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Objective To study the role of leucine rich repeat neuronal 3 (LRRN3) in the prolif-eration of non-small cell lung cancer and its possible mechanism of expression regulation. Methods The expression of LRRN3 in non-small cell lung cancer was detected by real-time quantitative polymerase chain reaction ( qPCR) , immunohistochemistry and bioinformatics retrieval;A lung cancer cell line A549-LRRN3 with stable over-expression of LRRN3 was established by lentivirus over-expression technology;The effect of LRRN3 on the proliferation of non-small cell lung cancer was detected by methyl thiazolyl tetrazolium ( MTT) assay;Bioinformatics search for changes in methylation of LRRN3 promoter region and treatment of lung cancer cells by methyltransferase inhibitors to detect the effect of methylation on the regulation of LR-RN3 expression; Finally, bioinformatics search analyzes the correlation between LRRN3 and lung cancer prognosis. Results The mRNA expression of LRRN3 in clinical tissues of non-small cell lung cancer (n=12) was significantly lower than that of adjacent normal tissues (n=12) (P=0. 0014). The results of immunohistochemistry showed that the protein expression level of LRRN3 in non-small cell lung adenocar-cinoma was lower than that in normal tissues (P=0. 001), and the expression in non-small cell lung squa-mous cell carcinoma was also lower than that in normal tissues (P=0. 003). Overexpression of LRRN3 in-hibited the proliferation of tumor cells (P<0. 01), and the hypermethylation of LRRN3 in the promoter re-gion inhibited its transcriptional expression. LRRN3 was positively correlated with the survival prognosis of lung adenocarcinoma (P=5. 2e-09;HR=0. 48). Conclusions Hypermethylation in the promoter region of LRRN3 inhibits its transcriptional expression, thereby promoting the proliferation of lung cancer cells.
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Objective To evaluate the role of etomidate post-conditioning on mitochondrial permeability transition pore (mPTP) in the rat cortical neurons subjected to oxygen-glucose deprivation and restoration (OGD/R) and the relationship with Robo receptors.Methods The cortical neurons obtained from Sprague-Dawley rats (＜ 24 h after birth) were cultured in vitro and seeded in 6-well plates (2 ml/well).The neurons were divided into 4 groups (n=24 each) using a random number table:control group (group C),OGD/R group,etomidate post-conditioning group (group E),and etomidate post-conditioning + Robo receptor blocker group (group ER).The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h.In E and ER groups,etomidate was added to the culture medium with the final concentration of 6 μmol/L immediately after onset of O2-glucose supply.In group ER,Robo blocker RoboN was added to the culture medium with the final concentration of 1 μg/ml at 6 h before O2-glucose deprivation.The neuronal apoptosis was detected using Hoechst/PI double staining,the viability of neurons was measured by MTT assay,and the amount of lactic dehydrogenase (LDH) released was measured using colorimetric method.The mitochondria were extracted,and mitochondrial permeability transition pore (mPTP) opening was detected.Results Compared with group C,the apoptosis rate,amount of LDH released,and mPTP opening were significantly increased,and the cell survival rate was decreased in OGD/R,E and ER groups (P＜0.05).Compared with group OGD/R,the apoptosis rate,amount of LDH released,and mPTP opening were significantly decreased,and the cell survival rate was increased in group E,and the apoptosis and amount of LDH released were significantly decreased,and the cell survival rate was increased in group ER (P＜0.05).Compared with group E,the apoptosis rate,amount of LDH released,and mPTP opening were significantly increased,and the cell survival rate was decreased in group ER (P＜0.05).Conclusion Etomidate post-conditioning mitigates OGD/R-induced damage to the cortical neurons through activating Robo receptors and inhibiting mPTP opening in rats.
