Background Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step.Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line (HLE-B3) and establish low-expressed Smac HLE-B3 line.Methods Based on the genebank and our previous study,siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GVll8 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR.Results GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp,and viral titer was 3.0× 108 TU/ml.At a MOI of 100,the infecting efficiency of the vector on HLE-B3 cells was about 82％ and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)％,(92.330±6.320)％ and (32.540±4.221)％ in the negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group,respectively,showing a significant difference among the three groups(F =32.871,P＜0.01),and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P =0.000).However,no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535).Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs.

Objective To explore the effect of treating chronic pelvic inflammatory disease with the combination of retention enema,acupuncture and microwave physical therapy.Methods 80 patients were randomly assigned into two groups:a treatment group(50 cases) treated with combination of retention enema,acupuncture and microwave physical therapy,and a control group (30 cases) treated with Fuyankang capsule.The efficiency was observed after the treatment.Results The total effective rate in the treatment group was 92.0%,showing significant difference (P＜0.05) compared with the control group.Conclusion The triple-combined therapy of retention enema,acupuncture and microwave physical therapy has sound effect in treating chronic pelvic inflammatory disease.

Objective To investigate the effect of transforming growth factor-?1 (TGF-?1) on cell cycle,cell proliferation,and invasive capacity of human colon cancer cells of the line SW480,for its role in colon carcinogenesis and development,as well as its application in gene therapy for colon cancer with RNA interference.Methods The recombinant expressing plasmids pEGFP-N1-TGF-?1 was constructed and transfected into SW480 cells by Lipofectamine 2000.The expression of TGF-?1 mRNA and protein in the transfected SW480 cells were detected by fluorescent microscopy,RT-PCR and Western blot analysis respectively.The cell proliferation of SW480 cells was tested by MTT assay,the cell cycle was analyzed by flow cytometry,and the invasion ability of SW480 cells were investigated by Transwell-matrigel invasion chambers.Results After transfected into SW480 cells,both the mRNA and protein levels of TGF-?1 were effectively increased (P

Objective To investigate the efficacy of efficacy of warm needling plus Yunnan Baiyao aerosol in treating ankylosing spondylitis. Method Ninety patients with ankylosing spondylitis were randomly allocated to three groups, 30 cases each. The treatment group received warm needling plus Yunnan Baiyao aerosol in addition to medication;control group 1, warm needling plus Yunnan Baiyao tincture in addition to medication; control group 2, routine medication for rheumatism. Result There was no statistically significant difference between the treatment group and control group 1 (P>0.05) and there was between the treatment group and control group 2 (P＜0.05), indicating that the therapeutic effect was better in the treatment group than in the control group. Conclusion Warm needling plus Yunnan Baiyao aerosol can effectively relieve the pain, improve joint function and activity, prevent joint stiffness and deformity and reduce the disability rate in ankylosing spondylitis patients.

Objective:To explore the effect of modified multifunctional dressing bag with glauber's salt for relieving swelling and pain in early patients with limb fracture.Methods:A total of 111 patients with limb fracture in Tongde Hospital of Zhejiang Province were divided into two groups according to the admission time. The control group consisted of 53 patients with limbs fracture using common mirabilite bag with glauber′s salt and ice bag for external application admitted from January to August 2018, and the observation group consisted of 58 patients with limbs fracture using modified multifunctional dressing bag with glauber′s salt admitted from September 2018 to January 2020. The degree of pain, the time of appearance of dermatoglyphia, the cases of severe swelling, waiting time for operation, compliance and comfort of the patients were observed within 72 hours after injury.Results:The degree of pain, the time of appearance of dermatoglyphia, the waiting time, the cases of severe swelling for surgery in the observation group were (1.8±0.7) points, (4.5±1.2) days, (5.7±1.2) days,1 case, respectively, which were all lower than those in the control group (2.2±0.8) points, (6.0±1.5) days, (6.5±1.4) days, 19 cases, the differences between the two groups were statistically significant ( t values were -2.6,-5.6,-3.3, χ2 value was 21.8, all P<0.05). The total compliance rate in the observation group was 82.76% (48/58), higher than that in the control group 56.60% (30/53), the differences were statistically significant ( χ2 value was 9.34, P<0.05). The comfort of the observation group was 82.76% (48/58), significantly higher than that of the control group 3.77% (2/53), the difference was statistically significant ( χ2 value was 69.8, P<0.05). Conclusions:The modified multifunctional dressing bag with glauber′s salt can reduce the swelling of limbs, reduce the appearance of skin blisters, stop bleeding and pain, and shorten the waiting time for operation, at the same time, it improves the compliance and comfort of the patients.

