BACKGROUND:Central nerve damage and peripheral nerve injury are common clinical problems that have no ideal treatment. Nerve growth factor has an important role in neuronal repairing and growth. But its local injections may have shorts of inactivation and loss. OBJECTIVE:To construct human nerve growth factor beta recombinant plasmids, which are transfected into bone marrow mesenchymal stem cells from the rabbit mandible by lentiviral vectors, and to investigate the bioactivity of human nerve growth factor beta. METHODS:pDC316-hNGFβ-mCMV-EGFP plasmids were constructed via lentiviral vectors using Hind III+Not I digestion. Bone marrow mesenchymal stem cells from the rabbit mandible were isolated and cultured, and then transfected by recombinant plasmids. The expression of human nerve growth factor beta in transfected cells was detected by ELISA method. RESULTS AND CONCLUSION:pDC316-hNGFβ-mCMV-EGFP plasmids were proved to be constructed successful y by gene sequencing and enzyme identification. The transfected cells under a fluorescence microscope emitted green fluorescence, and the fluorescence intensity had no change with incubation time. The expression of human nerve growth factor beta was maintained at a level of 25μg/L at 7 days after celltransfection, and the bioacitivty was increased significnalty.

Gas gangrene is an emergency condition, which usually develops after injuries or surgery. This study was designed to investigate clinical characteristics, appropriate therapy, and effective control of nosocomial cross-infection of gas gangrene in Wenchuan earthquake victims. Data on diagnosis, treatment, and prevention of confirmed, suspected, or highly suspected gas gangrene were collected. Sixty-seven (2.41%) cases of suspected gas gangrene were found, in which 32 cases were highly suspected of gas gangrene and 5 cases were confirmed by culture of Clostridium perfringens. Thereof, injury sites were mainly located on the limbs, and typical indications, including crepitation, severe localized pain, swelling, wound discoloration, dark red or black necrotic muscle, foul smell as well as different degrees of systemic toxic performance were common among them. After hospitalization, all patients were isolated and had surgery quickly to remove dead, damaged or infected tissue. The wounds were also exposed for drainage and washed or padded with 3% liquid hydrogen peroxide for disinfection before all diagnostic test results were available. Additionally, high doses of antibiotics (mainly penicillin) were given for the prevention of infection, and supportive therapy was applied for corresponding symptoms control. Among those cases, no fatality was reported. In summary, in post-disaster emergency relief, the diagnosis of gas gangrene should be primarily based on clinical manifestations; while patient isolation, wound debridement and disinfection, as well as antibiotics treatment, is the main measures for proper treatment and control of nosocomial infection for gas gangrene.

Gas gangrene is an emergency condition,which usually develops after injuries or surgery.This study was designed to investigate clinical characteristics,appropriate therapy,and effective control of nosocomial cross-infection of gas gangrene in Wenchuan earthquake victims.Data on diagnosis,treatment,and prevention of confirmed,suspected,or highly suspected gas gangrene were collected.Sixty-seven (2.41％) cases of suspected gas gangrene were found,in which 32 cases were highly suspected of gas gangrene and 5 cases were confirmed by culture of Clostridium perfringens.Thereof,injury sites were mainly located on the limbs,and typical indications,including crepitation,severe localized pain,swelling,wound discoloration,dark red or black necrotic muscle,foul smell as well as different degrees of systemic toxic performance were common among them.After hospitalization,all patients were isolated and had surgery quickly to remove dead,damaged or infected tissue.The wounds were also exposed for drainage and washed or padded with 3％ liquid hydrogen peroxide for disinfection before all diagnostic test results were available.Additionally,high doses of antibiotics (mainly penicillin) were given for the prevention of infection,and supportive therapy was applied for corresponding symptoms control.Among those cases,no fatality was reported.In summary,in post-disaster emergency relief,the diagnosis of gas gangrene should be primarily based on clinical manifestations; while patient isolation,wound debridement and disinfection,as well as antibiotics treatment,is the main measures for proper treatment and control of nosocomial infection for gas gangrene.

