Objective:To investigate the effect of bone mesenchymal stem cells derived exosomes (BMSC-Exo) on improving hippocampal microangiogenesis, energy metabolism, and behaviors in depression mouse models.Methods:(1) Mouse bone marrow mesenchymal stem cells were isolated and cultured to extract BMSC-Exo; BMSC-Exo morphology was observed by transmission electron microscopy, BMSC-Exo particle diameter ranges were determined by Zetaview analyzer, and expressions of CD9 and CD63 in BMSC-Exo were detected by Western blotting. (2) Depression models were established in 2 mice by chronic unforeseeable mild stress (CUMS); 24 h after stereotaxic injection of phosphate buffer solution (PBS) or DiR labeled BMSC-Exo, BMSC-Exo uptake was detected by in vivo imaging system. (3) Thirty-six mice were randomly divided into control group, model group and BMSC-Exo group ( n=12); CUMS was used to establish depression models in the latter 2 groups; brain stereotaxic injection of 1 μL BMSC-Exo was given to mice in the BMSC-Exo group after modeling, and same amount of PBS was given to the control group; behaviors were observed by forced swimming test (FST), tail suspension test (TST) and open field test (OFT); hippocampal microvascular length and number were detected by alkaline phosphatase staining; energy metabolism in the hippocampus was detected by micro positron emission tomography/computed tomography (mPET/CT); glucose transporter 1 (GLUT1) expression in the hippocampus was detected by Western blotting. Results:(1) BMSC-Exo had a typical disk-like vesicle-like structure with particle size of (100.5±1.4) nm; Western blotting confirmed that CD9 and CD63 expressed in BMSC-Exo. (2) In vivo imaging showed no fluorescence in the brain and liver after PBS injection, but obvious local fluorescence after BMSC-Exo injection. (3) Compared with the control group, the model group and BMSC-Exo group had significantly longer rest time in FST and TST and shorter movement distance and time in the central region of OFT ( P<0.05); compared with the model group, BMSC-Exo group had significantly shorter rest time in FST and TST and longer movement distance and time in the central region of OFT ( P<0.05). Compared with the control group, the model group and BMSC-Exo group had significantly decreased standard uptake value (SUV) of regions of interest, microvascular length and number, and GLUT1 expression in the hippocampus ( P<0.05); compared with the model group, the BMSC-Exo group had significantly higher SUV, microvascular length and number, and GLUT1 expression in the hippocampus ( P<0.05). Positive correlations were noted between hippocampal microvascular length and SUV and between microvascular number and SUV in the 3 groups ( r=0.540, P<0.001; r=0.600, P<0.001). Conclusion:BMSC-Exo could promote microangiogenesis energy metabolism in the hippocampus to improve depression-like behaviors in depression mouse models.

Objective:To investigate the effects of human umbilical cord mesenchymal stem cells exosomes (hucMSC-Exo) on depression-like behavior and polarization dynamic transition of hippocampal microglia in chronic unpredictable mild stress(CUMS) mice.Methods:The hucMSC was isolated, cultured, and the 5th generation hucMSC-Exo was extracted by ultracentrifugation and identified.Biological markers of CD9 and CD63 of hucMSC-Exo were detected by Western blot.A total of 12 SPF grade male C57BL/6J mice were randomly selected to establish depression model by CUMS method at the same time, and relevant behavioral experiments, including opening field test (OFT) and forced swimming test (FST) were used to detect changes of depression-like behavior in CUMS mice. After modeling, 12 mice were randomly divided into two groups, with 6 mice in each group. One group of mice was the exosome treatment group (CUMS+ Exo group) after stereotactic brain injection of hucMSC-Exo in the hippocampus, and the other group was the CUMS group. The expression of inflammatory signals in the hippocampus of mice in both groups were detected by micro PET/CT scanning technique. The proportion of M2-type microglia and M1-type microglia in the hippocampus of mice was detected by tissue immunofluorescence. β3-tubulin immunofluorescence staining was used to detect the length changes of neuronal axons in the depressed cell model constructed with corticosterone (CORT). EdU staining was used to detect neuronal proliferation. TUNEL staining was used to observe the apoptosis of neurons in the hippocampus of mice.Statistical analysis was conducted by GraphPad 8.0 software, and t-test was used for inter group comparison. Results:Under the electron microscope, hucMSC-Exo showed typical " double-layer cup-like" small vesicle-like changes and the particle diameter was about 100 nm.Western blot confirmed the expression of lconic proteins of CD9 and CD63. In the micro PET/CT scans, the uptake of [18F]DPA-714 in the CUMS+ Exo group was lower than that of the CUMS group, and the difference was statistically significant ((0.91±0.02)g/mL, (0.81±0.05)g/mL, t=4.54, P=0.001 1). In the opening field test, the percentage of central path length ((3.40±0.44)%, (5.17±0.90)%, t=4.33, P=0.001 5) and the time spent on the central path ((7.04±0.60)s, (10.22±1.41)s, t=6.02, P=0.000 1) in CUMS+ Exo group were higher than those in CUMS group, while the total distances of the two groups were not statistically significant ( t=0.48, P>0.05). In the forced swimming test, the immobility time in the CUMS+ Exo group was less than that in the CUMS group ((152.33±7.28) s, (133.50±4.32) s, t=5.45, P=0.000 3). In tissue immunofluorescence experiments, compared with the CUMS group, the proportion of M2-type microglia of hippocampus in the CUMS+ Exo group((0.33±0.04), (0.59±0.12), t=5.11, P=0.000 5)increased and the proportion of M1-type microglia ((0.56±0.06), (0.41±0.03), t=5.15, P=0.000 4) decreased in the CUMS+ Exo group. β3-tubulin-labeled immunofluorescence results showed an increase of neuronal axon length in the CORT+ Exo group compared with that in the CORT group((3.99±0.99) μm, (6.76±1.11) μm, t=6.10, P=0.000 1). The results of cell proliferation test showed an increase of proliferation rate in the CORT+ Exo group compared with that in the CORT group((0.74±0.07), (0.64±0.03), t=3.32, P=0.001 8). In the TUNEL staining experiment, the apoptosis rate of neurons in the hippocampal region of mice in the CUMS+ Exo group was lower than that of CUMS group ((0.24±0.04), (0.39±0.04), t=6.11, P=0.000 1). Conclusion:hucMSC-Exo can promote the conversion of M1 polarized microglia to M2 type microglia to alleviate depression-like behavior and reduce neuronal apoptosis in CUMS mice.

