Objective To culture human pituitary adenoma cells to provide some useful experimental foundations to practice pituitary adenoma cells as donor cells in the extracranial implantation. Methods Human pituitary adenoma cells obtained by operation were cultured in vitro, and their growth state was dynamically observed. Immunohistochemical staining and RIA were used to evaluate their functional status. Results The cultured pituitary adenoma cells still had secretory function in vitro. 6 cases of GH adenoma cells secreted a large amount of GH, 5 cases of PRL adenoma cells secreted a large amount of PRL, and 3 cases of pituitary adenoma cells without function could also secrete a small quantity of FSH, LH and PRL. Conclusion 14 cases of cultured human pituitary adenoma cells had secretory function in vitro, which provides the useful experimental data to perform extracranial implantation with pituitary adenoma cells as donor cells.

Objective To investigate the effect of neutral amino acid on preventing Parkinson disease.Methods Mice were injected with L-Valine,L-Pheylalanine,D-Valine or L-Lysine before or after paraquat administration,by which prakinsonian mouse model was constructed.The paraquat immunoreactivity was observed within nigral cell bodies.Then neurodegeneration and ?-synuclein aggregation were observed by immunohistochemistry and Western blot.Results Paraquat immunoreactivity was abolished by the administration of L-Valine,L-Pheylalanine before paraquat exposure.Pre-treatment with these two amino acids also protected the paraquat-induced loss of nigrostriatal dopaminergic cells and formation of thioflavine S-positive aggregates.In contrast, paraquat-induced toxicity was unaffected if animals were injected with these two amino acids after paraquat exposure or pre-treated with D-Valine or L-Lysine.Conclusions L-type neutral amino acids such as L Valine and L-Pheylalanine can prevent paraquat-induced neurodegeneration and a synuclein pathology through a competitive inhibition mechanism with stereospecificity in the central nervous system (CNS).Neutral amino acid could protect the dopaminergic neuron in substantia nigra and may prevent Parkinson disease.

Objective To explore the relationship between the genesis of dyskinesia and the degree of substantia nigra lesion in Parkinson disease(PD).Methods The hemi-parkinsonian rat model was established by injecting 6-OHDA stereotaxically to right medial forebrain bundle(MFB). Then the hemi-parkinsonian rat was injected intraperitoneally with levodopa methylester(25 mg?kg~(-1)?d~(-1),twice a day)for 21 days,the abnormal involuntary movements were estimated.After being sacrificed,the midbrain was removed,and the injured degree of dopaminergic neurons at substantia nigra was observed by tyrosine hydroxylase immunohistochemistry staining.The relationship between the abnormal involuntary movement scores and dopaminergic neurons loss at substantia nigra was evaluated by sigmoid equation analysis by using Excel software.Results The apomorphine-induced rotation rate above 7 r/min was found in 10 of 25 rats,those rats were regarded as successful hemi-parkinsonian model rats.After the treatment with levodopa methylester,8 of 10 rats displayed abnormal involuntary movements,including stereotype and contralateral rotation,the types of movements varied.Abnormal involuntary movements were appeared in the rats with dopaminergic neurons loss above 90%.The positive relationship was observed between the degree of lesion in substantia nigra and the severity of abnormal involuntary movements.Conclusions The severe loss of dopaminergic neurons at substantia nigra probably plays a role in the development of levodopa-induced dyskinesia in patient with Parkinson disease.

