Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro.MethodsHuman corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells.ResultsRT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells.ConclusionsThe expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.

Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.

Objective To investigate the influencing factors of strain ratio(SR) value in differential diagnosis of benign and malignant thyroid nodules by using real-time tissue elastosonography (RTE).Methods One hundred and seventy-one patients with a total of 171 thyroid nodules were analyzed retrospectively.Their images,including 2D ultrasound,color Doppler flow imaging (CDFI) and RTE were reviewed and conventional ultrasonic features (including the maximum diameter,composition,shape,magin,calcification,intranodular blood flow,depth) and SR value were recorded.Receiver-operating characteristic (ROC) curve was employed to assess the diagnostic efficiency of SR value in differentiating malignant nodules from benign ones.Firstly,the correlation between the aforementioned factors and SR value was assessed by using malignant lesions as the research subjects.And then,the multiple linear regressions (MLR) was employed to evaluate the influence of particular features which turned out to be an important disturbing factor affecting SR value of the lesion in the first step of analysis and pathological type in all nodules (benign and malignant) on SR value.Results With a cut-off point of SR value 3.67,the sensitivity and specificity of SR value in differential diagnosis of benign and malignant thyroid nodules was 85.6％ and 81.1 ％,respectively,and the area under ROC curve was 0.891.Correlation between the maximum diameter and calcification and SR value was significant(r =0.345 and 0.261 respectively,P ＜0.05).However,there was no significant correlation between other features(5 factors) and SR value(P ≥0.05).MLR indicated that the maximum diameter,calcification and the type of pathology of the nodule were associated with SR value (P ＜ 0.05).Among them,pathological nature was the most significant impact factor with a standardized coefficient 0.494).Conclusions SR value can be used to evaluate the hardness of thyroid nodules semi-quantitatively.Its value mainly depends on the pathological nature of the nodules.The maximum diameter and calcification are also the influencing factors of SR value.However,the composition,shape,margin,intranodular blood flow and depth have no obvious effect on SR value.

OBJECTIVETo explore the technique of induction of polyploidy in Salvia bowleyana by colchicine treatment.

METHODThe three kinds of explant of bud, leaf and calli were induced by colchicine treatment.

RESULTThe induction effects were better when the calli was treated by colchicines (15 mg x L(-1)) and the leaf was pre-cultured for one week. The doubling rate was 33.33%, while the majority were wholy doubled plants, and the leaves were thicker and broader, the color was darker, the root was thicker and the stoma size was obviously bigger than the diploid plants. The number of chromosome were 8 to 64. Isoenzyme analysis showed that the enzyme activities between the polyploid and the diploid plants were quite different.

CONCLUSIONInduction of polyploidy by colchicine treatment is efficacious. The part of the doubled plants were identified as homologmous tetraploids.

Chromosomes, Plant ; genetics ; Colchicine ; pharmacology ; Plant Leaves ; anatomy & histology ; genetics ; growth & development ; Plant Shoots ; anatomy & histology ; genetics ; growth & development ; Plants, Medicinal ; anatomy & histology ; genetics ; growth & development ; Polyploidy ; Salvia ; anatomy & histology ; genetics ; growth & development

The present study was to investigate the effect of Salvia miltiorrhiza Bunge. f. alba (SMA) pharmacological pretreatment on apoptosis of cultured hippocampal neurons from neonate rats under oxygen-glucose deprivation (OGD). Cultured hippocampal neurons were randomly divided into five groups (n = 6): normal plasma group, low dose SMA plasma (2.5%) group, middle dose SMA plasma (5%) group, high dose SMA plasma (10%) group and control group. The hippocampal neurons were cultured and treated with plasma from adult Wistar rats intragastrically administered with saline or aqueous extract of SMA. The apoptosis of neurons was induced by glucose-free Earle's solution containing 1 mmol/L Na2S2O4 and labeled by MTT and Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in control group, whereas the number of apoptotic cells was greatly increased in normal plasma group and low dose SMA plasma group. Both middle and high dose SMA plasma could protect cultured hippocampal neurons from apoptosis induced by OGD (P < 0.05). The protective effect of high dose SMA plasma was stronger than that of middle one (P < 0.05). Compared to control, normal plasma and low dose SMA plasma groups, middle and high dose SMA plasma groups both showed significantly higher levels of Bcl-2 (P < 0.05 or 0.01), whereas expressions of Bax was opposite. There were no significant differences of Bcl-2 and Bax expressions between middle and high dose SMA plasma groups. Number of Bcl-2- and Bax-positive cells had similar tendency. Bcl-2/Bax (number of positive cells) ratio was higher in high dose SMA plasma group than those of all the other groups (P < 0.05 or 0.01). These results suggest that pharmacological pretreatment of blood plasma containing middle and high dose SMA could raise viability and inhibit apoptosis of OGD-injured hippocampal neurons by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.
Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Female ; Glucose ; metabolism ; Hippocampus ; cytology ; Ischemic Preconditioning ; methods ; Male ; Neurons ; cytology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Reperfusion Injury ; prevention & control ; Salvia miltiorrhiza ; chemistry ; bcl-2-Associated X Protein ; metabolism

