The human wild-type Rb cDNA has been inserted into a retrovirus vector DOL and introduced into the human breast cancer cell MDAMB468,which has a large deletion of exons 3-27 of Rb genes,by electroporation transfection techniques.The exogenous Rb gene expresses the 110kd Rb protein.The morphology of the transfected cells is similar to that of the parent MDAMB468 cells.With the expression of Rb protein,the growth rate of MDAMB468 cells decreases by about 50%,and the colony formation ability in soft agaris repressed completely.After injection of 3times;106Rb+ cells and Rb-MDAMB468 cells into nude mice,the tumors formed from 106Rb+ cells are smaller than those from Rb-cells.The cell population of G1 and S phase of Rb+ MDAMB468 cells increases and the proliferation quotient decreases by about 50%.This result supports the former report the Rb protein. （Chin J Ocul Fundus Dis,1993,9:135-140）
Objective To explore the effect of exogenous estrogen receptor β1 (ERβ1) gene on the expression of p53 as well as the changes of apoptosis in MDA-MB-231 cell line and to investigate the biological role of ERβ1 in breast cancer. Methods Recombinant eukaryotic expressing vector containing ERβ1 cDNA was transfected into human breast cancer cell MDA-MB-231 by using cationic liposome LipofectamineTM 2000. The expression levels of p53 and ERβ1 in mRNA and protein were evaluated by real-time PCR and Western blot, respectively. Cell growth curve was used to detect the changes of cell proliferation ability. Cell apoptosis was detected by flow cytometry. Results After transfected with vector containing ERβ1 cDNA, proliferation ability of MDA-MB-231 cell decreased and the expression levels of both ERβ1 and p53 in both mRNA and protein increased (Plt;0.01). Rate of cell apoptosis increased in ERβ1 upregulation groups （Plt;0.01）. Conclusion ERβ1 can induce apoptosis and inhibit the growth of MDA-MB-231 cells by upregulating p53 expression.
Objective According to heparanase’s gene sequence of GenBank, to construct heparanase gene-targeted small interfering RNA (siRNA) and its expression vector and to observe its interference effect on the expression of heparanase gene in human malignant breast cancer MDA-MB-231 cell. Methods Heparanase gene-targeted hairpin siRNA was designed, two complementary oligonucleotide strands were synthesized and inserted into pGPU6/GFP/Neo vector, which was identified by sequence identify. Human malignant breast cancer MDA-MB-231 cell was transfected with the constructed vector with lipofectamine method. Fluorescence photograph was taken. Real-time PCR (RT-PCR) was performed to evaluate the level of heparanase mRNA expression. Results Four kinds of heparanase gene-targeted hairpin siRNA were designed, then were inserted into pGPU6/GFP/Neo vector after annealing. Sequencing indicated the construction was successful. Fluorescence photographs showed MDA-MB-231 cells were transfected successfully. RT-PCR showed that heparanase mRNA expression levels were inhibited significantly （Plt;0.05）. Conclusion The heparanase gene-targeted siRNA and its vector are successfully constructed and MDA-MB-231 cells are transfected successfully. Heparanase mRNA expression levels are significantly inhibited by siRNA vector, which provide a new method for the treatment of cancer.
ObjectiveTo explore changes on Electroencephalograph (EEG) and Evoked Potential (EP) changes in autoimmune encephalitis.MethodsEight cases with autoimmune encephalitis from Sichuan people's hospital during July 18th 2014 to July 18th 2016 were recruited. The inclusion criteria included:① The blood and cerebral spinal fluid (CSF) of patients were sent to Neurology Lab of Peking Union Medical College Hospital for autoimmunerelated antibody analysis and confirmed as autoimmune encephalitis.2 Patient had done at least 2 or more times of routine EEG or video EEG (VEEG). 1 or more times of auditory brainstem response (ABR), Visual evoked potential (VEP) and Somatosensory evoked potential (SEP) for both upper and lower limbs. 3 Patients had classical clinical manifestation of autoimmune encephalitis as abnormal psychomotor behaviors, seizures, memory loss, fever, headache, and even disturbance of consciousness or decreased ventilate function.ResulstOf 8 patients in this study, 5 were anti NMDA-R encephalitis, 2 were anti GABABR encephalitis, and 1 was positive for both antibodies. The EEG profile of 5 anti NMDA-R encephalitis:2 of them had β wave in early stage (about 10th day) and δ wave with fast wave even appeared as δ brush in middle stage (about 20th day). They all had severe symptoms and long hospitalization but negative MRI. Another 2 of them could be seen sparsely distributed sharp wave and sharp-slow wave in their EEG. Their EEG gradually turned to normal when their symptoms gradually disappeared. The last one had normal EEG during the whole disease course. The EEG profile of anti GABAB-R encephalitis as following. 1 was dominant by slow wave and EEG went normal after effective treatment and the other showed generalized α wave especially α wave in frontal region. The latter patient withdraw treatment. For the only 1 both antibodies positive patient, EEG showed slow wave and it turned to normal when symptoms disappeared. EP showed some abnormalities with wave amplitude and latency changes in some patients.EP (SEP、VEP) turned to normal when symptoms disappeared.ConclusionThe EEG present differently in different types of autoimmune encephalitis and change with stages of disease. EEG may be used as an indicator for prognosis as well. When EEG shows fast wave with the history of patient points to encephalitis, blood and CSF antibodies for NMDA-R should be checked routinely. Generalized α wave on EEG should also be an indicator for checking GABAB-R. More researches should be done for EP changes in autoimmune encephalitis for our study was based on a small patient number.