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Objective To investigate the effect of minocycline on the cognition and expressions of brain-derived neurotrophic factor (BDNF), apoptosis related factor Bcl-2 and Bax in hippocampus of rats with Alzheimer’s disease (AD). Methods The rat model was established by microinjection of Aβ25-35 into lateral ventricle. Thirty healthy male SD rats were randomly divided into three groups:control group, model group and minocycline treatment group. Normal saline 1 mL/(kg·d) was intraperitoneally injected in control group and model group. The minocycline treatment group was intraperitoneally injected with minocycline 50 mg/(kg · d) for 14 days. Morris water maze was used to detect the behaviors of animals. The expressions of BDNF, Bcl-2 and Bax in hippocampus were measured by Western blotting and enzyme linked immunosorbent assay (ELISA). The apoptosis of neurons was detected by TdT-mediated dUTP nick-end labeling (TUNEL). Results Minocycline greatly improved the behaviors of AD rats, up-regulated the expressions of BDNF and Bcl-2, and down-regulated the expression of Bax in hippocampus, and reduced cell apoptosis. Conclusion Minocycline plays a protective role in neural function by promoting the growth of neurons and inhibiting the neuronal apoptosis.
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ObjectiveTo determine the optimal timing of antenatal taurine supplementation to improve neuron and neural stem cell proliferation in fetal rats with intrauterine growth restriction.Methods Twenty-five pregnant SD rats were randomly divided into five groups (five rats in each group): group A was the control group, group B to E were the fetal growth restriction (FGR) model groups with low-protein diet during the experiment, group C, D, and E were supplemented with taurine [300 mg/(kg·d)] at day 9, 11 and 15, respectively. The birth weight of newborn rats was measured after natural delivery. The rats with body weight two standard deviations lower than the average weight in group A were diagnosed as FGR. There were five litters of newborn rats in each group, and two were randomly selected from each litter, resulting in ten newborn rats in each group. Proliferating cell nuclear antigen (PCNA) and fatty acid binding protein 7 (FABP7) positive cell expression in newborn rat brain tissues were detected by immunohistochemistry. Single factor analysis of variance, LSD tests were used for statistical analysis.ResultsThe average birth weight of newborn rats in group A, B, C, D and E were (6.61±0.45), (4.65±0.23), (5.37±0.17), (5.74±0.21), and (5.00±0.24) g, respectively. Average birth weight was lower in group B than in group A (t=2.447), higher in group D and E than in group B (t=2.306 and 2.306), higher in group D than in group C and E (t=2.306 and 2.306), and the differences were statistically significant (allP0.05). The IOD in group D was higher than that in group E, and the difference was statistically significant (t=4.182,P<0.05).ConclusionsAntenatal taurine supplementation can promote neuron and neural stem cell proliferation in rats with FGR. The effect is most obvious on the 11th day of pregnancy, and may lead to the promotion of brain development.
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Objective To investigate the expressions of Slit2 and Nogo-A in the ipsilateral hippocampus after cerebral hemorrhage in rats.Methods A total of 64 adult Sprague-Dawley (SD) rats was randomly divided into control and model groups.Collagenase Ⅶ was used to induce cerebral hemorrhage model.Immunohistochemistry was used to detect the expressions of Slit2 and Nogo-A in hippocampus at the cerebral hemorrhage side at the time points (24 h,7 d,14 d,and 21 d).Results Compared to the control group,the expressions of Slit2 and Nogo-A were significantly enhanced in model group (P ＜0.01),with the highest level at the 7 d.Conclutions Cerebral hemorrhage can significantly enhance expressions of Slit2 and Nogo-A with a positive correlation of Slit2 and Nogo-A,which might have an important effect on the recovery of brain injury.
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Objective To investigate the association between high-risk types of human papillomavirus (HR-HPV) load and the expression of nestin in different cervical lesions.Methods The hybridization capture Ⅱ (HC-Ⅱ) assay was used to test the HR-HPV load from 60 patients who were the first time to do the cervical cancer screening in Xiangya Hospital,and immunohistochemistry was used to detect the expression of nestin in those biopsy tissue samples.Results (1) The lgHPV level (logarithm of HR-HPV load) in the high level lesion group was higher than the low level one (P ＜0.05),and HR-HPV load was positively associated with the degree of cervical lesions (rs =0.269,P =0.037).(2) The expression of nestin in A group was weaker than groups B and C (H =7.271,22.843,P ＜0.01),and the expression of nestin in C group was stronger than B group (H =7.270,P ＜0.01),and the expression of nestin was positively associated with the degree of cervical lesion (rs =0.646,P =0.000).(3) The HR-HPV load was positively associated with the expression of nestin (P ＜ 0.05).Conclusions The HR-HPV load and the expression of nestin are closely related to the cervical lesions,and the joint detection has a referential value for early prevention of cervical lesions and prediction of progress of cervical precancerous lesion.It might guide the early prevention and treatment of cervical cancers.
