Objective To study the effects of high matrix metalloproteinase 9 (MMP 9 ) on the biological behaviors of fibroblasts,in order to clarify the possible mechanisms in the wound healing of diabetic foot.Methods Establish the cell model of skin fibroblast with high expression of MMP 9 by high glucose (22.0 mmol/L) and high homocysteine (100 μmol/L) co-culture.Control group was incubated with normal glucose (5.5 mmol/L).Realtime PCR,ELISA and gelatin zymography were used to detect the MMP 9 mRNA,protein expression and activity of MMP 9.Flow cytometry,CCK-8,ELISA assay,scratch test and transwell were used to detect cell proliferation,viability,collagen (hydroxyproline) secretion,horizontal migration and vertical migration of cells.Results Data were expressed as (x- ± s).Differences were considered significant if their P value was ＜ 0.05.Results The expression of MMP 9 mRNA,protein levels and the activity of MMP 9 in high MMP 9 group were 6.05 folds,4.12 folds and 1.58 folds higher than those in control group (P ＜ 0.01,respectively).The proportion of S phase cells,proliferation index,cell viability,collagen (hydroxyproline) secretion,6 h horizontal migration rate and the number of vertical migration cells in high MMP9 group decreased by 29.8 ％,18.1％,23.3 ％,68.7 ％,45.0 ％ and 21.4 ％ than those in control group (P ＜ 0.01,respectively ). Conclusions Fibroblast with high expression of MMP 9 have decreased proliferation,activity,collagen secretion and reduced migration,which suggests that MMP 9 may inhibit the biological behaviors of fibroblasts.

Objective To explore the value of prenatal genetical diagnosis by mutation analysis of achondroplasia (ACH) fibroblast growth factor receptor 3 (FGFR3) gene.Methods Genomic DNA from nine ACH patients and their parents in Gansu Maternal and Child Health Hospital from July,2010 to December,2014 was prepared for polymerase chain reaction.Direct sequencing revealed the samples were performed after amplification of exon 10 of FGFR3 containing the potential mutation.Fetal DNA was extracted from cells in both amniotic fluid and umbilical cord,and then exon 10 of FGFR3 was also tested.Three fetuses with short-limb dysplasia were also included and prenatal diagnosis was offered to them through amniocentesis or cordocentesis.Results Prenatal ultrasonography test showed shorter femoral length,which was less than 2-3 standard deviation of normal reference dysplasia fetal performance for femoral short.Femur length is lower than 2-3 standard deviation minus normal value,and discrepancy in biparietal diameter compared with fetuses at the same gestational age.In the four families with one ACH parent,c.1138G ＞ A heterozygous mutation was detected in all of the four mothers,while two fetuses among them showed c.1138G ＞ A heterozygous mutation mutation and the other two were normal.There were other two fetuses with c.1138G ＞ A heterozygous mutation from other two families,one's father had c.1138G ＞ A heterozygous mutations,but not the mother,the other had c.1138G ＞ A heterozygous mutations in both the mother and father.Among the three families with unaffected parents but each had a de novo c.1138G ＞ A mutation child,no mutation of c.1138G ＞ A genotype was detected in their fetuses,neither in the three fetus with short limb dysplasia.Four fetuses with a c.1138G ＞ A mutation and three with short-limb dysplasia were terminated.The other five fetuses whose genotype was normal were born and healthy with normal phenotype at one-year-old follow-up.Conclusion FGFR3 genetic analysis could provide information for genetic counseling and prenatal diagnosis for ACH parents or parents who had an ACH baby to prevent birth defect.

