Laser scanning confocal microscopy is a strong suppoR tool in the study of morphons,fluorescence positioning,quantitative analysis and function.It has been widely used in biomedical research in various fields.This paper summarizes the domestic research applications of laser scanning confocal microscope in glioma.

To observe the growth, expansion and differentiation of the cultured bone marrow stroma cell (BMSC) from Macaca Lrus, BMSC isolated from adult Macaca Lrus were cultured with the culture medium confected by ourselves and were induced with some cytokines such as LIF and bFGF. The results showed that the BMSC could proliferate and generate Nestin positive clones when they were cultured in vitro. After subculture, these cells could grow rapidly and differentiated into neuron like cells and astrocyte like cells further, which expressed GFAP or NSE antigen respectively. Therefore, these BMSC possess renewal and differentiation abilities. On the other hand, the culture method we used in this experiment is suitable for culture of BMSC in vitro. The BMSC might be used as the seed cells of the neural stem cells.

Objective To study prognosis and grading of somatosensory evoked potential(SEP)in long-term unconscious patients after severe traumatic brain injury(TBI).Methods Five prognostic factors including age,sex,injury mechanism,history of temporal craniotomy and SEP grading were selected and analyzed in 47 patients after severe TBI with a duration of unconsciousness longer than two weeks.The prognosis was judged by Glasgow Outcome Scale.Results Prognosis was closely associated with SEP grading(P=0.024).The accuracy of SEP in assessing the prognosis was 91.5%.About 95%-100% of patients with SEP at grade Ⅲ-Ⅲ ended up with severe disability,persistent vegetative state or death.However,43.75% of patients with SEP at grade Ⅰ had good prognoses.Conclusions The SEP grading can objectively and accurately evaluate patients' prognosis and demonstrate the brain function.

BACKGROUND: Researches indicated that bone marrow stem cells (BMSCs) could differentiated into neural stem cells in vitro, but what was the role of neural stem cells(NSCs) in the recovery of cortical injury,whether the NSC is capable of growing and migration in injured still remained unknown.OBJECTIVE: To explore the growing state of autograft NSC derived from crab-eating macaque BMSC transplanted in brain.DESIGN: Prospective case control study based on experimental animals.SETTING: Department of neurosurgery in a hospital of a military medical university.MATERIALS: This study was carried out at Center Laboratory of Neurological Research Institute, Zhujiang Hospital affiliated to the First Military Medical University of Chinese PLA. Six healthy adult crab-eating macaques were purchased from the South China Primate Animal Center.INTERVENTIONS: BMSCs harvested from six crab-eating macaques were cultured in vitro and induced to differentiate into neural stem cells, which then labeled by bromodeoxyuridine(BrdU) and autografted into brains.MAIN OUTCOME MEASURES: Brain tissues underwent hematoxylin and eosin(HE) staining and immunohistochemical staining before observed under optical microscope.RESULTS: The results of HE staining showed that the cell number in injured brain vas obviously higher in both instant and delayed transplanting groups than sham-transplanting group; moreover cells were proved reacting to BrdU by immunohistochemical staining in cortical injuries of both groups at 1-6 months following stem cells autograft, as well as at neighboring white matters at half year later, but no BrdU positive cells could be found in traumatic controls, sham-transplanting group and normal brains.CONCLUSION: NSCs derived from in vitro cultured BMSCs were proved capable of surviving, proliferating, differentiating and migrating in cortex after autograft, so that BMSCs is considered as replacing cells or the source of NSCs; moreover autograft stem cells could survive, proliferate and migrate in old cortical traumatic focus.

Objective To explore the effect of fibroblast growth factor receptor(FGFR)inhibitor BGJ398 on the vasculogenic mimicry(VM)formation of glioma cells. Methods The phosphor-FGFR(pFGFR)was de-tected by Western blot,the expressions of MMP2 and MMP14 were detected by Western blot and immunocytochem-istry;the VM formation of U87MG and U251MG was tested by tube formation assay;subcutaneously implanted tu-mor model in nude mouse was established and tumor sections were CD34/PAS double-stained to detect the forma-tion of VM in vivo. Results Western blot showed that pFGFR in the experimental groups decreased significantly (P < 0.05);western blot and immunohistochemical staining showed that the expression of MMP2 and MMP14 in the experimental groups decreased significantly compared to the control group. In the tube formation assay ,the tube formation of U87MG and U251MG cells were restrained. In the subcutaneously implanted tumor model ,the VM number of the experimental group(13.85 ± 3.96)was significantly lower than that in the control group(26.40 ± 5.06,P < 0.05). Conclusions In vivo and in vitro experiments confirmed that BGJ398 can inhibit the activa-tion of FGFR,and inhibit the VM formation of glioma cells. These indicate FGFR signaling pathway is involved in the formation of VM.

