Objective To explore the effects of prohibitin 2(PHB2)on sensitivity of non-small cell lung cancer cell line A549 to erlotinib(Erl)and its potential mechanisms.Methods RACK1-specific small interfering RNA was transfected in A549 cells for knocking-down of PHB2.The effects of PHB2 inhibition on cell proliferation and apop-tosis induced by Erl were observed.The colocalization of microtuble-associated protein light chain 3 alpha(LC3)and mitochondria was visualized by MitoTracker staining and green fluorescent protein-microtuble-associated protein light chain 3 alpha(GFP-LC3)transfection.Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine(EdU)staining.Cell colony formation was evaluated by colony forming assay.Apoptotic index of A549 cells was evaluated by TUNEL.Western blot was used to measure the expressions of PHB2 and LC3Ⅱ.Mitochondrial transmembrane potential,cytochrome c and respiratory chain complexⅠ/Ⅱ/Ⅴactivity were analyzed by the commercially availa-ble kits.Results Compared with the siPHB2 and siCtrl+Erl group,the EdU-positive A549 cells and the number of cell colonies decreased significantly(P＜0.05),while the TUNEL-positive A549 cells increased significantly(P＜0.05)in the siPHB2+Erl group.In addition,compared with the siPHB2 and siCtrl+Erl group,mitochondrial transmembrane potential and respiratory chain complexⅠ/Ⅱ/Ⅴactivity decreased significantly(all P＜0.05)and the levels of cytochrome c increased in mitochondrial fractions(P＜0.05)and decreased in cytosolic fractions(P＜0.05)in the siPHB2+Erl group.Conclusions PHB2 inhibition significantly improves sensitivity of A549 cells to Erl,which may be explained by inhibition of PHB2-mediated mitochondrial autophagy.

Objective:To explore the application effect of aerobic exercise combined with progressive muscle relaxation training in patients undergoing peritoneal dialysis.Methods:Using the convenient sampling method, a total of 90 patients undergoing peritoneal dialysis who were regularly followed up in Peritoneal Dialysis Center of Yancheng Third People's Hospital in Jiangsu Province were selected from July to December of 2020. According to the random number table method, they were divided into the intervention group and the control group, with 45 cases in each group. Patients in the control group were given routine nursing, while patients in the intervention group were given aerobic exercise combined with progressive muscle relaxation training for 12 weeks on the basis of routine nursing. Gastrointestinal Symptom Rating Scale (GSRS), Gastroesophageal Reflux Disease Questionnaire (GerdQ) and urea clearance index (Kt/V) were used to evaluate the intervention effects of the two groups.Results:After 12 weeks of intervention, the incidence of dyspepsia, constipation, reflux, diarrhea, abdominal pain, eating disorder and GerdQ score greater than 8 in the intervention group were lower than those in the control group, and the differences were statistically significant (χ 2=6.429, 5.421, 6.403, 6.559, 3.986, 7.647, 5.789; P<0.05). The total score of GSRS and the scores of each dimension of the intervention group were lower than those of the control group, and the differences were statistically significant ( Z=-5.829, -2.637, -2.434, -2.812, -2.447, -2.027, -2.9958; P<0.05). The Kt/V compliance ratio of the intervention group was higher than that of the control group, and the difference was statistically significant (χ 2=5.683, P<0.05) . Conclusions:Compared with conventional nursing methods, aerobic exercise combined with progressive muscle relaxation training can reduce the incidence of gastrointestinal symptoms in peritoneal dialysis patients, effectively alleviate gastrointestinal symptoms and improve adequacy of dialysis.

