Hepatocellular carcinoma cells containing hepatitis B virus X protein have enhanced invasive potential conditionally.

AIMS: To establish a sustaining hepatitis B virus X protein expressed Chang liver cell line and to explore their biological behaviours of invasive potential induced by hepatitis B virus X protein. METHODS: Polymerase chain reaction was used to amplify the HBx gene from the whole hepatitis B virus genome. The gene was then subcloned into the eukaryotic expression vector pcDNA3.1 to construct the pcDNA3.1-HBx plasmid. Gene transfection mediated by Lipofectamine was used to introduce the plasmid into the human liver cell line Chang, and stable expression of the HBx gene was detected. RESULTS: HBx gene was cloned from the transfected Chang liver cells by reverse transcription-polymerase chain reaction, and confirmed by electrophoresis. The stably transfected Chang cells expressing HBx with malignant characteristics were verified and compared with control cells in terms of their growth curves, clonogenicity, wound healing abilities, migration and metastasis. CONCLUSION: The stabilising human liver cell lines Chang liver containing HBx gene expression have been established successfully. The invasive potential of Chang cells was conditionally enhanced by HBx transfection.

Telomerase activity of peripheral blood lymphocytes in patients with chronic hepatitis B.

Chronic hepatitis B is the immunocompromising condition. The decrease of lymphocyte telomerase is linked to immunosenescence in hosts. To know whether telomerase activity of lymphocytes is involved in immunopathogenesis in patients with chronic hepatitis B, telomerase activity of peripheral lymphocytes was determined in such patients. The results showed that telomerase activity in resting peripheral lymphocytes of healthy subjects was detectable at low level, and obviously increased (P<0.001) after stimulation in vitro with phytohaemagglutinin (PHA). Telomerase activity of lymphocytes decreased with age in both groups with or without PHA stimulation. Telomerase activity of resting lymphocytes in patients with chronic hepatitis B was also observed at detectable level and markedly upregulated after PHA stimulation. The decreased telomerase activity of resting lymphocytes was found in patients with chronic hepatitis B (n=14, 0.32+/-0.27) compared to that in healthy subjects (n=17, 0. 52+/-0.28; P<0.05). However, there was no difference present between these two groups in telomerase activity of activated lymphocytes with PHA. In addition, no effect of recombinant human interleukin-12 (rhIL-12) on telomerase expression was observed in either the patient group or the healthy group. We concluded that the decreased telomerase activity of lymphocytes in chronic hepatitis B patients is present, which may be partly responsible for immunosuppressive condition in such patients.

Effect of IL-12 on T-cell immune responses in patients with chronic HCV infection.

As the host's immune responses may determine the outcome of hepatitis C virus (HCV) infection, and interleukin (IL)-12 plays an essential role in host defense against infectious diseases, we studied the antigen-specific and non-specific cellular immune responses in patients with chronic HCV infection. A proliferative response to phytohemagglutinin (PHA) was found in all 20 patients. Of the 20, 8 (40%) displayed a lymphocyte proliferation in response to HCV antigen c22, 2 (10%) to c33, 6 (30%) to c100-3, and 1 (5%) to NS5. The addition of rhIL-12 to cultures of peripheral blood mononuclear cells (PBMC) stimulated with PHA significantly enhanced the proliferative responses in normal controls as well as in HCV-infected subjects. The increased proliferation was also observed in HCV-infected patients when PBMC were co-cultured with HCV antigens c22 and c100-3 in the presence of rhIL-12. The production of interferon gamma (IFNgamma), IL-2, IL-4 and IL-10 was observed in 7 (58.3%), 5 (41.7%), 3 (25.0%) and 5 (41.7%) HCV-infected individuals stimulated with c22, and in 4 (33.3%), 2 (16.7%), 2 (16.7%) and 2 (16.7%) with c100-3, respectively. All HCV-infected individuals had increased production of cytokines IFNgamma, IL-2, IL-4 and IL-10 in supernatants of PBMC after stimulation with PHA. IL-12 significantly augmented Th1 cytokine production in HCV-infected individuals stimulated with PHA and with HCV antigens. In conclusion, deficient cellular immune responses are present in HCV-infected patients and IL-12 can enhance the immune responses in these patients.

[Study of SCE frequency before and after tubal sterilization with phenol mucilage].

