Hypoxia-inducible factor-1α and glucose transporter 1 in the malignant transformation of oral lichen planus.

Hypoxia-inducible factor-1α (HIF-1α) and glucose transporter 1 (GLUT1) are key factors in numerous physiological and pathological processes. However, studies on their involvement in the pathogenesis of oral lichen planus (OLP) and its progression toward oral squamous cell carcinomas (OSCC) are scarce. In this study, we examined the protein and gene expressions of both HIF-1α and GLUT1 in normal mucosa, nonatrophic OLP (OLPI), atrophic OLP (OLPII), and OSCC resulting from OLP. Tissues were obtained from 60 cases of OLP patients (n=36 for OLPI, n=24 for OLPII), 20 cases of OSCC patients and 30 healthy control individuals. In addition, in order to investigate if the pathological changes are due to hypoxia, we cultured keratinocytes under hypoxia conditions and measured the expression of HIF-1α and GLUT1. The results indicated that the expressions of HIF-1α and GLUT1 were gradually amplified from normal mucosa to OLPI, OLPII, and OSCC. The expression of both HIF-1α and GLUT1 in OLPII was significantly greater than OLPII. Likewise, the HIF-1α and GLUT1 expressions in OSCC were markedly higher compared to both OLPI and OLPII. Similar trends were obtained in real time PCR and Western blot analyses. A progressive increased micro-vessel density (MVD) was also recorded from normal mucosa to OLPI, OLPII, and OSCC. Moreover, the correlation analysis revealed significant positive correlations between HIF-1α and GLUT1 which were both correlated with MVD in the OLP and OSCC groups. Culture of keratinocytes isolated from OLP tissues under hypoxic and normoxic conditions showed a time-dependent inhibition of keratinocyte proliferation and increased expression of HIF-1α and GLUT1 under hypoxia conditions. In summary, we provided new evidence that hypoxia markers HIF-1α and GLUT1 are upregulated in OLP and are potentially involved in pathological changes leading to malignant transformation of OLP. Further characterization of these factors will provide new ideas for the diagnosis and treatment of OLP.

Low expression of glucocorticoid receptor a in oral lichen planus correlates with activation of nuclear factor κB: a preliminary study.

BACKGROUND: Emerging evidence indicates that the interaction between glucocorticoid receptor α (GRα) and nuclear factor κB (NF-κB) is a key pathogenetic cross talk in the autoimmune and inflammatory disorders. The objective of this study was to determine the GRα expression in patients with oral lichen planus (OLP) and investigate its correlation with NF-κB in OLP. METHODS: We compared the expression of GRα and NF-κB in oral biopsy specimens from patients with OLP(n = 32) against normal controls (n = 12) and investigated the correlation between the expression of GRα and NF-κB in OLP. RESULTS: Immunohistochemistry showed that GRα mainly expressed in the cytoplasm of keratinocytes of basal and spinosum layer of OLP. Both real-time quantitative PCR and Western blots revealed that the mRNA and protein expression levels of GRα were decreased compared with normal controls (both P < 0.001). Conversely, those levels of nuclear factor-kappa B (NF-κB) were increased compared with normal controls (both P < 0.001). Importantly, a significant inverse correlation between the GRα and NF-κB was found (P < 0.05). CONCLUSIONS: Our findings demonstrated that low expression of GRα in OLP correlates with activation of NF-κB, which indicates that the cross talk between GRα and NF-κB in OLP may become a new therapeutic target and represent a new approach to explore the pathogenesis of OLP.

PAMAM-triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity.

The excellent transfection efficiency and viability are essential for successful gene therapy. It suggested that when bound to its glucocorticoid receptor, glucocorticoid steroid can dilate the nuclear pore complexes and facilitated the transport of pDNA into the nucleus. In this research, the two different degrees of substitution of PAMAM-triamcinolone acetonide (PAMAM-TA) conjugates were synthesised for efficient translocation of pDNA into the nucleus. The physicochemical properties of the polyplexes were investigated by agarose gel electrophoresis, Zeta-sizer and TEM. They both could form nano-size polyplexes with pDNA. The polyplexes were very stable and showed excellent buffering capacities, facilitating endosomal escape, and no obvious difference was found between them. The TA-conjugated PAMAM-mediated transfection of luciferase and EGFP genes showed better transfer activity than native PAMAM and was comparable to the PEI 25K (polyethylenimine), and lower cytotoxicity in HEK 293 and HepG 2 cells. Even with 10% serum, their transfer activity was still high relatively. In addition, confocal microscopy examination confirmed that the enhancing mechanism for enhanced gene transfer activity of PAMAM-TA conjugate may involve the nuclear translocation of the polyplex. The low substituted degree of TA to 0.22 did not interrupt its nuclear localization potency. These findings demonstrated that the TA-grafted PAMAM dendrimer is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.

[Preparation and identification of monoclonal antibodies to staphylococcal enterotoxin I].