BACKGROUND:The high level of matrix metalloproteinase 9 (MMP9) is thought to slow down the healing of diabetic foot ulcers. Whether it can influence the biological behaviors of skin fibroblasts and affect wound healing is stillunclear. The present study aimed to observe changes in the biological behaviors of rat dermal fibroblasts induced by high expression of MMP9 and to clarify the possible mechanisms of wound healing for diabetic foot. METHODS:A cellmodel of skin fibroblast with high expression of MMP9 was established by co-culture of high glucose (22.0 mmol/L) and homocysteine (100 μmol/L). A control group was incubated with normal glucose (5.5 mmol/L). Realtime PCR, ELISA and gelatin zymography were used to detect the MMP9 mRNA, protein expression and activity of MMP9. Flow cytometry, CCK-8, ELISA assay, scratch test and transwellwere used to detect cellproliferation, viability, collagen (hydroxyproline) secretion, horizontal migration and vertical migration of cells. The data were expressed as mean±SD. P value less than 0.05 was considered statistically significant. RESULTS:The expression of MMP9 mRNA, protein levels and the activity of MMP9 were much higher in the high MMP9 group than in the control group (7.05±1.02 vs. 1.00±0.00, 206.9±33.6 pg/mL vs. 40.4±5.9 pg/mL, and 1.47±0.13 vs. 0.57±0.12, respectively,P<0.01). The proportion of S-phase cells, proliferation index, cellviability, collagen (hydroxyproline) secretion, horizontal migration rate and the number of vertical migration cells were lower in the high MMP9 group than in the control group (P<0.01). CONCLUSION:Fibroblasts with a high expression of MMP9 decreased proliferation, activity, secretion and migration of collagens, suggesting that MMP9 may inhibit the biological behaviors of fibroblasts.

Objective To observe the influence of coal burning fluorosis on learning and memory ability in rats and reveal its possible mechanisms. Methods Healthy 48 SD rats were divided into control, low-fluoride and high-fluoride group. All rats in fluoride exposed groups were fed with the eom polluted by drying processes with burning coal containing high level of fluoride obtained from the endemic fluorosis area to produce the animal model of fluorosis. The experiment period were 3,6 mouths, respectively. The ability of leaning and memory was measured by Morris test and cholinesterase activity detected by photometric method at 3 or 6 month after experiment, respectively. Results Fluoride contents signifieantlly influenced the escape latency, the numbers of crossing the platforms and the time of staying the platforms(the value of F was 29.29,6.47,6.50, respectively, P<0.01).In addition, the numbers of crossing the platforms and the time of staying the platforms were influenced by the exposed time(the value of F was 16.11,45.59, P<0.01). Furthermore, the fluoride contents and the exposed time had an interaction between the numbers of crossing the platforms and the time of staying the platforms (the value of F was 4.67,5.68, P<0.05 or<0.01). Three months after the experiment, the mean values of escape latency [(14.71± 4.85)s] of rats in highly fluoride exposed group were significantly prolonged as compared with controls [(9.28±4.22)s]; 6 month after the experiment, the mean values of escape latency[(12.42±8.03)s, (17.48± 8.05)s] of rats in both groups exposed to fluoride were significantly prolonged as compared to controls [(7.04± 3.29)s, P<0.05]. The decreased numbers of crossing the platforms[(1.62±0.87)number] and the declined time of staying the platforms[(16.70±5.02)s] were found in the rats exposed to high fluoride as compared to controls [(3.53±1.67 )number, (23.33±5.35)s, P<0.05]. The fluoride contents obviously influenced the activities of acetylcholinesterase and butylcolinesterase (the value of F was 12.83,13.27, P<0.01). On the other hand, the times of breeding also influnced the activities of butylcolinesterase (the value of F was 16.26, P<0.01). In 3 months of the experiment, the activities of butylcolinesterase [(0.55±0.12)kU/g] in low fluoride exposed group were significantly decreased in comparison with controls[(0.73±0.10)kU/g, P<0.05]. The activities of acetylcholinesterase[(0.62±0.42)kU/g] and butylcolioesterase[(0.58±0.10)kU/g] in high fluoride group were significantly decreased as compared to eontrois[(1.41±0.52), (0.73±0.10)kU/g, P<0.05]. The correlation analysis showed that there was a negative correlation between the cholinesterase and the escape latency(r=-0.68, P< 0.01), and a positive correlation between the cholinesterase and the time of staying the platforms(r=0.57, P< 0.01). Conclusions The ability of learning and memory in rats with coal buring fluorosis was decreased, which might be connected to the decreased activity of cholinesterase in a dose-effect correlation.