PIP: SCE frequency was compared in the chromosomes of lymphocytes before and after chemical occlusive tubal sterilization with phenol mucilage compound made by 2 manufacturers. 88 women were recruited in the study, 50% of the cases were injected with either of the 2 compounds. Blood samples were drawn both before and from 7-490 days the sterilization procedures. SCE frequencies were counted from lymphocyte cell cultures by 7 chromosome groupings. The results showed that differences in the mean and range of SCE frequency in each cell before and after sterilization were not statistically significant. The difference of SCE frequencies for different time spans between the 2 blood tests was not statistically significant either, nor were significant differences observed between the cases using the 2 different compounds. It was concluded that the dosage of phenol mucilage compound used for chemical occlusive sterilization did not induce any increase in SCE frequency. Therefore, these compounds did not cause damage to DNA primary structure in human lymphocytes. If the sterilization procedures are done properly, few complications or failures would occur. This procedure could, hopefully, become a widely used permanent method of contraception among rural women.^ieng

[Determination of 17-hydroxycorticosteroid by capillary zone electrophoresis].

OBJECTIVE: To detect and dissociate 17-hydroxycorticosteroids(17-OHCS) in urine by using high performance capillary zone electrophoresis(CZE) so as to solve the problem that phenolic compounds interfere with the determination of urine 17-OHCS by chromatographic-spectrophotometry. METHODS: Pigments were absorbed from samples by activated kaolin in which the internal standard was theophyline. Urine 17-OHCS was absorbed by neutral styrene-divinylbenzene resin. Thus pigments and phenolic compounds were separated roughly. Then 17-OHCS was washed down by alcohol and separated and detected by capillary zone electrophoresis. RESULTS: Urinary 17-OHCS was separated successfully. Urinary 17-OHCS had a good linearity in the range of 2-50 mg.L-1 (r = 0.994). The average recovery rate was 96.17%. The relative standard deviation was less than 5%. The consistence detection limit was 0.3 mg.L-1. CONCLUSIONS: Compared with chromatographic-spectrophotometry, the high performance capillary electrophoresis has much less interference and is more simple, rapid, and accurate.

[Inhibitory effect of yiqijiedu powder to telomerase and telomerase RNA in nasopharyngeal tumorigenesis of rats].

OBJECTIVE: Telomerase activation is a common event in malignant transformation, and is associated with tumorigenesis. This has been clarified in nasopharyngeal carcinoma. YIQIJIEDU powder (Chinese traditional medicine) is used to treated the patients with nasopharyngeal carcinoma (NPC) after radio-therapy and patients with precancerous lesion of NPC. We investigated whether YIQIJIEDU powder inhibits the telomerase activity in nasopharyngeal carcinogenesis. METHODS: Nasopharyngeal carcinomas in rats were induced by N, N' Dinitrosopiperazine (DNP), and some of them were treated with YIQIJIEDU powder. Telomerase and telomerase RNA in the rats' nasopharyngeal tissue were assayed by using PCR-ELISSA and Nested-PCR, and were pathologically diagnosed. RESULTS: YIQIJIEDU powder could inhibit telomerase activity and nasopharyngeal carcinogenesis in the nasopharyngeal tissue of the rats treated, but no telomerase RNA. CONCLUSION: The findings suggest that the anticancer effect of YIQIJIEDU powder links with their inhibitory effects on telomerase.

[Observation of the biological characterizations of nasopharyngeal epithelial cells by EB virus infection in early phase of immortalization].

The multi-stage cell model of the nasopharyngeal carcinoma development in vitro by Epstein-Barr virus transformation is beneficial for the elucidation of the mechanism of nasopharyngeal cancer. To observe the biological changes of primary human nasopharyngeal epithelial cells in early phase of immortalization, in this study, we have detected the morphological changes and the expression profile of senescence-associated beta-galactosidase (SA-beta-Gal) in primary culture. In addition, the expression of EB virus latent membrane protein 1 (LMP1) and the growth curve of primary cells were also detected. Our results showed a low percentage of cells infected with EB virus expressing SA-beta-Gal activity at the late primary culture. In morphology, the cells also formed multilayer foci, and the cell population doubling time was showed. These results demonstrated that the nasopharyngeal epithelial cells by EB virus infection have passed through the senescence and entered the early phase of immortalization. These cells have some of the transformed characteristics. Our results provided the data for further study on the mechanism of immortalization and the establishment of human nasopharyngeal epithelial cell line.