BACKGROUND: Dopamine is closely associated with occurrence of epilepsy and transmission in central nerval system, and its various functions are determined by specific receptors.OBJECTIVE: To establish temporal epilepsy model so as to probe into the influences of SCH23390, the antagonist of dopamine D1 receptors and haloperidol, the antagonist of dopamine D2 receptors injected in substantia nigra on temporal epileptic seizure induced by kainic acid and on electroencephalic activityDESIGN: Randomized controlled verified experiment.SETTING: Neurology Medicine Institute of Zhujiang Hospital Affiliated to Southern Medical University.MATERIALS: The experiment was performed in General Military Neurology Medicine Institute of Zhujiang Hospital Affiliated to First Military University of Chinese PLA from August to December 2004, in which, 30SD adult male rats were employed, massed varied from 250 to 300 g.METHODS: ① 30 rats were randomized into physiological saline (control) group (6 rats), kainic acid (KA) group (6 rats) and experimental group (18 rats). The experimental group was divided into 3 subgroups, named the antagonist of dopamine D1 receptors, SCH23390 + kainic acid group (D1 +KA group), the antagonist of dopamine D2 receptors,haloperidol + kainic acid group (D2+KA group) and physiological saline + kainic acid group (PS + KA group), 6 rats in each. In the control, physi ological saline 2 μL was injected in the right cerebral ventricle unilaterally. In KA group, kainic acid 2 μL was injected in the right ventricle. In each of experimental group, SCH23390, the antagonist of dopamine D1 re ceptors, haloperidol, the antagonist of dopamine D2 receptors and physio logical saline 1 μL for each was injected in substantia nigra on the right side successively and simultaneously, kainic acid 2 μL was injected in the right ventricle. ② Observed items: alters of EEG on the 0.5th 1st, 2nd, 6th and 24th hours after medication in each experimental group (compared with EEG of non-epileptic behavior, appearance of sharp wave, spike wave,sharp (spike) slow comprehensive wave and multi-spike slow wave determines epileptic activity) and changes in animal behaviors (0 grade: normal; Ⅰ grade: wet dog-like trembling, paroxysmal facial spasm, like winking,beard moving, rhythmic chawing; Ⅱ grade: rhythmic nodding; Ⅲ grade:paroxysmal spasm of anterior limbs; Ⅳ grade: paroxysmal spasm of bilateral anterior limbs when standing; Ⅴ grade: falling down, loss of balance and convulsion of four limbs). Cerebral hippocampal neural cell apoptosis was observed and the rats were sacrificed on the 5' day of medication. Cerebral hippocampal section was prepared and determined after in situ end labeling staining.MAIN OUTCOME MEAUSRES: ① Changes in behavior in rats before and after epilepsy and electroencephalogram (EEG) alters. ② Results of cerebra hippocampal neural cell apoptosis.RESULTS: Thirty rats entered result analysis. ① Epilepsy seizure: In the control group, there was no epilepsy attacked. In KA group, all of rats ap pear seizure, which attacked 10 minutes after KA injected in brain ventricle, reached the peak in 1 hour and stopped in 3 to 6 hours. ② EEG record: In the control group, there was not epileptic activity manifestations,like sharp wave, spike wave, spike slow comprehensive wave, etc. In KA group, epileptic wave presented in 10 minutes after injection, the seizure developed to the peak in about 1 hour, the wave amplitude was decreased in 3 to 6 hours, presenting paroxysmal slow and spike slow waves and no epileptic wave appeared after 12 hours. ③ Neuronal apoptosis: In the control group, few neural cell apoptosis was visible in hippocampus after injection.In KA group, neural cell apoptosis was visible obviously in hippocampus in 5 days after injection (P =0.00). With SCH23390, the antagonist of dopamine D1 receptors, hippocampal cell apoptosis was not reduced remarkably (P ＞0.05) and with haloperidol, the antagonist of dopamine D2 receptors injected in substantia nigra, hippocampal cell apoptosis was aggravated (P =0.00).CONCLUSION: Injection of SCH23390, the antagonist of dopamine D1 receptors in substantia nigra cannot block kainic acid inducing epilepsy and epileptic electroencephalic activity is not weakened remarkably. Injection of haloperidol,the antagonist of dopamine D2 receptors enhances epileptic electroencephalic activity in kainic acid induced epilepsy and increases cell apoptosis remarkably in cerebral hippocampal CA3 area.It is to explain that it is dopamine D2 acceptor that is involved in regulation of temporal epilepsy in substantia nigra rather than D1 acceptor.

