Objective: To determine if the hemagglutinin A (HagA) of Porphyromonas gingivalis could be involved in the adhesion and invasion in human gingival epithelial cells (HGEC). Methods:P. gingivalis 381 hagA mutant was constructed by conjugation method. The whole length of hagA gene was cloned into pYA292 in Salmonella typhimurium x4072 (S. typhimurium-hagA). The strains were used to test their ability of adhesion and invasion into HGEC using a standard antibiotic protection assay. S. typhimurium x4072 strains containing empty vectors were used as negative control. HagA expression in S. typhimurium-hagA was confirmed by Western blot. Results:Although there were no significant differences between P. gingivalis 381 hagA mutant and wild type in adhesion and invasion into HGEC, the adhesion values of S. typhimurium-hagA to HGEC were increased by 3 times compared to their respective controls, while the invasion ability of S. typhimurium-hagA was 4 times greater than that of the negative controls. Conclusion: These results suggest that HagA may participate in P. gingivalis adhesion and invasion into HGEC.

9β,11β-Epoxypregn-4-ene-17α,21-diol-3,20-dione 21-acetate (Ⅰ) is a substrate for the production of 9β,11β-Epoxypregn-1,4-diene-17α,21-diol-3,20-dione (Ⅳ),which is a key precursor for the production of many 9-fluoro-substituted corticosteroid hormones.By comparing whole cells catalysis and cellular lysates conversion,it was found that whole cells of Mycobacterium sp.MS136 could only convert Ⅰ to 9β,11β-Epoxypregn-4-ene-17α,21-diol-3,20-dione (Ⅱ),and Ⅰ can be effectively converted toⅣ by cellular lysates.The reaction order is that Ⅰ is spontaneously hydrolyzed to Ⅱ and Ⅱ undergoes C1,2-dehydrogenation reaction to Ⅳ.In order to improve the productivity of Ⅳ,the key genes kstD,kstD3 and kstDM encoding C1,2-dehydrogenase (KSTD) were overexpressed in Mycobacterium sp.MS136 to enhance the C1,2-dehydrogenation reaction rate,and the results showed that 1 g/L substrate Ⅰ can be converted by recombinant strain MS136-kstDM cellular lysates at pH 7.0,the productivity of Ⅳ reached 78.4％ after 45 h,which is 38.9％ higher than original strain.The reaction rate is enhanced by optimizing the pH,and the results showed that 1 g/L substrate (Ⅰ) can be converted by recombinant strain MS136-kstDM cellular lysates at pH 7.5,the productivity of Ⅳreached 92.8％ after 45 h,which was 63.4％ higher than original strain.

