It is meaningful to prompt the research-aimed experiments in the laboratory edu-cation of undergraduate students majoring in pharmacy,which means under the guidance of tutors, students collect information,design the experiment procedure and conduct the whole experiment independently. Under this novel mode of laboratory education, students’ abilities of indepen-dent-thinking and comprehensive-experimental conduction are largely improved. Meanwhile,the sense of team-work is enhanced. To sum up,the research-aimed experiments is significantly ben-eficial to training undergraduate students for future scientific researches.

BACKGROUND: An ideal marked molecule has not been found to detarmine bone marrow mesenchymal stem cells (MSCs) so as to make sure the homogenicity.OBJECTIVE: To verify the in vitro differentiation from BrdU-treced MSCs into osteoblasts.DESIGN, TIME AND SETTING: A cytological observation/n vitro was performed in Shanxi Medical University from January to October 2008.MATERIALS: Wistar rats aging 4 weeks old were provided by Experimental Animal Center of Shanxi Medical University.METHODS: MSCs were isolated and cultured by using density gradient cantrifugation combined with attachment culture method.At about 80% confluence, trypsin was used for passage and amplification. MSCs at density of 5×1010/L were inoculated in a 25-mm culture dish with L-DMDM culture medium containing dexamethasone, β -phosphoglycarol, vitamin C, and 10% fetal bovine serum. The third-passaged MSCs were labeled in vitro with 10 μmol/L BrdU tracer. Thereafter, 10 visual fields were randomly selected to calculate numbers of positive and negative ceils so as to obtain BrdU tracing rate under a fluorescence microscope (×200).MAIN OUTCOME MEASURES: Inverted microscope was used to observe cell morphology; flow cytometry was used to detect cell surface antigen, differentiation into osteoblasts, and BrdU tracing rate in vitro.RESULTS: The purified MSCs which were like fibroblasts were adherent and fusiform. The third-passaged cells were changed equidirectionally and whirlpool-arranged, and the survival rate was more than 95%. The seventh-passaged cells still grew rapidly.CD44, CD71, and CD105 expressions were positive, but CD45 expression was negative. Black particles were visualized in MSCs after Von kossa staining. BrdU tracing rate was more than 90%.CONCLUSION: Density gradient centrifugation combined with attachment culture method can effectively isolate and purify rat MSCs which are cultured in vitro for a long period and differentiated into osteoblasts. BrdU tracer is safe, effective, and convenient to successfully label MSCs.

Objective To investigate the influencing factors of opioid analgesics need within 48 hours after arthroscopic rotator cuff repair.Methods Clinical data of 90 consecutive arthroscopic rotator cuff repairs by the same operator from March 2017 to July 2022 were retrospectively analyzed.The patients were divided into opioid group(62 patients)and control group(28 patients)according to whether they used opioid analgesics within 48 hours after surgery.The control group consisted of patients who did not use analgesics or who had good analgesic effects with conventional analgesic regimens(non-steroidal anti-inflammatory drugs or non-opioid central analgesics)after surgery,and the opioid group consisted of patients who required opioid analgesics as assessed by the surgeon or who need opioid analgesics because of inefficacy of conventional analgesic regimens.The following factors were selected as independent variables:gender,age(whether≥65 years old),duration of disease(whether≥4 weeks),body mass index(BMI)(whether≥25),occupation(whether employed),with or without a history of smoking and alcohol consumption,hypertension,diabetes,heart disease,and trauma,duration of surgery(whether≤180 min),number of torn tendons(whether≥2),number of screws(whether≥2),large nodular osteophyte,and whether there was moderate-to-severe impingement.Single factor analysis was used to screen the factors influencing the need for opioid analgesics within 48 hours after arthroscopic rotator cuff repair.Results The results of univariate analysis showed that among the above independent variables,only the number of torn tendons≥2 was the factor affecting the need for opioid analgesics within 48 hours after arthroscopic rotator cuff repair(OR = 5.263,P = 0.007).Conclusions Patients with rotator cuff tears with≥2 tendons had more severe pain within 48 hours after rotator cuff repair,requiring opioid analgesics.Focus should be placed on postoperative pain in such patients,and a diverse analgesic regimen should be used early.

