OBJECTIVE To construct a lentiviral vector of RNA interference (RNAi) of MMP-9 gene and observe its inhibitive role on the invasion of laryngeal cancer cells.METHODS The effective sequence of siRNA targeting MMP-9 gene was confirmed.Both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pLVTHM vector,which contained H1 promoter and green fluorescent protein (GFP).The resulting lentiviral vector containing MMP-9 shRNA was called LV2 shMMP-9,and it was confirmed by PCR and sequencing.After that,MMP-9 shRNA was transfected into Hep-2 cells and western blot was used to test the expression of MMP-9.At last,Boyden Chamber was used to observe the invasion of the Hep-2 cells. RESULTS PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of MMP-9 (LV2shMMP-9) producing MMP-9 shRNA was constructed successfully. The titer of concentrated virus was 8?1010TU /L. Western blot showed that the expression of MMP-9 was negative in the MMP-9 siRNA transfected Hep-2 cells. And Boyden Chamber showed the invasive capability of Hep-2 cells transfected MMP-9 siRNA were obviously decreased.CONCLUSION The lentivirus RNAi vector of MMP-9 was constructed successfully,and MMP-9 silence can inhibit invasion of laryngeal cancer in vitro.

Aim To investigate the neuroprotective effects of osthole(Ost)on the primary cultured cortical neurons transfected with APP595/596 gene and its underlying mechanism.Methods Neonatal mouse cortical neurons were transfected with APP595/596 gene to establish AD cell models for the further study.Then,the cell viability was detected by CCK-8 assay,and the leakage of lactate dehydrogenase(LDH)was assayed by LDH kit to evaluate the injury degree.Transferase-mediated nick end labeling(TUNEL)was used to evaluate the cell apoptosis.The expression of β-amyloid peptide(Aβ)and β-site APP cleaving enzyme 1(BACE1)was measured by immunofluorescence,while the miRNA-107 was measured by RT-PCR.Results Compared to model group,Ost could significantly improve the neurons viability,decrease the LDH release and prevent the apoptosis.Ost also inhibited the expression of Aβ and BACE1 at protein level,while enhanced the expression of miRNA-107 at gene level.Conclusion Ost plays a neuroprotective role in neurons transfected with APP595/596 gene in part through up-regulating miRNA-107.

Objective To investigate the application effect of nasal gastrointestinal double lumen catheter tube placement with air insufflation in mechanically ventilated patients with intra-abdominal hypertension.Methods A total of 20 patients with intra-abdominal hypertension were randomly divided into control group and observation group (n=10 in each group).Patients in control group received indwelling nasogastric tube for decompression and indwelling nasal intestine tube by air insufflation for enteral nutrition support .Patients in observation group received the dual lumen gastrointestinal tube for decompression and enteral nutrition support.The time required for catheterization,changes in intra-abdominal pressure before and after air insufflation,catheter success rate at one attempt,pain scores of patients to catheter operation, duration of decompression,enteral nutrition start time and the duration of mechanical ventilation were compared between two groups.Results The time required for catheterization,catheter air insufflation volume in observation group were significantly lower than those in control group (P <0.01);pain scores of patients to catheter operation were significantly lower in observation group than those in control group (P <0.01);There was no significantly difference in once catheter success rate,changes in intra-abdominal pressure before and after catheterization,abdominal high pressure relief time ,enteral feeding start time, and duration of mechanical ventilation between the two groups (P >0.05 ).Conclusions The technique of air insufflation has higher success rate for Indwelling nasal intestine tube in mechanically ventilated patients with intra-abdominal hypertension,and this method is safe and reliable,dual lumen gastrointestinal tube can improve patients'comfort,shorter catheterization time and reduce catheter air insufflation volume.

