Objective
To investigate the tumor suppressor genes of phlegm DNA in smokers, and analyze the correlation between methylation level of tumor suppressor gene promoter and chronic mucus hypersecretion (CMH).
Methods
The study recruited the patients who were admitted in the respiratory department during 2013-2016 in this hospital, including 700 cases of urban smokers and 380 cases of rural smokers. Eleven genes commonly silenced by promoter methylation in lung cancer and associated with cancer risk were selected. Methylation specific PCR (MSP) was used in the sputum sample of 700 individuals in the urban smokers cohort. Replication was performed in 380 individuals from the rural smokers cohort.
Results
CMH was significantly associated with an overall increased number of methylated genes, with SULF2 methylation demonstrating the most consistent association. The association between SULF2 methylation and CMH was significantly increased in males but not in females both in the urban and rural groups (OR=2.73, 95%CI 1.53-4.93, P=0.001; OR=2.96, 95%CI 1.47-5.94, P=0.002, respectively). Furthermore, the association between methylation and CMH was more obvious among 139 male former smokers with persistent CMH compared with current smokers (SULF2, OR=3.64, 95%CI 1.57-8.35, P=0.002).
Conclusion
These findings demonstrate that especially male former smokers with persistent CMH have markedly increased promoter methylation of lung cancer risk genes and potentially could be at increased risk for lung cancer.
Objective To compare gene express difference ofkeloid and normal skin tissues by using the suppression subtractive hybridization (SSH) so asto find the differential express gene in keloid. Methods mRNA extracted fromkeloid and normal skin tissues was used as the template to synthesis cDNA of keoid and normal skin. The cDNA of keloid served as a tester, the cDNA of normal skin as a driver. cDNA was digested with RsaⅠ. Adaptor-ligated tester cDNA was prepared. Then first hybridization, second hybridization and PCR amplificationwere done. Differentially expressed cDNA was selectively amplified during thesereactions. After SSH, the PCR mixture was ligated with T-vector. The positive clones were selected and the insert gene fragments were analyzed. Southern hybridization identified the keloid differential express genes. The positive clones ofSouthern hybridization were selected, and these sequences were analyzed. The results were compared with that of GeneBank. Results Thirteen differential genes were found in keloid, of which 11 gene clones have been known their function, and 2 clones have not known their function. 〖WTHZ〗Conclusion Keloid differentially expressed gene was screened successfully by SSH.
ObjectiveTo identify mutations in NDP, FZD4, LRP5, TSPAN12 in Chinese families with familial exudative vitreoretinopathy (FEVR) and observe the clinical features.MethodsRetrospective case series study. The 9 patients (18 eyes) and 5 normal members from 4 unrelated families were included in the study. The patients medical history and family history were collected in detail. All patients underwent best corrected visual acuity (BCVA), slit-lamp biomicroscopy, fundus colorized photography, fundus fluorescein angiography (FFA). Genomic DNA were collected from all the patients. Mutations were detected by directly sequencing to the whole coding region and exon-intron boundaries of NDP, FZD4, LRP5 and TSPAN12 gene. Polyphen and SIFT programs were used to predict the effects on the structure and functional properties of mutant protein.ResultsThere were two affected individuals in the family 2 carried LRP5 gene mutation [c.1330C＞T (p.R444C )] in exon 6 by sequence analysis. A score of 0.882 was acquired by Polyphen program analysis. And the missense change was predicted to be pathogenic by SIFT. Fundus changes of the proband showed angioplasia, tortuosity of peripheral vessels. And temporal dragging of the optic disc, peripheral avascular zone, neovascularization were found in FFA. Brush-like and straight of peripheral vessels were found in Ⅰ1. No variant was found in NDP, FZD4 and TSPAN12 gene.ConclusionOur study supports the gene mutation c.1330C＞T (p.R444C) of LRP5 is pathogenesis of FEVR. Patients with the same mutation could have variable phenotypic characteristics.
【Abstract】Objective To explore the relation between the expression of telomerase and DNA ploidy with biliarypancreatic system cancer, so as to find a better way to diagnose and distinguish jaundice between malignance and benign disease.Methods Endoscopic retrograde cholangiopancreatography (ERCP) were performed before operation in patients with obstructive jaundice. The bile and pancreatice juice were collected before ERCP. Biopsy specimens from part of patients were obtained during ERCP. All cancer specimens were possessed once again during operation and were assessed by the activity of telomerase and DNA ploidy. Results ① Telomerase positive rate 〔87.50%(56/64)〕 of tissue specimens in malignant obstructive jaundice were higher than that in benign obstructive jaundice 〔3.33%(2/60)〕，P=0.000. ② Telomerase positive rate〔71.88%(46/64)〕of Bile and pancreatice juice in malignant obstructive jaundice were higher than that in benign obstructive jaundice 〔3.33%(2/60)〕， P=0.000, tissue specimens obtained by endoscopy with malignant obstructive jaundice had detectable telomerase activity, positive rate was 83.33%（20/24）. ③ The rate of DNA heteroploid with malignant obstructive jaundice was 62.50%（40/64）, that of diploid can be seen in all patients with benign obstructive jaundice, the difference was statistically significant (P=0.000). ④ The rate of telomerase positive and DNA heteroploid in high differentiation tumor were significantly lower than in middlelow differentiation tumor （P=0.028，P=0.001）.Conclusion Applying the duodenoscope we collected the bile and pancreatic fluid before operation and obtain biopsy specimens whose telomerase activity and DNA ploid were detected. This is simple, safe, quick method which can identify the malignant and benign obstructive jaundice.
