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Objective To screen the dual oxidase maturation factor 1 (DUOXA1) gene mutations in children with congenital hypothyroidism (CH) and thyroid goiter from Shandong Province,China,and to identify the gene mutation type and characteristics of DUOXA1 gene mutations in order to provide some evidence for gene diagnosis and therapy of CH.Methods A cohort of 52 cases of CH with thyroid goiter and 100 normal controls were selected according to neonatal screening system in Shandong Province whose genomic DNA was isolated from peripheral blood leukocytes with a standard phenol chloroform method.The whole coding sequence (CDS) of DUOXA1 gene was amplified with 8 pairs of sequence specific primers by using PCR.The PCR products were directly sequenced with Sanger sequencing to detect new mutations types of DUOXA1 gene.The sequencing data were compared to the DUOXA1 gene reference sequence(National Center for Biotechnology Information:RefSeq:NG_033105.1) to see if there was any mutation.Ax2 test was done for the gene frequency of discovered single nucleotide polymorphisms (SNP).Results There was no mutation in CDS of 52 CH patients with thyroid goiter and 100 normal controls.However,a SNP (rs75981505,c.398G ＞ T) which was an missense mutation and could lead to a change of the codon from CGC to CTC,was found in 9 CH patients with thyroid goiter and 11 normal controls in the exon 7.The corresponding amino acid arginine was replaced by histidine(p.Arg133His).There was no significant difference in the SNP rate between CH patients with thyroid goiter and normal controls (17.3％ vs 11.0％,x2 =1.24,P ＞ 0.05).Conclusion DUOXA1 gene mutation rate is very low which may not be the main cause of CH patients with thyroid goiter in the population of Shandong Province.
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Objective To study the thyroid hormone receptor β(TRβ)gene mutation types and characteristics in children with congenital hypothyroidism(CH)and thyroid dysgenesis(TD)from Shandong Province,and to provide theoretical basis for gene diagnosis and prenatal diagnosis. Methods Sixty cases of TD patients of which genomic DNA were isolated from peripheral blood leukocytes were selected by neonatal screening system in Shandong Province. The exon 6 to 12 of TRβ gene were amplified with 8 pairs of sequence specific primers using PCR and the first generation of sequencing method(Sanger method)to detect mutation. The sequencing results were compared with the TRβ gene reference sequence[National Center for Biotechnology Information(NCBI)Reference Sequence:NC 000003. 12]to see whether there was a mutation. Results Analysis of TRβ in 60 cases of CH patients with TD revealed no mutation was demonstrated in exons 6 - 12,but 2 single nucleotide polymorphism(SNP)( rs 3752874,c. 735C ﹥ T;rs79220627, c. 162G ﹥ A)were detected. Through the analysis,the 2 SNP were all synonymous mutations(Phe→Phe;Ser→Ser), without the change of the amino acids. Conclusions TRβ mutation rate is very low,which may not be the main mutation type in CH patients with TD in Shandong Province.
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Objective: To explore the effect of the fat-1 gene encoding n-3 fatty acid desaturase on the proliferation and apoptosis of human colon cancer cell line HT-29 and to explore its value in the gene therapy for colorectal cancer. Methods: The fat-1 gene was cloned into adenovirus shuttle vector pAd-CMV and then recombinated with backbone vector to con-struct a recombinant adenoviral vector pAd.GFP.fat1. The vector was transfected into 293 cells to get the recombinant virus infected human colon cancer cell line HT-29. Total RNA of the cells was analyzed by Northern blot. The effect of fat-1 gene on the proliferation and apoptosis of the infected cells was analyzed by flow cytometry. The content of n-6PUFAs/n-3PUFAs was analyzed by Gas Chromatography. The anti-cancer effect of fat-1 gene was studied on HT-29 xenografts in nude mice in vivo. Results: The high titer recombinant virus was obtained. Fat-1 gene can be highly expressed in human colon cancer call line HT-29. Compared with that of the control cells (Ad.GFP), proliferation of HT-29 cells was inhibited by fat-1 gene. Fat-1 gene can lower the ratio of n-6/n-3PUFAs. The growth of tumors in nude mice is also inhibited by fat-1 gene. Conclu-sion: Fat-1 gene is of great value in the gene therapy for colorectal cancer.
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Objective:To investigate the effects of soluble factors secreted by MCF-7 breast cancer cells on the differentiation,maturation and function of DCs.Methods:Mononuclear cells were cultured with the culture supernatants from primary MCF-7 cells.Combination of granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin 4(IL-4) and tumor necrosis factor-a(TNF-a) was used to stimulate the cultured mononuclear cells.Then DCs and CTL assays were analyzed.Results:MCF-7 cell supernatant-treated DCs resulted in low expression of CD80,CD83,CD86 and HLA-DR,and the inhibition rate of CTL was 17.35% significantly lower than 56.14% induced by the control DCs(P＜0.01).IL-12 secreted by DCs and IFN-γ produced by PBMNC were all significantly lower than those in the control group (P＜0.01).Conclusion:Experiments in vitro shows that the culture supernatants from MCF-7 breast cancer cells could inhibit the development and functions of DCs.