Aim To investigate the effects of osthol on cell apoptosis and inflammatory cell infiltration after brain stab wound injury in mice. Methods The mice underwent the stab wound injury by a needle, then were randomly divided into sham operation group, model group, osthol 10, 20, 30 mg · kg-1 treatment group. The main examinations included mice brain wa-ter content; the apoptotic cytokines Bax, Bcl-2, Caspase-3 mRNA expression were assessed by PT-PCR; immunohistochemistry staining was used to de-tect neutrophils (MPO) and microglia (Iba-1) infiltra-tion and Caspase-3 positive cell expression around in-jured lesions. Results Treatment with osthole 20, 30 mg·kg-1 group significantly reduced the water content in injured brain, improved the ratio of Bax/Bcl-2, and reduced the expression of apoptosis cytokine Caspase-3 mRNA. Osthole 30 mg·kg-1 treatment group obvious-ly reduced the infiltration of neutrophils and microglial cells and significantly reduced the number of apoptotic cells around the injured cerebral cortex. Conclusion Osthole has therapeutic effect on stab wound injury in mice, and the possible mechanism may be by reducing the infiltration of inflammatory cells and reducing apop-totic cells.

OBJECTIVE:To investigate the protective effects of osthole on the nerves in model mice with craniocerebral injury. METHODS:Mice models of craniocerebral injury were established by craniotomy drill. There was a sham-operation group(isomet-ric normal saline),a model group (isometric normal saline) and osthole high,mediu,low dose groups (30,20,10 mg/kg). The drugs were given to the mice 1 h after successful establishment of the models,ip,once a day,for consecutive 14 d. Neurological severity score was conducted for the mice 12 h,3 d,7 d,14 d and 21 d after the establishment of models;HE stain was conduct-ed 7 d and 14 d thereafter and the wounds areas of brain were observed by microscope;the activity of myeloperoxidase(MPO)in the homogenate of mice’s brain tissues were determined 1 d and 3 d after the establishment of models;immunohistochemical meth-od was adopted to determine the expressions of the brain-derived neurotrophic factors (BDNF) and neurotrophic factor (NT) 3 in the mice’s brain tissues 7 d after the establishment of models. RESULTS:Compared with model group,the neurological severity scores of the mice in osthole high dose group and medium dose group were decreased 3 d,14 d and 21 d after the establishment of models;that in osthole high dose group were decreased 7 d after the establishment of models. The wounds areas of brain in osthole high dose group were smaller 7 d after the establishment of models;those in osthole high dose group and medium dose group were smaller 14 d after the establishment of models. The activity of MPO in the brain tissue in osthole high dose group was decreased 24 h and 72 h after the establishment of models.The expressions of the BDNF and NT-3 in the brain tissue homogenate in osthole high dose group and medium dose group were increased 7 d after the establishment of models,with significant differences(P<0.01 or P<0.05). CONCLUSIONS:Osthole has certain protective effects on the nerves in mice with craniocerebral injury. The mechanism may be related to improving the mice’s neurological functions,promoting wound healing,inhibiting the production of inflammato-ry factors,increasing the expression of neurotrophic factors.

AIM:To explore the protective effect of osthole on the SH-SY5Y cells transfected with APP595/596 gene, and to investigate the molecular mechanism.METHODS:The SH-SY5Y cells were transfected with APP595/596 gene in vitro for establishing a cell model to study the pathogenic role of amyloid β-protein ( Aβ) .The cell viability was detected by CCK-8 assay.The release of lactate dehydrogenase ( LDH) was determined by the colour reaction of dia-phorase-INT.The cell apoptotic rate was analyzed by flow cytometry.The expression of β-site APP cleaving enzyme 1 ( BACE1) at mRNA and protein levels was detected by RT-PCR and Western blot.The expression of Aβwas measured by the technique of immunofluorescence cytochemistry and Western blot.RESULTS: Treatment with osthole inhibited the LDH release, and increased the viability of the cells.The percentage of apoptotic cells was also significantly decreased. Osthole also inhibited the expression of BACE1 at mRNA and protein levels and the protein expression of Aβ.CONCLU-SION:Osthole has protective effect on SH-SY5Y cells transfected with APP595/596 gene.The mechanism may be associ-ation with inhibiting the mRNA and protein expression of BACE1.

