Gene disruption by homologous recombination is a powerfu l tool for investigating gene function in yeast. Since 1980’s, it has been deve loped a lot. PCR-mediated gene disruption technique makes the manipulation easi er and it can be used to precisely delete genes in yeast. Multi-gene disruption technique can delete several genes successively in the same yeast strain. After the completion of the yeast Saccharomyces cerevisiae genome sequencing, the gene disruption technique for systematic analysis meets the need of the functio nal genomics in yeast. It also enlighten the study on human functional genomics.

9 cm in 66 cases and 8 cm in 4 cases.A vaginal speculum was inserted smoothly,showing a pinky,wet,and normal-tensioned vaginal mucous membrane.Of 44 cases with intercourse experience,there were 43 cases with satisfactory sexual life and 1 case with a complaint of too short vagina(vaginal length,8 cm).Conclusions Laparoscopic vaginoplasty of peritoneum has advantages of minimal invasion,simplicity of performance,and rapid recovery.The appearance and functions of "artificial vagina" after surgery are similar to those of normal vagina.

Objective To explore the influence of fluoxertin on level of IL-2 ,IL-6 and TNF-α in brain tissue and serum of rats with post-stroke depression(PSD) .Methods 60 rats were divided into sham operation group ,depression group ,stroke group ,PSD group and fluoxertin group ,12 cases in each group .ELISA was used to detect the level of IL-2 ,IL-6 and TNF-αin brain tissue and serum .Results The level of IL-2 ,IL-6 and TNF-αin depression group ,stroke group and PSD group were significantly increased and higher than that in sham operation group and fluoxertin group(P<0 .05) .There was no significant difference between sham op-eration group and fluoxertin group(P>0 .05) .Conclusion Fluoxertin could significantly decrease the level of IL-2 ,IL-6 and TNF-αand play an important role in therapy of PSD .

Objective To investigate the changes of cardiac function and myocardial apoptosis after limb ischemia reperfusion in rabbits.Methods Thirty rabbits were randomly divided into 3 groups:sham-operated control group (group SC),ischemia group (groupⅠ,8 hours of bilateral hind limb ischemia) and ischemia reperfusion group (group IR,4 hours of bilateral hind limb ischemia and 4 hours of reperfusion).Immunohistochemical studies and TUNEL were done to evaluate cell apoptosis.The SOD activity and MDA content in myocardial tissue and plasma LDH activity were determined. The left ventricular function were observed by physiology recording instrument.The cardiac histopathologic changes in experimental rabbits were observed.Results In group IR,the apoptosis index and expression of Bax were significantly increased compared with the other two groups.Similarly,ischemia reperfusion reduced SOD activity and enhanced LDH activity and MDA level,and the left ventricular function decreased in group IR.There was a positive correlation among the MDA content and the LDH activity,the expression of bax,the number of apoptosis cells.Under light microscope, myocardial impairment was found in group IR.Conclusion The myocardial injury occured and the left ventrieular function decreased after limb ischemia reperfusion,and they were associated with oxidative damage and apoptosis.

AIM: To explore the effects of fluctuant high blood glucose and stable high blood glucose on apoptosis and the expression of Bax and Bcl-2 in glomerular endothelial cells and renal tubular epithelial cells in diabetic rats. METHODS: 24 SD rats were divided into 3 groups: control group, stable high blood glucose group and fluctuant high blood glucose group. Diabetic rats were induced by intraperitoneal injection of STZ, and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of aspart and glucose at different time points every day. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), and immunohistochemistry was used to detect apoptosis associated gene bax and bcl-2 expression in kidney. RESULTS: After 4 experimental weeks, a significant increase in cell apoptosis, up-regulation of Bax protein expression in kidney tubular epithelial cell and down-regulation of Bcl-2 in glomerular endothelial cell in fluctuant high blood glucose rats were observed compared with stable high blood glucose rats.CONCLUSION: Fluctuant high blood glucose induces more apoptosis in renal tubular epithelial cells than that in stable high blood glucose diabetic rats.