BACKGROUND: Bone marrow mesenchymal cells, multiple-potential non-hematopoiefic stem cells adhering to the wall in vitro culture, can be induced to proliferate and differentiate towards neurons and glia cells.OBJECTIVE: To investigate the growth state of cat bone marrow mesenchymal cells in vitro culture, as well as the capability to differentiate towards neural stem cells.DESIGN: A randomized sampling study.SETTING: Department of Neurosurgery, Zhujiang Hospital, Southern Medical University.MATERIALS: This study was carried out at the Central Laboratory of the General Military Neurological Research Institute, Zhujiang Hospital, Southern Medical University between January and December 2002. Twenty healthy home-raised cats, aged 1.0 - 2.0 years and weighing 2. 5 - 4.0 kg, male and female in half, were provided by the Animal Center of the First Military Medical University of Chinese PLA.INTERVENTIONS: Bone marrows were randomly aspirated from the left or right hindlimbs in order to separate bone marrow mesenchymal cells, then the bone marrow mesenchymal cells single cell suspension was co-cultured with neural stem cell culture media in vitro so as to induce differentiation to neural stem cells with tretinoin. CK2 type inverted optical microscope(Olympus,Japan) was used to observe the growth of bone marrow mesenchymal cells in vitro culture, as well as 4, 12, 24, 48 hours of induction upon eliminating or not eliminating the wall-adhering cells. Bone marrow mesenchymal cells in stem cell stage were identified under Olympus optical microscope with modified immunohistochemical staining.MAIN OUTCOME MEASURES: The growth state and the immunocytochemical staining of living bone marrow mesenchymal cells exposed to experimental intervention were observed under the Olympus inverted optical microscope.RESULTS: Data from the 20 cats were analyzed without loss. Reversed microscopic observation revealed that cat bone marrow mesenchymal cells becrame larger when cultured in vitro, which were rich in plasmic granules with prominence projecting, adhering to the wall and forming cell clones. These cells were then successively cultured, and imnunohistochemical staining analysis suggested that the passaged bone marrow mesenchymal cells could express neural stem cells-specific antigen Nestin and differentiate towards glia-like cells and neuron-like cells.CONCLUSION: Cat bone marrow mesenchymal cells possess the characteristics of stem cells; they can be amplified into cell clones and induced to express the property of neural glia cells and neuron-like cells under proper condition.

Objective To optimize the preparation of nanoparticles encapsulating antisense oligodeoxynucleotides in a-butyleyanoacrylate carrier (ASODN in NP) and investigate their stability. Methods ASODN in NP were prepared by interfacial polymerization of butyleyanoacrylate (BCA). The formulation and technology of the prepared NP was optimized by using orthogonal design based on the single-factor experiment. The morphology of NP was examined by transmission electron microscope; The size and size distribution of NP were determined by Malvern laser granularity equipment;The encapsulation efficiency and drug loading were determined by HPLC; The ability of protecting oligodeoxynucleotides from serum was investigated on a 20% polyacrylamide-7 Murea sequencing gel (PAGE). Results The nanoparticles in the optimal conditions were of regular spherical surface and discrete. The average size was 97.1 nm,the average encapsulation efficiency and drug loading of ASODN in NP were 96.7% and 10.1% respectively; The oligonucleotides were more efficiently protected from degradation by nucleases than by oligonucleotides adsorbed into nanospheres.Conclusion ASODN in NP has good stability,encapsulation efficiency,drug loading and great potential for ASODN delivery.

Objective To construct a new recombinant immunotoxin expression vector by using human VEGF165 and a truncated pseudomonas exotoxin A ramification (PE38) gene, and explore the expression of the VEGF165-PE38 fusion protein in HEK293 cells. Methods VEGF165 was cloned by polymerase chain reaction (PCR). PE38 gene was gained from an vector plasmid pRB391 by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pIRES2-EGFP. After the eukaryotic recombinant vector pIRES2-VEGF165-PE38-EGFP was identified by restriction endonuclease digestion and sequence analysis, the vector was transfected into HEK293 cells by liposome protocol. RT-PCR and ELISA method was used to confirm the expression of the fusion gene in the HEK293 cells. Results Restriction endonuclease digestion and sequence analysis revealed the VEGF165-PE38 fusion gene was cloned into the eukaryotic expression plasmid vector pIRES2-EGFP successfully. The pIRES2-VEGF165-PE38-EGFP fusion gene could express in the HEK293 cells. Conclusion The result provide the basis for search of the targeted cytotoxic activity to tumor vascular endothelial cells and may have some potential value in clinical application.

In order to observe the growth, expansion and differentiation of the cultured bone marrow stromal cells (BMSC), we isolated the BMSC from adult SD rats and cultivated them with LIF and bFGF. Then, we cultured and induced the stem cells by using retinoic acid and the culture medium confected in our lab by ourselves. We found that the BMSC could expand and generate clones when they were cultured in vitro. These cells subcultured grew rapidly and differentiated into neuron-like cells and astrocyte-like cells. The results showed that BMSC have the abilities to self renew and differentiate, thus demonstrating the culture method we used is suitable for the culture of BMSC in vitro. The bone marrow stromal cell is not difficult to obtain; it is capable of expanding and differentiating in culture. If the culture condition is appropriate, it can differentiate into neuron and astrocyte. So, it is a kind of perfect seed cells.
Actihaemyl ; pharmacology ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fibroblast Growth Factor 2 ; pharmacology ; Neurons ; cytology ; Pilot Projects ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; Stromal Cells ; cytology ; drug effects ; Tretinoin ; pharmacology