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BACKGROUND: Chondroitin sulfate is the important component of cell matrix, it can accelerate the proliferation of tumor cells and restrain its ransfer.OBJECTIVE: To observe the effects of chondroitin sulfate on the proliferation and differentiation of HL60 cells under the action of adriamycin.DESIGN: An open experiment.SETTING: Department of Biochemistry and Molecular Biology, Medical College of Qingdao University.MATERIALS: The experiments were carried out in the Research Room of Biochemistry and Molecular Biology, Medical College of Qingdao University of September 2003 to December 2004. Experimental materials and reagents: HL60 cell strains, which were the cells from promylocytic leukemia, were purchased from Shanghai Cell Bank, Chinese Academy of Medical Sciences; Bovine cartilage chondroitin sulfate (Sigma) was also used.METHODS: ① After the passage and culture, the cells at the logarithmic proliferative phase were dispensed into cell suspension of 1×108 L-1 with RPMI1640 culture medium containing inactivated fetal bovine serum of 0.1in volume fraction, and then filled into the culture bottles with 4 mL in each bottle for a total of 45 bottles. ② Chondroitin sulfate was added to 15 bottles filled with cell suspension according to the concentrations of 0, 5,25, 50 and 75 mg/L respectively, and 3 bottles for each concentration, and 0.01 mol/L phosphate buffer (pH7.2) was added in the blank control group. Then the density of HL60 cells was determined by cell counting after treatment of chondroitin sulfate. ③ Thirty bottles filled with cell suspension were divided into chondroitin sulfate+adriamycin group and chondroitin sulfate group, 15 bottles in each group. Chondroitin sulfate of 0, 5,25, 50 and 75 mg/L was added to the two groups, and 3 bottles for each concentration, and 0.01 mol/L phosphate buffer (pH7.2) wass adokd in the blank control group. Then the survival rate of chondroitin sulfate treated HL60 was detected after adding adriamycin. ④ Chondroitin sulfate was added to 15 bottles filled with cell suspension according to the concentrations of 0, 5, 25, 50 and 75 mg/L respectively, and 3 bottles for each concentration, 0.01 mol/L phosphate buffer (pH7.2) was added in the blank control group. The activity of acid phosphatase was detected with enzymelinked immunosorbant assay (ELISA) in each group, and the effect of the cell differentiation was observed.MAIN OUTCOME MEASURES: ① Effects of chondroitin sulfate on the proliferation of HL60 cells; ② Effects of chondroitin sulfate plus adriamycin on the survival rate of HL60 cells; ③ Effect of chondroitin sulfate on the activity of acid phosphatase of HL60 cells.RESULTS: Totally 45 bottles of cell suspension were prepared, and all were involved in the analysis of results. ① As compared with the blank control group, the densities of HL60 cells at 24 hours after treated with chondroitin sulfate of different concentrations were all significantly increased (P ＜ 0.01), which were increased more obviously in the 50 and 75 mg/L chondroitin sulfate treated groups, and there was no significant difference between the two groups (P ＞ 0.05), which indicated that chondroitin sulfate within the range of concentration did not accelerate the growth of cells greatly. ② As compared with the blank control group, the survival rates of HL60 cells in the chondroitin sulfate+adriamycin groups were decreased to different extents after chondroitin sulfate of different concentrations were added, and it decreased obviously when the concentration of chondroitin sulfate was higher than 25 mg/L (P ＜ 0.01). ③ As compared with the blank control group, the A values of acid phosphatase of the HHL60 cells were all obviously increased in the 5, 25 and 50 mL chondroitin sulfate treated groups (1.268±0.038, 1.305±0.101, 1.321±0.021,1.354±0.013, P ＜ 0.01 or 0.05), especially that it reached 1.406±0.113 in the 75 mL chondroitin sulfate treated group, which was extremely and significantly different from that in the blank control group (P ＜ 0.001).CONCLUSION: Chondroitin sulfate with a proper concentration can accelerate the proliferation of HL60 cells, and it can increase the sensibility of HL60 cells to adriamycin, and promote the differentiation of HL-60 cells.
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Objective To investigate the effect of fat-1 gene encoding n-3 fatty acid desaturase on the proliferation and apoptosis of colon cancer cell HT-29. Methods fat-1 gene was transfected into HT-29 cells by liposomal reagent. The expression of fat-1 gene was detected by fluorescent micrographs and RT-PCR. Gas chromatography, MTT and flow cytometry were used to examine the change in n-6/n-3 PUFAs ratio, proliferation, cell cycle and apoptosis, respectively. Results After transfection of fat-1 gene, n-6/n-3 PUFAs ratio decreased significantly. Apoptosis of HT-29 cells was induced and cell cycle was changed. Apoptosis mainly appeared in the synthesis phase. Conclusion fat-1 gene encoding n-3 fatty acid desaturase can significantly decrease n-6/n-3 ratio. Consequently, apoptosis was triggered and cell cycle was changed. Tranfection of fat-1 gene into HT-29 cells may be a new potential treatment for colon cancer.