AIM: To study the effect of L-arginine (L-Arg) on function and structure of mitochondria in ischemia-reperfusion (MRI) myocardial cells. METHODS: Thirty rabbits were randomly divided into three groups (n=10 in each), control group, MIR group and MIR+L-Arg group. The mitochondrial respiratory function, Ca~(2+) concentration ([Ca~(2+)]m), malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Meanwhile, the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), total adenylic acid number (TAN) and energy charge (EC) in the myocardial tissue were respectively measured. Moreover, the ultrastructure changes in myocardial mitochondria were observed during MIR. RESULTS: The mitochondrial respiratory control rate (RCR), velocity 3 (V_3), SOD, surface density (Sv) and specific surface (?) in MIR+L-Arg group were higher than those in MIR group, velocity 4 (V_4), [Ca~(2+)]m, MDA, volume density (Vv), horizental diameter (Hd) were lower than those in MIR group. ATP, ADP, TAN and EC levels of myocardial tissue were higher than those in MIR group. There was no significant difference between MIR+L-Arg and control group in V_3, V_4, SOD, MDA, Vv, Sv, ?, Nv, Vd, AMP and TNA. CONCLUSION: It is suggested that L-Arg improves the function and structure of mitochondria in myocardial cells in the reperfusion injury after myocardial ischemia by decreasing oxygen free radical level and Ca~(2+) overload in the mitochondria. [

Objective To explore the effects of oscillating blood glucose on apoptosis in renal tubular epithelial cells of diabetic rats and the role of oxidative stress.Methods Renal tubular epithelial cells (HK-2) were cultured in vitro with stable high glucose or oscillating high glucose,and MTF assay was applied to the neasurement of cell proliferation.Streptozotocin induced diabetic model was established with SD rats and the oscillating high blood glucose animal model was induced by intraperitoneal injection of insulin and glucose at different time points every.day.12 weeks later 24 h urine protein (24hUP),blood urea nitrogen (BUN),serum creatinine (Scr),superoxide dismutase (SOD), and malondialdehyde (MDA)were determined. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL),and immunohisto-chemistry was used to detect apoptosis associated gene Bax,Bcl-2,and NOX-4 expression in kidney.Changes in ultrastructure were observed.Results Oscillating high glucose may inhibit renal cells proliferation obviously when comparing with stable high glucose.In the rats with oscillating blood glucose rather than those with stable high blood glucose,there was a significant increase of BUN,Scr,24hUP,and MDA and a decrease of SOD[ (21.50 ± 1.72 vs 12.50 ± 1.85 )mmol/L,(97.51 ± 7.84 vs 82.12 ± 11.48 ) μmol/L,( 1.57 ± 0.09 vs 1.04 ± 0.12 ) mg/24 h,( 23.50 ± 1.87 vs 14.82 ± 2.96) nmol/ml,( 17.22 ± 1.12 vs 21.11 ± 1.80) U/ml,all P＜0.05 ] ; cell apoptosis was intensified with the up-regulation of Bax and NOX-4 protein expression and down-regulation of Bcl-2 in glomerular endothelial cells; and more severe pathological damages were observed.ConclusionComparing with stable high blood glucose,oscillating high blood glucose induces more apoptosis and less proliferation of renal tubular epithelial cells and the mechanism may be related to the increased oxidative stress.

Objective To investigate the clinical effect of percutaneous transluminal angiography in diabetic infrapopliteal arterial disease patients and the influence of post-procedural intraluminal small dose urokinase infusion on infrapopliteal arterial blood flow.Methods From January 2011 to September 2013,37 limbs (16 left and 21 right) in 28 diabetic patients inflicted with infrapopliteal critical limb ischemia underwent endovascular recannalization at our institution and were retrospectively analyzed.Stenotic or occlusive lesions were demonstrated in 74 infrapopliteal vessels,including 30 anterior tibial arteries (ATA),22 posterior tibial arteries (PTA),and 22 peroneal arteries (PA).In 30 limbs,tandem lesions in iliac-femoral arteries were also diagnosed.Antegrade ipsilateral femoral access,retrograde contralateral femoral or brachial arterial access had all been adopted as well as both angioplasty and stenting.Case specific decisions were made based on pre-procedural computed tomographic angiogram (CTA).Ankle-brachial index (ABI) was recorded before and after each procedure.Urokinase was continuously infused through arterial sheath catheter into vessels of target limb from a microinfusion pump at 200 000 to 300 000 units per 24 hour for 48 hours after procedure.Angiogram was performed before and after thrombolysis therapy aiming to ascertain the number of frames of images obtained during the period of time it took blood flow to carry contrast medium from the level of tibial plateau to ankle,which was recorded as index frame count (IFC).Patients were followed up for at least 3 months.ABI and ultrasound or CTA were performed on each follow-up visit to validate patency.Quantitative data such as ABI value and IFC were analyzed using paired samples t-test.Results Thirty two limbs were radiographically recanalized by angioplasty or stenting.Technical success rate was 86.4％ (32/37).Average ABI of all limbs increased significantly from 0.70±0.31 to 0.90± 0.21 (t=10.734,P＜0.05).Of the 32 limbs recanalized,IFC decreased significantly from 6.3 ± 1.6 before thrombolysis to 4.7± 1.4 after thrombolysis (t=12.136,P＜0.05).Six rest pain patients reported significantly alleviated symptoms.Fourteen limbs presented with feet ulcers or gangrene.Of these patients after endovascular treatment,1 underwent ankle level amputation,3 underwent toe amputation and 3 patients who did not seek further treatment reported spontaneous autoamputation and wound healing.The remaining 9 patients reported wound healing within 1 to 3 months.Secondary angioplasty was needed for symptom recurrence in 3 limbs of 3 patients 3 to 24 months after first procedure.Conclusions Endovascular treatment of diabetic infrapopliteal arterial diseases exhibited significant short term effect and was safe to perform.Small dose urokinase infusion after recanalization procedure was safe and effective in helping to improve infragenicular blood flow.