Objective：Rat UREB1 protein coded by the gene UREB1 can specially bind to URE (upstream regulatory element) which is in the upstream of the promoter. It′ s reported that the protein of UREB1 promote the transcription of Dynorphin gene and inhibits p53 transactivation. This study was designed to clone human UREB1 gene and explore the relationship between UREB1 and the development of tumor. Methods： The artificial synthetic oligonucleotide was used as the probe to screen human brain cDNA library and human UREB1 gene was cloned. The antibody, which was produced using the recombinant UREB1 from E.coli as the antigen and immunizing the animals, was utilized for detecting the distribution of UREB1 in different tumor tissues. Results： The human UREB1 gene was cloned by using in situ hybridization for screening human brain cDNA library, and the nucleotide sequences and the deduced amino acid sequence of human UREB1 has 91％ homology with that of rat UREB1 identified previously. Western blot analysis revealed that the human UREB1 was present in all tumor tissues but the quantity of UREB1 in different tissues was not the same. Immunohistochemistry results shown that the human UREB1 distributes primarily in the cytoplasm and nuclear of tumor cells and nuclear UREB1 in carcinosarcoma is much more than that in adenoma. After analyzing the level of tyrosine phosphorylated UREB1 in a few tumor tissues, the result shown that the more malignant the tumor tissue was, the higher level the tyrosine phosphorylated of UREB1 was in that tumor tissues. Conclusion： Human UREB1 may be involved in the development of tumor and its tyrosine phosphorylation may affect the degree of tumor malignant.

OBJECTIVE: To clone the transmembrane (TM) domain sequence of EGFR gene and lay a good foundation for constructing the transmembrane expression vector of recombinant superantigens and cytokines. METHODS: A pair of primers special to the sequence encoding TM domain of EGFR gene were synthesized, TM domain fragment was cloned by RT-PCR, and the PCR product of TM domain sequence was ligated with the pGEM-T vector and confirmed by DNA sequencing. RESULTS: TM domain sequence was successfully cloned and verified by DNA sequencing. CONCLUSION: The successful cloning of TM domain sequence provides a basis for the construction of transmembrane fusion protein of Superantigen-TM or Cytokines-TM in cancer biotherapy.

OBJECTIVETo analyze the computed tomography (CT) and magnetic resonance imaging (MRI) findings of small hepatocellular carcinoma to improve the accuracy in the diagnosis.

METHODSThis retrospective analysis involved 41 patients with small hepatocellular carcinoma cases confirmed by pathological examination of the biopsy samples or follow-up. These patients were assessed for CT and MRI findings including lesion size, density or signal intensity, enhancement patterns, and presence of tumor capsules.

RESULTSOn unenhanced CT images, small hepatocellular carcinomas were displayed mainly as low-density masses, and the majority of tumors presented with low signal intensity on T1-weighted unenhanced MR images with increased signal intensity on T2-weighted images in comparison with the surrounding liver parenchyma. Most of tumors showed intense enhancement during the arterial phase (CT in 15 cases and MRI in 13 cases), but some appeared isointense to the liver parenchyma (CT in 4 cases and MRI in 4 cases). In portal and delayed phases, the tumors typically had lower signal intensity than that of the surrounding liver tissues (CT in 25 cases and MRI in 12 cases) with enhancement of the tumor capsules (13 cases).

CONCLUSIONDynamic enhanced scanning can be more informative of the pathology and blood supply of small hepatocellular carcinoma. Early and late arterial phase imaging may help in detecting the small lesions and in making differential diagnosis.

Adult ; Aged ; Carcinoma, Hepatocellular ; diagnosis ; diagnostic imaging ; Diagnosis, Differential ; Female ; Humans ; Image Enhancement ; Liver Neoplasms ; diagnosis ; diagnostic imaging ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Retrospective Studies ; Tomography, X-Ray Computed

OBJECTIVEFabry disease is a rare lysosome storage disease featuring X-linked recessive inheritance. The study was to explore potential mutations of alpha-galactosidase A (GLA) gene and their correlation with clinic manifestations in three Chinese pedigrees with Fabry disease.

