Objective To investigate the possibility of differentiation and spermiogenesis of spermatocytes under in vitro condition. Methods Testis biopsy was done in 11 cases with non-obstructive azoospermia.Cells were prepared from 9 samples with spermatocytes and cultured in medium containing follicle stimulating hormone 50 U/L and testosterone 1 μmol/L.Sperms and cells of other types were counted and the proportion of every cell type was calculated before and 24 hours after culture.Flow cytometry was conducted before and 24 hours after culture in 2 cases to analyze the ploidy of the cells. Results The proportion of sperm in 9 samples was ( 17.7 ± 8.9 ) ％ before culture and ( 25.6 ± 10.3 ) ％ after culture ( P =0.004).Sperm increased significantly after culture.Flow cytometry demonstrated that the diagram of 4 n,2 n and 1 n converted to 2 n and broader 1 n. Conclusion Meiosis of spermatocytes and the transformation of spermatid into sperm could arise in in vitro culture.

Objective To evaluate the role of the mitochondrial ATP-sensitive potassium (mito-KATP)channel in sevoflurane preconditioning-induced delayed cardioprotection against ischemia-reperfusion (I/R) injury in isolated rat hearts.Methods Seventy-two adult male Sprague-Dawley rats were randomly divided into 6 groups (n =12 each):control group (group CON),I/R group,sevoflurane control group (group SEVO),sevoflurane preconditioning group (group SWO P),5-hydroxydeconoate (5-HD) + sevoflurane preconditioning group (group 5-HD+ SWOP) and 5-HD control group (group 5-HD).The rats were exposed to 33％ pure oxygen for 1 h in groups CON and I/R.The rats were exposed to 2.5％ sevoflurane for 1 h in groups SEVO and SWOP.5-HD (a mito-KATP channel inhibitor) 10 mg/kg was injected intraperitoneally 30 min before sevoflurane preconditioning in group 5-HD + SWOP.5-HD 10 mg/kg was injected intraperitoneally in group 5-HD.The hearts were immediately removed and perfused in a Langendorff apparatus.The hearts were made globally ischemic for 30 min followed by 120 min reperfusion in groups I/R,SWOP,5-HD + SWOP and 5-HD.The expression of phosphorylated protein kinase C-epsilon (p-PKC-ε) and caspase-8 was measured by Western blot immediately before ischemia (T0) and at 120 min of reperfusion (T1).The myocardial infarct volume was measured by TTC staining.Results Compared with group CON,the myocardial infarct volume was significantly increased at T1 in groups I/R,SWOP,5-HD +SWOP and 5-HD,p-PKC-ε expression was up-regulated at T0 in groups SEVO and SWOP and at T1 in groups I/R,SWOP,5-HD + SWOP and 5-HD,and caspase-8 expression was down-regulated at T1 in group SEVO (P ＜0.05).Compared with group I/R,the myocardial infarct volume was significantly decreased at T1 in groups SWOP and 5-HD + SWOP,p-PKC-ε expression was up-regulated at T0 in groups SEVO and SWOP,and caspase-8 expression was down-regulated at T1 in group SWOP (P ＜ 0.05).Compared with group SWOP,the myocardial infarct volume was significantly increased,p-PKC-ε expression was down-regulated at T0,and caspase-8 expression was up-regulated at T1 in group 5-HD + SWOP (P ＜ 0.05).Conclusion The mito-KATP channel is involved in sevoflurane preconditioning-induced delayed cardioprotection against I/R injury in isolated rat hearts through upregulation of p-PKC-ε expression before ischemia and inhibition of cell apoptosis during reperfusion.

