Objective:To discuss the promotive effect of nerve growth factor(NGF),which is highly expressed in the adipose-derived stem cell(ADSC)-induced Schwann-like cells(SCLCs),on the growth of dorsal root ganglion(DRG)cell processes in the rats,and to clarify its mechanism.Methods:The ADSCs were extracted from the epididymal adipose tissue of the SD rats,and their multidirectional differentiation potential was identified through osteogenic,adipogenic,and chondrogenic induction.The ADSCs were induced to differentiate into the SCLCs,and the expression levels of glial fibrillary acidic protein(GFAP)and S100 calcium-binding protein β(S100β)protein in the ADSCs and SCLCs were detected by immunofluorescence staining and Western blotting methods.The DRG cells were isolated and cultured,and immunofluorescence staining was used to detect the βⅢ-tubulin expression in the DRG cells for identification.The SCLCs were co-cultured with the DRG cells(co-culture group),the single-culture DRG cells were regared as DRG group and toluidine blue staining was used to observe and measure the length of DRG cell processes under the optical microscope in co-culture group and DRG group.Small interfering RNA(siRNA)transfection was used to knock down NGF,and plasmid transfection was used to over-express NGF.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the NGF mRNA expression levels in the cells in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the NGF protein levels in the cell supernatants.The transfected SCLCs were co-cultured with DRG cells and divided into control group,siNC/vector group,NGF knockdown group(si-NGF group),and NGF over-expression group(oe-NGF group).The lengths of DRG cell processes in various groups were observed.Results:The primary ADSCs adhered within 24 h after seeding,with a small number of lipid droplets remaining.After 3 d of culture,the cells were mostly short spindle-shaped,fusiform,or polygonal,growing rapidly in a vortex pattern.After passaging,the cells exhibited a uniform morphology,appearing as long spindles arranged in a fish-school pattern.After 14 d of adipogenic induction,the cell morphology changed from spindle-shaped to flat-round,with translucent lipid droplets forming in the cytoplasm,which were stained red by Oil Red O.After 28 d of osteogenic induction,the cells appeared sand-like with blurred morphology,and calcified nodules were observed,which were stained red by Alizarin Red and deposited in the extracellular matrix.After 28 d of chondrogenic induction in a 3D culture system,millet-sized chondrogenic spheres formed.Frozen sections of the spheres were stained with Alcian Blue,and acidic mucopolysaccharides in the cartilage tissue were stained blue under the microscope.Under the fluorescence microscope,the third-passage purified ADSCs showed positive expression of CD29[fluorescein isothiocy anate(FITC)-labeled green fluorescence]and CD44(Cy3-labeled red fluorescence).The immunofluorescence staining results showed that GFAP was labeled with FITC(green fluorescence),and S100β was labeled with Cy3(red fluorescence).The Western blotting results showed that compared with ADSCs,the expression levels of S100β and GFAP proteins in the SCLCs were increased(P<0.05).The primary DRG cells began to adhere 6 h after conventional culture,and after 3 d,the cell bodies appeared round and bright,with two linear processes extending from them.Under fluorescence microscope,the cells positively expressed the neuron-specific marker βⅢ-tubulin,confirming that the isolated cells were DRG cells.Compared with the ADSCs,the NGF protein expression level in the SCLCs was increased(P<0.05).Compared with DRG group,the length of DRG cell processes in co-culture group was the highest when DRG cells and SCLCs were co-cultured at a 1∶2 ratio(P<0.05).The RT-qPCR results showed that compared with si-NC group,the expression levels of NGF mRNA in the cell supernatant in si-NGF-1,si-NGF-2,and si-NGF-3 groups were significantly decreased(P<0.05),with si-NGF-1 showing the highest knockdown efficiency,which was selected for subsequent experiments.The ELISA results showed that compared with si-NC group,the NGF levels in the cell supernatant of si-NGF-1,si-NGF-2,and si-NGF-3 groups were decreased(P<0.05).Compared with Vector group,the expression level of NGF mRNA and NGF protein level in the supernatant in oe-NGF group were increased(P<0.05).Compared with control group and siNC/vector group,the length of DRG cell processes in si-NGF group was decreased(P<0.05),while the length of DRG cell processes in oe-NGF group was increased(P<0.05).Conclusion:ADSCs can be directionally differentiated into SCLCs,and the differentiated cells highly express NGF.Knockdown or overexpression of NGF can affect the growth of DRG cell processes.