The present study was to investigate the effect of cerebrospinal fluid (CSF) from the rats with hypoxic preconditioning (HPC) on apoptosis of cultured hippocampal neurons in neonate rats under oxygen glucose deprivation (OGD). Adult Wistar rats were exposed to 3 h of hypoxia for HPC, and then their CSF was taken out. Cultured hippocampal neurons from the neonate rats were randomly divided into four groups (n = 6): normal control group, OGD group, normal CSF group and HPC CSF group. OGD group received 1.5 h of incubation in glucose-free Earle's solution containing 1 mmol/L Na2S2O4, and normal and HPC CSF groups were subjected to 1 d of corresponding CSF treatments followed by 1.5 h OGD. The apoptosis of neurons was analyzed by confocal laser scanning microscope and flow cytometry using Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in normal control group, whereas the number of apoptotic cells was greatly increased in OGD group. Both normal and HPC CSF could decrease the apoptosis of cultured hippocampal neurons injured by OGD (P < 0.01). Notably, the protective effect of HPC CSF was stronger than that of normal one (P < 0.01). Compared to OGD group, normal and HPC CSF groups both showed significantly higher levels of Bcl-2 (P < 0.01), and Bcl-2 expression level in HPC CSF group was even higher than that in normal CSF group (P < 0.01). Whereas the expressions of Bax in normal and HPC CSF groups were significantly lower than that in OGD group (P < 0.01), and the Bax expression in HPC CSF group was even lower than that in normal CSF group (P < 0.01). These results suggest that CSF from hypoxic-preconditioned rats could degrade apoptotic rate of OGD-injured hippocampal neurons by up-regulating expression of Bcl-2 and down-regulating expression of Bax.
Animals ; Animals, Newborn ; Apoptosis ; physiology ; Cell Hypoxia ; physiology ; Cells, Cultured ; Cerebrospinal Fluid ; physiology ; Female ; Glucose ; metabolism ; Hippocampus ; cytology ; pathology ; Hypoxia ; cerebrospinal fluid ; physiopathology ; Ischemic Preconditioning ; Male ; Neurons ; pathology ; Oxygen ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; bcl-2-Associated X Protein ; metabolism

The purpose of the present study was to explore the roles of Bcl-2 and Caspase-3 in mouse cortex in hypoxic preconditioning. Blb/c mice were randomly divided into three groups: control group, hypoxic group and hypoxic preconditioning group. Fluorescence intensity of Bcl-2 and Caspase-3 was observed and number of positive cells was counted in parietal cortex by immunofluorescence and confocal laser scanning microscope. Fluorescence intensity of Bcl-2 in the normal group, hypoxic group and hypoxic preconditioning group was 6.2±1.7, 68.5±13.1, 180.6±34.8, respectively, and number of Bcl-2-positive cells was 18.5±4.9, 52.3±10.5, 150.8±24.7, respectively. Fluorescence intensity of Caspase-3 in the control group, hypoxic group and hypoxic preconditioning group was 8.6±2.0, 40.2±8.2, 26.4±6.1, respectively, and number of Caspase-3-positive cells of was 4.3±1.2, 63.6±12.5, 45.7±9.8, respectively. The results showed that the expressions of Bcl-2 in both hypoxic group and hypoxic preconditioning group were significantly higher than that in the control group; and the expression of Bcl-2 in hypoxic preconditioning group was even higher than that in hypoxic group. The expressions of Caspase-3 in hypoxic group and hypoxic preconditioning group were also significantly higher than that in the control group; whereas the expression of Caspase-3 in hypoxic preconditioning group was significantly lower than that in hypoxic group. These results suggest that cortex cells are resistant to apoptosis via increased expression of Bcl-2 and lowered expression of Caspase-3 in the cortex and brain cells are thereby protected during hypoxic preconditioning.
Animals ; Animals, Newborn ; Apoptosis ; Caspase 3 ; metabolism ; Cerebral Cortex ; metabolism ; Hypoxia ; metabolism ; Ischemic Preconditioning ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