We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as nonribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.
Clostridium Infections ; diagnosis ; Clostridium difficile ; genetics ; Electrophoresis, Capillary ; Feces ; microbiology ; Genes, Bacterial ; Humans ; Polymerase Chain Reaction ; Ribotyping ; Sensitivity and Specificity

We analyze the epidemiology,clinical features,and outcome of the patients with Creutzfeldt-Jakob diseases (CJD) in Guizhou Province from 2010 to 2015.The epidemiology,clinical characteristics and follow-up data of CJD suspected patients obtained from Guizhou CJD surveillance network were analyzed.The testing results of cerebrospinal fluid (CFS) and blood from the patients were also collected and analyzed.Results showed that a total of 11 CJD cases was found from 23 reported CJD suspected patients in Guizhou from 2010 to 2015,including 8 probable sporadic CJD(sCJD) cases,2 possible sCJD cases and 1 genetic CJD(gCJD) case.In 11 cases,rapidly progressive dementia was the major initial symptom,following by mental symptoms,extrapyramidal symptoms,signs and cerebellum cortical blindness.Clinical symptoms of progressive dementia were the main symptoms,following by visual or cerebellar dysfunction,myoclonus,cone system/extrapyramidal dysfunction,and akinetic mutism.Most of cases were abnormal in MRI (45.45％) and 14-3-3 protein detection in CSF(70％).The 14-3-3 blood samples of prion gene 129 amino acids (PRNP)polymorphisms were M/M type,excepting for 1 case gCJD confirmed diagnosis cases with D178N mutation in PRNP gene.Eleven CJD cases did not show season and regional clusterings and vocational tendency.The majority of the cases were male,the median age was 65,and mainly were the Han nationality.For all cases of CJD reported during that year for follow-up,the lost-tofollow-up rate was 27％,and the majority of cases died within one year.The sCJD cases were the majority in CJD cases of Guizhou Province,2010-2015.The epidemiological characteristics were similar to the national monitoring cases in the same period.

Objective To monitor ventilator-associated event (VAE) for the first time in an intensive care unit (ICU) in China,understand the applicability,incidence,and clinical significance of VAE in China.Methods Targeted monitoring on VAE was performed among patients ≥18 years and with mechanical ventilation (MV)＞2 days in the ICU of a hospital between January 2014 and September 2015,incidence of VAE was calculated,and patients were grouped according to whether or not they had VAE,prognostic factors were analyzed statistically.Results A total of 1 004 patients were monitored,the total hospital stay was 13 795 days in patients who used ventilator,307 (30.58％) cases of VAE occurred,incidence of VAE per 1 000 ventilator-days was 22.25.Univariate analysis showed that patients with VAE had longer length of ICU stay and MV,and higher mortality rate than patients without VAE when they moved out of ICU (all P＜0.05).Multivariate logistic regression analysis showed that VAE was independent risk factor for length of ICU stay,duration of MV,as well as mortality when patients moved out of ICU(all P＜0.05).Conclusion Judgment of VAE is based on MV parameters,it is more objective and accurate.There is a high incidence of VAE among ICU patients,it may lead to poor clinical outcomes,and has good values for the targeted monitoring on ICU patients in large comprehensive hospitals of China.