ObjectiveTo investigate the inhibitory effect of short hairpin RNA (shRNA) mediated contactin-1 (CNTN1) gene silencing on growth of human breast cancer cell line MDA-MB-468 transplanted tumors in nude mice.MethodsEighteen nude mice (4-week-old male BALB/c) were randomly equally divided into three groups: blank control group, empty vector group, and silencing group. The MDA-MB-468 cells (blank control group), MDA-MB-468 cells transfected by nonsense shRNA (empty vector group), and MDA-MB-468 cells transfected by shRNA (silencing group) were collected in the logarithmic growth period, respectively. The subcutaneous tumor models of nude mice were prepared by the subcutaneous injection of the different group cells. The tumor growth was observed and the expressions of CNTN1 and Ki-67 proteins in the transplanted tumor were detected by the immunohistochemistry.ResultsThe xenograft models of human breast cancer cells were established successfully. The tumor growth in the silencing group was significantly slower than that of the other two groups at every 3 d point (P<0.05). The tumor volume and the tumor weight in the silencing group were significantly smaller or slighter than those of the other two groups at day 18 (P<0.05). The positive rates of CNTN1 and Ki-67 protein expressions in the tumor tissues of the silencing group were lower than those of the other two groups (P<0.05), respectively.ConclusionSilencing expression of CNTN1 gene might inhibit growth of breast cancer cell line MDA-MB-468 transplanted tumors in mude mice.
ObjectiveTo analyze the protective mechanism of spinal cord ischemia-reperfusion injury mediated by N-methyl-D-aspartate (NMDA) receptor.MethodsA total of 42 SD rats were randomly assigned to 4 groups: a non-blocking group (n=6), a saline group (n=12), a NMDA receptor blocker K-1024 (25 mg/kg) group (n=12) and a voltage-gated Ca2+ channel blocker nimodipine (0.5 mg/kg) group (n=12). The medications were injected intraperitoneally 30 min before ischemia. The neural function was evaluated. The neuronal histologic change of spinal cord lumbar region, the release of neurotransmitter amino acids and expression of spinal cord neuronal nitric oxide synthase (nNOS) were compared.ResultsAt 8 h after reperfusion, the behavioral score of the K-1024 group was 2.00±0.00 points, which was statistically different from those of the saline group (5.83±0.41 points) and the nimodipine group (5.00±1.00 points, P<0.05). Compared with the saline group and nimodipine group, K-1024 group had more normal motor neurons (P<0.05). There was no significant difference in glutamic acid concentration in each group at 10 min after ischemia (P=0.731). The nNOS protein expression in the K-1024 group was significantly down-regulated compared with the saline group (P<0.01). After 8 h of reperfusion, the expression of nNOS protein in the K-1024 group was significantly up-regulated compared with the saline group (P<0.05).ConclusionK-1024 plays a protective role in spinal cord ischemia by inhibiting NMDA receptor and down-regulating nNOS protein expression; during the reperfusion, K-1024 has a satisfactory protective effect on spinal cord function, structure and biological activity of nerve cells.
