The Long Culture Of Human Peripheral Blood Mononuclear Cell And The Establishment Of The Cell Model Of Hepatitis C Virus

Background: Hepatitis C virus (HCV) is the major cause of transfusion-associated non-A, non-B hepatitis and Hepatitis C is the most prevalent liver disease in the world. Acute HCV infection leads, in more than 50-85% of the patients, to the development of chronic hepatitis, and then cirrhosis and hepatocellular carcinoma. According to the American Centers for Disease Control, 20% to 30% of people with chronic Hepatitis C will eventually face life-threatening symptoms. Over the next 10-20 years chronic HCV is predicted to become a major burden on the health care system as patients who are currently asymptomatic with relatively mild disease progress to end-stage liver disease and develop hepatocellular carcinoma. Hepatitis C virus was first definitively identified by molecular cloning of the virus genome in 1989. But there is currently no suitable cell model for growing HCV in vitro. Despite increasing knowledge of genome structure and individual viral proteins, studies on virus replication and pathogenesis have been hampered by the lack of reliable and efficient cell culture systems. And there is no vaccine and no completely effective treatment! This virus is however capable of infecting cells of lymphocytic origin, especially human peripheral blood mononuclear cells (PBMC). But PBMC was normal somatic cells and their lifespan are limited. So they are no suitable for long-term research. To establish a perfect cell culture model with long-term replication in the cell, we must prolong the lifespan of PBMC.In each eukaryotic cell the genotype is stored in chromosomes, whose end structures are called telomeres. Whereas the telomeres of embryonic cells are long, senescent cells have much shorter ones. In most human somatic cells telomeres progressively shorten with each cell division eventually leading to chromosomal instability and cell senescence. In germline and tumor cells the enzyme telomerase causes elongation of telomeres, making them potentially immortal. In a recently published experiment, scientists succeeded in transfecting telomerase genes into cultured human cells to give them an unlimited capacity for cell divisions.In this study, we employed PBMC to test its susceptibility to telomerase genes and established cell models that can support HCV long-term replication in vitro. The presence of both plus- and minus- strand of HCV RNA were demonstrated.Objective: To extend lifespan and replicative potential of human peripheral blood mononuclear cells by transfected by ectopic human telomerase reverse transcriptase (hTERT) gene and to establish stable and perfect cell culture models of hepatitis C virus.Materials and Methods: a.) Primary human peripheral blood mononuclear cells were separated from a healthy volunteer and cultivated in RPMI1640 medium, which contained 10% calf blood serum and was placed in a constant temperature oven of 37℃ and 5% CO2. After 1 week the cells were transfected with ectopic human telomerase reverse transcriptase gene. Expression of hTERT mRNA in the cells were detected by RT-PCR. The biological characteristics of the cells were analyzed: inverted microscope was used to abserve morphologic change of the cells, cellular growth curve was drawn and the phenotype of the cells was detected by Trypsin-Giemsa dye. b.) After PBMC were cultivated in vitro for 1 week the cells were transfected with hTERT gene. Then continuous inoculation of anti-HCV positive and HCV RNA RT-PCR positive sera was used to infect the cells. The plus- and minus-strain of HCV RNA in the cells were detected by RT-PCR and the biological characteristics of the cells were analyzed after inoculation, c.) Primary peripheral blood mononuclear cells were separated from a hepatitis C patient and cultivated in RPMI1640 medium, which contained 10% calf bloodserum and was placed in a constant temperature oven of 37℃ and 5% CO2. After cultivated for 1 week ,patient' s PBMC were transfected with hTERT gene. Then the expression of hTERT mRNA in the cells were detected by RT-PCR and hTERT protein were detected by indirect immunofluorescence. The biological characteristics of the cells were analyzed. The plus- and minus-strain of HCV RNA in PBMC and supernate were also detected by RT-PCR.Results: a.) After PBMC were transfected by plasmid pCIneo-hTERT, stable expression of hTERT mRNA were detected in the cells. The cells had extended lifespan and proliferative potential. The biological characteristics of the cells were normal: the cells had normal morphologic features and normal phenotype (46,XY). b.) After transfected with hTERT gene and inoculated with anti-HCV positive and HCV RNA RT-PCR positive sera, the expression of hTERT mRNA were detected in PBMC. hTERT gene enhanced the prolifetation and division of PBMC. HCV RNA plus-strain was detected continuously for 4 passages in the postinoculated cells transfected by plasmid pCIneo-hTERT, and minus-strain was detected intermittently. While in the cells transfected by plasmid pCIneo , HCV RNA plus strain was detected only in two passages. No minus-strain was detected in PBMC of the control group. C.) After transfection, both stable expression of hTERT mRNA and hTERT protein could be detected in the patience's PBMC. The cells also had extended lifespan and proliferative potential. The phenotype of PBMC is normal (46,XX). HCV continuously existed in the cultivated supernate of experimental group cells for 8 weeks, while the HCV only existed in the cultivated supernate of control group cells for 3 weeks. HCV RNA plus and minus-strain was detected continuously in the cells transfected by plasmid pCIneo-hTERT for 8 weeks. While HCV RNA only could be detected for 2 weeks in the control group cells.Conclusions: a.) Ectopic hTERT gene can transfect human peripheral blood mononuclear cells and the expression hTERT gene is capable of extending the replicative lifespan. After transfection the cells maintaining the normal biological characteristics: normal morphologic features and phenotype. b.) After inoculated with anti-HCV positiveand HCV RNA RT-PCR positive sera, the transfected PBMC can be infected by HCV. HCV exist and replicate stably in PBMC for a relatively long time. A normal and stable HCV cell culture model can be established. C.) HCV may also exist and replicate in hepatitis C patient' s PBMC transfected by human telomerase reverse transcriptase gene and cultured in vitro. It is a stable and natural cell line with HCV replication in vitro.