Objective To evaluate the diagnostic value of different sequences of magnetic resonance imaging (MRI) in repaired meniscus.Methods From September 2002 to December 2008,118 patients (130 menisci) underwent arthroscopic meniscus suture in our hospital,including 94 males and 24 females,aged from 15 to 50 years (average,25.7t7.5 years).All patients underwent MRI and second-look arthroscopy postoperatively.Different sequences of MRI were taken to evaluate the grade of meniscal signal at repaired site and the slices involved by grade 3 signal.The diagnostic sensitivity,specificity,accuracy,positive predict value (PPV) and negative predict value (NPV) were calculated for each sequence by using second-look arthroscopy as the gold standard.Results The total healing rate was 80.8％ (105/130) by second-look arthroscopy,which was higher than that by different sequences of MRI.The integrated T2 sequence held the highest diagnostic value,and the sensitivity,specificity,accuracy,PPV and NPV were 76.0％,71.4％,72.3％,38.8％ and 92.6％,respectively.According to the second-look arthroscopy result,the menisci were divided into the healed group and unhealed group.In the healed group,28.6％ of cases (30/105) showed grade 3 signal in MRI,which was less than that (76.0％) in the unhealed group.The rate of the new grade 3 signal (8.6％) and the slices involved by grade 3 signal (0.8±1.0) in the healed group were less than those (16.0％ and 3.0±2.0) in the unhealed group.Conclusion The diagnostic value of the integrated T2 sequence is encouraging with high sensitivity,specificity and accuracy.The new grade 3 signal in the repaired meniscus usually implies that the meniscus is not healed.

Objective To establish a sensitive, rapid and simple high-performance liquid chromatography (HPLC) method for the determination of amphotericin B (AraB) and itraconazole (ITZ) in human cerebrospinal fluid (CSF). Methods Solid-phase extraction (SPE) was involved in sample preparation in this method, followed by reverse-phase separation under two different chromatographic systems. Results The tinearity for both AraB and ITZ ranged from 5 ng/mL to 100 ng/mL and the inter-day and intra-day RSD were both below 7.6% at the concentrations of 20,50 and 100 ng/mL. Conclusions The method we established has been applied to the therapeutic drug monitoring on cryptococcal-meningitis-infected patients who were given comedication of these two drugs.

Objective To investigate the therapeutic effects of repeated transcranial magnetic stimulation (rTMS) at high and low frequency for the rigidity and tremors of Parkinson's disease (PD).Methods Sixty-nine patients with PD received rTMS at 5 Hz or 0.5 Hz.The efficacy was assessed using the unified Parkinson's disease rating scale (UPDRS),motor function tests and motor evoked potentials (MEPs).Results Thirty-two patients with rigidity from PD received 5 Hz rTMS therapy,and their UPDRS total scores decreased significantly.Their motor function test results improved significantly and their MEP relaxed motor thresholds were significantly elevated.Thirtyseven patients with tremors from PD received 0.5 Hz rTMS therapy,and their UPDRS total scores were significantly decreased.Their motor function test results improved significantly and their MEP relaxed motor thresholds were significantly elevated.Conclusion High frequency rTMS may improve the symptoms of patients with PD rigidity and low frequency rTMS may improve the symptoms of patients with PD tremors.

Objective To explore the antitumor mechanism of xianhe yanling recipe( XHYLR ). Methods The mechanism of action of 241 compounds contained in the Chinese medicine, XHYLR was studied by using computational pharmacological. Methods including molecular docking,biotechnology network,chemical space technology and the prediction of absorption,distribution,metabolism,excretion and toxicity( ADME/T). Results Most of the compounds in XHYLR had good drug-like properties. Distribution of 241 compounds in chemical space also revealed that these compounds showed similar anti-tumor activity. It suggested that the prescription of XHYLR was reasonable. The phenomena that one molecular interacted with multiple target proteins and several molecules interact with one target protein were observed in XHYLR. For example,the COX-2 interacted with 40 micromolecules,and catechin interacted with five targets including COX-2. Conclusion These results are helpful for understanding the complicated mechanism of anti-tumor activity of XHYLR.

Objective:To construct a recombinant eukaryotic expression vector containing gene encoding 18 000 outer membrane protein from human Helicobacter pylori(H.pylori) and be expressed in COS-7 cell,and lay the foundation for the exploiting DNA vaccine of Hp.Methods:The target genes encoding 18 000 outer membrane protein were acquired from the prokaryotic expression vector pET32a(+)/528 by XholⅠ,KpnⅠ digestion simultaneously,in the same way,the pcDNA3.1 was digested by XhoⅠ,KpnⅠ digestion simultaneously,and the objective genes and pcDNA3.1 were extracted out of agarose electrophoresis with gel kit,and connected by T4 ligase.The recombinant vector pcDNA3.1/528 was used to select and transform,meanwhile express in COS-7 cell.The expressions of recombinant eukaryotic vector in COS-7 cell were investigated by reverse transcriptive-polymerase chain reaction and Western.Results:The gene had inserted into eukaryotic vector was the target gene encoding 18 000 outer membrane protein by enzyme digestion analysis,and objective gene was amplified from COS-7 cell transfected with pcDNA3.1/528 by RT-PCR,the Western showed that recombinant eukaryotic vector could be expressed in COS-7 cell.Conclusion:The recombinant eukaryotic vector pcDNA3.1/528 was constructed and expressed in COS-7 cell successfully.The results obtained lay the foundation for research on development of H.pylori DNA vaccine.