Enrichment of breast cancer stem cells using a keratinocyte serum-free medium / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Keratinocyte serum-free medium (K-SFM) is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells (CSCs) using K-SFM.</p><p><b>METHODS</b>A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum (FCS) was used as a control. CSCs were identified with flow cytometry using CD44(+)/CD24(-) as molecular markers. The expression of a variety of CSC markers (Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin) was analyzed with real-time PCR.</p><p><b>RESULTS</b>Much higher percentage of CSCs was achieved with K-SFM: 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin.</p><p><b>CONCLUSION</b>K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.</p>

Effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma, and to explore the intrinsic factors influencing H101 sensitivity.</p><p><b>METHODS</b>Stable human esophageal cancer cell line EC9706 cells with lower (EC9706-SCEA) and higher CEA expression (EC9706-CEA) were chosen, thawed and cultured, and then to analyse the influence of CEA expressed at different levels on cell growth. The cytotoxic effect of H101 was assayed by in vitro and nude mouse in vivo.</p><p><b>RESULTS</b>The cell growth experiment showed that the population doubling time of EC9706-SCEA, EC9706-CEA and EC9706 cells were (30.9 ± 2.0) h, (31.1 ± 2.5) h and (29.1 ± 2.6) h, respectively, showing no significant difference among them (P > 0.05). The cytotoxic activity of H101 was higher on EC9706-SCEA than on other four groups, when MOI was ≥ 0.01 PFU (P < 0.05). The mouse experiment showed that H101 inhibited the growth of transplanted tumors in all experimental groups. Its effect on CEA-silenced tumors (inhibition rate was 61.5% to 74.5%) was significantly higher than that on CEA-overexpression tumors (32.3% to 38.5%) and control EC9706 transplanted tumors (35.5% to 44.8%). There was a significant difference between them (P < 0.05).</p><p><b>CONCLUSIONS</b>The results in vitro and in vivo experiments show that H101 can enhance the cytotoxic effect on EC9706 cells with lower CEA expression. To silence the expression of CEA may provide a novel strategy for target gene therapy of esophageal carcinoma.</p>

Construction of a recombinant lentiviral expression vector carrying carcinoembryonic antigen gene and its expression in dendritic cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a lentiviral expression vector of human carcinoembryonic antigen (CEA), and identify its expression in dendritic cells (DCs).</p><p><b>METHODS</b>Human CEA-encoding sequence was amplified, purified, ligated with lentiviral vector plasmid pLentiGFP and verified by sequencing. The verified recombinant vector plasmid (pLentiGFP-CEA), the packaging plasmid p 8.2 and pVSV-G were transfected into 293T cells by Lipofectamine(TM) 2000 reagent. The supernatant of the cultured 293T cells was collected to infect the DCs. The expression of CEA in the transfected DCs was assayed by RT-PCR and Western blotting.</p><p><b>RESULTS</b>CEA lentiviral vector was highly expressed in the transfected DCs as observed using fluorescence microscope 48 h after the the transfection. The human CEA gene was successfully amplified by RT-PCR with a length of about 2100 bp. Western blotting also showed CEA expression in the transfected DCs.</p><p><b>CONCLUSION</b>The human CEA lentiviral expression vector has been successfully constructed and the functional CEA protein can be expression in the transfected DCs. This facilitates further studies of the function of CEA at the molecular level.</p>

Preliminary study of DNA polymerase beta gene silencing by small interfering RNA in human gastric cancer BGC-823 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the influence of DNA polymerase beta (polbeta) gene silencing by small interfering RNA on biological behavior of human gastric cancer cell line BGC-823.</p><p><b>METHODS</b>The siRNA eukaryotic expression vectors targeting polbeta gene were constructed and transfected into BGC-823 cells by liposome. Stable cell lines were screened with G418. The expression levels of polbeta mRNA and protein were detected by real time PCR and Western blot in the cells of each group. The proliferation of each group was detected by flow cytometry and tumorigenicity was determined in nude mice.</p><p><b>RESULTS</b>The siRNA expression vector targeting polbeta gene was successfully constructed. The expression levels of polbeta mRNA and protein were significantly reduced in the experimental group transfected with siRNA expression vectors targeting polbeta, and the silencing effect of pRNAT-U6.1-sipolbeta2 (suppression degree was 83%) was stronger than that of pRNAT-U6.1-sipolbeta1 (depression degree is 56%). Compared with irrelevant siRNA control group, empty vector control group and untransfected group, the ratio of G0/G1 cells was increased, proportion of S phase cells and cell proliferation were decreased in the experimental group 1 cells transfected with pRNAT-U6.1-sipolbeta1 (P < 0.05). On the contrary, the ratio of G1/G0 was decreased, proportion of S phase cells and cell proliferation was increased in the experimental group 2 cells transfected with pRNAT-U6.1-sipolbeta2 (P < 0.05).</p><p><b>CONCLUSION</b>The siRNA expression vectors targeting DNA polymerase beta gene can significantly inhibit the expression of polbeta mRNA. Neither high nor extremely low expression of polbeta is beneficial to maintain the cellular physiological functions. The expression of polbeta silenced to a proper level by siRNA may play an important role in inhibiting tumorigenesis.</p>