Objective To investigate the genotypes and encoding resistance genes differences of Acinetobacter baumannii and analyze their interrelations with multi-drug resistance.Methods A total of 77strains Acinetobacter baumannii were collected random from the second Xiangya Hospital during September 2008 to September 2009.The K-B method which was WHO recommended was adopted to Acinetobacter baumannii drug sensitivity test to 15 kinds of antibiotics to establish susceptibility spectrum.At the same time,random amplified polymorphic DNA(RAPD)technique was used to establish DNA fingerprinting.The genes of β-lactamase(TEM-1,IMP,OXA-23,OXA-24,AmpC),aminoglycoside-modifying enzymes[aac(3')-Ⅰ ,aac(6')-Ⅰ ,ant(3")-Ⅰ]and 16S rRNA methylase(armA,rmtA,rmtB)were detected by PCR and sequenced,and find the relationship between the gene encoding and multi-drug resistance.In addition,we compared the rates of resistance genes of Acinetobacter baumannii and the relations with the genotype and the multi-resistance.Results Thirty-one sensitive strains and 46 multi-drug resistance strains(10 Pan-drug resistances)were isolated.Seventeen types from A to Q were separated using RAPD technique.E genotype widely popular in the ICU was the advantage type in multi-drug resistance strains,and the rate was 47.1%.While the various types scattered in sensitive strains.The positive rates of TEM-1,IMP,OXA-23,OXA-24,AmpC,aac(3')-Ⅰ ,aac(6')-Ⅰ ,ant(3")-Ⅰ ,armA in the multi-drug resistance strains and the sensitive strains were 95.7%,39.1%,84.8% ,54.3%,87.0%,89.1%,84.8%,45.7%,63.0% and 58.1%,9.7%,32.3%,48.4%,48.4%,29.0%,45.2%,12.9%,9.7%,respectively,and there was significant difference except for OXA-24 using the X2 test(P ＜ 0.05).All isolates were negative for rmtA gene and rmtB gene.Drug susceptibility analysis showed that the resistant rate was significantly higher of the strains carrying resistant genes than that of the resistance negative strains.When the strains were resistant to gentamicin and amikacin,the rate of three aminoglycoside genes positive was 34.8%.The trains containing all the measured β-lactamase genes were all resistant strains.Conclusion Compared with the sensitive Acinetobacter baumannii strains,a broad resistance spectrum and a high drug resistance rate were showed in multidrug resistance strains isolated from clinic,which harboring many kinds of β-lactamase genes and aminoglycosides genes with a high separation rate,and the same clone of multiple drug-resistant strains may be transmitted in and among wards.

Objective To investigate the prevalence of 16S rRNA methylase gene armA and to analyze their effect on the drug resistance in multi drug-resistant strains of Acinetobacter baumannii . Methods A total of 72 Acinetobacter baumannii isolates were collected from the Second Xiangya Hospital from Jan. 2008 to Dec. 2008. The size of inhibitory zone of these strains to gentamycin, tobramycin and amikacin were determinate using Kirby-Bauer( K-B) method. The 16S rRNA methylase genes armA were detected by PCR. PCR products were purified and sequenced. Then we used randomly amplified polymorphic DNA method (RAPD) genotyping technology for the establishment of DNA fingerprinting. In addition, we compared drug sensitivity test with RAPD technology. Results Twenty isolates of 72 strains were armA positive and the resistance rates of the strains with armA gene to gentamycin, tobramycin, amikacin were 90.0% , 90.0% and 90. 0% , respectivily. armA positive stains were divided into 7 types using RAPD technology. A genotype was the advantage type. Conclusion The study showed that 16S rRNA methylases gene armA was prevalent in Acinetobacter baumannii which could lead to resistant to almost all aminoglycosides at a high level. And the main form of armA gene prevalence in our hospital was the spread of the same clone strain inside and outside of clinic department.

Objective:To test the expression of Minichromosome maintenance complex component 7(MCM7) protein in hepato-cellular carcinoma(HCC) of different species including human, rat and tree shrew (tupaia) by cross-species oncogenomics approach, and to investigate the relationship between the expression of MCM7 and the development of hepatocellular carcinoma and its clinical significance. Methods:Western blot and Immunohistochemistry were applied to detect the expression levels of MCM7 protein in HCC tissues,corresponding HCC-adjacent liver tissues and normal liver tissues collected from different species including human, rat and tree shrew, respectively. The clinicopathologic factors were also analyzed with the results of Immunohistochemistry. Results:Western blot analysis showed that the expression of MCM7 protein in HCC tissues of human and rat were higher than that in corresponding HCC-ad-jacent liver tissues and normal liver tissues, respectively and significantly (P<0.05). However, the expression of MCM7 protein in HCC tissues of tree shrew were also higher than that in corresponding HCC-adjacent liver tissues and normal liver tissues, but no significant difference was found among three types of tissues (P>0.05).There was also no significant difference between HCC-adjacent liver tis-sues and normal liver tissues in three species (P>0.05). Immunohistochemical analysis showed that MCM7 protein was mainly ex-pressed in nucleus of HCC cells, and the positive rate of MCM7 protein in HCC tissues of human, rat and tree shrew were significantly higher than that in corresponding HCC-adjacent liver tissues and normal liver tissues, respectively (P<0.05). However, no significant difference was found between HCC-adjacent liver tissues and normal liver tissues (P>0.05). Moreover, the protein level of MCM7 was intimately related to patient's HCC stage, extrahepatic metastases and postoperative recurrence (P<0.05). Conclusion:MCM7 protein might play a pivotal role in hepatocarcinogenesis. In addition, it was probably related to patient's HCC stage, extrahepatic metastases and postoperative recurrence. It seems very likely that MCM7 may be applied as a new molecular target in HCC prevention and treat-ment.

