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Objective To compare the three kinds of methods for in vitro primary culturing of rheumatoid arthritis synovial fibroblast-like cells (RASFs), in order to get fast and effective culture methods. Methods Synovial tissue from RA synovial arthroscopic resection were treated by collagenase digestion method, modified tissue culture method, double enzyme digestion method respectively. By using an inverted phase contrast microscope, cell morphology and growth characteristics were observed and identified with vimentin staining. Trypan blue was used to count the number of living cells after culturing for 14d. Results The three primary methods could successfully isolate and culture RASFs, and RASFs met the morphological characteristics of vimentin-positive cells>95%, namely, the proportion of RASFs cell confluence was 70% after 16-20days by the collagenase digestion method,whose cell confluence proportion reached 95%after 4 weeks;and the cell confluence proportion was above 70%after 10-14days by modified tissue culture method,and the cell confluence proportion reached 85%after 4 weeks by the double enzyme digestion method. The comparison of the viable cells number cultured same number of synovial tissue by the three methods show the viable cells number cultured by the modified tissue culture method were (1.60±0.08) ×106, those by the collagenase digestion method were (1.41±0.08) ×106, those by the double enzyme digestion method were (1.19 ±0.05) ×106, which were with significant difference among them (P<0.05) .The comparison of incubation time of RASFs primary cells showed it took (267.50±16.58) mins by the collagenase digestion method, (183.75 ±11.08) mins by the double enzyme digestion method, and 149.10 ±13.71mins by the modified tissue culture method, with significant differences (P<0.05) .Conclusion Modified tissue culture for RASFs is an efficient and fast culture method, the number and purity of RASFs can meet the requirements for biology experiments.
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Objective To construct a bicistronic expression vector containing HPV type 6b L1 gene, to express the recombinant vector in mammalian cells, and to establish a cell strain stably expressing HPV6b L1 gene. Methods After endonuclease digestion and purification, the gene fragment of HPV6b L1 was cloned into the eukaryotic expression vector pIRES2-enhanced green fluorescent protein (EGFP). The identification of the recombinant was realized via endonuclease digestion and sequence analysis. Then, the recombinant plasmid pIRES2-HPV6bLl-EGFP was transfected into NIH3T3 (a mouse embryonic fibroblast cell line) cells. Subsequently, the expression of EGFP was observed by fluorescent inverted microscopy, and HPV6b L1 mRNA expression by reverse transcription (RT)-PCR. Results The recombinant plasmid pIRES2-HPV6bLl-EGFP was successfully constructed, transfected into N1H3T3 cells, and selected by G418. The expression of EGFP was seen under an inverted fluorescence microscoy. RT-PCR proved the expression of HPV6b LI mRNA in transfected cells. Conclusions The recombinant plasmid pIRES2-HPV6bLl-EGFP was successfully constructed and transfected into NIH3T3 cells. Inverted fluorescent microscopy and RT-PCR confirmed the successful expression of HPV6b L1 in NIH3T3 cells.
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Objective To investigate the expressions of survivin, cyclooxyenase-2 (COX-2) and vascular endothelial growth factor (VEGF) and their relationship with angiogenesis in condyloma acuminatum (CA) tissues. Methods Immunohistochemistry using PowerVision staining kit was performed to detect the expression of survivin, COX-2 and VEGF protein in 60 CA tissue samples from patients and 21 normal skin samples from the foreskin of human controls. At the same time, the microvessel density was determined in CA tissues by staining blood vessel endothelium with anti-CD105 monoclonal antibody. Results The positivity rate of survivin and COX-2 expression was 56.67% and 63.33%, in CA tissues, 9.52% and 0 in normal skin tissues, respectively. There was a significant difference between the two groups of tissue samples in the positivity rate and intensity of survivin and COX-2 expression (all P < 0.05). VEGF was expressed in all of the CA tissues and normal skin tissues, while the intensity of VEGF expression was statistically different between the two groups of tissue samples (P < 0.05). The MVD was 16.38 ± 5.46 and 0.62 ± 0.44 in CA tissues and normal skin tissues, respectively (P < 0.05). There was a significant positive correlation between the expressions of survivin, COX-2 and VEGF, as well as between MVD and the expressions of survivin and COX-2 in CA tissues. Conclusion The expression levels of survivin, COX-2 and VEGF are significantly higher in CA tissues than in normal skin tissues.
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In order to explore the role of p15(INK4B) gene with highly methylated CpG island in the pathogenesis of leukemia, the expression levels of p15(INK4B) gene was detected in patients with AML and CML. Methylation-specific PCR (MSP) assay was employed in the experiments. The methylation incidence was 83.9% (26/31) in AML and 0% (0/28) in CML. The results showed that methylation of p15(INK4B) gene was the one of the major ways for inactivation of the gene, and the methylation could be appeared in clinical development of the disease and patients condition worsened. Methylation of p15(INK4B) did not occur and its function probably is normal in CML.