BACKGROUND:Previous studies have demonstrated that hepatocyte growth factor (HGF) gene transfection can improve the effectiveness of bone marrow mesenchymal stem cel transplantation, but the mechanism is stil unclear. OBJECTIVE:To observe the effects of HGF gene transfection on c-MET, Bax, Bcl-2, Caspase-3 of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions. METHODS:(1) Bone marrow mesenchymal stem cel s were isolated and amplified in vitro by differential adhesion method. The infection efficiency of recombinant adenovirus Ad-HGF in bone marrow mesechymal stem cel s was tested by x-gal staining. (2) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 0, 3, 6, 9, 12 hours. RT-PCR and western blot assays were used to evaluate the expression of Bax, Bcl-2, Caspase-3. (3) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 6 hours, and RT-PCR and western blot assays were adopted to detect HGF, c-Met, Bax, Bcl-2 and Caspase-3. (4) Cel scratch test was used to detect the effect of HGF transfection on the migration of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions for 6 hours. RESULTS AND CONCLUSION:(1) Transfection efficiency of bone marrow mesenchymal stem cel s was increased with multiplicity of infection in a dose-dependent manner. When the multiplicity of infection was 150, the transfection efficiency was 96.4%. (2) Expressions of Bax and Bcl-2 were gradual y increased with hypoxia time (P<0.05). The Bax/Bcl-2 ratio and Caspase-3 expression reach the minimum at 6 hours of hypoxia (P<0.05). (3) Compared with the control and Ad-LacZ groups, the expressions of HGF, c-Met, Bcl-2 increased, and the expressions of Bax and Caspase-3 decreased in the Ad-HGF group after 6 hours of culture under hypoxia and serum-free conditions (P<0.05). There was no significant difference between the control and Ad-LacZ groups. (4) The mobility of bone marrow mesenchymal stem cel s was higher in the Ad-HGF group than the control group and Ad-LacZ groups after 6 hours of culture under hypoxia (P<0.05). These findings indicate that transfection of HGF in bone marrow mesenchymal stem cel s can increase the expression of c-Met, Bcl-2 and decrease the expression of Bax, Caspase-3 under hypoxia and serum-free conditions, which also enhance the mobility of bone marrow mesenchymal stem cel s under hypoxia and serum-free conditions.

BACKGROUND:Adrenomedulin gene transfection can strength the anti-apoptotic ability of bone marrow mesenchymal stem cels under ischemia and hypoxia, but its mechanism is not yet clear.
OBJECTIVE:To investigate the effect of adrenomedulin on the expression of apoptosis-related proteins, Bax, Bcl-2 and Caspase-3, in bone marrow mesenchymal stem cels under hypoxia and ischemia.
METHODS:Bone marrow mesenchymal stem cels of Sprague-Dawley rats were isolated, cultured and purified, and then cultured in serum-free medium under hypoxic condition for 0, 3, 6, 9, 12 hours. Then, western blot assay was employed to detect the expression of Bax, Bcl-2 and Caspase-3 so as to determine the optimal hypoxia time that was determined at 6 hours of hypoxia. Depending on whether adrenomedulin pretreatment was done, the cels were divided into control group (with no adrenomedulin pretreatment before hypoxia and ischemia) and adrenomedulin groups with different concentrations (1, 10, 100 μg/L). Afterwards, the expression of Bax, Bcl-2 and Caspase-3 was detected by using western blot assay.
RESULTS AND CONCLUSION:(1) After cultured in serum-free medium under hypoxia for 0, 3, 6, 9, 12 hours, the expression of Bax, Bcl-2 and Caspase-3 in bone marrow mesenchymal stem cels were increased (P < 0.05);at 6 hours of hypoxia, the Bax/Bcl-2 ratio and Caspase-3 expression reached the minimum value (P < 0.05). (2) At 6 hours of hypoxia, the expression of Bax and Caspase-3 protein as wel as Bax/Bcl-2 ratio became the lowest in the 100 μg/L group compared with the 1 and 10 μg/L groups, but the expression of Bcl-2 protein reached the peak (P < 0.05). These findings indicate that adrenomedulin can reduce the expression of Bax/Bcl-2 ratio and Caspase-3 protein in bone marrow mesenchymal stem cels cultured in serum-free medium under hypoxic conditions, which is in a dose-dependent manner.