Aim To investigate the effects of midazolam on porcine isolated coronary artery rings pre-contracted by potassium chloride(KCl)and the possible mechanism.Methods The vessel tension recorder system was used.Isotonic tension of porcine isolated coronary artery rings precontracted by KCl(30 mmol·L-1)was recorded.The vasorelaxing action of midazolam and effects of various drugs were observed in the rings.Results Midazolam(3×10-6～1×10-4 mol·L-1)respectively concentration-dependently reduced the contraction induced by KCl,and there was significant difference between the rings with intact and denude endothelium(P<0.05).On KCl-induced precontraction,midazolam′s relaxation was depressed by L-NAME and the blend of L-NAME and L-Arg(P<0.05),but was not affected by Indo,L-Arg and 1400W.The contraction was not prevented by pretreatment with the inhibitor of Na+/Ca2+ exchanger(KB-R7943).The inhibitor of KATP(Gli)restrained the diastolic function of midazolam(P<0.05),while the inhibitor of BKCa(TEA),Kir(BaCl2),KV(4-AP)had no obvious effect.Conclusions Midazolam produces remarkable vasodilatation on KCl pre-contracted porcine isolated coronary artery rings.Its relaxtion effect is via concentration-dependent and endothelium-dependent mechanisms and relevant to the production of NO.Na+/Ca2+ exchanger is not involved midazolam′s vasodilatation on KCl pre-contracted porcine coronary artery rings.The relaxant mechanism of midazolam may be concerned with KATP.The Kir,BKCa and KV may be not involved.

Objective: To observe and compare the clinical efficacy and safety of rifapentine and rifampicin in the treatment of tuberculosis. Methods: From January 2015 to January 2018, 398 tuberculosis patients admitted to Tuberculosis Clinic of the Traditional Chinese Medicine Hospital of Cixi were selected and randomly divided into observation group and control group by complete Random Distribution Method, with 199 cases in each group.The two groups were given basic treatments such as isoniazid, pyrazinamide and ethambutol.On this basis, patients in the control group were treated with rifampicin, and the observation group was treated with rifapentine.The treatment time of both two groups was 6 months.The sputum bacterial conversion rate, lesion absorption rate and cavity closure rate were observed, and the clinical treatment effect and adverse reactions were compared between the two groups. Results: The sputum bacteria conversion rate (77.89%), lesion absorption rate (71.86%) and cavity closure rate (74.87%) in the control group were significantly lower than those in the the observation group(96.98%, 91.96%, 95.98%) (χ²=31.03, 27.13, 35.60, all P<0.01). The total effective rate of the observation group was 93.97%, which was higher than 75.88% of the control group, and the difference was statistically significant (χ²=38.37, P<0.01). The incidence rate of adverse reactions in the observation group was 12.56%, which was significantly lower than 29.56% in the control group, the difference was statistically significant (χ²=17.59, P<0.01). Conclusion Compared with rifampicin, rifapentine is effective in treating pulmonary tuberculosis patients with high safety and effective improvement of clinical symptoms, which is worthy of further application.