Objective:To observe the effects of adipose-derived stem cells(ADSC)combined with acellular scaffold(AS)on the ultrastructure of dorsal root ganglion and the protein and mRNA expression levels of ciliary neurotrophic factor(CNTF),Janus kinase 2(JAK2),phosphorylated JAK2(p-JAK2),signal transducer and activator of transcription 3(STAT3)and phosphorylated STAT3(p-STAT3)in the rats with sciatic nerve injury(SNI),and to clarify the protective effect of ADSC combined with AS on dorsal root ganglion in the SNI rats and its possible mechanism.Methods:The rat ADSCs were isolated and cultured and their multidirectional differentiation potential was detected.The AS of rats was prepared,and ADSCs were injected into the AS to construct tissue-engineered nerve.A total of 36 rats were randomly divided into control group,model group,AS group,and ADSC+AS group.The rats in control group were routinely fed,and the rats in other groups were used to establish the SNI models by resecting 10 mm of right sciatic nerve.The rats in model group received no further treatment,while the rats in AS group and ADSC+AS group were bridged with AS and the constructed tissue-engineered nerve at the two ends of the injured nerve,respectively.At 6 weeks after surgery,transmission electron microscope was used to observe the ultrastructure of dorsal root ganglion of the rats in various groups;immunofluorescence method was used to detect the protein expression levels of CNTF,p-JAK2,and p-STAT3 in dorsal root ganglion of the rats;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of CNTF,JAK2,and STAT3 in dorsal root ganglion of the rats in various groups.Results:After 7 d of primary ADSC culture,a large number of large and long spindle-shaped cells were observed under the inverted microscope,arranged in clusters or whirlpools;red lipid droplets were observed with oil red O staining under microscope,and calcified nodules were observed with Alizarin red staining under microscope,indicating that the isolated and cultured cells had multidirectional differentiation ability.Compared with normal nerve tissue,the level of DNA in AS of rats was significantly decreased(P<0.05).Compared with control group,the nuclear membrane of dorsal root ganglion cells in model group was uneven and serrated,the number of organelles in the cytoplasm was decreased,mitochondria were swollen with broken or missing cristae and unclear structure;the CNTF protein and mRNA expression levels were significantly decreased(P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly increased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly increased(P<0.01).Compared with model group,the serrated change of nuclear membrane of the dorsal root ganglion cells in AS group was significantly alleviated,the number of organelles in the cytoplasm was increased,and mitochondrial swelling was reduced;in ADSC+AS group,the nuclear membrane of dorsal root ganglion cells tended to be intact,the number of organelles was increased,and mitochondrial swelling and vacuolization were significantly reduced;the CNTF protein and mRNA expression levels in the dorsal root ganglion in AS group and ADSC+AS group were significantly increased(P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly decreased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly decreased(P<0.01).Compared with AS group,the CNTF protein and mRNA expression levels in ADSC+AS group were significantly increased(P<0.05 or P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly decreased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly decreased(P<0.01).Conclusion:The application of ADSC combined with AS can improve the ultrastructure of dorsal root ganglion in the SNI rats,and the mechanism may be related to the increased CNTF expression and decreased activation of the JAK2/STAT3 signaling pathway in the dorsal root ganglion by ADSC combined with AS application.

