Objective To investigate the apoptosis of cell line HL-60 induced by matrine and its possible mechanism. Methods CCK-8 assay was used to observe the proliferation inhibitation of HL-60 cells treated with matrine. FCM was applied to detect apoptotic cells and mitoehondrial transmembrane potential.Spectrophotometry was used to detect the activity of Caspase-9. Results Matrine (0.25-2.0 mg/ml) could significantly inhibit proliferation of HL-60 cells (F=67.83, P ＜0.05). Ater 48 hours, the apoptosis rate increased obviously in dose dependence (t = 4.685, 6.300. 9.641, 6.786, P ＜0.05). Within 48 hours, the mitochondrial transmembrance potential of HL-60 cells treated with matrine (1.0 mg/ml) gradually decreased (F = 54.83, P ＜0.05), and the activation of Caspase-9 gradually decreased in time dependence (F = 72.31,P ＜0.05). Conclusion Matrine can induce apoptosis of HL-60 cells by reducing mitochondrial transmembrance potential and activating Caspase-9.

Objective To observe the therapeutic effects and side effects of FLAG regimen(fludarabine, Ara-C, and granulocyte-colony stimulating factor( C-CSF) for refractory or relapsed acute myeloid leukemia( AML). Methods Twenty-six AML were treated with fludarabine plus intermediate-dose Ara-C and G-CSF,of whom 15 cases belonged to refractory and 11 cases belonged to relapsed. Results After two courses of treatment, 14 cases were completely relieved (53. 8% ) and 5 cases were partially relieved (19.2% ). The overall effective rates was 73.1%. The main side effects were severe myelosuppression and non-hematological toxicity was mild. Conclusion FLAG regimen was very effective for refractory or relapsed acute myeloid leukemia and was well tolerated. The treatment-related mortality rate was low,so it provided a treatment choice for these patients.

AIM: To establish the method of determining cinobufagin and resibufogenin in Xinkening Capsules(Venenum Bufonis,etc.). METHODS: HPLC was applied to determining cinobufagin and resibufogenin.The determination was carried out using an ODS column with a solvent system: methanol-water(50∶40).The flow rate was 1.0 mL/min,detection wavelength was at 296 nm. RESULTS: The linear ranges of cinobufagin and resibufogenin were 3.5 ?g-0.28 ?g and 3.75 ?g-0.30 ?g,respectively.The average recoveries of cinobufagin and resibufogenin were 99.86%(RSD=1.8%,n=5),and 99.84%(RSD=1.2%,n=5),respectively. CONCLUSION: This method is simple,accurate with strong specificity and can be used to control the quality of Xinkening Capsules.

Objective To evaluate the effect of Da Vinci surgical system for the treatment of hepatopancreatobiliary diseases.Methods The clinical data of 29 patients with hepatopancreatobiliary diseases who had undergone operations with Da Vinci surgical system from March to November 2009 at the General Hospital of PLA were retrospectively analyzed.Results The operations were successfully done on 28 patients,except 1 patient was converted to open pancreaticoenterostomy.The total operation time was(339±149)minutes,and the time for operations done with Da Vinci surgical system was(285±117)minutes.The postoperative bowl movement recovery time was(33±21)hours,and the length of postoperative hospital stay was(8±6)days.No blood transfusion was needed.Three patients had postoperative complications and were cured by conservative treatment.Conclusion Laparoscopic operations for hepatopancreatobiliary diseases can be applied with the help of the threedimensional imaging system and flexible surgical instruments of the Da Vinci surgical system,and its superiority is more obvious when applied for intractable hepatopancreatobiliary diseases.

Objective To investigate and cure major complications after percutaneous transhepastic biliary drainage(PTBD).Methods The clinical data of 13 major complications after PTBD were retrospectively analyzed,5 complications were acute and the other 8 complications were delayed.Two cases were dealed with intervention.and operations were performed for the other 11 patients immediately.Results Among the 7 patients who received one-stage operation,3 patients were accompanied with acute kidney failure,and 2 patients were died.Two patients who received the second-stage operation recovered snccessfully.Two patients who surrendered were surrived 3 and 8 months respectively.Conclusions It is difficult to deal with the major complications after PTBD,and the main cause of postoperative death is acute kidney failure.It will be helpful to deal with the primary disease on the second-stage.

Objective: Cell cycle has recently become more appealing as a new target of anti-carcinogen-ic agent. Diallyl disulfide (DADS) inhibits growth and induces call cycle G_2/M arrest in human gastric cancer BGC823 cells. Cell division cycle protein 25C (Cdc25C) and CyclinB1 expression are involved in G_2/M arrest.However, mechanisms of G_2/M arrest are not yet fully understood. The aim of this study was to elucidate the mechanism of cell cycle G_2/M arrest in human gastric cancer BGC823 cells induced by DADS. Methods: The expression of chk1 and Chk2 mRNA associated with cell cycle arrest of BGC823 cells after the induction with DADS for 1 or 2 days was detected by RT-PCR. The protein expression of cycle-related proteins ATM-RAD3-related gene (ATR), checkpoint kinase1 (Chk1), checkpoint kinase 2 (Chk2), P-ATR, P-Chk1 and P-Chk2 was measured by Western blot. Interaction between Chk1/2 and Cdc25C was analyzed by immuno-precipitation. Results: After the cells were treated with 15 mg/L DADS for 1 or 2 days, the expression of Chk1 and Chk2 mRNA was not significantly different from that in untreated cells (P>0.05). Western blot analysis showed that the expression of total Chk1 and Chk2 treated with 15 mg/L DADS was not significantly different from that in untreated cells. But phospho-chk1 showed a significant increase after stimulation with 15 mg/L DADS for 2h to 12h and continued to increase gradually as time went on (P<0.05). Phospho-Chk2 showed a eak expression and a weaker expression after stimulation with DADS, but the changes were not statistically significant (P>0.05). Addition of 15 mg/L DADS to BGC823 cells for 15 rain to 120 min resulted in an increase in phospho-ATR expression, whereas no changes were found in ATR expression (P<0.05). The Chk1 Ab in-creasingly precipitated Cdc25C in BGC823 cells treated with DADS (P<0.05). In contrast, Chk2 Ab failed to change precipitation with Cdc25C by DADS (P>0.05). Conclusion: Activation of chk1 was involved in cell cy-cle G_2/M arrest in BGC823 cells treated with DADS. Cell cycle G_2/M arrest by DADS is associated with phos-phorylation of several cell cycle regulatory proteins including ATR and Chk1 which regulate expression of Cdc25C.