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Objective To investigate the potential therapeutic efficacy of lentivirus-mediated artemin (ARTN) gene modified bone marrow mesenchymal stem cells (MSCs) transplantation on the rat model of Parkinson' s disease (PD) and the effects on expression of brain-related proteins.Methods MSCs were isolated and cultured in vitro,transfected by recombinant lentiviral vectors carrying ARTN gene.The PD rat model established by 6-hydroxydopamine (6-OHDA) was randomly divided into 5 groups:Sham group,PD group,MSCs group,MSCs transfected with empty lentiviral vectors transplanted (LV-MSCs)group and MSCs transfected with recombinant lentiviral vectors carrying ARTN gene transplanted (LVARTN-MSCs) group.The MSCs,LV-MSCs and LV-ARTN-MSCs groups were transplanted into the left striatum of each rat model of PD and ethology tests in every group were made with intraperitoneal injection of apomorphine (APO) 2,4,6,8 weeks after transplantation.The expression of tyrosine hydroxylase (TH) protein in substantia nigra (SN) was measured by Western blotting and immunohistochemistry,and immunofluorescence showed ARTN gene modified MSCs expression in rat brain tissue.The levels of dopamine (DA),dihydroxy-phenylacetic acid and homovanillic acid in striatum of each group were detected by high performance liquid chromatography.Results After injection of APO,rotation frequency decreased in LV-ARTN-MSCs group,i.e.(179.33 ± 10.74) circles/30 min vs (235.83 ± 18.95),(203.67 ±11.50) and (206.33 ± 11.86) circles/30 min in PD,MSCs and LV-MSCs groups (q =8.828,P ＜ 0.01;q =3.802,P ＜ 0.05;q =4.219,P ＜ 0.05).The percentage of TH-positive cells in SN after cell transplantation was increased significantly in LV-ARTN-MSCs group (64.05％ ± 5.49％) when compared with PD group (34.18％ ±3.35％),MSCs group (52.59％ ±4.48％) and LV-MSCs group (50.57％ ± 4.41％),respectively (q =13.280,5.135,6.028,all P ＜0.01).At the same time,TH protein in SN after cell transplantation was also increased obviously in LV-ARTN-MSCs group.ARTN gene modified MSCs can survive for at least 6 weeks in the rat brain of PD,mainly concentrated in the transplantation side of striatum.Eight weeks later,the levels of DA in striatum after cell transplantation were elevated significantly in MSCs group (2.34 ± 0.54),LV-MSCs group (2.28 ± 0.45) and LV-ARTN-MSCs group (2.28 ± 0.45)when compared with PD group (0.87 ± 0.07) (q =5.233,P ＜ 0.05;q =5.020,P ＜ 0.01;q =20.190,P ＜ 0.01),and LV-ARTN-MSCs group showed the most significant improvement.Conclusion ARTN gene modified bone marrow MSCs transplanted into the striatum of brain may have therapeutic effects on rat models of PD.
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Neuroglobin (Ngb) is an oxygen-carrying globin that specifically expresses in brain tissue.It is involved in energy metabolism,mitochondrial function,as well as cell survival and proliferation of signaling pathway regulation.Under physiological conditions,Ngb presents as a form of ferrous deoxy hexacoordination,which has stronger oxygen affinity.During ischemia and hypoxia,the expression of Ngb is upregulated in brain tissue and interacts through a variety of proteins of its downstream,and plays a protective role for the damaged brain tissue.
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Objective To report the clinical and genetic characteristics of a dentatorubralpallidoluysian atrophy (DRPLA) pedigree with an onset of cognitive impairment.Methods Clinical data of this pedigree was collected.The numbers of CAG repeats in the exon 5 of atrophin-1 (ATN1) gene were analysed in the proband and the other 4 healthy family individuals.The polymerase chain reaction (PCR) products of the proband underwent cloning-sequencing using an original TA cloning kit.Results There were 5 patients in this family,4 with onset in adult and one in childhood.The proband had an onset manifestation of cognitive impairment,while the other 3 adult patients presented with ataxia.The two-year-old child in the pedigree had myoclonic epilepsy.The proband had 61 CAG repeats in the exon 5 of ATN1 gene.After TA cloning-sequencing of the proband ' s PCR products,there were 2 different numbers of CAG repeats,including 61 and 64.Conclusions The clinical manifestations of DRPLA can have obvious heterogeneity in one family.Some patients present with cognitive impairment.It is very important to test the numbers of CAG repeats of ATN1 gen for DRPLA diagnosis.Somatic mosaicism may be also observed in Chinese DRPLA patients.