Objective To observe the protective effect of hypoxic preconditioning against cerebral ischemic injury induced by acute cerebral infarction in mice. Methods Blb/c mice were randomized into normal control, sham-operated, acute cerebral infarction (CI), and hypoxic preconditioning with CI (HP+CI) groups. In the latter two groups, acute cerebral cortical infarction was induced photochemically by cold light exposure of the brain tissue following intravenous Rose Bengal injection. The mice in the sham-operated group received only cold light exposure without Rose Bengal injection. Behavioral test, immunofluorescence assay and confocal laser scanning microscopy were performed to evaluate the neurological deficits of the mice and assess the cell apoptosis, and the cerebral infarction volume was measured. Results No infarct was found in the normal control and sham-operated groups, whereas obvious isehemic infarction foci were found in CI and HP+CI groups, but the infarction volume was significantly smaller in HP+CI group (P<0.05). The mice in the normal control and sham-operated groups presented with no neurological impairment, but in CI and HP+CI groups, obvious symptoms of neurological impairment were observed with lowered neurological scores. The neurological scores were significantly higher in HP+CI group than in CI group (P<0.05). Occasional apoptotic cells were found in the normal control and sham-operated groups, while in the other two groups, the TUNEL-positive cell number increased significantly. The apoptotic cell number was significantly smaller in HP+CI group than in CI group (P<0.05). Conclusion Hypoxic preconditioning may protect against cerebral ischemic injury induced by acute cerebral infarction in mice possibly by reducing the cerebral infarction volume and cell apoptosis.

Objective To observe the effect of brain homogenate (BH) extracted from hypoxia-preconditioned mice on the viability and apoptosis of rat embryonic hippocampal neurons with hypoxia/reoxygenation-induced injury. Methods Rat embryonic hippocampal neurons primarily cultured for 8 days in 96 well tissue culture plate were divided into 5 groups, namely the normal control group (treated with PBS), H<,4>R<,48> group (with hypoxia for 4 h followed by reoxygenation for 48 h and PBS treatment), H0 group (with BH from normal mice prior to hypoxia/reoxygenation), H1 group (with BH from acute hypoxia-preconditioned mice and hypoxia/reoxygenation), and H4 group (with BH from hypoxia-preconditioned mice and hypoxia/reoxygenation). The viability and apoptosis of the cells in the 5 groups were observed by MTT assay and flow cytomertry, respectively. Results The cell viability was significantly higher in the normal control group than in H<,4>R<,48> group. In H0, H1, and H4 groups, the cell viability increased significantly as compared with that in H<,4>R<,48> group, and the cells in H4 group showed the highest viability. Apoptotie cells were scarcely observed in the normal control group, but were numerous in H<,4>R<,48> group. Compared with H<,4>R<,48> group, H0, H1, and H4 groups showed obviously reduced apoptotic cells, and the reduction was the most conspicuous in H4 group. Conclusion BH extracted from hypoxia-preconditioned mice may offer protection against hypoxic injury of rat embryonic hippocampal neurons challenged with hypoxia-reoxygenation by promoting the cell viability and decreasing cell apoptosis.

PURPOSE: The role of IL28B gene variants and expression in hepatitis B virus (HBV) infections are not well understood. Here, we evaluated whether IL28B gene expression and rs12979860 variations are associated with HBV outcomes. MATERIALS AND METHODS: IL28B genetic variations (rs12979860) were genotyped by pyrosequencing of DNA samples from 137 individuals with chronic HBV infection [50 inactive carriers (IC), 34 chronic hepatitis B (CHB), 27 cirrhosis, 26 hepatocellular carcinoma (HCC)], and 19 healthy controls. IL28A/B mRNA expression in peripheral blood mononuclear cells was determined by qRT-PCR, and serum IL28B protein was measured by ELISA. RESULTS: Patients with IL28B C/C genotype had greater IL28A/B mRNA expression and higher IL28B protein levels than C/T patients. Within the various disease stages, compared to IC and healthy controls, IL28B expression was reduced in the CHB, cirrhosis, and HCC cohorts (CHB vs. IC, p=0.02; cirrhosis vs. IC, p=0.01; HCC vs. IC, p=0.001; CHB vs. controls, p<0.01; cirrhosis vs. controls, p<0.01; HCC vs. controls, p<0.01). When stratified with respect to serum HBV markers in the IC and CHB cohorts, IL28B mRNA and protein levels were higher in HBeAg-positive than negative individuals (p=0.01). Logistic regression analysis revealed that factors associated with high IL28B protein levels were C/C versus C/T genotype [p=0.016, odds ratio (OR)=0.25, 95% confidence interval (CI)=0.08-0.78], high alanine aminotransferase values (p<0.001, OR=8.02, 95% CI=2.64-24.4), and the IC stage of HBV infection (p<0.001). CONCLUSION: Our data suggest that IL28B genetic variations may play an important role in long-term development of disease in chronic HBV infections.
Adult ; Aged ; Alanine Transaminase/blood ; Asian Continental Ancestry Group/*genetics ; Biological Markers/blood ; Carcinoma, Hepatocellular/genetics ; Case-Control Studies ; China ; DNA, Viral/blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Genotype ; Hepatitis B virus/genetics ; Hepatitis B, Chronic/ethnology/*genetics/immunology/*virology ; Humans ; Interleukins/blood/*genetics/metabolism ; Leukocytes, Mononuclear ; Liver Cirrhosis/blood ; Liver Neoplasms/genetics ; Male ; Middle Aged ; RNA, Messenger/*genetics ; Reverse Transcriptase Polymerase Chain Reaction