| ObjectiveTNF-related apoptosis inducing ligand(TRAIL), originally identified in 1994, is a member of the TNF(Tumor necrosis factor) superfamily. It exists wildly in human tissues, takes part in adjusting body immune surveillance and selectively induces apoptosis of tumor cells while leaving normal cells intact.It is reported that the reduced expression of TRAIL was possiblely related to chronic Hepatitis B, so we suppose to find out whether TRAIL can restrain HBV transcription and replication, as well as the relationship between the dose of sTRAIL and sTRAIL-induced apoptosis, through researching on hepatoma cell models of HepG2 and HepG2.215.Methods1. To study the sTRAIL's effect on HBV transcription, replication and expression. HepG2 and HepG2.215 were choosed as cell models in vitro for they have the same genetic background. Firstly, the pHBV4.1 was transfected into HepG2 by calcium phosphate method. Secondly, HepG2.215 and HepG2 were divided into 2 groups respectively, and each group contained an EG(experimental group) and a CG(control group). Concerning all the EGs, Group 1 of HepG2.215 and both two group of HepG2 were exposed to sTRAIL at a final concentrations of 10ng/ml in 48 hours, while Group 2 of HepG2.215 were treated by different final concentrations of 2.5～20ng/ml in 0～5 days. Thirdly, the total RNA and the replication intermediates of HBV were extracted from one group of HepG2, and the latter were extracted from Group 1 of HepG2.215. Then the transcription levels of 3.5kb, 2.4kb, 2. 1kb and 0.7kb HBV RNA were analyzed by Northern blot hybrization, and the level of HBV DNA replication intermediates was detected by Southern blot hybridization analysis. Fourthly, the expression of HBsAg and HBcAg in the other group of HepG2 was detected by immunohistochemistry, while every day's(0～5th day)expression of HBsAg and HBsAg by EIISA.2. Observe the sTRAIL-induced apoptosis of HepG2 which were transfected by pHBV4.1. After the transfection, HepG2 were exposed to sTRAIL of different final concentrations (10,20,100,200,1000ng/ml) in 48 hours. Then HepG2' chromosomal DNA were extracted by LADDER Kit after 48h. Finally, these DNA were detected by agarose electrophoresis.Results1. The results of Southern and Northern blot hybridization showed, after 48h's effect, sTRAIL of 10ng/ml could inhibit HBV DNA replication approximately 3- to 20-fold and reduce transcription of 3.5kb HBV RNA approximately 5- to 20-fold, respectively.2. ELISA assay prompted, sTRAIL began reducing the expression of HBsAg and HBeAg significantly at final concentration of 2.5 and 5 ng/ml respectively(P＜0.01 or P＜0.05). The more(among 0～20ng/ml) and longer(among 0～5th day)sTRAIL effected, the lower HBsAg would express. But inhibition of HBeAg didn't show this tendency.3. Immunohistochemistry hinted, after 48h's effect, sTRAIL of 10ng/ml could reduce the expression of HBsAg and HBcAg.4. sTRAIL-induced apoptosis of HepG2 was concentration-dependent. LADDER strap could be observed after 48h with 100ng/ml sTRAIL effecting HepG2 and turn brighter with 200ng/ml, while the same phenomenon could not be seen under 100ng/ml sTRAIL.ConclusionThis study showed that after 48h's effect, sTRAIL at final concentration of 10ng/ml could inhibit HBV transcription and replication, as well as the expression of virus protein among 2.5～20ng/ml in five days. Meanwhile, sTRAIL-induced apoptosis of HepG2 started being seen under 100ng/ml sTRAIL effecting after 48 hours. It hinted that sTRAIL's inhibition towards HBV did not depend on sTRAIL-induced apoptosis, at least not totally. |
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