OBJECTIVE: To investigate the effects of subacute systemic inhalation exposure of 1,2-dichloroethane(1,2-DCE) on learning and memory in NIH mice. METHODS: Forty-five specific pathogen free healthy 7-week-old NIH mice were randomly divided into control,low-dose and high-dose groups with 5 female mice and 10 male mice in each group. The mice were exposed to 1,2-DCE at dosages of 0. 00,100. 00 and 350. 00 mg/m3 for 6 hours per day for consecutive 28 days by dynamic systemic inhalation. The neurobehavioral tests of mice were performed before and after the first to fourth weeks of exposure using the Morris water maze test. RESULTS: There was no significant difference in body weight and swimming speed among the three groups of mice( P > 0. 05). The navigation experiment results showed that the escape latency of mice in both low-and high-dose groups were longer than that of the control group at the same time point(P < 0. 05) during 1-4 weeks after exposure. In the control group,the escape latency was shorter than that of the same group before exposure( P < 0. 05). The escape latency of high-dose group prolonged with the increase of exposure time,and in the 4 th week the escape latency was significantly higher than that of the same group before exposure( P < 0. 05).The experiment results of space exploration indicated that the first time of crossing platform in low-and high-dose groups were longer than that of the control group at the second to the fourth week( P < 0. 05). The target quadrant retention time and the number of crossing the platform in the low-and high-dose groups were lower than those in the control group( P <0. 05). CONCLUSION: Subacute inhalation exposure of 1,2-DCE can impair the learning and memory ability of NIH mice.The high-dose exposure may reduce learning ability in mice in a time-effect manner.

OBJECTIVE: To study the effect of aquaporin 4(AQP4) in regulating the permeability of blood-brain barrier(BBB) induced by subacute 1,2-dichloroethane(1,2-DCE) inhalation. METHODS: Specific pathogen free healthy CD-1 male Aqp4 genetically engineered mice(Aqp4~(+/+)and Aqp4~(-/-)) were randomly divided into control and low-, medium-and high-dose groups. The mice were exposed to 1,2-DCE at the dosages of 0.00, 100.00, 350.00 and 700.00 mg/m~3 for 6 hours per day for consecutive 28 days by systemic dynamic inhalation. After the end of 1,2-DCE exposure, the BBB permeability was evaluated by Evans blue staining. Real-time fluorescence quantitative polymerase chain reaction method was used to detect the mRNA expression of genes related to BBB tight junction protein(Tjp)1, Tjp2, Tjp3, claudin(Cldn)3, Cldn5, Cldn11, occludin(Ocln), matrix metalloproteinase(Mmp)2, Mmp9 and Na-K-Cl cotransporter-1(Nkcc1). RESULTS: The BBB permeability in mice showed significant change with 1,2-DCE dose and Aqp4 genotype(P<0.01). The BBB permeability of Aqp4~(+/+) genotype mice was higher in low-, medium-and high-dose groups than that of control group(all P values were <0.05). The permeability of BBB was lower in Aqp4~(+/+) genotype mice in the control group than that of Aqp4~(-/-) genotype mice in the same group(P<0.05), but BBB permeability was higher in Aqp4~(+/+) genotype mice in the three dose groups than that of Aqp4~(-/-) genotype mice in the same group(all P values were <0.05). The Cldn3 and Olcn mRNA relative expression in the brain cortex had statistical difference in mice with different genotype(all P values were <0.01). The mRNA relative expressions of Cldn3 and Olcn in the brain cortex were higher in Aqp4~(-/-) genotype mice than that of Aqp4~(+/+) genotype mice(all P values were <0.01). The relative mRNA expression levels of Tjp1, Tjp2, Tjp3, Cldn5, Cldn11, Mmp2, Mmp9 and Nkcc1 in the cerebral cortex of mice were not statistically significant in aspect of 1,2-DCE exposure dose and genotype(all P values were >0.05). CONCLUSION: Exposure to 1,2-DCE can increase BBB permeability in mice, and the mechanism may be associated with 1,2-DCE-induced down-regulation of Aqp4 and up-regulation of mRNA expression of the cerebral cortex TJP-related molecules Cldn3 and Ocln.