Objective:To explore the clinicopathological characteristics, immunophenotype, diagnosis and differential diagnosis of renal mucinous tubular and spindle cell carcinoma (MTSCC), and to explore the all-exon mutations, microsatellite stability and tumor mutational burden (TMB) in MTSCC cases.Methods:The data of 5 patients with MTSCC that were submitted to the Department of Pathology, First Affiliated Hospital of Soochow University, China from January 2008 to May 2020, were reviewed and analyzed. The whole exome sequencing (WES) was conducted in all patients, while 3 of them were subject to the analyses of microsatellite stability and TMB.Results:Among the 5 patients, 3 were males and 2 were females. They were 37-76 years old. The maximum diameter of the tumor was 3.5-6.0 cm. The borders of the tumors were well defined. Microscopically, MTSCC was characterized by tubular structure, spindle cell and mucinous stroma, and the nuclear grade of tumor cells was overall low. The average follow-up was 15 months, and no recurrence or metastasis was found. Immunohistochemistry showed that all 5 cases were positive for broad-spectrum cytokeratin (CKpan), cytokeratin (CK)7, CK19, vimentin, PAX8, and P504s (varying expression levels), and the Ki-67 positive index was low. The WES of 5 cases showed that NF2 and PTPN14 exhibited higher mutation rates, which were 3/5 and 2/5, respectively. The microsatellite stability analysis indicated that the 3 cases were all microsatellite stable, and the TMB analysis showed that the TMB of the 3 cases were all <9 mut/Mb.Conclusions:MTSCC is a unique, low-grade pleomorphic kidney tumor. The WES analyses suggest that NF2 and PTPN14 have a higher mutation rate, indicating that the occurrence and development of MTSCC may be closely related to the Hippo pathway. The analysis of microsatellite stability indicates that there is no significant relationship between microsatellite stability and MTSCC, and the TMB analysis suggests that MTSCC patients may not benefit from immunotherapy.

OBJECTIVE： To establish method for simultaneous determination of 7 related substances in solifenacin succinate raw material. METHODS： HPLC method was adopted. The determination was performed on Thermo Hypersil ODS C18 column with mobile phase consisted of 0.02 mol/L KH2PO4 （0.02％ triethylamine， pH＝3.0）-acetonitrile （gradient elution） at the flow rate of 1.2 mL/min. The detection wavelength was set at 210 nm， and column temperature was 40 ℃. The sample size was 20 μL. The regression equation of solifenacin succinate and impurity A， C， D， I， J， K， L were drawn. Correction factors of impurities to solifenacin succinate were calculated with slope. The contents of impurities A， C， D， I， J， K and L were determined in 3 batches of solifenacin succinate raw material. RESULTS： The linear ranges of impurity A， C， D， I， J， K and L were 0.148 1-0.740 3， 0.142 9-0.714 5， 0.141 1-0.705 6， 0.148 9-0.744 6， 0.152 0-0.759 9， 0.137 9-0.689 6， 0.020 0-0.100 0 μg/mL （r＝0.999 8， 0.999 9 or 1.000 0）， respectively. The relative correction factors were 0.51， 0.40， 0.41， 0.91， 0.47， 0.85， 1.23. The limits of detection were 0.049 3， 0.047 6， 0.047 0， 0.048 1， 0.050 7， 0.046 0， 0.006 7   μg/mL. The quantification limits were 0.148 1， 0.142 9， 0.141 1， 0.148 9， 0.152 0， 0.137 9， 0.020 0 μg/mL， respectively. RSDs of precision， stability （24 h） and reproducibility tests were all lower than 5.0％ （n＝6）. Average recoveries were 101.09％， 97.58％， 93.77％， 98.56％， 99.68％， 97.07％ and 93.54％； RSDs were 0.75％ ， 0.51％， 0.47％， 0.84％， 0.70％， 0.75％， 1.21％ （n＝9）. The contents of impurity I in 3 batches of solifenacin succinate raw material were 0.015％-0.018％， other impurities were not detected. CONCLUSIONS： The method is sensitive， accurate and reliable， which can be used to determine the related substances of solifenacin succinate raw material.