BACKGROUND: Neural axon regeneration is one of the difficulties that must be overcome in treatment of injury of central nerve system. Significant therapeutic effects have been obtained in transplantation of neural stem cells (NSCs), embryonic stem cells (ESCs) and Schwann cells. But the bottleneck situation of insufficiency of cell provider has limited the development on it.OBJECTIVE: To observe directional-differentiation of retinoic-acid induced ESCs so as to find optimal condition for neuronal differentiation.DESIGN: Non-randomized controlled experiment was designed.SETTING: Teaching-Research Room of Histology and Embryology, Department of Basic Medicines. Third Military Medical University of Chinese PLAMATERIALS: The experiment was performed in Staff Room of Histology and Embryology, Third Military Medical University of Chinese PLA from January to May 2000. Eighteen Kunming mice in disoestrus were employed, of which. 12 mice were female and 6 mice male. They were placed in same cage at ratio of 2:1 for mating. The date of pregnancy was recorded. MESPU35 ESC line was prepared.METHODS: Removed head. internal organs and four limbs, feeder-layer Feeder-layer adherent culture was used to proliferate MESPU35 ESCs.Classic 4-/4+ method [The embryoid body (EB) grew naturally for 4 days,without retinoic acid added. In the coming 4 days, retinoic acid was added to induce neural EB of high proportion] was applied to induce the directional differentiation of the nerve. EB was cultured with serum of different concentrations. Phase contrast microscope was used to observe nerve-like EB in serum of different concentrations and to count numbers. ②Immunocytochemical technique was used to observe cellular morphological charac ters at various differentiating phase spots (5th. 9th, 14th days) and with retinoic acid at various concentrations. Flow cytometer (FCM) was used to count the proportion of differentiated neurons.MAIN OUTCOME MEASURES: ①Estimated measurement of the length of process and cell body during formation of neural EB after retinoic-acid induced differentiation of MESPU35 ESCs. ② Observation of cell morphology with immunocytochemical staining and proportion of differentiated cells assayed with FCM.RESULTS: ①It was discovered with phase contrast microscope that serum of different concentrations affect neural directional differentiation after EB formation to certain extent. Excessively high and low concentrations of serum reduced the proportion of neural differentiation of EB. The differentiating proportion is high in serum with 5% concentration. ② It is observed with immunocytochemical technique that the proportions of NF200 positive cell and glial fibrillary acidicprotein (GFAP) positive cell in differentiation of MESPU35 ESCs induced by retinoic acid were increased with phase spots in differentiation and increased concentration of retinoic acid. NF200 positive cell is transformed as multipolar neurons from absence of process in morphology. The processes of GFAP positive cell became longer and linked among each other as reticular pattern finally. ③ It was assayed with FCM that the proportion changes of GFAP positive cell and NF200 positive cell manufactured in differentiation were similar to immunocytochemical one.CONCLUSION: Retinoic acid in combination with proper concentration of serum and differentiating phase spots can induce neural-differentiation of MESPU35 ESC at high proportion and its differentiating regulation is in the patterns of concentration dependence and time dependence.

BACKGROUND: NOV protein encoded by nephroblastoma overexpression gene(NOV) is IGF(insulin-like growth factor) -binding protein. What is its impact on human neural stem cell(hNSC) proliferation and differentiation?OBJECTIVE: To investigate the impacts of NOV protein on hNSCs proliferation and differentiation.DESIGN: A single factor analysis of variance experimental study using cells as subjectsSETTING: Department of histology and embryology, and department of neurobiology in a military medical university.MATERIALS: Study was conducted in the Department of Histology and Embryology of the Third Military Medical University of Chinese PLA. Subjects were hNSCs cultured from 10 to 14 weeks human embryo cerebral cortex.INTERVENTIONS: COS-7 cells were transfected by NOV gene recombined plasmid. COS-7 cell and COS-7 cell modified by NOV gene conditioned culture media(COS-CM and NOV-CM) were collected and reacted with the cultured HNSCs.MAIN OUTCOME MEASURES: hNSCs proliferation was detected by 3H-TdR scintillation analysis, and hNSCs differentiation was detected by immunocytochemistry and flow cytometer(FCM).RESULTS: Both COS-CM and NOV-CM could significant promote the intake of 3H-TdR by HNSCs, of which the 1/minute of NOV-CM group was significantly higher than that of COS-CM group(P ＜ 0.05), which indicated that NOV-CM contained component that could facilitate hNSCs proliferation, and moreover, there was certain dose-effect relationship in NOV-CM' s facilitation of cellular proliferation. The results of immunocytochemistry and FCM revealed that there were more NF-200 positive cells in NOV-CM group, while many glial fibrillary acidic protein positive cells could be seen in COS-CM group.CONCLUSION: NOV protein might have facilitative effects on hNSCs proliferation and differentiation into neurons.

Neurobiology is to study the structure,function,development and regeneration of the nervous system at molecular,cellular and whole level.The objective of neurobiological teaching is to explore the importont and difficult points in teaching' the present article attempts to design a project pattern in practical course of neurobiology,which is suited to learning mode of medical college students.

