Viral Etiology Relationship between Human Papillomavirus and Human Breast Cancer and Target of Gene Therapy / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the viral etiology of human breast cancer to determine whether there are novel molecular targets for gene therapy of breast cancer and provide evidence for the research of gene therapy and vaccine development for breast cancer.</p><p><b>METHODS</b>PCR was used to screen HPV16 and HPV18 oncogenes E6 and E7 in the SKBR3 cell line and in 76 paraffin embedded breast cancer tissue samples. RNA interference was used to knock down the expression of HPV18 E6 and E7 in SKBR3 cells, then the changes in the expression of cell-cycle related proteins, cell viability, colony formation, metastasis, and cell cycle progression were determined.</p><p><b>RESULTS</b>HPV18 oncogenes E6 and E7 were amplified and sequenced from the SKBR3 cells. Of the patient samples, 6.58% and 23.68% were tested to be positive for HPV18 E6 and HPV18 E7. In the cell culture models, the knockdown of HPV18 E6 and E7 inhibited the proliferation, metastasis, and cell cycle progression of SKBR3 cell. The knockdown also clearly affected the expression levels of cell cycle related proteins.</p><p><b>CONCLUSION</b>HPV was a contributor to virus caused human breast cancer, suggesting that the oncogenes in HPV were potential targets for gene therapy of breast cancer.</p>

Inhibition of HIV-1 in vitro by combination of vpr and tat specific short hairpin RNA via lentiviral vectors / 病毒学报

Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.

One case of correlation between the human herpesvirus 7 DNA load and the hemo hagocytic syndrome / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To research the relationship between human herpesvirus 7 (HHV-7) viral Load and the etiopathogenisis of hemophagocytic syndrome, in order to provide evidence for the clinical diagnosis of hemophagocytic syndrome and anti-virus therapy.</p><p><b>METHODS</b>Peripheral blood of patient with hemophagocytic syndrome during different treatment periods, extracted DNA, Syntheticed the primers of HHV-7, gene sequence of PCR amplified fragments detected, determined HHV-7 viral Load by Real-time fluorescent quantitative PCR and the ferritin concentration in peripheral blood detected by chemiluminescence.</p><p><b>RESULT</b>The sequence result indicated that PCR amplified fragment was a part of HHV-7 gene, the ferritin concentration viried with the load of HHV-7.</p><p><b>CONCLUSION</b>The occurrence of hemophagocytic syndrome is connetted with the load of HHV-7.</p>

Transient expression and identification of gene P and NP of NDV LaSota strain in two different cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To Construction of P and NP genes eukaryotic expression vectors of Newcastle Disease Virus LaSota strain,study its reverse genetics and functional genome of NDV.</p><p><b>METHODS</b>P, NP genes were amplified and cloned into pGEM-T easy vector and then subcloned into pcDNA3.1 (+) expression vector respectively, the recombinant plasmids were named pcDNA3.1 (+)-P and pcDNA3.1 (+)-NP, Recombinant plasmids were transfected into 293 and BHK-21 cells respectively and were detected using IE and Western blot analysis.</p><p><b>RESULTS</b>Expression of P, NP genes were detected and confirmed by the IE and WB analysis.</p><p><b>CONCLUSION</b>The recombinant eukaryotic plasmids pcDNA3. 1(+)-P, pcDNA3.1 (+)-NP were expressed in 293 and BHK-21 cells successfully. This research may be helpful for further study of reverse genetics and functional genome of NDV.</p>

The correlation between the polyomavirus DNA load and the dose of immunosuppressant / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the correlation between the polyomavirus DNA load and the dose of immunosuppressant in patients with allogene bone marrow transplantation (allo-BMT) for preventing the development of post-transplantational hemorrhagic cystitis.</p><p><b>METHODS</b>Serial blood and urine samples from 122 cases of allo-BMT recipients were obtained and DNA was extracted from urine samples. Polyomavirus DNA-specific probe was synthesized and Fluorescence quantitative polymerase chain reaction was used for detecting the polyomavirus DNA loads and Fluorescence polarization immunoassay (FPIA) was performed for determining the dose of immunosuppressant Cyclosporin A (CsA) in blood.</p><p><b>RESULTS</b>The altered polyomavirus DNA load in urine was followed by concentration of CsA in blood. When the concentration of CsA in blood was higher than 86-105 ng/ml, the positive rate of polyomavirus DNA load was significantly increased and both presented the linable correlation.</p><p><b>CONCLUSION</b>In immunosuppression condition, polyomavirus DNA load correlated to the dose of immunosuppressant, which increased the risk of post-transplantational hemorrhagic cystitis.</p>