Objective To investigate cellular and behavioural effects of 5-HT1A receptor agonist 8- OH-DPAT in a rat model of levodopa-induced motor complications.Methods The hemi-parkinsonian rat model was produced by stereotaxically injecting 6-OHDA to right medial forebrain bundle(MFB).Two sets of experiments were performed.First,rats were intrapefitoneally treated with levodopa 50 mg/kg plus benserazide 12.5 mg/kg twice a day for 22 days.On day 23,rats intraperitoneally received either 8-OH- DPAT(1 mg/kg)or 8-OH-DPAT plus WAY-100635(0.1 mg/kg)or dissolvent with each levodopa dose as controls.In the second set,rats were intraperitoneally treated either with levodopa(50 mg/kg)plus 8-OH- DPAT(1 mg/kg)or levodopa 50 mg/kg plus dissolvent,administered twice daily for 22 consecutive days. Rotational duration and frequency of off period were estimated.After sacrificed,subcellualr distribution of GluR1 and GluR1Ser845 phosphorylation was observed by Western blot.Results 8-OH-DPAT,reversing the shortened rotational duration induced by levodopa,prolonged the rotational duration by 27.8%?6.1% and reduced the frequency of failures to levodopa by 7.2%?1.7%.Co-administration of WAY-100635,a 5-HT1A receptor antagonist,with 8-OH-DPAT eliminated the effect of 8-OH-DPAT on motor complications, indicating that the observed 8-OH-DPAT responses were probably mediated via the 5-HT1A autoreceptor. Moreover,8-OH-DPAT could regulate subcellular distribution of GluR1 and reduce hyperphosphorylation of GluR1 Ser845 by 22.1%?3.5%,which was closely associated with levodopa-induced motor complications. Conclusions These results suggest that pharmaceuticals stimulating 5-HT1A receptors could be useful in the treatment and prevention of the motor complications in parkinsonian patients.

Objective To establish a method focusing on regulation of CNN3 gene in the rat hippocampus and help to explore the role of CNN3 gene played in the brain physiology and pathology.Methods One cDNA sequence and three shRNAs targeting CNN3 gene were designed and synthesized.The recombinant lentivirus-mediated expressing and three short hairpin RNA ( shRNA) vectors targeting CNN3 gene in the rats were constructed with engineering technology.All recombinant vectors were intravenously injected into rats hippocampi guided by stereotaxic apparatus.Western blot was performed to explore the best shRNA and to study the changes of CNN3 gene in the rat hippocampus after transfection with the silence and over-expressed vectors.Results The lentivirus-mediated vector expressing CNN3-OE and three shRNA vectors targeting CNN3 gene were successfully constructed.Within eight weeks after transfection, the vectors of CNN3-OE and three CNN3-shRNAs changed the expression of CNN3 gene in the rat hippocampus, in particular, all the protein levels of calponin-3 encoded by CNN3 gene were significantly down-regulated along with the time, with the highest inhibitory rate of 73.6%in the CNN3-shRNA2 group.Significant up-regulation of calponin-3 protein level by 93.88%, was found only on the 14th day after transfection.Conclusions Lentivirus-mediated vectors of CNN3-OE and CNN3-shRNAs may regulate in vivo the CNN3 gene level in the local brain region of rats via stereotactic injection.The study lays a foundation for disease prevention and treatment in the future.

Objective To explore the activition and polarization of microglia in the epileptic rats induced by lithium chloride-pilocarpine. Methods One hundred male SD rats were randomly divided into five groups: control group and different time points model groups including 1d,3d,7d and 14d. Epilepsy models were established by lithium chloride-pilocarpine intraperitoneal injection. The control group was given the same dosage of normal saline. The morphology change was detected by immunofluorescence,and the expressions of iNOS and Arg-1 were determined by IHC at respective time points. Results Compared the model groups with control group,microglia was activated,synapsis was shorten,volume got bigger,most of them seemed as amoebocyte,the expression of iNOS increased and Arg-1 decreased,especially at 3d.ConclusionThe results from this study indicated that microglia was activated and polarized in epileptic rats induced by pilocarpine.