Objective To investigate the safety and feasibility of peripheral vascular intervention via the ipsilateral transulnar access(TUA)due to failure of transradial access(TRA)puncture.Methods The clinical data of 2546 peripheral vascular interventions via TRA,which were performed at authors'hospital between January 2019 and December 2021,were retrospectively analyzed.Among the 2546 interventions,TRA puncture failed in 37 procedures,and in 27 of these patients the ipsilateral TUA puncture had to be adopted.The puncture success rate,surgical success rate and puncture approach-related complications of TUA of the 27 patients receiving ipsilateral TUA puncture were analyzed.Results The success rate of ipsilateral TUA puncture after TRA puncture failed was 96.3％(26/27),and in one patient transfemoral access(TFA)puncture had to be substituted because of the ulnar artery spasm.The total success rate of interventional procedures was 96.3％(26/27).No serious complications occurred,and the incidence of minor complications was 19.2％(5/26).Conclusion Preliminary results indicate that for the experienced TRA operators,using ipsilateral TUA puncture due to failure of TRA puncture is a safe and feasible strategy choice.

Objective To evaluate the accuracy and clinical application value of the fluorescence quantitative PCR melting curve meth-od for detecting fungal nucleic acid.Methods 460 suspected or confirmed patients with respiratory fungal infections were enrolled in the study.The fluorescence quantitative PCR melting curve method was used as the test method,and the fungal 26S rRNA gene nucleic acid detection kit combined with Sanger sequencing was used as the reference method.Sputum samples from each study subject were collected and detected by the test method and reference method,respectively.The Kappa value of the two methods was calculated to evaluate the consistency of the results.Results Compared with the reference method,the overall conformity rate of the test method was 92.83%(427/460).Compared with the reference method,the positive conformity rates,negative conformity rates,and overall conformity rates of the test method for detecting 8 fungi,including Candida albicans,Candida glabrata,Candida krusei,Candida trop-icalis,Candida parapsilosis,Cryptococcus neoformans,Candida guilliermondii,and Aspergillus,were 97.34%(183/188),97.06%(264/272),and 97.17%(447/460),100.00%(33/33),99.77%(426/427),and 99.78%(459/460),100.00%(16/16),99.55%(442/444),and 99.57%(458/460),98.11%(52/53),99.75%(442/444),and 99.57%(458/460),95.08%(58/61),99.50%(397/399),and 98.91%(455/460),100.00%(9/9),99.56%(449/451),and 99.57%(458/460),85.00%(17/20),99.32%(437/440),and 98.70%(454/460),and 97.59%(81/83),97.88%(369/377),and 97.83%(450/460),respectively.The Kappa values for the consistency evaluation of the two methods'detection results were both greater than 0.8.Upon retesting the inconsistent re-sults of the two methods,it was found that 53.7%(22/41)of the detection results were consistent with the test method,and the others were consistent with the reference method.Conclusion The fluorescence quantitative PCR melting curve method can simultaneously detect 8 kinds of fungi,and the detection results are highly consistent with the reference method.It has unique advantages in fungal de-tection and important clinical application value.

Objective:To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds.Methods:Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full-thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results:Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 ( P>0.05). On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)%, (19.7±1.2)%, (53.6±3.5)%, and (62.2±5.1)%, respectively, which was significantly lower than (11.0±0.9)%, (47.8±2.8)%, (86.1±7.1)%, and (89.7±9.0)% in PBS group ( t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points ( P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions:Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound, reduce inflammation and collagen protein expression, and promote the expression of MMP-9, thus improving the survival quality of skin grafts.

Objective To investigate the distribution patterns of mosquitoes and mosquito-borne viruses in Dehong prefecture,Yunnan province,China. Methods Mosquito samples were collected using the mosquito traps from five counties of Dehong prefecture on July,2007 and 2010. Mosquitoes were cell cultured for viral isolation,and positive isolates were identified using RT-PCR and sequence analysis. Results A total of 43 634 mosquitoes comprised of 29 species representing six genera were collected. Culex tritaeriorhynchus and Anopheles sinensis comprised 78.69% and 14.77% of the total. Six strains of viruses were isolated from the mosquito pools. RT-PCR and phylogenetic analysis revealed three strains from Cx. tritaeriorhynchus,identified as genotypeⅠJapanese encephalitis virus(JEV). One strain was identified from Cx. tritaeriorhynchus,as Getah virus (GETV). Two strains isolated from Cx. tritaeriorhynchus and Anopheles vagus were identified as Culex pipiens pallens Densovirus(CppDNV). Conclusion Cx. tritaeriorhynchus had been the major species of mosquitoes and mainly transmitting vector of mosquito-borne viruses in Dehong prefecture. GenotypeⅠJEV,GETV and CppDNV were the vectors causing transmission of mosquitoe-borne diseases in this area. Data from phylogenetic analysis showed that these newly discovered isolates seemed to have had close relationship with those viruses previously circulating in Yunnan and other provinces of China.