BACKGROUND: Cytokine is a poly-functional and effective regulator factor to regulate growth of multiple cells. Researches suggest that, as an inflammatory medium, cytokines play a key role in inflammatory reaction of eye; however, there are rare studies on dynamic changes after traumatic cataract.OBJECTIVE: To investigate the relationship among inflammatory reaction and dynamic changes of interleukin 1 (IL-1),IL-6 and tumor necrosis factor alpha (TNF-α) in aqueous humor of rabbits after extracapsular cataract extraction.DESIGN: Randomized controlled animal study.SETTING: Shenzhen Municipal Ophthalmology Hospital, Shenzhen Municipal Ophthalmology Center Affiliated to Medical College of Jinan University.MATERIALS: The experiment was carried out in Institute of Shenzhen Municipal Ophthalmology Center Affiliated to Medical College of Jinan University from March 2004 to March 2006. Fifteen healthy adult New Zealand rabbits of 30 eyes,general grade, weighting 2.5-3.0 kg, of either gender, were provided by Animal Center of the Second Clinical Medical College of Jinan University [certification: SYXK (yue) 2005-006]. Eyes of rabbits were normal before experiment. All rabbits were randomly divided into normal control group, traumatic control group and operative group with 5 in each group and in total of 10 eyes in each group. IL-1, IL-6 and TNF-α kits were provided by Shenzhen Yawei Biotechnology Company Limited.METHODS: Rabbits in traumatic control group and operative group were totally anesthetized with intravenous injection of 10% 1 mi/kg urethan, and then, 5# needle was punctured from corneal limbus to anterior chamber to scarify anterior membrane of lens about 5 mm to establish animal models of traumatic cataract of oculus uterque. Rabbits in normal control group were fed normally. After successful modeling, common antibiotic eyedrops was used to clean conjunctival sac of rabbits in traumatic control group and operative group 3 times a day. On the 3rd day of successful modeling, rabbits in operative group were totally anesthetized with intravenous injection of 10% 1 mL/kg urethan, and then, they undertook extracapsular cataract extraction of oculus uterque: horizontally intercepting bladder or waterly separating with breakage of anterior bladder membrane, expulsing nucleus of lens, washing lens cortex and suturing incisions. On the operative day and on the 1st, 3rd, 7th and 14th days after operation, inflammatory reaction of anterior chamber in traumatic control group and operative group was measured and 0.2 mL aqueous humor was extracted from rabbits in three groups to count and classify cells; meanwhile, expressed level and dynamic changes of IL-1, IL-6 and TNF-α in cytokines of aqueous humor were measured with double-antibodies ELASA technique.MAIN OUTCOME MEASURES: Total numbors of leucocytes and contents of IL-1, IL-6 and TNF-α in aqueous humor of rabbits in three groups after operation.RESULTS: ① On the 1st, 3rd, 7th and 14th days after operation, numbers of leucocytes were (2.4±0.7)×106/L, (2,2±0.5)×106/L, (2.8±0.8)×106/L and (2.0±0.5)×106/L in aqueous humor; (19.7±7.3)×106/L, (28.1±9.6)×106/L, (14.2±5.6)×106/L and(8.4±3.8)×106/L in traumatic control group; (65.3±14.5)×106/L, (79.8±12.7)×106/L, (21.7±8.2)×106/L and (12.4±4.1)×106/L in operative group. In addition, numbers of leucocytes were more in traumatic control group and operative group than those in normal control group (F =22.5, 27.9, 11.6, 8.4;P＜0.05). ② Within 1-14 days after operation, contents of IL-1, IL-6and TNF-α in aqueous humo were higher in traumatic control group and operative group than those in normal control group (P＜0.05), and there was a significant difference between traumatic control group and operative group (P＜0.05);however, there was no significant difference among three groups on the operative day (P＞0.05). ③ Contents of cytokines reached peak on the 7th day after operation, decreased gradually, and reached the lowest value on the 14th day.Contents in traumatic control group and operative group were higher than those in normal control group (P＜0.05).CONCLUSION: The intraocular inflammation after lens extraction is closely related to the dynamic changes of IL-1, IL-6and TNF-α levels in aqueous humor. Cytokine may be one of crucially inflammatory agents in the eyes after traumatic cataract.

Objective To explore the effects of chronic fluorosis on neurons in the cerebral cortex of rats,and to provide some morphological evidence of damage in the central nervous system induced by chronic fluorosis.Methods Male Wistar rats 40 days after birth were fed with high fluoride contented water(100 mg/L)for inducing chronic fluorosis.Immunocytochemistry and in situ hybridization were used to detect c-fos and Caspase 8 at cerebral cortical neurons respectively.Results c-fos positive cells rate and gray scale in the cerebral cortex of chronic fluorosis were 35.8%and 0.2756±0.0241,respectively,and that of control group were 32.1%and 0.2774±0.0331with statistical difference(χ2=0.305,t=0.826,P＞0.05).Caspase 8 positive cells rates of fluorosis group and control group were 18.7%and 14.1%,respectively,the difference being statistically significant(χ2=0.419,P＞0.05).The gray scale of fluorosis group and control group were 0.3874±0.0329 and 0.3884±0.0323,respectively,the difference being statistically significant(t=0.641,P＞0.05).Conclusion Chronic fluorosis had no significant influence on apoptosis of cerebral cortical neurons.

OBJECTIVETo observe the changes of electrocardiogram (ECG) and pregnancy outcome of the late pregnancy women.