Objective:To explore whether esketamine (ESK) can inhibit the proliferation and induce apoptosis of breast cancer cells, and explore the mechanism.Methods:CCK-8 assay was used to detect the inhibitory effect of ESK on the proliferation of breast cancer cells. Annexin V/PI staining was used to detect the morphological changes of cells; Apoptosis and reactive oxygen species were detected by flow cytometry. Western blot was used to detect apoptosis and pathway expression.Results:CCK-8 experiment results proved that ESK could inhibit the proliferation of breast cancer cells in a time-dependent manner. The survival rate of MDA-MB-468 cells treated with ESK at 20 μM was (35.47±2.61) %, which was statistically different from that treated with vinorelbine at the same concentration ( P<0.05). The IC50 value of ESK on MDA-MB-468 cells was (14.54±2.12) μM. After treatment with ESK, the mitochondrial membrane potential was significantly reduced. In the protein level, the expression of Cytochrome C, Bax and Caspase-3 was up-regulated, and the expression of Bcl-2 was down regulated, which induced the mitochondrial dependent apoptosis of MDA-MB-468 cells. ESK could up regulate the level of reactive oxygen species in MDA-MB-468 cells and regulate the expression of PI3K/AKT signaling pathway. Conclusions:ESK can inhibit the proliferation and migration of breast cancer cells and induce them to play a mitochondrial dependent apoptosis. Its mechanism is achieved by up regulating the level of ROS in breast cancer cells, thereby regulating the PI3K/AKT signaling pathway, which provides a theoretical basis for the development and utilization of Aln.

Objective:To explore the application effect of the teaching ward-round workshop on the teaching ability training for clinical teachers.Methods:From July to October 2019, 83 clinical teachers from The Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University included in the study were divided into 8 groups for the training of teaching ward-round workshop. After the training, the evaluation results of clinical teaching ward-round, the satisfaction of clinical teaching ward-round, and the satisfaction of the workshop teaching model were compared. Chi-square test and t test were performed by SPSS 22.0. Results:The trainees were highly satisfied with the training mode of the teaching ward-round workshop. After the training, the clinical teaching ward-round assessment scores [(96.83±1.77) points] were higher than the average scores of the same period from April to June in 2019 [(91.25±2.86) points], with statistical significance ( P<0.05). In terms of satisfaction with clinical teaching ward rounds, the scores of 7 dimensions including preparation before ward rounds, highlighting the key and difficulties, and standard physical examination after the training were all higher than those before the training, with significant differences ( P<0.05). Conclusion:The training model of the teaching ward-round workshop helps to enhance the training effect, promote the teaching ability of clinical teachers, and improve the clinical teaching ward-round assessment results and satisfaction, which provides new ideas and references for the training of clinical medical professionals.

Objective:To evaluate the effectiveness of medical practical training with a multi-level comprehensive model.Methods:We randomly selected 100 medical undergraduates who received practical training in The Affiliated Huai'an No.1 People's Hospital of Nanjing Medical University from 2017 to 2019, 20 medical education experts, and 30 teachers for a questionnaire survey using self-designed questionnaire with 3 first-level items, 8 second-level items, and 31 third-level items. Data processing and analysis was made by multi-level comprehensive model.Results:The comprehensive evaluation data (0.176 4, 0.512 3, 0.252 5, 0.058 8) obtained by the multi-level comprehensive model showed that the proportions of medical undergraduates achieving excellent, good, moderate, and poor effects of medical practical training were 17.64%, 51.23%, 25.25%, and 5.88% respectively. According to the principle of maximum membership, the final comprehensive evaluation result of the effectiveness of medical practical training was "good".Conclusion:This research has demonstrated the scientificity and feasibility of using the proposed multi-level comprehensive model to evaluate the effectiveness of medical practical teaching. In the comprehensive evaluation, the quantitative processing of qualitative indices can generate the matching score of each index in the multi-level index system. The evaluation results are intuitive and easy to analyze, thus providing the basis for the targeted improvement of medical practical teaching effect.