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Objective To discuss clinical,electroencephalogram(EEG) and PRRT2 gene mutation by reporting a febrile seizure (FS) with paroxysmal kinesigenic dyskinesia (PKD) family.Methods Detailed clinical data of the family were collected.The proband (Ⅳ1) and another 4 patients (Ⅲ1,Ⅲ4,Ⅳ2,Ⅳ3)were studied through clinical examinations.Clinical symptoms of Ⅳ2 were not typical,who was diagnosed as a suspected case.Mutation analysis of PRRT2 gene was screened by polymerase chain reaction (PCR) and DNA direct sequencing in 5 patients (Ⅳ1,Ⅲ1,Ⅲ4,Ⅳ2,Ⅳ3) and 4 unaffected family members (Ⅱ2,Ⅲ2,Ⅲ5,Ⅳ4).Results PKD patients had brief involuntary movements in the limbs or trunk induced by sudden voluntary movement when patients were in the stationary state since the teenagers.Two cases (Ⅲ,Ⅲ4) were accompanied by FS.Three cases(Ⅳ1,Ⅲ1 and Ⅲ4)had abnormal EEG records.The PRRT2 gene mutation (c.649dupC mutation) was identified in a healthy member (Ⅳ4) and 4 patients (Ⅳ1,Ⅲ1,Ⅲ4,Ⅳ3).Conclusions FS with PKD family has a PRRT2 gene mutation.The diagnosis is mainly based on family history,typical clinical manifestations and genetic test.This kind of disease may have pre-symptomatic patients.
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OBJECTIVE: Given the ability of mood stabilizers and antipsychotics to promote cell proliferation, we wanted to determine the effects of these drugs on neuronal markers previously reported to be altered in subjects with psychiatric disorders. METHODS: Male Sprauge-Dawley rats were treated with vehicle (ethanol), lithium (25.5 mg per day), haloperidol (0.1 mg/kg), olanzapine (1.0 mg/kg) or a combination of lithium and either of the antipsychotic drugs for 28 days. Levels of cortical synaptic (synaptosomal associated protein-25, synaptophysin, vesicle associated protein and syntaxin) and structural (neural cell adhesion molecule and alpha-synuclein) proteins were determined in each treatment group using Western blots. RESULTS: Compared to the vehicle treated group; animals treated with haloperidol had greater levels of synaptosomal associated protein-25 (p<0.01) and neural cell adhesion molecule (p<0.05), those treated with olanzapine had greater levels of synaptophysin (p<0.01) and syntaxin (p<0.01). Treatment with lithium alone did not affect the levels of any of the proteins. Combining lithium and haloperidol resulted in greater levels of synaptophysin (p<0.01), synaptosomal associated protein-25 (p<0.01) and neural cell adhesion molecule (p<0.01). The combination of lithium and olanzapine produced greater levels of synaptophysin (p<0.01) and alpha-synuclein (p<0.05). CONCLUSION: Lithium alone had no effect on the neuronal markers. However, haloperidol and olanzapine affected different presynaptic markers. Combining lithium with olanzapine additionally increased alpha-synuclein. These drug effects need to be taken into account by future studies examining presynaptic and neuronal markers in tissue from subjects with psychiatric disorders.