Genetically engineered intestinal microbes could be powerful tools to detect and treat intestine inflammation due to their non-invasive character, low costs, and convenience. Intestinal inflammation is usually detected along with an increasing concentration of thiosulfate and tetrathionate molecules in the intestines. ThsSR and TtrSR are two-component biosensors to detect the presence of thiosulfate and tetrathionate molecules, respectively. In real-life intestinal inflammation detection, sophisticated instruments are needed if using fluorescent proteins as reporters. However, chromoproteins and other colored small molecules, which can be seen by the unaided eye, could extend the use of ThsSR and TtrSR biosensors to detect intestine inflammation. The feasibility of ThsSR and TtrSR systems was tested by monitoring the fluorescence intensity of sfGFP in response to the concentration of thiosulfate and tetrathionate, followed by the incorporation of the two systems into Escherichia coli Top10 and E. coli Nissle 1917. The potential for the real-life application of the two systems was further corroborated by substituting sfGFP with a series of chromoproteins and a protoviolaceinic acid synthesis cassette as reporter genes. The results indicated that signal expression of the new systems had a positive correlation with the concentration of tetrathionate and thiosulfate molecules. Thus, the modified ThsSR and TtrSR system may potentially be applied in the human body for the detection of intestinal inflammation.
Escherichia coli ; Humans ; Inflammatory Bowel Diseases ; Intestines ; Thiosulfates

OBJECTIVE: To investigate the effect and mechanism of lead exposure on hypothalamic inflammatory factors in mice fed with high-fat diet. METHODS: Specific pathogen free healthy male Kunming mice were randomly divided into control group, high-fat diet group, lead exposure group, and combined exposure group, with 8 rats in each group. The control group and the lead exposure group were given regular diet, while high-fat diet group and combined exposure group were given high-fat diet. The lead exposure group and combined exposure group were given water with 250 mg/L lead acetate. The control group and high-fat diet group were given double distilled water. Continuous lead exposure was given for 9 weeks, 7 days per week. Body weights of the mice were measured every other week. After 9 weeks of exposure, the behavioral changes of mice were detected by open field test. The levels of triglyceride(TG), low density lipoprotein(LDL) and high density lipoprotein(HDL) in serum were detected by microplate reader. Western blotting was used to detect the relative protein expression of interleukin(IL)-1β, IL-6, IL-17 A, IL-22, tumor necrosis factor-α(TNF-α) and transforming growth factor-β(TGF-β) in the hypothalamus of mice. The relative expression of mRNA of IL-1β, IL-6, IL-17 A and TNF-α mRNA was detected by real-time fluorescence quantitative polymerase chain reaction. RESULTS: Beginning from the first week, the body weights of mice in the high-fat diet group and the combined exposure group were higher than that in the control group and the lead exposure group(P<0.05). The numbers of standing in the lead exposure group and the combined exposure group were lower than that in the control group and the high-fat diet group(P<0.05). The distances of central area activity in the high-fat diet group, the lead exposure group and the combined exposure group were lower than that in the control group(P<0.05). The total distances in the high-fat diet group and the combined exposure group were lower than that in the control group(P<0.05). The serum levels of TG and LDL in the combined exposure group increased(P<0.05), and the HDL level decreased(P<0.05), when compared with the control group and the lead exposure group. The relative protein expression of IL-1β, IL-6, IL-17 A and IL-22 in the hypothalamus of the high-fat diet group and lead exposure group was higher than those of the control group(P<0.05). The relative protein expression of TNF-α and TGF-β in the hypothalamus of the lead exposure group was higher than that in the control group(P<0.05). The relative protein expression of IL-1β, IL-6, IL-17 A, TGF-β in the hypothalamus of the combined exposure group was higher than the other 3 groups(P<0.05). The relative protein expression of IL-22 in the hypothalamus of the combined exposure group was higher than that of the control group(P<0.05), while the relative protein expression of TNF-α was higher than that of the control group and the high-fat diet group(P<0.05). The relative expression of IL-1β, IL-6, IL-17 A, and TNF-α mRNA in the hypothalamus of the high-fat diet group, the lead exposure group and the combined exposure group was higher than that in the control group(P<0.05). The above indicators of mice in the lead exposure group were higher than that in the high-fat diet group(P<0.05). The above indicators of mice in the combined exposure group were higher than those in the high-fat diet group and the lead exposure group(P<0.05). CONCLUSION: Lead exposure can promote neurobehavioral changes and hypothalamic inflammatory damage in high-fat diet mice. IL-1β, IL-6, IL-17 A, TGF-β and TNF-α might involve in the process of synergistic effect of lead and high-fat diet exposure on inflammatory hypothalamic injury.