METHODSLate pregnancy women were divided into two groups by age: over 35 group and under 35 group. The incidence of abnormal electrocardiogram was recorded when the patients were subjected to routine ECG examination. Then the pregnancy, delivery outcome and if there's low birth weight newborn were recorded later.

RESULTSThe incidence of abnormal ECG in over 35 group was significantly higher than that in under 35 group (P < 0.05). And the incidence of ST segment changes, arrhythmia in the group of former was higher than that in the group of latter (P < 0.05). Among the different type of arrhythmia, the incidence of sinus bradycardia and ventricular premature beat in the group of former were higher than those in the group of latter (P < 0.05). But the incidence of sinus tachycardia in the former group was obviously lower than that in the latter group (P < 0.05). The incidence of pregnancy loss in over 35 with abnormal ECG group was significantly higher than that in under 35 with normal or abnormal ECG groups (P < 0.05). The incidence of premature birth in over 35 with abnormal ECG group was significantly higher than that in over 35 with normal ECG group (P < 0.05). The incidence of low body weight in over 35 with abnormal ECG group was significantly higher than that in under 35 with normal ECG group (P < 0.05).

CONCLUSIONThe late pregnancy women with the age of over 35 are more likely to have ECG abnormalities, such as arrhythmia, myocardial ischemia and so on. The older pregnant women with abnormal ECG easily suffer from pregnancy losing, premature birth and having a low birth weight baby.

Adult ; Age Factors ; Arrhythmias, Cardiac ; epidemiology ; Electrocardiography ; Female ; Humans ; Pregnancy ; Pregnancy Outcome ; epidemiology

Objective: To study the role of activating transcription factor 2 (ATF-2) in the growth of mandibular condyle cartilage. Methods: Primary chondrocytes of condyle were cultured. Expression plasmid of ATF-2 and plasmid bcl-2 promoter were transfected into chondrocytes. Luciferase assay and Western blot were used. Results: The absence of ATF-2 in mandibular condyle chondrocytes resulted in a decline in bcl-2 promoter activity, reduction in bcl-2 protein level. Conclusion: The results strongly imply that ATF-2 is required for adequate bcl-2 expression, and play a significant role in controlling growth plate chondrocyte progression.

Objective To investigate the effects of microRNA-21-5p (miR-21-5p) on hyperoxic acute lung injury (HALI) in rats and provide a theoretical basis for HALI gene therapy. Methods One hundred and sixty Sprague-Dawley (SD) rats were randomly divided into four groups with number table:hyperoxia control group, phosphate buffer saline (PBS) group, blank virus group and miRNA-21-5p group (each, n = 40). The rats in hyperoxia control group were fed directly in the hyperoxia box (oxygen concentration > 90%); in the other three groups, 200 μL PBS, 200μL slow virus and 200μL miRNA-21-5p slow virus were dropped into the nose respectively, and then they were fed in the hyperoxia box. The rats were exposed to hyperoxia in the boxes for 0, 24, 48 and 72 hours in all the groups, and at each time point, 10 rats were taken randomly from each group to perform arterial blood-gas analysis, calculate oxygenation index (OI) and respiratory index (RI). Afterwards the rats were sacrificed by blood-letting from carotid artery under intra-peritoneal anesthesia, and the lung tissues were obtained to measure the left lung wet/dry weight (W/D) ratio, hemotoxylin-eosin (HE) staining was made and the pathological changes of the right lung were observed under light microscope and the pathological score was measured. Results At 0 hour, the OI, RI, lung W/D ratio and the lung tissue pathology score in rats with hyperoxic injury had no statistically significant differences among the four groups (all P>0.05). With the extension of time, the level of OI was gradually reduced, and the levels of RI, pathologic score and W/D ratio of lung tissues were gradually increased. Compared with the hyperoxia control group, in miRNA-21-5p group, the levels of OI were increased significantly at 24, 48 and 72 hours after the exposure to hyperoxia [mmHg (1 mmHg = 0.133 kPa): 24 hours 358.10±29.25 vs. 306.19±37.23, 48 hours 336.67±29.27 vs. 269.70±29.00, 72 hours 323.81±19.05 vs. 203.81±43.40, all P < 0.05], whereas the levels of RI were decreased significantly (24 hours 0.23±0.05 vs. 0.31±0.06, 48 hours 0.28±0.07 vs. 0.38±0.06, 72 hours 0.30±0.04 vs. 0.46±0.07, all P <0.05), the pathologic scores were decreased significantly (24 hours 0.60±0.52 vs. 0.90±0.74, 48 hours 1.30±0.95 vs.2.90±1.20, 72 hours 1.90±0.88 vs. 4.70±1.57, all P < 0.05) and the levels of W/D ratio were decreased obviously (24 hours 3.77±0.38 vs. 4.14±0.46, 48 hours 3.83±0.31 vs. 4.56±0.34, 72 hours 3.89±0.31 vs. 5.32±0.27, all P<0.05). Compared with the hyperoxia control group, the index results of the PBS group and the blank virus group after staying in the box had no statistical significant differences at each time point (all P>0.05). Under the optical microscope, along with the prolongation of exposure to hyperoxia, the structure of alveoli was gradually disturbed, their walls fractured and damaged, alveolar septa widened, edematous, infiltrated with inflammatory cells and in part of the rats a small amount of red blood cell exudates could be seen, but the degree of lung pathological injury in miRNA-21-5p group was much milder than that of the other groups. Conclusion The rat persistently exposed to hyperoxia for 24 hours can establish the rat model of HALI successfully, and the miRNA-21-5p can protect the lung tissue from the damage to some degrees in HALI rats.