ObjectiveTo explore the effects and related mechanisms of modified Shuyuwan on the decline of learning and memory in Alzheimer's disease （AD） mice. MethodForty 5-month-old SPF APP/PS1 mice were randomly divided into model group， Donepezil group， modified Shuyuwan group， modified Shuyuwan+ chloroquine （CQ） group， 10 mice in each group， the same background wild type C57BL/6J ten mice were set as the normal group. Among them， the modified Shuyuwan group was given the modified Shuyuwan decoction （10 g·kg^-1）， the Donepezil group was given the Donepezil hydrochloride solution （0.45 mg·kg^-1）， the modified Shuyuwan + CQ group was CQ （10 mg·kg^-1） was injected intraperitoneally on the basis of the modified Shuyuwan group， and the normal group and the model group were given the same amount of normal saline intragastrically， once a day， for a total of 35 days. After the administration， Morris water maze experiment and new object recognition experiment to detect the spatial memory ability of mice. TdT-mediated dUTP Nick-End Labeling（TUNEL） staining to detect the apoptosis level of mouse hippocampal CA1 neurons， biochemical detection of reactive oxygen species （ROS） and superoxide in mouse hippocampal neurons dismutase （SOD） levels. transmission electron microscopy to observe the ultrastructure of neuronal mitochondria in the CA1 region of mouse hippocampus. Western blot to detect mouse hippocampal mitochondrial autophagy adaptor protein （p62） and microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ）， PTEN-induced kinase 1 （PINK1）， E3 Ubiquitin Ligase（Parkin）protein expression level. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） detection of mouse hippocampal mitochondrial forkhead transcription factor O1 （FoxO1）， PINK1， Parkin mRNA expression level. ResultCompared with the normal group， the escape latency of the model group mice increased significantly， the number of crossing platforms and the retention time in the target quadrant decreased significantly， the relative resolution index decreased significantly， and the ability to recognize new objects was weakened （P<0.05）， neurons in the hippocampus CA1 area decreased. The number of dead cells increased significantly （P<0.05）， the level of ROS was significantly increased （P<0.01）， and the level of SOD was significantly decreased （P<0.01）， the morphology of hippocampal mitochondria was severely damaged， the expression of p62 and LC3Ⅱ proteins increased （P<0.01）， Parkin protein expression decreased （P<0.05）， and PINK1 protein expression increased （P<0.05）， FoxO1， PINK1， Parkin mRNA expressions all decreased （P<0.05）. Compared with the model group， the mice's escape latency was significantly shortened after the intervention of the modified Shuyuwan， the number of crossing platforms and the proportion of residence time in the target quadrant increased significantly， the relative resolution index increased significantly， and the ability to identify new objects was enhanced （P<0.05）. Apoptotic cells were significantly reduced （P<0.05）. ROS levels were significantly reduced （P<0.01）， and SOD levels were significantly increased （P<0.05， P<0.01）， mitochondrial morphology and various structures were significantly improved， p62， LC3Ⅱ protein expression decrease （P<0.05，P<0.01）， PINK1， Parkin protein expression increased （P<0.01）. FoxO1， PINK1， Parkin mRNA expression increased （P<0.05， P<0.01）. Compared with the modified Shuyuwan group， the evasion latency of mice in the modified Shuyuwan + CQ group increased significantly， the number of crossing platforms and the proportion of residence time in the target quadrant decreased， and the relative resolution index decreased （P<0.05）， the SOD level was significantly reduced （P<0.01）. The damage of mitochondrial morphology and structure increased again， the expression of p62 and LC3Ⅱ protein increased （P<0.05， P<0.01）， and the expression of PINK1 and Parkin decreased significantly（P<0.05， P<0.01）. FoxO1， PINK1， and Parkin mRNA expression was significantly reduced （P<0.05， P<0.01）. ConclusionModified Shuyuwan can effectively improve the oxidative stress damage and learning and memory ability of AD mice. The mechanism may be related to up-regulating the expression of FoxO1， PINK1， and Parkin factors， promoting mitochondrial autophagy， reducing oxidative stress， and protecting neuronal damage.