Correlation between load of polyomavirus and hemorrhagic cystitis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the correlation between polyoma virus load and hemorrhagic cystitis after allogeneic stem cells transplantation for prevention of hemorrhagic cystitis.</p><p><b>METHODS</b>Blood and urine specimens were collected from 40 healthy persons, 40 patient with stem cells transplantation and 20 cases complicated with hemorrhagic cystitis for determination of VP1 gene of polyomaviruses BK virus (BKV)/Jamestown Canyon virus (JCV) and simian virus 40 (SV40) by polymerase chain reaction (PCR) and EvaGreen stain fluorescence quantitative assay.</p><p><b>RESULTS</b>In the peripheral blood, all genes of BKV/JCV and SV40 were negative, while BKV gene in urine and blood from healthy persons and patient with stem cells transplantation was 15% (6/40) and 100% (40/40), respectively. The gene of JCV was positive in 10% (4/40) and 12% (5/40), the gene of SV40 was negative.</p><p><b>CONCLUSION</b>Genes of BKV and JCV was detectable in urine specimens of healthy persons and there was a correlation between the load of polyomavirus and incidence of hemorrhagic cystitis.</p>

The promotive effects of N-nitrosopiperidine on the malignant transformation of the immortalized esophageal epithelium induced by human papillomavirus / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Study on the promotive effects of N-nitrosopiperidine on carcinogenesis process was performed, based on the immortalization of human fetal esophageal epithelium induced by human papillomavirus (HPV) 18E6E7 genes.</p><p><b>METHODS</b>The immortalized esophageal epithelium SHEE was induced by HPV18E6E7. The cells at 17th passages were cultured in 50 ml flasks. The N-nitrosopiperidine (NPIP) 0, 2, 4, 8 mmol/L added to the cultured medium of SHEE cells for 3 weeks. The morphology, proliferation and apoptosis of the cells were studied by phase contrast microscopy and flow cytometry. Modal number of chromosomes was analyzed by standard method. Tumorigenicity of the cells was assessed by soft agar colony formation and by transplantation of cells into nude mice. Expression of HPV was detected by Western blot.</p><p><b>RESULTS</b>When cells were exposed to high concentration (8 mmol/L) of NPIP, cell death was increased, leaving a few live cells. In normal cultural medium instead of NPIP proliferative status of the cells restored after 4 weeks and the cells progressed to the proliferation stage with continuous replication and atypical hyperplasia. At the end of the 8th week, the cells appeared with large colonies in soft-agar and tumor formation in transplanted nude mice. When the cells were cultured in 2, 4 mmol/L NPIP the doubling passage was delayed and without tumor formation in transplanted nude mice. Modal number of chromosomes was 61-65, in 8 mmol/L NPIP group and control group, 56-61. Expression of HPV18 appeared in experimental and control groups.</p><p><b>CONCLUSION</b>NPIP promotes malignant change of the immortalized esophageal epithelial cells induced by HPV18E6E7. HPV18E6E7 synergy with NPIP will accelerate malignant transformation in esophageal epithelium.</p>

Study on the role of PML-RARalpha and RARalpha fusion proteins in NB4 cell apoptosis induced by arsenic trisulfide / 中国实验血液学杂志

In order to study the role of PML-RARalpha fusion gene and its expression product during apoptotic process in NB4 cells induced by arsenic trisulfide (As(2)S(3)), the apoptotic effects of NB4 cells were observed by cell morphology, flow cytometry and DNA electrophoresis. The change of PML-RARalpha fusion gene and its expression product were also assayed by chromosomal G banding, RT-PCR and Western blot. The results showed that arsenic trisulfide induced apoptosis of NB4 cells, during this process, PML-RARalpha fusion gene had no significant changes, but the expression of PML-RARalpha fusion protein and wild-type RARalpha were all reduced. It is concluded that arsenic trisulfide can induce apoptosis of NB4 cells, the degradation of PML-RARalpha fusion protein and wild-type RARalpha may play an important role during apoptotic process.

Construction of human papilloma virus type 18 E6E7 genes in adeno-associated virus expression vector and checking its activity for malignant transformation / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To construct human papillomavirus type 18 (HPV18 E6E7) adeno-associated virus (AAV) for studying the role of HPV E6E7 in the development of human cancer.</p><p><b>METHODS</b>HPV18 E6E7 genes were inserted into adeno-associated virus expression vector and then infected 293 cell line. The expression of HPV18 E6E7 genes were confirmed by using RT-PCR/Southern blot assay.</p><p><b>RESULTS</b>There was HPV18 E6E7 genes in the malignantly transformed cell line. The 293TL cells compared with the parent cells transformed cells grew more rapidly, lost their contact inhibition and formed more and large colonies in soft agar.</p><p><b>CONCLUSIONS</b>HPV18 E6E7 AAV was successfully constructed and could induce malignant transformation. HPV18 E6E7 AAV can be use for studying the immortalization and malignant transformation of human normal epithelial cells.</p>