Objective To investigate the effect of taurine transporter in the process of protection of brain edema in rats with severe traumatic head injury. Methods A total of 24 Male Sprague-Dawley rats were randomly divided into 4 groups. Except the control rats (Group Sham), all other three groups were subjected to lateral fluid percussion head injury. The TBI (Traumatic brain injury) models (Group TBI) and surgical control rats (Group Sham) were injected with saline through caudal vein after surgery, while the Taurine prevention and Taurine treatment models (Group Pre Tau and Group Tau) were injected with 120 g/L taurine solution before or after surgeries respectively. Water content in each brain, mRNA and protein expres?sion of aquaporin 4 and taurine transporter in the injured rat brain hemispheres were all evaluated over the time course of the study (7 d) in each group. Results Compared with rats in Group Sham, water content in each brain increase, mRNA tran?scription and protein expression of AQP4 were both up regulated but the mRNA transcription and protein expression of TauT were both down-regulated in rats in TBI group. Compared with rats in TBI group, brain water content, mRNA transcription and protein expression of AQP4 all decrease while mRNA transcription and protein expression of TauT all increase in rats in Pre tau and Tau groups. There is no statistical difference of TauT expression between rats in pre-tau group and Tau group. Conclusion Taurine exert its neuron protection role through draining water content from brain and down regulating expres?sion of AQP4 but rising expression of TauT after TBI.

Objective To investigate effect of taurine on respiration chain enzyme activity of mitochondria 24 hours after severe traumatic brain injury (TBI) in rats.Methods Fifty-six SD rats were divided into sham group,TBI group,taurine treatment group,and taurine prevention group according to the random number table,with 14 rats per group.Fluid percussion brain injury models were used.Via the caudal vein,normal saline was administered to animals in sham and traumatic brain injury groups immediately after injury,while taurine (200 mg/kg)was administered to animals in taurine treatment group after injury and in taurine prevention group 4 days before injury.Brains were harvested 24 hours postinjury for assays of HE staining and electron microscopy.Mitochondrial respiratory chain complex Ⅰ-Ⅴ activities were detected.Results TBI group presented swelling neurocytes,cell loss,karyopyknosis,shortened even vanished process,and inflammation cell infiltration at the edge of necrosis in HE staining.By contrast,morphological improvement was significant in taurine treatment group but only some neurons were intact in taurine prevention group.Swelling mitochondria and broken or vanished mitochondrial crests were seen in TBI group under the electron microscope.However,normal or minor swelling mitochondria was seen in taurine treatment group and cytoplasm slightly porous and absence of mitochondrial crests were seen in taurine prevention group.Activities of complex Ⅰ,Ⅱ and Ⅴ were significant lower in TBI group (32.52±2.41,4.68 ±0.15,2.49 ±0.73) compared to those in sham group (34.03 ±0.46,5.04 ±0.29,3.20±0.68) and in taurine treatment group (33.95±0.85,5.12-±0.23,3.53 ±0.48) (P＜0.05).And complex Ⅰ in taurine prevention group was significantly enhanced as well (34.44 ± 0.36,P ＜ 0.05).Conclusion Taurine may protect the brain tissues and mitochondrial structure from impairment in TBI rats by improving mitochondrial enzymes activity and reducing secondary energy loss.

Objective To investigate effect of mild hypothermia on changes of somatosensory evoked potential (SEP) and synaptophysin mRNA level after traumatic brain injury (TBI) and determine hypothermia-induced neuroprotection.Methods Forty-five SD rats were allocated into mild hypothermia group,TBI group and sham operation group with 15 rats per group according to the random number table.Left-side fluid percussion impact was performed to induce models of TBI.Rats were exposed to hypothermia environment (32-35℃) for 6 hours in mild hypothermia group after TBI.Rats in sham operation group were treated by only drilling on left side of the head,rather than hitting.To evaluate function outcome,modified neurological severity score (mNSS),SEP and synaptophysin mRNA level were measured at 6 hours,24 hours and 7 days postinjury.Results The mNSS in mild hypothermia group lowered compared with TBI group,especially at 24 hours and 7 days (P ＜ 0.05).SEP in mild hypothermia group was significantly shortened at 6 and 24 hours compared with TBI group (P ＜ 0.05),but SEP revealed no significant difference among the 3 groups at 7 days (P ＞ 0.05).Level of synaptophysin mRNA in mild hypothermia group increased at 6 hours postinjury compared with TBI group [(0.08 ± 0.02) vs (0.12 ±0.04)],with further increase at 7 days postinjury[(0.06 ± 0.01) vs (0.33 ± 0.10)] (P ＜0.05).Conclusion The shortage of nerve conduction time of the injured side and promotion of nerve regeneration suggest the neuroprotective role of mild hypothermia following TBI.

