| Objective: Investigate the effects of dengue type 2 virus(DENV-2)on the apoptosis and autophagy of primary human hepatic sinusoidal endothelial cells(HHSECs) and the expression of ICAM-1 and Beclin-1 at m RNA level and to analyze the possible pathogenic mechanism of DENV-2. Methods: HHSECs were identified by factor Ⅷ and CD31, detected by IHC and FCM, respectively. C6/36 was infected with DENV-2 and identified by RT-PCR and HindⅢ. The expression of NS1 gene in C6/36 cells infected with DENV-2 was detected by PCR and the expected fragment size was 413 bp. Further identification of DENV-2 NS1 gene by Hind Ⅲ and the expected restriction fragment size was 228 bp and 185 bp. TCID50 was used to determine the titration of DENV-2. HHSECs were infected with DENV-2 and identificatied by Real-time florescence quantitative PCR. Levels of DENV-2 NS1 m RNA was detected by real-time PCR. CCK8 was used to determine cytotoxicity of DENV-2 in HHSECs. The levers of Beclin1 and ICAM-1 m RNA were detected by Real-time PCR. Changes of apoptosis and the expression of LC3 B and ICAM-1 were detected by FCM. Results: The adherent cells were spindle, grew well and the contour of which was clear, which was in accordance with the growth characteristics of HHSECs. Test group cells were stained brown by DAB in compared with control group. The rate of CD31 expression detected by FCM is 87.1%. DENV-2 NS1 gene was identified by PCR and length of products was about 400 bp, and further identification of DENV-2 NS1 gene by HindⅢ then the expected restriction fragment size was about 200 bp, consistent with the expectation. TICD50 of C6/36 cells infected with DENV was 10-6.845/ml. Uninfected group did not express NS1 gene. After infected with DENV-2, the level of NS1 m RNA in HHSEC was intensely increased at36h(at 12 h as control, 2-△△Ctwas 11.33±1.77), with statistical significance(P<0.05).Cytotoxicity of DENV-2 were measured by CCK8. Compared with control group,HHSECs of test group were suppressed. At 12 h, 24 h, 36 h and 48 h, rate of suppression respectively were 10.9%±1.24%, 16.4%±0.42%, 17.0%±0.46% and 29.6%±0.26%,with statistically significant(P<0.05). The apoptosis rate was detected by flowcytometry. Compared with control group, HHSECs infected with DENV-2 seems with higher rate at 12h(13.17%), then which were reduced, but it is not obvious with each other(4.31%, 3.909% and 2.975%). Through the detection of FCM, the LC3 B expression in HHSECs infected with DENV-2 was 35.5% at 36 h, which was intensely higher than the expression in control group. The expression of ICAM-1 was detected by flow cytometry. Compared with control group, HHSECs infected with DENV-2increased and rate of expression were respectively 9.17%, 14.7%, 12.7%, 11.6%, at12 h, 24 h, 36 h and 48 h. HHSECs were Infected with DENV-2, the levels of Beclin-1,ICAM-1 was detected by Real time PCR. It was found that there was a difference between the experimental group and the control group, the levels of Beclin-1 m RNA and ICAM-1 m RNA intensely increased at 24h(2-△△Ctwas 46.77±2.55 and40.97±4.91, respectively), and then the two results decreased. Conclusion: HHSECs was susceptible to DENV-2, and the DENV-2 killed HHSECs. DENV-2 can induce ICAM-1 upregulation and activation of HHSECs. HHSEC can be used as a model for the pathogenesis of DENV infection and to study the possible mechanism of dengue virus pathogenesis. The expression of Beclin1 and LC3 B in HHSECs were affected while the cells were infected with DENV-2, and all increased. Apoptosis of HHSECs cells can be induced by DENV-2 infection in early stage. Autophagy and apoptosis are suggested to be involved in the process of DENV infection, and the specific mechanism remains to be studied. |
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