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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Objective To evaluate the therapeutical effect of dopaminergic neurons induced by transplantation on Parkinson's disease (PD) rats. Methods Mesencephalic nerve stem cells (NSCs) were induced by striatal extracts to differentiate into tyroxine hydroxylase (TH) positive dopaminergic neurons. The differentiated cells were transplanted into the striatum of PD rats. The survived cells were detected by TH immunocytochemical staining. The therapeutical effect was observed using apomorphine induced rotation. Results Mesencephalic NSCs could be induced to differentiate into dopaminergic neurons which could survive in the host for long time after cell transplantation, and could improve the apomorphine induced rotation. Conclusion The induced mesencephalic NSCs have the obvious therapeutical effect on PD.
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Objective To observe the expression changes of noggin mRNA and BMP4 mRNA in the hippocampus and frontal cortex of rats at different stages. Methods The expressions of noggin mRNA and BMP4 mRNA were analyzed by the method of reverse transcriptase polymerase chain reaction (RT PCR).Results It was revealed that the level of noggin mRNA in the frontal cortex decreased significantly in P1W rats but high level of BMP4 mRNA was detected in P1M and P3M rats. The expressions of noggin mRNA and BMP4 mRNA in the hippocampus showed the opposite expression pattern. The peak of noggin mRNA expression in the hippocampus was found in E13 and E16 rats. The expression of noggin mRNA decreased gradually but that of BMP4 mRNA in hippocampus increased gradually during the developmental stage. The peak of the expression of BMP4 mRNA was found in P1M rats. Conclusion There are expressions of noggin mRNA and BMP4 mRNA in the frontal cortex and hippocampus in rats at different developmental stages. The expression level is closely correlated with the developmental age. This indicates that noggin and BMP4 play important roles in the development of rat frontal cortex and hippocampus.
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Objective To investigate the effect of noggin on BrdU labeled cells in the adult rat hippocampus. Methods The expressions of noggin and bone morphogenetic protein 4 (BMP4) in rat hippocampus were detected using in situ hybridization histochemistry (ISHH) and reverse transcription polymerase chain reaction (RT PCR). By using antisense technique combined with bromodeoxyuridine!(BrdU) labeling, the effect of noggin on hippocampal neurogenesis in adult rats was explored. Results The number of noggin mRNA positive cells in the adult rat hippocampus decreased significantly after treatment with antisense noggin but no change was found in the number of BMP4 mRNA positive cells. In addition, the number of BrdU labeled cells decreased significantly in the adult rat hippocampus after treatment with antisense noggin, but the sense noggin had no such effect. Conclusion Noggin can promote proliferation of neural precursor cells in adult rat hippocampus.
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Objective To observe the change of nitric oxide(NO) content and explore its cell source following brain ischemic injury. Methods We established the model of transient global brain ischemia/reperfusion (IR) in rat. The concentration of NO and its cell source were investigated by detection of NO content, NDP histochemical staining, double label technique of immunofluorescence and stereological analysis after brain ischemia. Results (1)The content of NO increased at 12 h and peaked on days 1～3 after IR. The content of NO decreased gradually on the 5th day after IR. (2)The density of NDP positive cells increased and peaked on the 1st day after IR. The positive cells distributed mainly in the temporal cortex, hippocampus and periventricular zone after IR. The positive cells were found to reduce gradually or disappear on the 14th day after IR. (3)Few NDP positive and GFAP immunofluorescence positive cells were found to coexist at the early period of IR(1～3 d). The percentage of coexisting cells was 10%～15%. Conclusion The content of NO increases at the early period after ischemic brain injury. The main cell type to produce NO is neuron.
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Objective To measure the estrogen concentration of estrogen (E 2) in the culture medium of rat astrocytes (ASTs). Methods Astrocytes in brain cortex of the 2-day-old neonatal rats were collected and cultured. The number of astrocytes was counted and the concentration of estrogen was measured by ELISA method at 0, 7, 14, and 21 d after culture. Results The cell counts were 1?10 4/ml, 1.1?10 6/ml, 1.4?10 6/ ml, and 1.5?10 6/ml, respectively. The concentrations of E 2 were: 0 pg/ml, (117.03?21.32) pg/ml, (266.91?22.03) pg/ml, and (252.62?27.99) pg/ml, respectively. No estrogen was detected in the primary culture medium. The concentration of estrogen increased in a time-dependent manner and reached the peak at 14 d, and then decreased gradually but remained at a certain level. Conclusion E 2 is secreted by astrocytes in the brain cortex of the 2-day-old neonatal rats.