Objective : To analyze the effects of transglutaminase 2(TGM2) gene interference on the malignant proliferation and movement of lung cancer cells . Methods : A549 cells in logarithmic growth phase were divided into control group , shRNA⁃NC group and TGM2⁃shRNA1 group . After transfection , expression levels of TGM2 mRNA A549 cells in logarithmic growth phase were divided into control group , shRNA⁃NC group and TGM2⁃shRNA1 group . After transfection , expression levels of TGM2 mRNA clone formation assay . The proliferation activity of cells was detected by CCK⁃8 . The apoptosis rate was detected by flow cytometer. The invasion of cells was detected by Transwell . The number of tubule formation in each group was observed under microscope . The expression of E ⁃cadherin (E⁃cad) , N ⁃cadherin (N⁃cad) , fibronectin (FN) and vascular endothelial growth factor (VEGF) in each group was detected by Western blot . Results : Compared with shRNA⁃NC group , the mRNA level and protein expression of TGM2 in TGM2⁃shRNA1 group were decreased (P <0. 05) , and the rate of clone formation , proliferation activity , number of invasive cells and number of tubules formation were decreased ( P < 0. 05) . The apoptosis rate and the expression of E ⁃cad protein were increased ( P <0. 05) , while the expression of N ⁃cad , FN and VEGF protein were decreased (P < 0. 05) . Conclusion TGM2 gene silencing can inhibit the expression of VEGF protein , inhibit the malignant proliferation and invasion of A549 cells , and promote apoptosis .

Objective : To analyze the effects of miR⁃346 on alleviating cardiac function damage and oxidative stress in rats undergoing myocardial ischemia/reperfusion (I/R) . Methods : A total of 48 rats were randomly divided into control group , I/R group , I/R + agomiR⁃NC group and I/R + agomiR⁃346 group. I/R model was replicated by the ligation of left anterior descending coronary artery. The recombinant adeno⁃associated virus miR⁃346 was injected through the tail vein for overexpression intervention. After 120 minutes of reperfusion , heart rate (HR) , left ventricular ejection fraction (LVEF) , fractional shortening (FS) and left ventricular wall thickness (LVWT) , levels of serum creatine kinase MB (CK⁃MB) , myoglobin (Mb) , lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) , and levels of superoxide dismutase ( SOD) , glutathion peroxidase ( GSH⁃Px ) and malondialdehyde (MDA) in myocardial tissues were detected. The apoptosis of myocardial tissue cells was detected by TUNEL staining. The expression of B⁃cell lymphoma/leukemia⁃2 (Bcl⁃2) gene , Bcl⁃associated x protein (Bax) , cysteine protease 3 ( Cas⁃3 ) and Cas⁃9 in myocardial tissues was detected by Western blot. Results : HR , LVEF , FS and LVWT in I/R group were lower than those in control group , the levels of serum CK⁃MB , LDH , Mb and cTnI were higher than those in control group , the expression of MDA , Bax/Bcl⁃2 , cleaved Cas⁃3/Cas⁃3 and cleaved Cas⁃9/Cas⁃9 proteins in myocardial tissues was higher than that in control group , the levels of SOD and GSH⁃px were lower than those in control group , and the apoptosis rate of myocardial tissue cells was higher than that in control group (P < 0. 05) . HR , LVEF , FS and LVWT in I/R + agomiR⁃346 group were higher than those in I/R + agomiR⁃NC group , the levels of serum CK⁃MB , LDH , Mb and cTnI were lower than those in I/R + agomiR⁃NC group , the expression of MDA , Bax/Bcl⁃2 , cleaved Cas⁃3/Cas⁃3 and cleaved Cas⁃9/Cas⁃9 proteins in myocardial tissues was lower than that in I/R + agomiR⁃NC group , the levels of SOD and GSH⁃px were higher than those in I/R + agomiR⁃NC group , and the apoptosis rate of myocardial tissue cells was lower than that in I/R + agomiR⁃NC group ( P <0. 05) . Conclusion The miR⁃346 overexpression can reduce oxidative stress level in I/R rats , alleviate myocardial tissue damage , and improve cardiac function.