Signal Transduction Pathways Of Apoptosis Induced By Ppv Infection In Porcine Placenta Trophoblast Cells

Porcine parvovirus(PPV) is one of major infectious pathogen of reproductive failure in swine, which can break through the placental barrier and spread from mother to fetus by vertical transmission. Placental trophoblast cells are important for component placental barrier, which provide essential physiological and immunologic protection for placentation and embryonic development. However, little is known about the interaction mechanism between PPV and placenta tissue of pregnant swine. To provide a solid theoretical foundation and technical support, we firstly used PK-15 cells as a model cells line, detected a series of morphological and molecular changes in PK-15 cells for underlying the molecular mechanisms of apoptosis induced by PPV infection. Secondly, we isolated and immortalized trophoblast cells from porcine placenta for investigating the signal transduction mechanism of PPV induced apoptosis in porcine placental trophoblast cells. We further compared and verified the mechanism of PPV induced apoptosis in PTCs by animal experimentation. The purpose of this study is to illuminate the signal transduction pathways and its regulation mechanisms of apoptosis induced by PPV infection, and provide theoretic reference for researching pathogenesis and the pathogenic mechanism of porcine parvovirus disease. The results are as follows:1. PK-15 cells infected with 1.0 MOI PPV for 24 h represented a series of typical apoptotic morphology characteristics, including chromatin condensation, nuclear fragmentation and apoptotic body formation. DNA fragmentation assay result showed that DNA of PK-15 cells infected with 1.0 MOI PPV were introduced into 180~200 bp or the initial stages of fragmentation at 36 h p.i.. Annexin V and PI staining assay showed that the apoptosis rate was significantly increased in PPV-infected cells when compared with mock-infected population. The results of western blot demonstrated that the cleavage of PARP, a the specific substrates of caspase-3, was detected at 12 h p.i., the expression of Fas, FasL, Bid and tBid did not appear significant changes in the process of PPV infection, nevertheless, the expression of Bax was gradually increased at 6 h p.i., Bcl-2 protein was decreased correspondingly, followed by the translocation of cytosol Bax and release of Cyt c. PPV infection promoted the accumulation and phosphorylation of p53 at at serine 15, 20 and 46. Pretreatment of PK-15 cells with Pifithrin-a(PFT-a), a specific inhibitor of p53 signaling, partially decreased with the expression and translocation of Bax,attenuated Cyt c release and caspase-3 activity. These results suggested that PPV induced apoptosis in PK15 cells through mitochondria signal transduction pathway with p53 regulattion.2. Primary porcine placental trophoblast cells(PTCs) were isolated from healthy gilts at Day 30 to Day 50 of gestation through collagenase digestion and percoll gradient centrifugation, which expressed trophoblast-specific marker cytokeratin 7 when tested by immunohistochemistry and grew as epithelia-like monolayers. To provide stable and immortalized PTCs, primary PTCs were transfected with human telomerase reverse transcriptase(hTERT) gene. The results of RT-PCR and Telo TAGGG Telomerase PCR ELISA assay revealed that ectogenic hTERT could be stably expressed, keeping PTCs a relatively high telomerase activity and proliferative capacity during long term culture. The results of karyotype, cell colony formation in soft agar and teratoma formation in the female athymic nude BALB/c mice demonstrated that the immortalized PTCs at passage 50 retained normal karyotype, did not lose the property of anchorage-independent growth and did not give rise to solid tumors in vivo. The ultrastructural features of porcine placental trophoblast cells were observed by transmission electron microscopy, hTERT-PTCs at passage 50 maintained the original polarized phenotype in comparison to primary PTCs. The results of Western blot and RT-PCR showed that hTERT-PTCs at passage 50 expressed E-cadherin but without class I antigens. A slight secretion of chorionic gonadotrophin β-subunit and placental lactogen in hTERT-PTCs were revealed by radioimmunoassay(RIA), which was similar to primary PTCs. These results demonstrated that the immortalized PTCs achieved an extended replicative lifespan without malignant transformation, and retained major phenotype and functional characteristics. The immortalized hTERT-PTCs can be used as a model cells line to study the interaction mechanism between PTCs and PPV.3. In order to detect the infectivity and the induction mechanism of apoptosis of PPV, primary PTCs and hTERT-PTCs were infected with 1.0 MOI PPV for indicated times. The result of AO/EB staining showed that primary and immortalized PTCs presented typical apoptotic morphology characteristics at 24 h p.i.. The results of PCR and immunofluorescence revealed that primary and immortalized PTCs maintained the sensitivity to viral infection. The result of flow cytometry showed that the apoptosis rate of primary PTCs and hTERT-PTCs were significantly increased when infected with PPV infection at 1.0 MOI, with the activation of caspase-8, caspase-9 and caspase-3 and the cleavage of PARP. The result of western blot showed that PPV infection did not affect the expression of Fas, but increased the level of FasL expression at 6 h p.i., caused the cleavage of Bid to tBid, increased the level of Bax significantly whereas decreased Bcl-2 expression, promoted the translocation of Bax between cytosol and mitochondria, accompanied by Cyt c release. The changes of these apoptosis-associated factors were regulated by the upstream signal molecular of p53. These results demonstrated that PPV infection induces apoptosis in PTCs through p53 signal pathway, Fas and Fas ligend pathway and mitochondrial pathway.4. Animal experimental results showed that virus particles of PPV distributed in trophoblast cells of early pregnant sows infected with 1 mL of 107TCID50/0.1 mL post infection 4 weeks. DNA fragmentations were presented PPV-infected PTCs. The results of western blot showed that the level of Fas, Fas L, Bax and p53 expression in PPV-infected PTCs were higher than control group. These results suggested that PPV infection in early pregnant sows induced apoptosis in PTCs through regulating the expression of Fas, Fas L, Bax and p53 to induce apoptosis in porcine placental trophoblast cells. This study revealed mechanisms of apoptosis in PTCs induced by PPV infection through the cellular and animal experiments. These results may contribute to shed light on the molecular pathogenesis of reproductive failure diseases caused by PPV infection.