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Animals , Humans , Male , Rats , alpha-Synuclein , Antipsychotic Agents , Benzodiazepines , Cell Adhesion , Cell Proliferation , Haloperidol , Lithium , Nerve Tissue Proteins , Neural Cell Adhesion Molecules , Neurons , Neurotransmitter Agents , Proteins , Qa-SNARE Proteins , SNARE Proteins , SynaptophysinABSTRACT
Objective To investigate the effect of ketamine anesthesia in early pregnancy on expression of hippyragranin (HGN) mRNA in hippocampus in offsprings of rats.Methods One hundred and twenty pregnant Sprague-Dawley rats at 5-7 days of gestation,aged 7 weeks,weighing 270-300 g,were randomly divided into 4 groups (n =30 each):2 h anesthesia with ketamine group (group K1),4 h anesthesia with ketamine group (group K2),8 h anesthesia with ketamine group (group K3) and control group (group C).Ketamine 20 mg/kg was injected via the caudal vein,followed by continuous intravenous infusion at 130 mg· kg-1 · h-1 after loss of righting reflex,and the infusion lasted for 2,4 and 8 h in groups K1-K3,respectively.The equal volume of normal saline was given instead of ketamine in group C.The learning ability and memory of the offsprings were tested by Morris water maze test on postnatal day 30.The offsprings were sacrificed on 2nd day after the end of Morris water maze test,their brains were removed and hippocampi were isolated for determination of the expression of HGN mRNA by RT-PCR.Results Compared with group C,the escape latency was significantly prolonged,the frequency of crossing the original platform was decreased,the time of staying at the original platform quadrant was shortened,and the expression of HGN mRNA was up-regulated in groups K1-K3 (P ＜ 0.05).There was no significant difference in the escape latency,frequency of crossing the original platform and time of staying at the original platform quadrant among groups K1-K3 (P ＞ 0.05).The expression of HGN mRNA was significantly higher in group K3 that in group K1 (P ＜ 0.05).There was no significant difference in the expression of HGN mRNA between group K2 and group K3 (P ＞ 0.05).Conclusion The mechanism by which ketamine anesthesia in the early pregnancy induces impairment of learning ability and memory of the offsprings is related to up-regulation of HGN mRNA expression in hippocampus in offsprings of rats.
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ObjectiveTo observe the expression of nerve growth factor(NGF)and its receptors (TrkANGFRand P75NTR) in hepatocytes and to explore the biological effects of exogenous P75NTR protein on hepatocytes.MethodsL02 hepatocytes were cultured in vitro.The expression of NGF,TrkANNGFR and P75NTR in L02 cells were assessed by immunocytochemistry and fluorescent quantitation polymerase chain reaction (PCR). The effects on L02 cell proliferation by exogenous P75NTR,NGF,NGF+ P75NTR,anti-TrkANGFR and anti-P75NTR were detected by XTT assay.The effect of exogenous P75NTR on L02 cell apoptosis was measured by flow cytometry (Annexin V/PI) and the effect of exogenous P75NTR on L02 cell cycle was determined by flow cytometry (PI).ResultsL02 cell proliferation was promoted by P75NTR and in dose-dependent manner.The A value of NGF group and NGF+ P75NTR group was 0.4916±0.0565 and 0.5839 ± 0.0733,respectively,and there was statistical significance compared with control group (0.3601 ± 0.0310,P＜0.05).The A value ot anti-TrkANGFR group was 0.2689±0.0229,and there was statistical significance compared with control group (P=0.003).The A value of anti P75NTR was 0.3524 ± 0.0312,and there was no statistical significance compared with control group (P=1.000). Exogenous P75NTR had anti-apoptosis effect on L02 cells,the antiapoptosis effect was strongest when 100 ng/ml P75NTR was added and the expression quantity was 3.70 ±0.26.However there was no statistical significant compared with control group (4.10 ± 0.62,P=1.000).P75NTR affected the cell cycle S phase of L02 cells and in dose-dependent manner,which was inverted U shaped curve.The effect was strongest when the concentration was 100 ng/ml (25.60 ±0.40) and there was statistical significance compared with control group (20.10 ±1.00,P=0.000).Exogenous NGF,P75NTR and NGF + P75NTR up regulated the gene expression of NGFmRNA,TrkANGFRmRNA and P75NTR mRNA in L02 cells and there was statistical significance compared with control group (P＜0.05).There was no significant difference in the gene expression of NGFmRNA,TrkANGFFR mRNA and P75NTR mRNA between anti-TrkANGFR,anti-P75NTR groUp and control group (P＞0.05).Conclusion NGF and its receptors TrkANGFR and P75NTR were expressed in L02 cells.The appropriate dose of exogenous P75NTP protein promoted L02 cells proliferation via TrkANGFR/P75NTR heterodimer signal pathway.