OBJECTIVE：To investigate the effects of Periplaneta americana extract on the proliferation and apoptosis of human non-small cell lung cancer A 549 cells as well as its possible mechanism. METHODS ：The dry bodies of P. americana were soaked with 90％ ethanol and eluted with gradient water-methanol by polyamide column chromatography. The 20％，30％，40％， 50％，60％，70％，80％，90％ methanol elution sites （YS-A-H）were obtained. MTT method was used to screen the active site ， and the inhibition rate of different doses of active site was detected. Flow cytometry was adopted to detect cell apoptosis ，cell cycle and mitochondrial membrane potential of cells after treated with different doses of active site. RESULTS ：Half inhibition concentrations of YS-A-H were （95.25±8.42），（129.93±7.24），（221.28±12.68），（275.39±14.87），（276.76±16.32），（31.90± 5.34），（163.15±6.97），（122.81±8.36）μg/mL，respectively. YS-F had the strongest activity. After treated with 3，9，27，81 μg/mL YS-F for 24，48，72 h，cell proliferation inhibitory rate was increased significantly at different time points ；after treated for 48，72 h，that was significantly higher than same group after treated for 24 h；after 72 h treatment ，that was significantly higher than same group after 48 h treatment （P＜0.01）. There was no significant effect of 24 h treatment of 3 μg/mL YS-F and 72 h treatment of 9 μg/mL YS-F on the percentage of cells in the late stage of necrosis，24 h treatment of 3 μg/mL YS-F on the percentage of cells in G2/M phase and 48 h treatment of 3 μg/mL YS-F on the reduction rate of mitochondrial membrane potential（P＞0.05）. The percentage of cells in the early stage of apoptosis ，the late stage of apoptosis and the early stage of necrosis ，the late stage of necrosis，as well as the percentage of cells in the Sub-G 0/G1 and S phase at each time point were significantly increased in other different doses groups ，while the percentage of cells in G 0/G1 and G 2/M phase was decreased significantly （P＜0.01）. In each dose group，the percentage of cells in the early stage of apoptosis ，the late stage of apoptosis and the early stage of necrosis ，the late stage of necrosis （except for the percentage of cells in the late stage of necrosis treated with YS-F 9 μg/mL for 72 h）and the percentage of cells in Sub-G 0/G1 phase，G2/M phase （except for YS-F 27，81 μg/mL for 48 h）after treated for 48，72 h were significantly higher than same group after 24 h of treatment ；the percentage of cells in G 0/G1 phase，S phase and G 2/M phase （except for YS-F 9 μg/mL for 48 h）after treated for 48，72 h were significantly lower than same group after 24 h of treatment （P＜0.01）；the percentage of cells in the early stage of apoptosis ，the late stage of apoptosis and the early stage of necrosis ，the late stage of necrosis （except for the percentage of cells in the late stage of apoptosis and early stage of necrosis when treated with YS-F 27 μg/mL for 72 h，the percentage of cells in the late stage of necrosis when treated with YS-F 3，9 μg/mL for 72 h were decreased significantly ）and the percentage of cells in S phase （except for YS-F 3 μg/mL for 72 h）and Sub-G 0/G1 phase after treated for 72 h were significantly higher than same group after 48 h of treatment ，while the percentage of cells in G 0/G1 and G 2/M phase were significantly lower than same group after 48 h of treatment （P＜0.01）. After treated with YS-F 9，27，81 μg/mL for 48 h，the reduction rate of cell mitochondrial membrane potential was increased significantly ；YS-F 27，81 μg/mL groups were significantly higher than YS-F 9 μg/mL group，and YS-F 81 μg/mL group was significantly higher than YS-F 27 μg/mL group. CONCLUSIONS：YS-F can inhibit the proliferation and promote the apoptosis of A 549 cells by preventing cell transformation from S phase to G 2/M phase ，and reducing mitochondrial membrane potential ，in time-dependent or dose-dependent manner.

