发表于 2009-7-23 19:05 ：

cccDNA的检测其实里面问题很多。国内，包括国外很多方法都值得商榷。楼主用的是什么方法？引物探针位于哪个位置？

cao1976

davay发表于 2009-7-23 20:02 ：

恩,ccDNA不怎么好检测的,金标准是southern blot!

cao1976

dudiaojiangxue 发表于 2010-3-22 12:27 ：
可以检测到，我用的就是real-time PCR方法，而且测序正确。real-time PCR方法和southern是公认的两种最靠谱的检测，2004一篇外文综述这么写道。

就我掌握的资料来看：
1、2.2.15原始文献里提到，他们的数据支持该细胞有cccDNA。
2、有一偏研究cccDNA形成核小体的文章里面用的细胞是2215.
3、有一篇发在JV的文章里面用southern检测到了cccDNA。
4、有不少中文文章也检测到了cccDNA。