ObjectiveTo study the effect of hydrogen peroxide (H2O2) in inducing apoptosis of typeⅡalveolar epithelial cell (AECⅡ) after overexpression by adenoviral transfection of micro RNA-21-5p (miR-21-5p), and to explore the mechanism of its anti-apoptosis.Methods Subculture AECⅡ were randomly divided into four groups: normal control group (normal saline), H2O2 challenge group ( 0.5 mmol/L H2O2), miR-21-5p overexpression group (miR-21-5p adenovirus+ 0.5 mmol/L H2O2), miR-21-5p negative transfection group (adenovirus void+0.5 mmol/L H2O2). Transmission electron microscopy and flow cytometry were used to detect apoptotic morphology and early apoptotic rate. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-21-5p in AECⅡ, and Western Blot was used to detect the protein expressions of Bcl-2, Bax, and caspase-3 at the highest transfection efficiency at different time points (6, 12, 24, 48 hours).Results ① AECⅡ identification: fluorescence microscopy showed the presence of characteristic structure of AECⅡ, i.e. microvilli and osmiophilic lamellar bodies.② Apoptotic morphology: transmission electron microscopy showed cytoplasmic retraction, chromatin condensation, margination, lack of cell surface microvilli, and emptying of osmiophilic lamellar bodies in AECⅡ.③ The expression of miR-21-5p in AECⅡ: the highest transfection efficiency was found at 48 hours. The expression of miR-21-5p in miR-21-5p overexpression group was significantly higher than that of the normal control group, H2O2 challenge group and miR-21-5p negative transfection group (A value: 1.54±0.02 vs. 1.02±0.02, 0.56±0.03, 0.58±0.02, allP< 0.05).④ The rate of early apoptosis: compared with normal control group, the early apoptotic rates in H2O2 challenge group, miR-21-5p negative transfection group and miR-21-5p overexpression group were gradually elevated with the prolongation of injury time. The early apoptotic rate in miR-21-5p overexpression group was significantly lower than that of the H2O2 challenge group and miR-21-5p negative transfection group at all time points except 6 hours [12 hours: (10.73±2.80)% vs. (16.26±0.59)%, (16.04±0.70)%; 24 hours:(16.00±3.44)% vs. (23.29±2.78)%, (23.58±2.31)%; 48 hours: (31.30±3.55)% vs. (50.53±2.17)%, (49.41±1.97)%, allP< 0.05]. There was no significant difference in early apoptotic rate between miR-21-5p negative transfection group and H2O2 challenge group at each time point.⑤ Protein expression: the expressions of Bax and caspase-3 in miR-21-5p overexpression group were significantly lower than those of the H2O2 challenge group and miR-21-5p negative transfection group [Bax (A value): 0.07±0.01 vs. 0.18±0.01, 0.13±0.01; caspase-3 (A value): 0.07±0.01 vs. 0.23±0.01, 0.12±0.01, allP< 0.05], and Bcl-2 protein expression was significantly higher than that of the H2O2 challenge group and miR-21-5p negative transfection group (A value: 0.26±0.01 vs. 0.06±0.01, 0.10±0.01, both P< 0.05).Conclusions① miR-21-5p has the function of anti-apoptosis of AECⅡ.② Adenoviral vector is a successful gene transfer vector when transfected with AECⅡ.③ The anti-apoptosis of AECⅡ by miR-21-5p may be associated with reduced Bax and caspase-3 protein levels and raised expression levels of Bcl-2 protein.