ObjectiveTo retrospectively study the acute epileptic seizures of patients with anti-N-methyl-D-aspartate receptor (anti-NMDAR)and anti-leucine-rich glioma-inactivated 1(anti-LGI1)encephalitis. The characteristics and short-term prognosis provide reference for early clinical diagnosis and treatment.MethodsThe patients with anti-NMDAR and anti-LGI1 encephalitis who were admitted to the Department of Neurology of Sichuan Provincial People’s Hospital from January 2018 to June 2020 were continuously included. The general information, clinical manifestations, acute seizures and types of seizures were reviewed and analyzed.To evaluate the difference between the characteristics of two kinds of autoimmune encephalitis in the acute phase of seizures and the short-term prognosis.ResultsA total of 75 patients with anti-NMDAR encephalitis and anti-LGI1 encephalitis (41 males and 34 females) were included, of which average ages are(32.8±17.9)years, average courses are(1.8±1.1)months.59 and 16 are respectively positive for anti-NMDAR and anti-LGI1 antibodies, respectively. Of the 75 cases, 56 cases (74.7%) had seizures in the acute phase. Among the 56 cases of seizures, 38 cases (67.8%) were accompanied by disturbances of consciousness, 5 cases (8.9%) of autonomic dysfunction, and 24 cases of decreased oxygenation capacity. (42.9%) and 20 cases (35.7%) who were admitted to NICU, there was a significant statistical difference compared with the seizure-free group (P<0.05). The median age of anti-NMDAR encephalitis in the acute stage of seizures was 23 years, and that of anti-LGI1 encephalitis was 56.5 years (P<0.05). Anti-NMDAR encephalitis and anti-LGI1 encephalitis are common in the acute phase of epileptic seizures (55.9%vs.53.8%). Anti-NMDAR encephalitis has more frequent seizures and status epilepticus in the acute phase (P<0.05). After early and rational use of anti-epileptic drugs(AEDs) and immunotherapy and other symptomatic and supportive treatments, 70% of 56 patients were effectively controlled for seizure. Follow-up 3 months later, 18 patients (32.1%) stopped using anti-epileptic drugs (AEDs), While 30 patients (53.5%) continued to receive AEDs treatment, of which 25 patients (44.6%) had no seizures.ConclusionBoth anti-NMDAR encephalitis and anti-LGI1 encephalitis have a higher risk of seizures in the acute phase. Patients with seizures are more likely to have disturbances in consciousness, decreased oxygenation capacity, and higher rates of admission to NICU. Anti-NMDAR encephalitis is more common in young people around 30 years old, and anti-LGI1 encephalitis is more likely to develop around 60 years old. Patients with anti-NMDAR encephalitis are more likely to have abnormal electroencephalograms, have a longer average hospital stay, and are more likely to have recurrent seizures and status epilepticus in the acute phase. After timely diagnosis and intervention treatment, most patients' seizures can be well controlled. After the acute phase, AEDs can be withdrawed in one third of patients.
摘要:目的:探讨加兰他敏对急性酒精中毒大鼠海马神经元N甲基D天冬氨酸(NMDA)·R2B的影响。 方法:将60只大鼠分为对照组、酒精组及加兰他敏组,每组各20只。酒精组以50％(v/v) 酒精12 mL/kg灌胃两次/日,共7d。加兰他敏组酒精(浓度、剂量同上)灌胃的同时腹腔注射加兰他敏2mg/kg一次/日,共7d。对照组以等量生理盐水灌胃。实验第8天取大鼠海马区做苏木精伊红（HE）染色,观察海马区的病理学变化;免疫组织化学采用SABC法,观察海马区神经元NR2B的表达。 结果: 病理学观察结果：对照组海马区神经细胞排列整齐,胞质淡染,无变性、坏死;酒精组神经细胞层次不清、排列松散、细胞数量减少,部分细胞变性;加兰他敏组神经细胞层次较清、排列较密,细胞数目较酒精组增; 免疫组织化学结果：酒精组与对照组比较NR2B阳性表达细胞数量明显减少(Plt;0.01);加兰他敏组与酒精组比较NR2B阳性表达细胞数量明显增高(Plt;0.05);加兰他敏组与对照组比较NR2B表达细胞数量无明显差异(Pgt;005)。 结论: 急性酒精中毒与海马区神经细胞的NR2B表达下调有关;加兰他敏具有保护急性酒精中毒导致的大鼠海马区神经细胞毒性的作用,其机制可能与加兰他敏上调NR2B的表达有关。Abstract: Objective: To study the effects of galanthamine on NmethylDaspartic acid receptor 2B (NMDAR2B, NR2B) in the hippocampus (HIP) of acute alcoholism rats. Methods: Total of 60 wistar male rats were randomly divided into control group, ethanol group and glanthamine group, and there were 20 rats in each group. The rats in ethanol group were given by intragastric administration with 50% alcohol (v/v) on the dose of 12 ml/kg twice per day, in control group were given by same dose of saline, and in galanthamine group were treated by intragastric administration with the same concentration and dosage of alcohol as in ethanol group and peritoneal injection with 2 mg/kg of galanthamine once per day for 7 days. In eighth day of experiment, the rats were sacrificed under etherization, and pathological changes of HIP’s zone of rat were observed by HEstaining, and expression of NR2B in neurons of HIP’s zone by immunohistochemical SABC method. Results: The results observed by histopathology showed that in control group, neurons of HIP’s zone lined up in order, cytoplasm had faint staining, and were no degeneration and necrosis; in ethanol group, nerve cells’ layer was unclear, structure was loose, cell number reduced and part of cells degenerated; in galanthamine group, layer of neurons was comparatively clear and arrangement was comparatively dense, and the cell number increased obviously more than ethanol group. The results detected by Immunohistochemistry for NR2B showed that the cell number with expression of NR2B in the HIP’s zone decreased significantly in the ethanol group than in the control group (Plt;0.01), increased in the galanthamine group than in the ethanol group (Plt;0.05), and had no difference between the galanthamin and control group (Pgt;0.05). Conclusion: Acute alcoholism may relate to down regulation of expression of neuron’s NR2B in HIP’s zone;The galanthamin has role of protection for neuron in HIP’s zone induced by toxicity of acute alcoholism, and its mechanism may relate to galanthamin upregulation NR2B expression.