Construction of a siRNA vector targeting human MTA1 gene and the gene-silencing effect / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells.</p><p><b>METHODS</b>The siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively.</p><p><b>RESULTS</b>The double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA (481-499), which induced almost complete silencing of MTA1 protein expression.</p><p><b>CONCLUSION</b>The siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.</p>

Effect of CTL on K562 cell induced by exosomes and in combination with CPG OND / 中国实验血液学杂志

To investigate the specific anti-leukemia effect of cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) activated by exosomes alone or in combination with CpG ODN in vitro and the feasibility of exosomes as remedial vaccine, the DCs induced from normal volunteer PBMNCs were divided into 7 groups. Three groups of them were added with the exosomes: Kexo (exosomes derived from K562 cells) or DCexo (exosomes derived from DCs induced from K562 cells) or FTexo (exosomes derived from DCs induced from K562 cells and pulsed by freeze-thawing antigen of K562 cells) as experimental groups (Kexo, DCexo and FTexo). The other three groups were added with CPG ODN while added the exosomes (Kexo, DCexo and FTexo), and were used as experimental groups also (Kexo+CpG, DCexo+CpG and FTexo+CpG). The seventh group DCs was added with nothing as blank control. These DCs above mentioned were cultured continuously for 72 hours. The T lymphocytes were co-cultured with DCs for another 72 hours to generate CTL. Then, the killing effects of them on K562 cells were determined by MTT assay. The results showed that all experimental groups pulsed by exosomes displayed stronger killing effect, compared with control group (p<0.05). DCexo and FTexo displayed stronger killing effect too, compared with Kexo (p<0.05). CPG ODN as an adjuvant could enhance the killing effect (p<0.05). It is concluded that the special killing effect on K562 cells can be induced by exosomes, CPG ODN as an adjuvant can enhance the killing effect. Exosome is hopeful as a remedial vaccine to be used for the leukemia therapy.

Effects of novel human chemokine-like factor superfamily 8 on proliferation and EGFR expression of HL-60 cells / 中国实验血液学杂志

This study was aimed to investigate the effect of chemokine-like factor superfamily 8 (CKLFSF8) on proliferation and expression of epidermal growth factor receptor (EGFR) of HL-60 cells. Expression of CKLFSF8 mRNA on HL-60 cell line was assayed by RT-PCR; the target gene was transfected into the cells by lipid vector, cell proliferation was determined by MTT assay, while expression of EGFR in HL-60 was determined by immunocytochemical technique. The results indicated that expression of CKLFSF8 existed in HL-60 cells. After transfection, cell proliferation was inhibited (P < 0.05) and the expression of EGFR in HL-60 cells was also discovered to be inhibited (P < 0.05). It is concluded that the proliferation and expression of EGFR in HL-60 cells can be inhibited by transfection of CKLFSF8. The novel chemokine may provide a new approach in the treatment of leukemia.

cDNA microarray-based study of gene expression profile changes in human esophageal squamous cell carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the differentially expressed genes between human esophageal squamous cell carcinoma (ESCC) and normal esophageal mucosa and explore an effective method with high throughput for screening the molecular markers closely correlated with the development, invasion and metastasis of ESCC.</p><p><b>METHODS</b>With cDNA microarray and laser capture microdissection, T7-based amplification were used to detect the mRNA from both the primary carcinoma and the corresponding esophageal epithelium in 15 ESCC cases, and the results were analyzed by bioinformatics methods.</p><p><b>RESULTS</b>Among the 886 target genes, 110 (12.42%) genes were differentially expressed commonly at least twice in all the 15 samples, including 56 (6.32%) up-regulated by at least 2 folds and 54 (6.09%) down-regulated by at least 0.5 folds.</p><p><b>CONCLUSION</b>Many ESCC-associated genes were screened by the high-throughput gene chip method, and functional study of these genes may help to identify the key genes or pathways involved in the pathogenesis and development of ESCC.</p>