METHODSAll exons and flanking sequences of GLA gene were amplified with PCR. Potential mutations were detected with bidirectional DNA sequencing. Correlation between particular mutations and clinic features were analyzed.

RESULTSA unreported missense mutation, c.797A>C (D266A) in GLA exon 5 was identified in pedigree 1. Also in exon 5, a missense mutation c.644A>G (N215S) was found in pedigree 2. In pedigree 3, a nonsense mutation c.355C>T (Q119X) was found in exon 2. The c.797A>C mutation was not detected in 200 unrelated male controls. The probands of pedigrees 1 and 3 had presented mainly with skin damage and chronic renal insufficiency, whilst the proband of pedigree 2 had presented with hypertrophic cardiomyopathy.

CONCLUSIONThe unreported c.797A>C (D266A) mutation is the sixth missense type mutation of the 266th codon of GLA gene, and all other 5 missense mutations reported previously had been confirmed to be responsible for Fabry disease. The c.797A>C mutation, not found in 200 unrelated male controls, may be the causative mutation in pedigree 1. The c.644A>G and c.355C>T mutations were first detected in Chinese patients. Variable phenotypes of Fabry disease may be in part attributed to the natures of particular mutations of GLA gene.

Adult ; Fabry Disease ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; alpha-Galactosidase ; genetics

AIMTo investigate the relationship between glutathione S-transferases gene polymorphism and susceptibility response to hypoxia.

METHODSIn the case-control study, the gene polymorphisms of glutathione S-transferases were tested in Tibetan mountaineers and sea-level Han Chinese by multiple-PCR and PCR-RELP.

RESULTSThe frequency of GSTT1 null genotype was significant different between Tibetan mountaineers and sea-level Han Chinese (P < 0.05), OR = 1.86 (95% CI = 1.01-3.39), and also for GSTP(1-105) mutant genotype in two groups (P < 0.01), OR = 2.19 (95% CI = 1.16-4.13). There was significant difference between A allele and G allele of GSTP(1-105) groups (P < 0.01). There was no difference for GSTM1 null genotype between two groups (P > 0.05), OR = 0.78 (95% CI = 0.43 - 1.42).

CONCLUSIONGSTT1 and GSTP(1-105) genotype may be associated with susceptibility response to altitude hypoxia.

Adult ; Alleles ; China ; Genotype ; Glutathione S-Transferase pi ; genetics ; Glutathione Transferase ; genetics ; Humans ; Hypoxia ; genetics ; Male ; Mountaineering ; Polymorphism, Genetic ; Reactive Oxygen Species ; metabolism ; Young Adult

Wuzhuyu Tang is a classical formula for treating migraine, but its' pharmacological ingredients is unclear yet. Present study employed the everted intestinal sac model to collect the absorption samples of 10 kinds of Wuzhuyu decoction, and then analyzed the contents of 9 ingredients in Wuzhuyu Tang and absorption samples quantitatively or semi-quantitatively by HPLC-DAD method. Reserpine was used to establish the mice model of migraine, and then the contents and activities of 5-hydroxytryptamine, noradrenaline, dopamine, nitric oxide and nitricoxide synthase in brain tissues and serums were determined respectively after oral administration of Wuzhuyu Tang. Using the partial least squares regression method to correlate the total absorption quantity of 9 ingredients and pharmacodynamics. The result shows that limocitrin-3-O-beta-D-glucoside, ginsenoside Rg1 and Rb1, rutaevine, limonin, evodiamine and rutaecarpine are the main ingredients influenced the effects in absorption samples in everted intestinal sacs, especially ginsenoside Rg1, rutaevine, evodiamine and rutaecarpine among them have obvious improving effects to most pharmacodynamics index, might be the pharmacological ingredients influenced the therapeutical effects of Wuzhuyu Tang treating migraine.
Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Intestinal Absorption ; drug effects ; Intestines ; drug effects ; Male ; Mice ; Mice, Inbred ICR ; Migraine Disorders ; drug therapy ; Rats ; Rats, Wistar