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Musashil is an evolutionarily conserved RNA binding protein. It plays an important role in the aspects of maintaining stem cell state, differentiation and tumorigenesis. Notch signal pathway plays an important role in the proliferation of neural stem cells after cerebral ischemia. This article briefly introduces the structure and molecular characteristics of Musashil and Notch, and reviews the interaction in the neurogenesis of Musashil and Notch signaling pathway after cerebral ischemic reperfusion.
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Objective To investigate the significance and mechanism of intracerebroventricular injection viper venom nerve growth factor (Vngf) in rat neural plasticity after cerebral ischemia reperfusion injury.Methods Ninety Wistar male rats were randomly assigned into Vngf-25 U group (n = 18), Vngf-50 U group (n = 18), Vngf-100 U group (n = 18), ischemia reperfusion group (n = 18) and sham operated group.The expression of candidate plasticity-related gene 15(cpg-15) Mrna and nuclear factor of kappa B ( NF-Κb ) Mrna in rat brain tissues which were collection at 2,7,14 days after surgery were evaluated by the real time PCR.Results The expression of cpg-15 Mrna and NF-Κb Mrna began to increase after surgery( the F value of cpg-15:70.43, 34.11, 31.89, the F value of NF-Κb: 27.47, 34.56, 31.89,P＜0.01).At the same time, expression of cpg-15 Mrna and NF-Κb Mrna in the Vngf groups was significantly different from the I/R group and the sham operated group (the F value of cpg-15:48.18, 55.93, 78.43, the F value of NF-Κb: 45.92, 55.72, 50.49, P ＜0.01).The more Vngf were injected, the more cpg-15 Mrna and NF-Κb Mrna were expressed in Vngf groups.Conclusions The Vngf could accelerate neural plasticity and restore neurofunctional defect through up-regulated the expression of cpg-15 and NF-Κb.
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Objective To discuss the possible enteric nervous pathogenesis in gastritis related GI motor disorders on the expression changes of protein gene product9. 5 in neurons from the gastric walls of gastritis rat model. Methods 35 clean grade Wister rats were randomly divided into 3 groups, which included gastritis group A (n =10), gastritis group B(n =15) and control group(n =10). Rats in gastritis group A and B received gastric perfusion of HP and the mixture of 2% aspirin and 0. 6N hydrochloric acid respectively. The control group only received gastric perfusion of saline. All of the rats were killed and the gastric mucosal tissues were obtained for the pathological and HP examination. After immunohistochemical pretreatment, the tissues were stained with PGP9. 5 and at the same time the maximum diameter (Dmax, μm), mean area(μm2) and mean optical density (nm) of the neurons from the gastric walls were compared among the groups with Image-Pro Plus professional image analysis system. Results In gastric group A, HP could be found sparsely in the mucous layer or gastric pits, and all of the rapid urease tests were positive. In the other two groups, HP could not be found, and all of the rapid urease tests were negarive. In both gastric group A and B, different grades of inflammatory cell infiltration with active inflammation signs could be found in the deep layers of mucosa, while the control group was normal. The expression mean area, mean optical density of neurons from the gastric wall of rat in group A[(77. 10 ±48. 46) μm2, (53. 25 ±41.40) nm] or B [(73. 92 ± 39. 60) μm2, (45.33 ± 33.20) nm] was obvious lower than control group [(143.51 ± 29. 84) μm2, (85. 00 ± 14. 32) nm], while there was no significant difference between gastric group A and B (P ＞0. 05) (table 1). Conclusions Hp and NSAIDs might cause gastritis and decrease the PGP9. 5 expression of Neurous from gastric walls. The decrease of PGP9. 5 expression of neurons from the gastric wall might contribute to the pathogenesis of GI motor disorders or symptoms of functional dyspepsia.