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Objective To investigate the localization of Smad2, Smad3, Smad4, and Smad7 proteins and their expression changes in the 5/6 subtotally nephrectomized rat kidney. Methods The rat model of chronic renal failure was established by performing 5/6 subtotally nephrectomy (SNx) and rats in the control group underwent sham-operation. The rats were sacrificed at 4, 8, and 12 week after operation. The sites and levels of expressions of Smad2, Smad3, Smad4, and Smad7 proteins were examined by immunohistochemical staining. Renal fibrosis was assessed by measuring tissue hydroxyproline. Results Immunohistochemical staining indicated that Smad2, Smad3, and Smad4 proteins were mainly expressed in glomeruli and renal tubular cells, while Smad protein 7 was expressed in glomeruli, but rarely in proximal renal tubular cells. Expressions of Smad2, Smad3, and Smad4 proteins in glomeruli were significantly increased during 4-12 weeks after 5/6 nephrectomy, but the expression level of Smad protein 7 was significantly decreased, but accompanied increase of hydroxyproline content in the renal tissues. Conclusion These results indicate that TGF-?/Smad signaling is involved in the progress of chronic glomerulosclerosis. The high level expressions of Smad2, Smad3, and Smad4 proteins and the down-regulation of Smad7 protein may be the major cause of the glomerulosclerosis in this model.
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Objective To explore the effects of maternal exposure to dibutyl phthalate ( DBP) ,an environmental estrogen on the proliferation and differentiation of neural stem cells ( NSCs) in the neural tube of neonatal rats. Methods A total of 40 male and 40 female SD rates at age of 4 to 5 months,were matched,and the morning when vaginal plugs were found was designated as E0. Then the 40 pregnant rats were randomly divided into 4 groups,3 DBP exposure groups ( 25,75,and 225 mg/kg) and control group. DBP at corresponding doses were dissolved in corn oil,and administrated by intragastrically injection once per day from E0 till delivery,while those of control group were given corn oil. Brain tissue of newborn rats was collected for primary culture of NSCs. Then the cell colony formation was counted to detect the NSCs proliferation. Then 10% fetal bovine serum ( FBS) was added into the culture medium to induce the NSCS to differentiate. Neu-N and GFAP immunofluorescence stainings were used to detect neurons and astrocytes,and their morphological changes were observed. Results The proliferation of NSCs was decreased significantly after DBP administration. Compared with the control group ( 40. 53 ? 4. 65) % ,the colony formation rate of middle-and high-dose DBP exposure groups was significantly reduced and in a dose-dependent manner ( 30. 96 ? 3. 80) % ,( 15. 35 ?5. 29) %,( 6. 58 ?1. 43) %,P
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Objective To investigate the change of P2X7 receptor expression in neonatal rats after hypoxic-ischemic brain damage(HIBD).Methods Twenty SD rats were randomized into 2 groups,control group and hypoxic-ischemic(HI) group.Western blotting and immunohistochemistry was used to detect the protein expression of P2X7 receptor in cerebal cortex,subcortical white matter and hippocampus before and after hypoxia-ischemia.Results In comparison to the sham operation controls,a significant down-regulation of the P2X7 receptor protein was observed in ischemic cerebral cortex,subcortical white matter(P
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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.
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Objective To detect the expression changes and location of the mouse developmental regulation brain protein(Dbn1)in the developmental mouse brain.Methods Monoclonal antibodies against drebrin protein were used to assess Dbn1 by immunohistochemistry and Western blot.The paternal expression of Dbn1 in developmental mouse brain(E14,P1,P7 and adult)was initially investigated by Western blot.Dbn1 was shown at various developmental stages(E14,P1 and P7)as well as in adult in different brain area of developmental mouse brain by immunohistochemical.Results Dbn1 protein was detected in developmental brain but a little in adult brain by Western blot,high at E14,decreased at P1,gradually increased at P7 and lowest in adult.Immunohistochemistry confirmed as follows:Dbn1 expressed mostly in cortex,hippocampus and ependyma areas at E14,and the positive signal was distributed at cells border;The expression of Dbn1 was decreased at P1,mainly distributed at the verge of cells or dendrites;The peak expression of Dbn1 appeared at P7,Dbn1 located at nucleus of hippocampus and cortex,and the positive signal located within cytoplasm and dendrites;Only a little positive Dbn1 cells were found in adult mouse brain.Conclusion Dnb1 may be involved in regulating the differentiation and migration of neurons during the development of mouse brain.