ObjectiveTo investigate the effect of intermittent theta burst stimulation (iTBS) of the multi-target cerebral cortex after stroke on functional recovery of the upper limb of the hemiplegic side. MethodsFrom November, 2019 to August, 2020, 40 stroke patients in Gansu Provine Hospital Rehabilitation Center were included and randomly divided into single-target stimulation group (n = 20) and multiple-target stimulation group (n = 20). Both groups underwent basic neurorehabilitation drug therapy and conventional rehabilitation exercises. The single-target stimulation group received repetitive transcranial magnetic stimulation (rTMS) (iTBS mode) only in the primary motor cortex (M1) of the affected side. The multi-target stimulation group received rTMS (iTBS mode) in the cerebellar cortex of the healthy brain and M1 of the affected side, once a day, six days a week, for four weeks. Before and after treatment, the scores of Fugl-Meyer Assessment-Upper Extremities (FMA-UE), Action Research Arm Test (ARAT) and modified Barthel Index (MBI), and the latency and amplitude of somatosensory-evoked potentials N20 were compared. ResultsNo serious adverse reaction occurred during treatment. After treatment, the scores of FMA-UE, MBI and ARAT, and the amplitude and latency of N20 improved in both groups (|t| > 3.478, |Z| > 2.243, P < 0.05); and the scores of FMA-UE and ARAT, and the amplitude of N20 were better in the multiple-target stimulation group than in the single-target stimulation group (t > 2.939, Z = -2.697, P < 0.01). ConclusionMulti-target stimulation is superior to single-target stimulation for improving upper limb motor function and N20 amplitude in the hemiplegics after stroke.

OBJECTIVE: To predict the sensitizing potency and optimal sensitization dose of trichloroethylene( TCE) by an in vitro skin sensitization test on a human acute monocytosis cell line( THP-1).METHODS: THP-1 cells were cultured in vitro and exposed to 2,4-dinitrochlorobenzene( DNCB),sodium dodecyl sulfate( SDS),tert-butylhydroquinone( tBHQ)and TCE for 24 hours.Flow cytometry was used to detect the expression of cell surface marker such as cluster of differentiation( CD) 86 and CD54,and the optimal dose range for sensitization detection was determined.With the relative fluorescence intensity( RFI),CD86 ≥ 150 and CD54 ≥ 200 as the standard,the sensitizing potency and optimal sensitization dose of TCE were predicted.RESULTS: The concentration range of reagents for sensitization test on THP-1 cells was the dose range at which the relative cell survival rate reached 75.0%-100.0%.DNCB at the doses of 20.83,25.00 and 30.00 μmol/L,tBHQ at the dose of 5.80 μmol/L,TCE at the doses of 8.33,10.00 and 12.00 mmol/L,can cause sensitivity.SDS was recognized as a negative sensitizer.The expression of CD86 and CD54 was the highest when the concentration of TCE was 8.33 mmol/L,which was considered as the best sensitization dose.CONCLUSION: The optimum sensitization dose of TCE is 8.33 mmol/L,which can provide the basis for dose design in future study of TCE sensitization pathways.

OBJECTIVE: To explore the molecular mechanism underlying 1,2-dichloroethane(1,2-DCE) induced apoptosis by screening differentially expressed proteins in human astrocytes( HAs). METHODS: HAs were cultured in complete medium with 1,2-DCE at various concentrations of 0-80 or 0-40 mmol/L. After 24 hours,apoptosis of HAs was evaluated using flow cytometry and staining with annexin Ⅴ-fluoresce in isothiocyanate and propidium iodide. An AAH-APO-1-2 protein chip was used to screen differentially expressed proteins and quantitative real-time polymease chain reaction(qRT-PCR) was used to verify related differentially expressed genes(DEGs). RESULTS: At 1,2-DCE concentrations of0-80 mmol/L,the total apoptosis rate of HAs increased with 1,2-DCE concentrations in a dose-dependent manner( P <0. 01). Seven different kinds of proteins were screened out by apoptotic protein chip. Among them,the expression of insulin-like growth factor-binding protein( IGFBP)-1,IGFBP-4 and cytochrome C( Cyto C) were up-regulated,while the expression of P27,cysteine aspartic acid specific protease-3( Caspase-3),B-cell lymphoma-2 interacting mediator of cell death( BIM) and BH3 interacting domain death agonist( BID) were down-regulated compared with the control group. The result of DEGs verified by qRT-PCR showed that the expression of mRNA of IGFBP-1,IGFBP-4 and Cyto C at 1,2-DCE concentrations of 40 mmol/L was up-regulated. This result was in consistent with the trend of target expression in the protein chip. The mRNA expression of Caspase-3,BIM and BID was also up-regulated. CONCLUSION: 1,2-DCE induces apoptosis of HAs through mitochondrial pathway.