Objective
To construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits.
Methods
Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule.
Results
The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 108 TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% ± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm.
Conclusion
Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.
Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.
Objective To clone the genes of nogo-66 and NEP1-40 from spinal cord of rat and to realize the expression of its protein in vitro. Methods The nogo-66 and NEP1-40 genes were cloned from the spinal cord of juvenil rat by use of RT-PCR techniques, and the objective genes were bonded to T vector through gene coupled action, recombinant plasmid were sequencing, and the genes were cloned into PQE30-GST vector, then the recombinant plasmids were induced by isopropylthiogalactoside(IPTG) to express the proteins. The two proteins were purified by Ni-column and detected by using Westernblot test. Results The Nogo-66 and NEP1-40 genes were successfully cloned from rat, which were 215 bp and 137 bp for each one when add the enzyme site. No gene mutations were detected in the two genes after sequencing. The expression plasmids were cut by the two enzyme (BamH Ⅰ and Hind Ⅲ), the target bands were seen on the results of electrophoresis. The expression plasmids were induced by IPTG and got the purified GST fusion protein nogo-66 and NEP1-40, which relative molecular weight were 33.2×103 and 30.3×103 respectively. The results of Westernblot test confirmed that the antigenicity of the two proteins was precise. Conclusion Nogo-66 and NEP1-40 proteins can be expressed in a high efficiency in vitro using genetic engineering, so it provides a good basis for further research on its function and vaccine for spinal injury.
Objective To study the expression and clinic significance of nm23 gene (product of uncleoside ciphosphate kinase) in the pancreatic adenocarcinoma tissues. MethodsSP immunohistochemical method was used to examine the expression of nm23/NDPK in 40 pancreatic adenocarcinoma and 14 normal pancreas tissues.ResultsTwentysix of 40(65%) pancreatic adenocarcinoma showed b immunoreactivity for NDP kinase, whereas 4 of 14 (28.5%) normal pancreatic tissues showed weak immunoreactivity. Significant difference was found between the two groups (P＜0.05). The nm23/NDPK expression levels in pancreatic adenocarcinoma of lower differentiation was higher than those in pancreatic adenocarcinoma of higher differentiation (10/11，90.9%; 2/8, 25%; P＜0.05). Positive staining was associated with higher incidence of lymph node metastasis (10/14, 71.4%) than negative staining (6/19,31.5%, P＜0.05). These results suggested that nm23/NDPK expression was positively associated with lymph node metastasis and aggressiveness. They also suggested that nm23/NDPK expression had negative correlation with the extent of histologic differentiation. Conclusion nm23/NDPK can serve as a marker for malignant potentiality and indicate the prognosis of pancreatic adenocarcinoma.
【Abstract】ObjectiveTo investigate the effects of As2O3 on expression of NF-κB p65, survivin and caspase-3 in human breast infiltrating duct carcinoma xenograft model on nude mice. Methods A human breast infiltrating duct carcinoma model on nude mice was established and the nude mice were divided randomly into three groups: control group, DDP group and As2O3 group （1.5 and 3.0 mg/kg concentrations）. The expression of survivin mRNA was detected with the method of in situ hybridization and the expressions of NF-κB p65, survivin and caspase-3 protein were measured with immunohistochemistry. ResultsThe positive rates of NF-κB p65 and survivin expression were higher in the control group than those in the DDP group and the As2O3 groups, but that of caspase-3 was on the opposite way (P＜0.01). The positive rates of NF-κB p65 and survivin in As2O3 group were negatively related with the concentrations of As2O3 (P＜0.01), but that of caspase-3 was on the opposite way (P＜0.01). The expressions of NF-κB p65 and survivin protein were positively correlated with that of survivin mRNA, but any of them was negatively correlated with the expression of caspase-3 protein. ConclusionAs2O3 inhibites survivin probably by inhibiting the activity of NFκB p65 and subsequently activates caspase-3, which induces apoptosis of human breast infiltrating duct carcinoma cells and is in a dose-dependent manner.
OBJECTIVE: To study the stimulating effects of basic fibroblast growth factor(bFGF) on fibroblast function and its ability to expression of c-fos gene. Furthermore, to explore the possible network action between bFGF and oncogene in modulating wound healing. METHODS: Cultured rat fibroblasts were divided into bFGF stimulating group and control group. Fibroblasts in bFGF stimulating group were treated with bFGF in a dosage of 40 ng/culture hole, while the control fibroblasts were treated with the same vehicle without bFGF. The morphology, cell vitality and their ability to express c-fos gene in the fibroblasts in both groups were studied with MTT and immunohistochemical methods. RESULTS: All fibroblasts in bFGF treated groups were enlarged and showed increased vitality with MTT method. C-fos gene expression in bFGF stimulating group was increased, especially in nucleus when compared with those in control group. CONCLUSION: The results show that the function and the ability to express c-fos gene in bFGF treated fibroblasts are enhanced. Combined with our previous studies, it may make a conclusion that there is a network regulation mechanism between growth factors and some oncogenes.