OBJECTIVE： To study the improvement effects of Bee venom（BV） plastics on experimental cerebral thrombosis in rats. METHODS： Totally 96 SD rats were randomly divided into sham operation group （normal saline）， model group （plastics blank matrix）， Nimodipine group （positive drug， 4.00 mg/kg） and BV plastics low-dose， medium-dose， high-dose groups （1.67， 3.33， 6.67 mg/kg）， with 16 rats in each group. Rats in sham operation group and Nimodipine group were given medicine intragastrically， while rats in model group and BV plastics groups were given medicine by transdermal smearing. After 5 days of continuous administration， the experimental cerebral thrombosis model was established by ligating the right external carotid artery and pterygomandibular artery， and injecting compound thrombus inducer into the internal carotid artery. The wet mass ratio of right brain to left brain was measured to investigate the degree of brain edema on the infarcted side. The content of Evans blue （EB） in the left and right hemispheres of rats was determined by ultraviolet spectrophotometry to investigate the cerebral vascular permeability. Blood rheology and coagulation function indicators of rats were measured. The pathological changes of brain tissue in rats were observed by HE staining， and the number of survival neuron cells was counted. RESULTS： Compared with the indexes of sham operation group， the cerebral thrombosis model was established successfully. Compared with model group， the area of blue staining in the right brain （infarcted side） of rats in BV plastics groups was significantly reduced， and the right brain/left brain wet mass ratio and the content of EB in the right brain tissue were significantly reduced （P＜0.05 or P＜0.01）. The whole blood viscosity and Casson viscosity of rats in BV plastics groups， and the plasma viscosity of rats in BV plastics medium-dose and high-dose groups decreased significantly （P＜0.01）. PT and APTT of rats were prolonged significantly in BV plastics medium-dose group （P＜0.01）. The pathological changes of brain tissue in rats in BV plastics groups were significantly alleviated. The arrangement of neuron cells was more orderly， the shape and structure of cells were clear， the nucleolus was clear， the membrane was intact， and the number of survival neuron cells was significantly increased （P＜0.01）. CONCLUSIONS： BV plastics can alleviate brain edema， inhibit cerebral vascular permeability， improve hemorheology and coagulation function indicators of rats after the formation of cerebral thrombosis， and alleviate nerve cell injury after ischemia.

Objective To systematically review the evidence for the effect of vitamin D supplementation on insulin resistance and blood glucose in people who are obese or people with abnormal glucose metabolism. Methods We searched databases including Pubmed, Elsevier, Web of Science, and WANFANG Database etc. for randomized controlled trials comparing vitamin D or analogues with placebo. We extracted data on insulin resistance and blood glucose, including homeostasis model assessment of insulin resistance( HOMA-IR), fasting blood glucose, HbA1C (% ). A1l data were analyzed using Review Manager 5. 0. Results Nine studies involving 867 participants were included. The results of meta-analysis showed that: ( 1 ) For people who are obese and with abnormal glucose metabolism, meta-analysis showed a small improvement in HOMA-IR(SMD -0. 34,95% CI -0. 61 to -0. 06, P<0. 05) and a small effect on fasting glucose (SMD -0. 41 mmol/ L, 95% CI -0. 68 to -0. 15, P<0. 05),while such effects were not seen in people who are obese but with normal blood glucose. (2) No serious adverse events were associated with the administration of vitamin D. Conclusion vitamin D supplementation may be benefit for improving insulin resistance and fasting blood glucose in people who are obese and with abnormal glucose metabolism, but has no effect on obese people with normal blood glucose.

Objective To investigate the change of genomic DNA of liver and spleen tissue for different age of the elderly,and provide the experimental data for aging-related research. Methods 35 livers and 33 spleens of autopsied samples preserved in refrigerator at-80 ℃ were divided into 3 groups according to age:age 65y to 79y,age 80y to 89y,age≥90y. The content of DNA in liver and spleen was determined by ultraviolet absorbent method. Results Compaired with age 80y to 89y (0. 310 ± 0. 286)mg/mL,the content of DNA in liver was significant higher at age 65y to 79y (1.464 ±0.488)mg/mL and age ≥90y(1.147 ±0.333)mg/mL(P<0.05);Compared with age 80y to 89y(0. 938 ± 0. 589)mg/mL,the content of DNA in spleen was significant higher at age 65y to 79y(1. 723 ± 0. 726)mg/mL and age≥90y(1. 688 ± 0. 963)mg/mL(P<0. 05). The content of DNA was significant lower in liver (0. 856 ± 0. 658)mg/mL than that in spleen (1. 414 ± 0. 852)mg/mL. Conclusion The content of DNA in human liver and spleen tissue may be decrease along with aging. The content of DNA in the group at age≥90y may be increase. There were some differences between different viscera tissue in content of DNA.

Gastrointestinal Stromal Tumors ; genetics ; pathology ; Genotype ; Humans