Objective To investigate the effect of short hairpin RNA(shRNA) eukaryotic expression vector‐mediated silen‐cing of the survivin‐gene on proliferation and apoptosis of human umbilical vein endothelial cells(HUVEC) .Methods The shRNA vector targeting the survivin gene and negative control vector were transfected into human umbilical vein endothelial cells(HUVEC) incubated with 50 ng/mL of recombinant VEGF in vitro by lipofectamine 2000 .Transfection after 48 h ,the expression of survivin mRNA and protein was detected by quantitative real‐time PCR and Western blot ,respectively .HUVEC proliferation was assayed by four methylthiazolyl tetrazolium(MTT) and cell apoptosis was detected by TUNEL .Results (1)Transfection with survivin‐shR‐NA vector significantly down‐regulated the expression of survivin mRNA and protein as compared with the control group ,after transfection of 48 h(P<0 .05) .(2)After survivin‐shRNA vector transfected ,the proliferation of HUVEC decreased significantly . After transfection 24 ,48 ,72 h ,the growth inhibition rate were (13 .53 ± 3 .91)% ,(38 .97 ± 1 .82)% ,(65 .75 ± 1 .83)% respective‐ly ,at 72 hours after transfection was the most significant .(3)The apoptosis rate of experimental group was (28 .07 ± 1 .71)% , which was higher than the negative control group (11 .45 ± 1 .52)% and blank control group (10 .04 ± 1 .46)% (P<0 .05) .Conclu‐sion The shRNA‐mediated mediated silencing of the survivin‐gene could significantly inhibit proliferation and promote the apopto‐sis of rheumatoid arthritis synovial fibroblasts by regulating survivin expression .

Objective To study the targeting survivin small interfering RNA ( siRNA ) to inhibit proliferation and apoptosis survivin gene expression in rheumatoid arthritis synovial fibroblasts ( RASFs) .Methods RA patients were isolated and cultured in vitro synovial fibroblasts ( RASFs) , designed and synthesized siRNA targeting survivin and negative control, by liposome transfection RASFs cell; real-time quantitative polymerase chain reaction (PCR) and Western blot RASFs detect mRNA expression and protein levels of survivin.Tetrazolium blue (MTT) assay of cell proliferation;TUNEL assay apoptosis.Results The experimental group compared with the negative control siRNA group and control group, 48h after transfection of synovial fibroblasts survivin mRNA and protein expression levels were significantly decreased ( P<0.05 ) .The experimental group compared with the negative control siRNA group and control group, synovial fibroblast proliferation after transfection significantly decreased ( P<0.05 ) . After the experimental group transfected 24h, 48h, 72h growth inhibition rates were (11.5 ±2.6)%, (26.2 ±3.4)%, (47.6 ±4.1)%, at 72 hours after transfection most significant.The rate of apoptosis in experimental group (23.87 ±1.6)%, significantly higher than the negative control group (9.72 ± 1.15)% and the control group (8.70 ±1.09)% (all P<0.05).Conclusion siRNA targeting survivin expression levels through reducing survivin, inhibit synovial fibroblast proliferation and promotes apoptosis.