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Objective To develop urine AD7C-NTP diagnostic kit,analyze and evaluate its application value on AD.Methods Immunogenicity AD7C-NTP peptide fragments had synthesized by solidphase methods.The animals immunized to prepare antibodies.After matching screening.mouse antibody was uesed as coating antibody.biotin-labeled rabbit antibody wag used as testing antibody,and horseradish peroxidase was labeied with avidin.The urine AD7C-NTP ELISA detective method was established.The AD7C-NTP levels in morning urine samples of 121 AD patients and 118 age-matched controls were collected.Results AD7C-NTP antibodies were identified.Mouse anti-AD7C-NTP antibody titer in ELISAwas 1:8 000,and rabbit anti-AD7C-NTP antibody titer in ELISA was 1:32 000:WB was uesd to detect human brain specimens and there was a single band with molecular weight of 41 000.The lowst detection limit of ELISA methodology was 0.5μg/L The linear range was 0-10μg/L,normal reference value ≤1.5μg/J,the average recovery rate was 100.2%.The intra and inter of CV were 3.8%,4.5%,7.6%,6.8% respectively.The AD7C-NTP levels[2.25(0.43-8.62)μg/L]of urine in AD group was higher than those in contorl group[0.82(0.47-2.77)μg/L,P<0.01].The positive rates in AD group and control group were 89.3% and 15.3% respectively.The sensitivity Was 89.3%and specificity was 84.7%.Conclusions The animals are immunized with the self-designed synthetic peptide fragment to prepare AD7C-NTP antibodies successfully.The established ELISA method for detection of urine AD7C-NTP with high sensitivity,and precision can be used as an assistant examination in clinical diagnosis of AD.
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Objective To screen and analyze nucleotide variants in 5'-untranslated region(5'-UTR)in voltage-gated sodium channel α1-subunit gene(SCN1A)in patients with Dravet syndrome and to evaluate the association of the variants with disease.Methods Peripheral blood of 24 patients with Dravet syndrome and 100 unrelated normal persons were collected and genomic DNA was extracted.PCR-sequencing of SCN1 A 5'-UTR in these DNA was performed.To evaluate the possibility of mutation inducing disease,bioinformatics analysis was applied to analyze the conservation of the sequences around the mutation site and predict the potential transcription elements.Results The nucleotide variant of 166.642.520G→A in exon 2 was identified in two patients,but not in normal controls.The mutation was a de novo mutation in a patient with early-onset.In the second proband,the mutation was also carried by his clinically asymptomatic mother.The nucleotide site 166.642.520 was moderately conserved in mammals(62.5%).The average nucleotide identity rate between human and other mammals species in the region adjacent to 166.642.520 was 88.5%.Two potential transcription regulatory elements were predicted on the sequence with the mutation of 166.642.520G>A,and only one on the sequence with wild-type.Conclusions The mutation 166.642.520G>A may be associated with Dravet syndrome and further studied should be performed to verify it and demonstrate its pathogenic mechanisms.
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Objective Cloning-sequencing is a common method to detect the number of trinucleotide repeats.The aim of the present study is to discuss its reliability.Methods One clinically diagnosed SCA1 patient was recruited in the study.The numbers of CAG repeats in ATXN1 gene were estimated via polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis (DPAGE).To verify accuracy of CAG numbers estimated, the PCR products were electrophoresed on a 2.5% agarose ge] and separated bands were excised for direct sequencing.Also, the longer separated band underwent cloning-sequencing using a TA cloning kit.Results The patient was identified as SCA1 by DPAGE.After direct sequencing, the numbers of CAG repeats were 26 and 47 in the shorter and longer bands, respectively.However, after cloning-sequencing of the longer band, there are 10 different numbers of CAG repeats, including 50, 47, 46, 41,32, 28, 27, 26, 25 and 24.Furthermore, there are other kinds of trinucleotide repeats, such as CCG, CGG, CTG, CAA and TAT scattered among the CAG repeats.Conclusions It is not reliable to identify the number of trinucleotide repeats by cloning-sequencing alone.To improve the reliability, it is better to combine cloning-sequencing with other methods.
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Objective To explore the inheritance characteristics of SCN1A gene in familial severe myoclome epilepsy in infancy.Methods The clinical information and blood of the patients and their relatives who had febrile seizure(FS)or epilepsy history were collected.Blood genome DNA were extracted.All exons of SeN1A gene were PCR amplified and screened with denaturing high Performance liquid chromatography(DHPLC)technology,and sequence analysis was performed.Results Fourteen SME patients had FS or epilepsy family history.Five were found positive history in first class relatives and 2 of them had inherited mutations of SCN1A(C.4284+2T>C and e.1216G>T):Other9 were found positive history in second class relatives and 2 of them had de novo mutations of SCN1A.Condusions SCN1A is the pathogenic gene for SME.The same muatation of SCN1A gene can be related to different clinical phenotypes.SME patients whose first class relatives with FS or epilepsy history should be taken as the focus of SCN1A inherited mutation screening.