Objective To investigate the relationship between HBV genotypes and YMDD motif mutations or BCP mutations in Xiangtan of Hunan Province. Methods HBV genotypes, YMDD motif mutations and BCP mutations were analyzed in 952 HBV infected patients. Results HBV genotyping showed that 698 HBV type B patients and 115 HBV type C patients accounted for 73.32% and 12.08% respectively of all the participants. The rest 139( 14.60% )were genotype B and C mixed infection( B + C ). The analysis of YMDD motif mutations showed that 844 YMDD wild-type which accounted for 88.66% of all the subjects and the remainder were YMDD mutation types, of which 54( 5.67% ) carried YVDD, 53( 5.57% ) YIDD,and 1 YVDD and YIDD mixed infection. Basic Core Promoter mutations showed that 1762A/1764G ( wild type )accounted for 70.59% and 1762T/1764A( mutant ) accounted for 19.75%. The rest 92 patients were 1762T/1764A and 1762A/1764G mixed infection. This study showed no significant difference in the rate of YMDD mutation( 10.04% vs 10.43% ,χ2 =0.017,P＞0.05 ) ,but a significant difference in the types of YMDD mutation(χ2 = 4.836, P ＜ 0.05 )between HBV types B and C. The YVDD mutation was more commonly seen in genotype C( 9.57% ) than in genotype B( 5.88% ). The BCP mutation rate showed a significant difference( 27.36% vs 46.09%, χ2 = 16.478, P ＜ 0.01 ). Genotype C was more frequent than genotype B. The BCP mutation rate showed no significant difference between YMDD Wild-type and YMDD mutation types( 28.67% vs 35.51%, χ2 = 2.139, P ＞ 0.05 ), but most of BCP mutations happened in YVDD mutant type( 61.11% ). Conclusions ( 1 ) The predominant HBV genotypes in Xiangtan were genotype B and genotype C, the major genotype was type B, which display the characteristics of epidemiology in Southern China. ( 2 ) Determination of HBV genotypes before lamivudine therapy was probably not an important pretreatment investigation to predict antiviral responses. ( 3 ) Detection of HBV genotypes, YMDD motif mutations and BCP mutations will contribute to the correct evaluation of prognosis and timely proper management of HBV patients.

OBJECTIVEImmortal cell line of human embryonic esophageal epithelium (SHEE) was induced by E6E7 genes of human papillomavirus (HPV) type 18 in our laboratory. To identify the fully malignant transformation at its 85th passage (SHEE85), the malignant phenotype, tumorigenesis and invasive potency were studied.

METHODThe cultured SHEE85 cells were observed under the light and the electron microscope (EM) for cell morphology, analyzed by flow cytometry for cell cycle. The tumorigenesis was assayed by plating cells in soft-agar and transplanting cells into the nude mice and SCID mice. To detect invasive potency, cells were cultured on amniotic membrane in vitro and transplanted into peritoneal cavity of mice in vivo.

RESULTSSHEE85 cells were crowded in cultivation with different sizes and shapes under light microscope, and displayed proliferative morphology under EM. Proliferative index was 47% with 12% hyperploidy cells in determination of DNA histogram. Many large colonies grew in soft-agar (4%) and the transplanted tumors were found in all 4 nude and 4 SCID mice, with strong invasive potency demonstrated in vitro and in vivo.

CONCLUSIONThe immortal esophageal epithelial cell line induced by HPV18 E6 E7 is derived from a fully malignant transformation with a strong invasive potency at the 85th passage. It is also a reliable model for studying the cellular and molecular mechanisms of carcinogenesis of the esophageal carcinoma.

Animals ; Cell Division ; genetics ; Cell Transformation, Neoplastic ; Cell Transformation, Viral ; genetics ; Cells, Cultured ; Epithelial Cells ; cytology ; ultrastructure ; virology ; Esophagus ; cytology ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mice, SCID ; Microscopy, Electron ; Neoplasm Transplantation ; Neoplasms, Experimental ; pathology ; Oncogene Proteins, Viral ; genetics ; Papillomaviridae ; genetics ; Ploidies ; Transplantation, Heterologous