Objective To explo the antioxidant effect and molecular mechanism of gastrodin (Gas) in epilepsy (EP) rats induced by LiCl-pilocarpine (PILO) . Methods Eighty male SD rats were randomly divided into 5 groups: sham group, EP group, therapy groups (pretreated with 60 mg/kg, 90 mg/kg, 120 mg/kg of gastrodin respectively) . The EP model was esteblished by peritoneal injection of LiCl-PILO. Therapy groups were pretreated with various concerntration of Gas. The control group was given the same dosage of normal saline. The alteration of behavior was observed, the concentration of catalase (CAT), glutathion (GSH), superoxide dismutase (SOD), glutathion reductase (GR), total antioxidtion (T-AOC) and malondialdehyde (MDA) in rats brain cortex were detected by chemical colorimetric method, phosphorylation of p38 was determined by western blot. Results There was no EP seizure in sham group,and the EP seizure degree in therapy groups (gas pretreated groups) was significantly decreased,and had statistically significant difference with EP group (P<0.05) . The EP model rats exhibited a significant decrease in the concentration of endogenous antioxidants (CAT, GSH, SOD, GR and T-AOC), while an increase of the concentration of MDA and phosphorylation p38 protein as compared to sham group (P<0.05) . After treatment of the Gas,treatment group rats attenuated the seizure degree,exhibited a significant increase of the concentration of endogenous antioxidants (P<0.05),while a decrease in concentration of MDA and phosphorylation of p38 as compared to model group (P<0.05) . Conclusion Gas may have a neuroprotective role in central nervous system of epileptic rats modle by down-regulateing the seizure degree and the activity of p38 kinase and up-regulateing the content of endogenous antioxidants.

BACKGROUND: There are still few effective methods to repair injured myocardium after myocardial failure and pathologically rebuild reverral myocardium. As a new therapy, normal myocytes and therapeutic gene to interfere injured myocardium have advantageous effects in improving heart function.OBJECTIVE: To observe the efficiency and stability of adenovirus-medicated gene transferred into different passages of bone marrow mesenchymal stem cell (MSC) and investigate the effect of MSC-based sarcoplasmic reticulum Ca2+ ATPase gene (SERCA2a) gene therapy for rats with chronic heart failure. To compare the effects of gene therapy, cell transplantation and MSC-based SERCA2a gene therapy for chronic heart failure. DESIGN: Randomized controlled study.SETTING: Department of Senile Angiocardiopathy, General Hospital of Chinese PLA; Department of Biochemistry, Beijing Medical University. MATERIALS: Male Sprague-Dawley (SD) rats with 4 weeks old, clean grade and weighing 45-50 g provided by the Animal Experimental Center, Peking Medical University were used as donators of bone marrow. Other female SD rats of 12 weeks old, clean grade and weighing 200-250 g were used as receptors of cell transplantation and gene therapy. Sry gene of Y chromosome in male rats was used to evaluate whether transplanted cells of donators lived in myocardium of receptor rats. Ad-SERCa2a and Ad-EGFP were constructed by Doctor Lu Xiao-chun; MSC in the 3rd and 8th generations was isolating cultured on its own. METHODS: The experiment was carried out in the Zhou CY Laboratory (BSL-2), Department of Biochemistry, Beijing Medical University from July 2004 to December 2005. Thirty female SD rats received ligation at the left coronary artery to make models with chronic cardiac failure following acute myocardial infarction. And then, 29 rats were randomly divided into four groups, including gene therapy group (n=7), MSC group (n=7), gene-modified MSC group (n=8) and control group (n=7). Rats in the four groups were given the interventions of SERCA2a gene, MSC transplantation, MSC+Ad/SERCa2a and empty adenoviral vector, respectively. MSCs were separated and cultured, and then Ad-SERCA2a-GFP was used to transfer MSC in the 3rd and 8th generations.MAIN OUTCOME MEASURES: Ad-SERCA2a-GFP transfection rate of MSC was measured by using flow cytometer. Before and at 14 and 21 days after treatment, cardiac function was evaluated by ultrasonic echocardiogram. Expression of cytokine Ⅷ was tested by immunohistochemical staining. SERCA2a gene and protein expression were evaluated by RT-PCR and Western blot respectively, as well as SERCA2a enzyme activity. RESULTS: ① Transfection rate: The infection efficiency of adenovirus-medicated gene into different passages of MSC was over 80%, and there was no difference between passage three (P3) MSC and P8 MSC (P > 0.05). ② Heart function: Left ventricle wall was thickened obviously in group MSC and group MSC+Ad/SERCa2a on the 21st day after treatment, while volume was shortened and gradually rounded. Compared to control group, ejection fraction (EF) and shortening fraction (FS) of group Ad-SERCa2a, group MSC and group MSC+Ad/SERCa2a were elevated significantly on the 14th day after therapy (P < 0.01). While the elevation values of EF and FS began to reduce in group Ad-SERCa2a on 14th day after therapy, it continued to increase in both group MSC and group MSC+Ad/SERCa2a (P < 0.01). Improvement rate of EF at 21 days after therapy (EF D21) increased in group MSC and group MSC+Ad/SERCa2a respectively, but decreased in group Ad-SERCa2a. Compared to group Ad-SERCa2a, peak systolic flow velocity of anterior wall and interventricular septum in group MSC+Ad/SERCa2a increased significantly on the 21st day after therapy, and peak diastolic flow velocity of anterior wall and interventricular septum elevated in group MSC+Ad/SERCa2a, too (P < 0.01). ③ SERCA2a gene, protein expression and enzyme activity in group MSC+Ad/SERCa2a were significantly stronger in group MSC and control group. Parts of MSC transplanted into scar zone expressed Ⅷ.CONCLUSION: ① MSC is an effective platform for the targeted delivery of therapeutic gene. It suggests that different passages of MSC from P3 MSC to P8 MSC are regarded as high-effectively gene vehicles. MSC-based SERCA2a gene therapy showed much strong and lasting beneficial effect on exhausted myocardium. ② Effect of MSC transplantation on improving heart function may be related to promoting vascular neogenesis.