In order to further study mouse embryonic stem cells(ES cells),lentiviral vector PLL-IRES-Nanog-Neo was constructed.Mouse ES cells overexpressed nanog by mediation of lentiviral were cultured on mouse fetal fibroblast feeders after 2 weeks under G418 media and examined according to gowth characteristics. Results were showed that 918 bp nanog fragments were expressed in mouse ES cells mediated by lentiviral vector PLL-IRES-Nanog-Neo,mouse nanog-ES cells were taken on mass-like image and positve with alkaline phosphatase staining and Oct4 and SSEA1 immunocytochemistry under no LIF condition in the media. It is concluded that mouse ES cells Elevated nanog gene expression by mediation of lentiviral were constucted and cultured.

Aim To establish a model of the vascular endothelial cell (VEC) injury in SDrats.Methods SD rats were randomly divided into the control and the modelgroups. The model rats were injected with adrenaline diluted to 2. 5 times 0. 05 mg?100 g-1 (tid) for 5 d continously. From the 4th d, they were irritated for 5 min in the0℃ cold-water in the middle between adrenaline injections.The control rats weregiven 0. 9% NS as above. At 6th d, blood samples were taken from carotid arteries ofthe rats and the CEC counts, t - PA、PAI activities, 6-keto-PGF1? concentrations andthe platelet aggregation rate(max) were detected respectively. Results In the modelgroup, as compared with those in the control group, t - PA activity and 6-keto-PGF1?concentration decreased significantly(P

Objective To observe the behavior changes and synaptic reconstruction of brain and the impact of gastrodin in rats with epilepysy induced by pentylenetetrazole.Methods Fifty Wistar rats in growth(50-70 g) were randomly divided into 5 groups:control group,Pentylenetetrazole (PTZ) group,gastrodin high dose group,gastrodin low dose group and sodium valproate group,each group had 10 rats.According to Racine classification,the behavior changes of rats and the frequency were recorded.Conventional method was adopt to perfuse cordis, fix and extract the brain,and the immunohistochemistry was used to detect synaptophysin(P38) expression in the temporal lobe and hippocampus of the brain after 4 weeks.Results 1.Four weeks after pentylenetetrazole ignited the growth period rats,the attack-level of each experimental group through comparison with each other,there was statistically significant difference(?2=35.83 P0.05).Conclusions There are mossy fiber sprouting and the formation of synaptic reconstruction in temporal lobe and hippocampus of the growth period rats repea-tedly ignited by pentylenetetrazole.Gastrodin may play a role in the formation of antiepileptic obviously,and through decreasing the expression of P38,which can inhibit the formation of synaptic reconstruction,as control seizures indirectly.

OBJECTIVETo study the effects of Tongxinluo on the expressions of matrix metalloproteinase-3 (MMP-3), MMP-9 and peroxisome proliferators-activated receptor gamma (PPARgamma) in atherosclerotic rabbits and explore the mechanism of its anti-atherosclerotic effect.

METHODSTwenty-four rabbits were randomized equally into control group, atherosclerotic model group (fed with high-fat diet for 14 weeks) and Tongxinluo group. The expressions of MMP-3, 9 and PPARgamma in the 3 groups were observed by means of immunohistochemistry.

RESULTSThe expressions of MMP-3, 9 and PPARgamma in the model group and Tongxinluo group were significantly higher than those in the control group (P<0.01). After high-fat diet feeding for 14 weeks, Tongxinluo group showed significantly lower expressions of MMP-3 and 9 but higher expression of PPARgamma than the model group (P<0.01).

CONCLUSIONTongxinluo can inhibit the expression of MMP-3 and 9 and increase the expression of PPARgamma, which might be the mechanism of its anti-atherosclerotic effect.

Animals ; Atherosclerosis ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Male ; Matrix Metalloproteinase 3 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; PPAR gamma ; genetics ; metabolism ; Rabbits ; Random Allocation

Objective To explore link of change of plasma C-reactive protein (CRP) and atrial fibrillation (AF). Method The hsCRP enzyme-linked immunosorbent assay (ELISA) methods were used to measure plasma CRP levels in 21 patients with lone paroxysmal AF (LPAF) during AF and 7 days after return to sinus rhythm ;28 patients with lone sustained atrial fibrillation(LSAF) and 27 patients with rheumatic heart disease and chronic AF(RHD).Levels of plasma CRP were compared to levels in 32 patients with paroxysmal supraventricular tachycardia(PSVT) and 20 voluntary healthy subjects. Results Patients with LPAF,LPAF and RHD had high levels of CRP when compared with PSVT and the healthy subjects.No significant changes of CRP levels were found between the onset AF patients and those after 7 days of recovery.Patients with lone persistent AF and rheumatic heart disease with persistent AF had higher levels of CRP than those with LPAF,but have no significance.There were positive correlation between the duration of AF and the levels of CRP.But there were no relation with left atrial size on echocardiography,sex and ages. Conclusion These results indicate that elevated CRP levels in AF patients may play an important role in the occurrence and sustainment of AF.