Aim To investigate the effects of osthole ( Ost) on the ability of proliferation and differentiation in APP transduced neural stem cells( NSCs) , and neu-ronal apoptosis, in order to find related mechanism. Methods A model of Alzheimer′s disease( AD) cells was successfully established by transducing APP gene into NSCs in vitro. The ability of proliferation and dif-ferentiation was tested by staining. The viability of NSCs was determined by using CCK-8 assay. The cell apoptosis was tested by Hoechst 33258 staining. The expression of GSK-3β and β-catenin mRNA was deter-mined by RT-PCR. The expression of GSK-3β and β-catenin protein was determined by Western blot. Re-sults The ability of proliferation had increased by 10 . 24% with Ost treatment, compared with APP group. The ability of differentiation had increased by 6 . 74%with Ost treatment, compared with APP group. The vi-ability of NSCs had increased and cell apoptotic rate had decreased significantly. From the results of RT-PCR and Western blot, we could find the expression of GSK-3βmRNA and protein had decreased, and the ex-pression of β-catenin mRNA and protein had increased significantly, compared with APP group. Conclusion Ost could enhance the ability of proliferation and dif-ferentiation into more neurons of NSCs transducing APP gene, and reduce neuronal apoptosis. It might be relat-ed with activiting Wnt/β-catenin signaling pathway.

Aim To investigate the effect of osthole on neuron synapses infected APP gene and its underlying mechanism. Methods The neurons were divided into three groups:GFP, APP, APP+Ost groups. The neu-rons were infected APP gene with containing mutational site in vitro for mimicking the characterstics of Alzhei-mer’ s disease ( AD) . The cell viability was assessed by CCK-8 , the expression of synapsin-1 was deter-mined by immunofluorescence, and the concentration of PSD-95 and SYP were detected by ELISA. The ex-pressions of Aβ1-42 , CAMKK2 , phoshorylated AMPKα1 , AMPKα1 protein were determined by West-ern blot. Results Strong APP staining was visible in neurons infected with APP and abundant expression of Aβ1-42 , a neurotoxic oligomer. Compared with APP group, APP+Ost group significantly increased cell vi-ability, promoted the expression of synapsin-1, up-reg-ulated the concentration of PSD-95 and SYP, and de-creased the expressions of CAMKK2 and p-AMPKα1 . Conclusions Ost can protect the neuron synapses a-gainst infected with APP gene. Its neuroprotective effect may be related to inhibiting the CAMKK2/AMPK signal pathway.

Objective To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluo-rescence labeling for mitochondrial DNA (mtDNA) SNP typing. Methods Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided in-to 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood sam-ples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three ran-dom samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. Results Distinct electropherograms of 200 blood samples were obtained suc-cessfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10μL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. Conclusion AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.

BACKGROUND:Neural stem cells have self-renewal and multidirectional differentiation potential, but under normal circumstances, the number of neural stem cells is less, and most cells are in the resting state. Thus, to promote the proliferation of neural stem cells is the key to the treatment of neurodegenerative diseases. OBJECTIVE:To investigate the effects of osthole on the proliferation of neural stem cells cultured in vitro, and to analyze its mechanism underlying promoting the proliferation. METHODS:Neural stem cells were cultured in vitro, and passage 3 cells were cultured with different concentrations of osthole(10, 50 and 100μmol/L). After 24 hours, cellvitality was determined by cellcounting kit-8. After 3, 5, 7 days of further culture, the radius of neurospheres was measured, and Ki67-positive cells were counted by immunofluorescence staining. Meanwhile, after 3 days of further culture, the gene expression of Notch 1, Hes 1 and Mash 1 in neural stem cells was detected by RT-PCR. RESULTS AND CONCLUSION:Compared with the control group, 50, 100μmol/L osthole could obviously promote the proliferation ability of neural stem cells. 100μmol/L osthole had the most significant effect and increased the expression of Notch 1 gene, Hes 1 gene, but it had no effect on Mash 1 gene. These results suggest that osthole can promote proliferation of neural stem cells cultured in vitro and its mechanism may be associated with activation of Notch 1 gene and Hes 1 gene in Notch signaling pathway.