Objective To evaluate the efficiency of normal sacral nerve root transposition in repair of the sacral plexus root avulsion. Methods A total of 30 adult SD rats were chosen and divided into three groups,ie,group A(the sciatic nerve received no repair),group B(the autologous sacral plexus root nerve was bridged with the right L6 nerve root by the translocation of the left L6)and group C (the right L5 nerve root nerve was bridged by the translocation of the left L6),10 rats per group.The left side of the rats was used as the control side and the right one as the experimental side.Twelve weeks after operation,the rats in each group were selected for the histomorphological observation of the nerves under the microscope and the electron microscope.The models were evaluated by observing the survival rates of the rats,BBB scores,electron microscope weight and muscle fiber CSA(cross section area)of double biceps femoris,triceps surae and tibial muscle. Results Twelve weeks after operation,the BBB scores in groups B and C was higher than that in group A,with statistical difference(P<0.01)between three groups.A remarkable improvement was found in the ratio of weight and muscle fiber CSA of double biceps femoris,triceps surse and tibial muscle.The repair efficiency in the group C was better than that in the group B.In the group B,the biceps femoris,triceps surae and tibial muscle recovered at different degrees.The biceps femoris recovered the best,when a great deal of myelinated nerve fiber regeneration was observed under the microscope and the electromicroscope.Electromyography revealed the volatility in the muscles of three groups,with larger peak value for the proximal biceps femoris and the triceps muscle but smaller peak value for the distal anterior tibial muscle. Conclusions L6 transposition combined with auto-graft of nerve root or without the auto-graft can reconstruct the partial function of the sciatic nerve in the paraplegia rats,when the latter has the better effect.

AlM: To introduce a new color pediatric visual acuity chart and its clinical application.
METHODS:The color pediatric visual acuity chart was designed based on principle of visual angle. The optotype on the color chart had graphics. The progression rate of optotype size between 2 lines was 10 10 and 1. 2589. A regular geometric progression of optotype sizes and distribution was employed to arrange 8 lines with 11 optotype on the color chart. The testing distance was 3m. The visual acuity score could be recorded as logarithm of the minimum angle of resolution notation or decimal notation. The reliability of naked distant measurements with this new chart was tested in one eye of 100 children (4 ~ 6 years old) taking the Chinese national standard logarithm visual acuity chart standard.
RESULTS: The color pediatric visual acuity chart and logarithmic chart controls, visual acuity test results that in the two groups had no significant difference (t=1. 2671, P> 0. 05 ). Two sets of vision data existed positive correlation (r= 0. 924, P<0. 01). Cooperation rate was 100%, the recognition rate was more than 90%.
CONCLUSlON:Children are easier to accept used new color pediatric visual acuity chart to inspect vision. New chart is reliability and apply to children's vision screening.