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To study the action, characteristics and expression of high methylation of p15(INK4B) and p16(INK4A) genes in multiple myeloma (MM), the sensitive methylation specific PCR method was employed to detect the hypermethylation of p15(INK4B) and p16(INK4A) in 24 patients with MM. Results showed that the methylation incidence of p15(INK4B) and p16(INK4A) genes were 70.8% (17/24) and 58.3% (14/24) in the MM patients, with the products of 148 bp and 150 bp fragments, respectively. The methylation of p15(INK4B) and p16(INK4A) genes were simultaneously happened in MM patients of plasmocytoma type with two cases at II phase and two cases at III phase. The simultaneous non-methylation of p15(INK4B) and p16(INK4A) genes were founded in five cases of MM patients, all of the tumor cells were of small plasmocyte type with mature differention. Conclusion suggested that there were high incidence of methylation of p15(INK4B) and p16(INK4A) genes in patients with MM. Hypermethylation can be detected in the early stage of disease, which was associated with its progress. It indicated a bad prognosis when methylation happended simultaneously in the two genes. Methylation of p15(INK4B) and p16(INK4A) genes may be related to the pathogeny of MM.
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AIM: To illustrate the expression of hypermethylation p15 gene in 53 non-Hodgkin’s lymphoma (NHL). METHODS: The methylation of p15 gene in 53 cases of non-Hodgkin’s lymphoma was detected by using methylation-specific PCR (MSP) technique. RESULTS: 18.9% (10/53) in NHL were methylation in p15 gene. p15 gene was frequently in high malignant NHL patients (27.3%) compared with in low malignant patients (0%). CONCLUSION: The result suggested that the hypermethylation of p15 may play an important role in non-Hodgkin’s lymphoma.
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AIM: IL-12 acts upon T lymphocytes and activates its receptor complexes of ?1/?2 ,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of T cells and expression and signal transduction of Fas/FasL during T cell apoptosis. METHODS: The apoptosis of T cells was detected by Annexin V staining cytometry and the expression of Fas/FasL under different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic T cell line(TIB-152) and the human lymphoma T cell line(HTB-176) and the normal human T cells to undergo apoptosis. The FasL expression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasL expression induced by IL-12 was inhibited by PKC inhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce T cells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of T cells. FasL participates in the progression of T cell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasL expression may be related with PKC pathway.
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AIM: To study the effect of IL-12 on T lymphocytes apoptosis, the expression of Fas/FasL and TNFR/TNF?. METHODS: Terminal dUTP nick end labeling(TUNEL) and Annexin V assay were used. Anti-TNFR were labeled with FITC, anti-CD95 was labeled with PE and Anti-FasL with biotin. Three kinds of T cells (HTB176,TIB152 and human normal T cells) were analysed through flow cytometry. RESULTS: At 1st hour after being treated with IL-12, the expression of FasL protein and FasL mRNA in HTB176 and TIB152 began to increase and reached peak value in 24 hours. In the normal T cells, FasL just began to increase in 1 hour and maintained stability in 6, 12 and 24 hours through the later experiment period. All three kinds of T cells displayed no change in the expression of CD95 and TNFR/TNF? under the stimulation of IL-12. CONCLUSION: Expression of such apoptosis regulating factors were different in the apoptosis of T cells induced by IL-12.
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AIM: To investigate the variation and distribution of abnormaly methylated p15 INK4B in myelodysplastic syndrome (MDS) and its subgroups. METHODS: The abnormal methylation of p15 INK4B in 32 cases with MDS was studied using methylation-specific PCR (MSP). RESULTS: The positive rate of abnormal methylation of p15 INK4B was about 50% in MDS. The ratios in subtypes were: 0% (0/6) in RA,20% (1/5) in RA-S,57.1% (4/7) in RAEB,74.1% (5/7) in CMML,85.7% (6/7) in RAEB-t, respectively.It was worth noticing that 4 cases represented abnormal methylation of p15INK4B during their transformation and progression into AML. CONCLUSION: The abnormal methylation in p15 INK4B might be one of the causes of MDS, which was related to pathologial process of MDS.Every subtype was not solitary classification completely. Abnormal methylation of p15 INK4B was apt to occur accompanying the progression and transformation of the subtypes.
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AIM: IL-12 acts upon T lymphocytes and activates its receptor complexes of ?1/?2 ,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of T cells and expression and signal conduction of Bcl-2 during T cell apoptosis. METHODS: The apoptosis of T cells was detected by Annexin V staining cytometry and the expression of Bcl-2 under different inhibitors were detected by the method of semi-quantitative PCR. RESULTS: IL-12 can induce the human leukemic T cell line(TIB-152) and the human lymphoma T cell line(HTB-176) and the normal human T cells to undergo apoptosis. The Bcl-2 expression at 6 hours of treatment with IL-12 increased aparently,and reached the max at 24 hours. But IL-12 did influence Bcl-2 expression. IL-12 can induce T cells to undergo apoptosis which is characterized by early membrane changes. CONCLUSION: The inducing effect is correlated with the concentration of IL-12 and the maturation of T cells. Bcl-2 takes part in the progression of T cells' apoptosis as a apoptosis mediator.