Production of specific CTL induced by exosomes derived from K562 cells / 中国实验血液学杂志

The aim of this study was to investigate whether exosomes derived from K562 cells and human monocyte-derived dendritic cells (DCs) transfected with total RNA of K562 cells are capable of inducing antigen-specific cytotoxic T lymphocytes (CTL) responses in vitro. DCs were generated from peripheral blood mononuclear cells (PBMNC) of healthy volunteers in the presence of GM-CSF and IL-4, and then were transfected with K562 RNA by using DOTAP lipofection. Exosomes was extracted from the supernatant of DCs and K562 cells. The T cell were activated to be tumor specific CTL after DCs and exosomes were co-cultured with autologous T cells derived from healthy volunteers' PBMNC. The effect of CTL on K562 cells was detected by MTT assay. The results showed that treatment of T cells with exosomes derived from K562 cells or DCs transfected with total RNA of K562 cells could significantly promote their killing ability on K562 cells as compared with untreated T cells (P < 0.05). The killing ability of T cells treated with exosomes on K562 cells was stronger than on HL-60 cells (P < 0.05). It is concluded that the specific CTL immune response to leukemia cells can be induced by exosomes derived from K562 cells.

Study on the association of polymorphisms in homocysteine metabolism related enzymes with deep venous thrombosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the significance of gene mutation of methylenetetrahydrofolate reductase (MTHFR) C677T, methionine synthase (MS) 2756 AG and cystathionine beta-synthase (CBS) 844ins68 in the development of deep venous thrombosis.</p><p><b>METHODS</b>One hundred and three cases of deep venous thrombosis (DVT group) and 250 healthy subjects (control group) were recruited in the study. The polymorphisms of MTHFR C677T, MS A2756G and CBS 844ins68 were detected by PCR-restriction fragment length polymorphism(PCR-RFLP).</p><p><b>RESULTS</b>The prevalences of TT genotypes of MTHFR (C677T) between DVT group and normal control group had significant difference (27.2% vs 17.2%, P< 0.05), the prevalence of AG genotypes of MS A2756G in the DVT group was less than that in the control group (9.7% vs 19.2%, P< 0.05). The prevalence of 677T-2756A haplotype in the DVT group was higher than that in the control group (P< 0.05), the prevalence of 677C-2756A haplotype in the DVT group was less than that in the control group (P< 0.05). There were no significant differences in the prevalences of CBS 844ins68 mutation.</p><p><b>CONCLUSION</b>The homozygote of MTHFR C677T (TT) may be a risk factor of DVT. MS A2756 G(AG) genotypes may reduce the development of DVT. The 677T-2756A haplotype may be a risk factor of DVT. The 677C-2756A haplotype may be a protective factor of DVT. The prevalence of gene mutation of CBS 844ins68 might vary with different ethnic group or geographic regions.</p>

Rapid detection of the genotyping of hepatitis C virus using DNA chip with coloration methods / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To develop a new DNA chip with coloration, which can be used for rapid and economical detection of the genotyping of hepatitis C virus (HCV).</p><p><b>METHODS</b>Probes and primers were designed according to the sequence of HCV 5' non-coding region (5' NCR) to fabricate DNA chip. Experimental group consisted of 60 positive serum samples and control group consisted of 20 negative serum samples. To obtain the aimed gene, then they were hybridized with DNA chip. Finally, the results showed in a nylon film. The results of DNA sequencing of samples were used as the control in double blind experimental.</p><p><b>RESULTS</b>Using DNA chip, HCV was detected in positive of all serum specimens of experimental group and negative in control group. The determination of HCV genotype by DNA chip showed corresponding rate of 96.7% with those by sequence assay.</p><p><b>CONCLUSION</b>It showed higher specialty and sensitivity using DNA chip to detect the genotype of HCV. It would be valuable for the clinical genotyping of HCV</p>

Studies on the activation of oncogenes by alternariol in human fetal esophageal epithelium / 中国病理生理杂志

This paper reported that the activation of oncogenes in human fetal esopha geal epithelium treated by alternariol (AOH). It was found that NIH/3T3 cells were transformed via transfeetion of DNA extracted from human fetal esophageal epithelium which was cultured and treated by 10?g/ml AOH in a short term in vitro. The efficiency of primary loci was 0.17 focus per ?g of DNA. In the secondary transfection, the efficiency was 0.58 focus per ?g of DNA (P