OBJECTIVE: To prepare and identify monoclonal antibodies against staphylococcal enterotoxin I (SEI). METHODS: Spleen cells obtained from mice immunized with the SEI protein were fused with the myeloma cells (SP2/0). Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA) and the stable monoclonal hybridomas were isolated by limiting dilution at least three times. The characters of purified monoclonal antibodies were identified by indirect ELISA and Western blotting. RESULT: The monoclonal antibodies secreted by two hybridomas 8F7 and D8 belonged to IgG(2b) and IgG(1) subtypes. Both had high titer and specificity with no cross reaction to SEG, SEE and SEC. CONCLUSION: The monoclonal antibodies against SEI has been successfully prepared and identified in this study.

[Preparation and application of antibody against staphylococcal enterotoxin C2].

The filtrate of Staphylococcus aureus culture has been used in an ampule form named as staphylococcal enterotoxin C injection for cancer therapy in clinic for ten years in China and proved to be effective. The active constituent of three kinds of injections is claimed to be staphylococcal enterotoxin C2 (SEC2), and the content of SEC2 is used as quality control. However, the correct content of SEC2 was not known and the relative amount of SEC2 was very low because of the complicated components of the filtrate. In this research, we established a proper ELISA system for the detection of SEC2 in staphylococcal enterotoxin C injection, which will improve the quality control of the injection. We produced and identified polyclonal and monoclonal antibodies of SEC2 and established BA-ELISA method based on the method of sandwich ELISA. It was found that the BA-ELISA method had good specificity, sensitivity and reproducibility, and being able to detect SEC2 at concentration from 2 to 20 ng x mL(-1), with an average CV value of 5.08%. The SEC2 content in staphylococcal enterotoxin C injection was calculated. There is some difference between the actual and labeled contents in the injections.

[Expression and bioactivity of the cloned staphylococcal enterotoxin O].

This study is to clone the gene of staphylococcal enterotoxins O, obtain recombinant protein (rSEO) and investigate its activity on mice lymphocyte. Staphylococcus aureus O gene is cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEO was used to transform E. coLi BL21, where the GST-SEO fusion protein was expressed efficiently. Then SEO was purified by Glutathione Sepharose 4B affinity column and digested with thrombin. The bioactivity of SEO was analyzed by MTT assay on mice lymphocyte and tumor cells. The nucleotide sequence was confirmed to code for the protein correctly, and soluble SEO was expressed efficiently in E. coli BL21 with pGEX-4T-SEO. The protein purified by affinity chromatography resulted to be one single band by SDS-PAGE detection. The MTT assay of the purified rSEO demonstrated that its abilities of stimulating T cells and inhibiting the proliferation of K562, K562-ADM and B16 cells were equivalent to that of SEC in vitro. The expression plasmid pGEX-4T-SEO was constructed and the recombinant superantigen was expressed successfully, which may provide a foundation for the further research of the anticancer activity of SEO.

[Expression and bioactivity analysis of staphylococcal enterotoxin C2].

AIM: To clone the gene of staphylococcal enterotoxin C2 and express it in the form of a soluble fusion protein in E. coli. Then the activation of SEC2 on mice lymphocyte and its lethal effects on tumor cells were studied. METHODS: Staphylococcus aureus SEC2 gene was cloned into GST gene fusion vector pGEX-4T-1. The resultant plasmid pGEX-4T-SEC2 was used to transform E. coli BL21, where the GST-SEC2 fusion protein was expressed efficiently. The rSEC2 protein was purified with Glutathione Sepharose 4B affinity column and digested with thrombin. The in vitro culture system was utilized to observe the activation of the SEC2 on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte. RESULTS: The proper gene of SEC2 was cloned and purified rSEC2 was obtained. The MTT results indicated that rSEC2 have strong ability to stimulate mice lymphocyte to proliferate with a dose-dependent manner. With the proliferation of mice splenic lymphocyte, rSEC2 has a strong lethal effect on tumor cells B16, K562 and K562-AD. CONCLUSION: In this study, the gene of SEC2 was cloned and the rSEC2 protein was obtained, which had strong lethal effect on tumor cells B16, K562 and K562-AD.

[Construction and expression of recombinant adenovirus containing human thymic stromal lymphopoietin gene].

AIM: To construct and express recombinant adenovirus bearing human thymic stromal lymphopoietin (TSLP) gene. METHODS: TSLP gene amplified from human fetal lung cells was first cloned into eukaryotic expression vector pcDNA3.1, and then subcloned into shuttle vector pShuttle. The resultant plasmid was subsequently cotransformed into E. coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1. The recombinant adenovirus plasmid containing TSLP was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. The TSLP gene of the recombinant virus was detected by PCR, and its expression was analyzed by Western blot. RESULTS: Recombinant adenovirus vector bearing human TSLP gene was constructed by homologous recombination in E.coli, and recombinant adenovirus was obtained by transfecting HEK293 cells with this infectious DNA. PCR test indicated that TSLP gene was successfully integrated into the adenoviral genome, and the titer of the recombinant Ad reached 1 x 10(11) pfu/L. Meanwhile, expression of TSLP in the infected Hela cells was confirmed by Western blot. CONCLUSION: The construction of recombinant adenovirus bearing human TSLP gene and its expression mediated by this adenovirus pave a foundation for the study on the biological function of this novel cytokine.