| Part 1:Effect of Morphine on the Replication of HIV-1 in MT2 cells and MacrophageObjective To determine whether Morphine has the ability to enhance HIV-1 replication in MT2 and Macrophage in vitro. Methods MT2 cells and Macrophages were random assigned into Morphine treatment group, Morphine+Naloxone co-treatment group, Naloxone treatment group and Control. The MT2 cells and Macrophages except Control were treated with Morphine and/or Naloxone for 24 hours, and then infected with HIV-1 B’substyle strain. p24 antigen in MT2 cell culture supernatants collected on days 3,4,5 and 6 after infection were determined, while culture supernatants for p24 antigen assay in Macrophage were collected on days 4,6,8 and 10. Results When MT2 cells infected with HIV-1 on the 3rd day,4th day,5th day and 6th day, the expression of p24 antigen of Morphine treated group were higher than Control (P<0.01). There was no significant difference in p24 expression between Morphine+Naloxone co-treatment group, Naloxone treatment group and Control after HIV-1 infected MT2 cells on days 3,4,5 and 6 (F=0.1,0.1,0.47 and 3.5, P>0.05). The p24 antigen expression enhanced multiple of morphine treatment group compared to Control was increased by HIV-1 infected MT2 cells time with a rising trend, trend analysis of repeated measurements showed statistic time effect (F= 45.72, P<0.01). When Macrophage cells infected with HIV-1 on the 4th day,6th day,8th day and 10th day, the expression of p24 antigen in Morphine treated group were higher than Control (P<0.05). There was no significant difference in p24 expression among Morphine+Naloxone co-treatment group, Naloxone treatment group and Control after HIV-1 infected Macrophage on days 4,6,8 and 10 (F=0.51,0.05,0.04 and 0.14, P>0.05). The p24 antigen expression enhanced multiple of morphine treatment group compared to Control was increased by HIV-1 infected time with a rising trend, trend analysis of repeated measurements showed statistically significant time effect (F=135.58, P<0.01). Conclusion Morphine has the ability to enhance HIV-1 of China replication in MT2 cell and Macrophage. This Morphine-mediated increase of p24 antigen expression was blocked by Naloxone.Part 2:Effect of Morphine on the Antiviral of Lamivudine against HIV-1Objective To determine whether Morphine has the ability to influence the antiviral effect of 3TC in vitro. Methods MT2 cells were random assigned into Morphine+3TC treatment group, Morphine+Naloxone+3TC treatment group, Naloxone+3TC treatment group,3TC Control, and Virus Control. The corresponding treatment groups were treated with opiates antagonist (Naloxone) for 0.5 hours before 24 hours Morphine treatment, next all of groups were infected with equal amounts of cell-free HIV-1ⅢB strain, and then all of groups were treated with equal dose of 3TC. The p24 antigen expression in culture supernatants collected on days 3,4,5 and 6 after infection was tested and the inhibitions of 3TC anti-HIV-1 p24 antigen of all treatment groups except Virus Control were calculated. Results The inhibition of 3TC anti-HIV-1 p24 antigen of Morphine+3TC treatment group was the lowest when HIV-1 infected cells on 3rd and 4th day and has significantly difference(P<0.05) compared with 3TC Control, while there was no statistically significant (P>0.05) when virus infected cells at 5th and 6th day. The difference of 3TC anti-HIV-1 p24 antigen inhibition between Morphine+Naloxone+3TC treatment group and Naloxone +3TC treatment group was no statistic difference (P>0.05). Similar results were obtained when these two groups were compared to the 3TC Control (P>0.05), respectively. The 3TC anti-HIV-1 p24 antigen inhibition of each treatment group reduced as infection time prolongation, resulted in a significant and time-course effect. Conclusion The effectiveness of 3TC antiviral was reduced by Morphine in the early infection, and this effect can be blocked by Naloxone.Part 3:Effect of Morphine on the Lamivudine Inducing HIV-1 resistanceObjective To investigate the influence of Morphine on the process of inducing HIV-1 wild type strain of china into resistance strain, according to cell-virus-3TC co-culture in vitro, and to induce stable passage resistance strain of china. Methods MT4 cells were random assigned into Morphine+3TC treatment group (Mo+3TC), Morphine treatment group (Mo),3TC treatment group (3TC), Virus Control (V Control). The MT4 cells of Mo+3TC and Mo were treated with three dose of Morphine in 10-10 mol/L,10-8 mol/L and 10-6 mol/L for 2 hours, and then infected with B’subtype HIV-1 strain of 100 TCID50 that isolated from AIDS patient in Henan of China. Maintain the same concentration of morphine and 3TC passaged (6 days for one generation,6 days one week) culture, and passed to the next generation of viruses, each experiment were cultured with the same concentration of 3TC and Morphine contained for 8 to 10 weeks. Supernatants were collected every week for Virus RNA extraction, and nested RT-PCR was employed to amplify nucleic acid fragments encoding reverse transcriptase, and the amplified products were subjected to DNA sequencing. Sequences obtained were then analyzed with Stanford HIV drug resistance algorithm for informations about mutations and levels of drug resistance, and then resistance mutations difference were compared among different treatment groups with Student-/test. Result Mo+3TC treatment group induced M184I, V118I/V, T69N, T215I, D67G and V90I mutaition; M1841 and V1181 mutation were induce by 3TC treatment group,and V1181, V90I and T215I mutation occurred in Mo treatment group while Virus Control induced none mutation. The timing of M184I emergence in 10-10mol/L of Morphine+3TC treatment group was significantly shorter than Virus Control, that was 15±7.75 days compared to 33.00±10.4 days (P<0.05). There was no significant time difference between Morphine+3TC treatment group and 3TC treatment group to induce V118I/V resistance mutation (P>0.05). The shortest emergencing time of M184I in Morphine+3TC treatment group was 13.5±7.55 days, and was significantly shorter than Virus Control that was 33.0±10.4 days. There was no significant difference between Morphine+3TC treatment group and 3TC treatment group in the shortest emergencing time to induce V118I/V mutation (P>0.05). Virus cultured for 8 to 10 genarations,10-10 mol/L and 10-8 mol/L of morphine+3TC treatment group maintained M184I mutation time were longer than the 3TC Control treatment group; V118I/V mutation maintained in 10-10 mol/L and 10"8 mol/L of morphine+3TC treatment group were longer than the 3TC Control group. Conclusion Morphine shorters the time of 3TC inducing M184I mutation, which is NRTIs main resistance mutation, and assistance the replication of M184I and V118I/V resistance strain. 3TC induce more mutation with Morphine action. |
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