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Objective To investigate the migration pathway and feature of neuronal precursors in normal adult rats and provide theory and experiment basis for the neuronal migration under pathological condition. Methods The adult rat brains were cut into 20 ?m coronal and sagittal sections on a freezing microtome. Immunohistochemistry was applied to observe the migration pathway and feature of DCX-expressing cells. Results There were two migration pathways with the neuronal precursors in normal adult rats. One was the rostral migratory stream (RMS) from the subventricular zone to the olfactory bulb, another appeared to travel in a chain along the interface between cortex and corpus callosum. The DCX-positive cells in the RMS had the fusiform somata with a single leading process and the process orientated to the olfactory bulb, while the DCX-positive cells around the corpus callosum had similarly round somata and the size, number and orientation of process were of diversity.Conclusion The study of neuronal precursors migrating not only contributes to identify the migration mechanisms, but also contributes to the control of neuronal migration and designs some effective therapy strategies.
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Objective To obtain eukaryotic expression vectors containing coding region of nephroblastoma overexpression gene (NOV) and detect its expression in COS-7 cells. Methods A 1 165-bp cDNA fragment was amplified from the total RNA of normal rat brain tissue by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1/Myc-His(+)/lacZ. The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes HindⅢ and BamHⅠ. The recombinant plasmid was transfected into COS-7 cells with liposome. The expression of NOV gene was detected by Western blotting and immunocytochemistry. Results Eukaryotic expression vectors containing 1 165 -bp coding region of NOV gene was constructed. COS-7 cells transfected with the recombinant plasmid expressed high level of NOV protein in cytoplasm. Conclusion That eukaryotic expression vectors containing coding region of NOV gene was constructed can provide a strong molecular tool for the studies of effect of NOV gene.
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<p><b>BACKGROUND</b>To evaluate the relationship between estrogen receptor (ER) expression and level of serum sexual hormones in male patients with lung cancer.</p><p><b>METHODS</b>The levels of serum estradiol (E 2), testosterone (T), follicle stimulating hormone (FSH) and lutenising hormone (LH) were measured in 25 patients with lung cancer and 30 healthy men by enzyme immunoassay magnetic solid phase (IEMA), the ER expression was detected in 25 cancer tissues, 25 paracancerous tissues, and 11 benign pulmonary tissues by immunocytochemistry (ICC).</p><p><b>RESULTS</b>The level of plasma E₂ in male patients with lung cancer was significantly higher than that in normal controls [(22.4±15.7) ng/L [WTBX]vs ( 12.6±4.8) ng/L, P=0.001 9] while the level of T was significantly lower while the level of T was significantly lower [(2.9±1.3) μg/L [WTBX]vs (4.1±1.5) μg/L, P= 0.003 0]. The ratio of E₂/T of male patients with lung cancer was also remarkably higher than that of control . The ratio of E₂/T of male patients with lung cancer was also remarkably higher than that of control [(9.7±10.0) ×10⁻³[WTBX]vs ( 3.4±1.6)×10⁻³, P=0.004 6]. The expression rate of ER in lung cancer tissue samples was 60.0% (15/25), but no ER expression was found in the paracancerous tissues and benign pulmonary tissues. The level of E₂ had positive correlation with the expression of ER in male patients with lung cancer (. The expression rate of ER in lung cancer tissue samples was 60.0% (15/25), but no ER expression was found in the paracancerous tissues and benign pulmonary tissues. The level of E₂ had positive correlation with the expression of ER in male patients with lung cancer (r=0.916 7, P < 0.001).</p><p><b>CONCLUSIONS</b>There are disorders and imbalances of sexual hormone metabolism in male patients with lung cancer, and these imbalances relate to the expression of ER. The elevation of E₂ level in peripheral blood might be related to the overexpression of ER.</p>