BACKGROUND: Cell apoptosis is one of the important pathological changes in ischemic-reperfusion (IR) injury. As the key factor involved in cell apoptosis regulation, interleukin (IL)-iβ converting enzyme, when activated, leads to cell apoptosis via protein degradation.OBJECTIVE: To investigate the relationship between the expression of IL-1β converting enzyme and cell apoptosis in cerebral IR injury and explore the role of this enzyme in post-ischemia cell apoptosis.DESIGN: Randomized controlled experiment.MATERIALS: The experiment was performed at the Center of Neuroscience of the Third Military Medical University between March 1996 and December 2000. Totally 64 adult healthy Wistar rats were randomized into two groups, namely IR group (n=56) and sham operation group (n=8). In IR group, the rats were subjected to four vessel occlusion to mimic whole brain IR injury, and reperfusion was carried out after 30 minutes of ischemia for 3, 6, 12, 24, 48, 72 hours and 7 days, respectively (8 rats at each time point). Only separation but not occlusion of the bilateral common carotid artery was performed in sham operation group.METHODS: Four rats were randomly selected from IR group at each time point and 4 from the sham operation group for immunohistochemical study and in situ hybridization, with the other 4 rats for in situ end-labeling assay.MAIN OUTCOME MEASURES: Protein and mRNA expression of ILlβ converting enzyme and neural cell apoptosis in the brain.RESULTS: Totally 64 rats were used in this study and all data were statistically analyzed. In the sham operation group, IL-1β converting enzyme protein and mRNA were expressed in small amount in most of the normal brain tissues, and their expressions were also detected in the neurons and small glial cells in IR group localized mainly in the cerebral cortex, cerebellum Purkinje's cells, hippocampal and subcortical white matters. The expression of IL-lβ converting enzyme began to increase at IR 12 hours, reaching the peak level at 48-72 hours followed by declination since 7 days after the operation. Cell apoptosis occurred 12 hours after IR (49.4±6.8) /section and peaked at 72 hours (228.6±29.8)/section, showing significant correlation with the temporal expression of IL-1β converting enzyme protein and mRNA (r=0.89, 0.68, P ＜ 0.05).CONCLUSIONS: Expressions of IL-1β converting enzyme protein and mRNA increased after IR in close correlation with post-ischemia cell apoptosis, and their temporal expression pattern supports the presumption that IL-1β converting enzyme is an important factor in cell apoptosis.Apoptosis is mostly likely to occur in the cerebral cortex, hippocampus and basal ganglion in IR injury, where IL-1β converting enzyme is highly expressed, further demonstrating that post-ischemia expression of IL-1β converting enzyme might be involved in cell apoptosis regulation.

BACKGROUND: Cyclin-dependent kinase-5 (CDK-5) is one of the members in cyclin-dependent protein kinase family. The attention has being drawn by researchers on the relationship between the expression and distribution of CDK-5 mRNA and its protein in the brain during brain development and neural degeneration in thought-cognition.OBJECTIVE: To probe into the influence of CDK-5 on neurogeny and neural degeneration during cerebral development.DESIGN: Single factor analysis of variance.SETTING: Histological and Embryological Department and Neurobiological Department in Third Military Medical University of Chinese PLA.MATERIALS: The experiment was performed in Histological and Embryological Department and Neurobiological Department in Third Military Medical University of Chinese PLA. Twenty-five Wistar rats of 5 phases were employed, named embryonicphase (E8-E21), neonatal phase (P0-P15),childhood (P16-2 months), grown-up phase (＞ 2 months) and senile phase (＞ 8 months), 5 rats in each group.METHODS: In situ hybridization histochemistry (ISH) and immunohistochemistry (IHC) staining was adopted in brain sections from embryonic phase to senile phase.MAIN OUTCOME MEASURFS: Distribution and expression of positive cells of CDK-5 mRNA and protein in various brain areas.RESULTS: Twenty-five rats entered result analysis for all. ① The expression of CDK-5 mRNA presented in entire development from E14 to P350and was in tendency of stability after growth-up. CDK-5 mRNA localized mainly in neurons and positive regions distributed mainly in cerebral cortex, hippocampus, thalamus, hypothalamus, cerebellum and a part of nerve nuclei. ② The expression of CDK-5 was strong after birth and it was weaker in embryonic and senile rats. Positive regions concentrated mainly in peripheral ventricle, hippocampus, cerebellum and a part of nerve nuclei.The expression only presented in hippocampus and Purkinje cellular layer of cerebellum in senile rats.CONCLUSION: CDK-5 in brain runs through entire phases of neural development, it expresses more significantly in neonatal phase and childhood and declines after growth-up, especially in senile phase. The declined expression of CDK-5 in hippocampus of senile rats is closely associated with decline of learning and memory in senility probably.

Objective To observe the postnatal development and perinatal electrophysiological characteristics of oligodendrocyte precursor cell (OPC) in rats. Methods Immunohistochemistry and Western blot were applied to determine the expression of NG2 OPC in cerebral cortex and hippocampus at various developmental stages of SD rats. Electrophysiological characteristics of OPC were also recorded in slices of 7-day rats. Results The majority of hippocampal and cerebral OPC exhibited stellate shape,a small cell body with a few processus. Total population of the NG2 immunopositive OPC was numerous at P7d in cerebral cortex and hippocampus. OPC expressed in adult rats with slightly more quantity. Moreover,OPC in hippocampus of P7d rats typically exhibited small inward sodium current and weak active responses,whereas only outward potassium current and inactive responses were recorded in white matter OPC of P7d rats. Conclusion Total population of OPC and relative optical density of NG2 are the highest in P7d rats at the postnatal developmental stages. OPC in cerebrum and hippocampus of P7d rats displays electrophysiological heterogeneity.