乙醇抑制原代大鼠星形细胞神经营养因子的基因表达

为了阐明乙醇中毒对神经系统损害的机理 ,用0 .5- 5.0 g· L-1浓度的乙醇处理原代培养的 CD新生大鼠大脑皮层星形细胞 ,用反转录 PCR和Northern印迹杂交方法同时测定神经营养因子( NGF)的基因表达水平 .结果显示 0 .5g·L-1乙醇在染毒 2 4 h后 ,NGF m RNA水平开始降低 ,5.0 g· L-1在染毒 48或 72 h后 ,NGF m RNA水平与对照组比较降低了 50 % - 58% .结果表明 ,乙醇中毒在体外可降低新生大鼠星形细胞 NGF m RNA的生成 ,这种作用可干扰神经元与胶质细胞间的正常交互作用并可能与胚胎发育期乙醇接触导致的神经病理改变有关 .

Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro

AIM: To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (MSCs) into hepatocytes and to find a new source of celltypes for therapies of hepatic diseases. METHODS: MSCs were isolated by combining gradient density centrifugation with plastic adherence. The cells were cultured in osteogenic or adipogenic differentiation medium and determined by histochemical staining. MSCs were plated in plastic culture flasks that were not coated with components of extracellular matrix (ECM). When MSCs reached 70% confluence, they were cultured in low glucose Dulbecco's modified Eagle's medium supplemented with 10 mL/L fetal bovine serum, 20 ng/mL hepatocyte growth factor (HGF) and 10 ng/mL fibroblast growth factor-4 (FGF-4). The medium was changed every 3 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Glycogen store of hepatocytes was determined by periodic acid-Schiff staining.RESULTS: By combining gradient density centrifugation with plastic adherence, we isolated a homogeneous population of cells from rat bone marrow and differentiated them into osteocytes and adipocytes. When MSCs were cultured withFGF-4 and HGF, approximately 56.6% of cells became smallround and epithelioid on d 24 by morphology. Compared with the control, levels of AFP increased significantly from d 12 to 15.5±1.4 μg/L (t = 2.31, P＜0.05) in MSCs cultured with FGF-4and HGF, and were higher (46.2±1.5 μg/L)ond 21 (t = 41.926, P＜0.01), then decreased to 24.8±2.2 μg/L on d 24 (t = 10.345, P＜0.01). Albumin increased significantly on d 21 (t= 3.325, P＜0.01) to 1.4±0.2 μg/mL,and to 2.1±0.7 μg/mL on d 24 (t= 3.646, P＜0.01). Urea(2.3±0.4 mmol/L) was first detected on d 21 (t = 6.739, P＜0.01), and continued to increase to 2.6±0.9 mmol/Lon d 24 (t= 4.753, P＜0.01). Glycogen storage was first seen on d 21.CONCLUSION: The method combining gradient density centrifugation with plastic adherence can isolate MSCs. Rat MSCs may be differentiated into hepatocytes by FGF-4 and HGF. Cytokines may play a more important role in differentiation from rat MSCs into hepatocytes.

Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.METHODS: Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMARTTM PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder,cDNA xProfiler, Digital GENE Expression Displayer (DGED),and Digital Differential Display (DDD) were used.RESULTS: Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed.CONCLUSION: Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocardnoma.

Matrix metalloproteinases and their expression in mammary gland

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events. The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation, and is controlled by growth factors, cytokines, hormones, as well as interactions with the ECM macromolecules. Furthermore, the activity of the MMPs is regulated by their natural endogenous inhibitors, which are members of the tissue inhibitor of metalloproteinases (TIMP) family. In the normal mammary gland, MMPs are expressed during ductal development, lobulo-alveolar development in pregnancy and involution after lactation. Under pathological conditions, such as tumorigenesis, the dysregulated expression of MMPs play a role in tumor initiation, progression and malignant conversion as well as facilitating invasion and motastasis of malignant cells through degradation of the ECM and basement membranes. Matrix metalloproteinases and their expression in mammary gland

Expression of gamma-aminobutyric acid A receptor subunits aα_1,β_1,γ_2 mRNA in rats with hepatic encephalopathy

AIM To investigate the mRNA expression of gammaaminobutyric acid A (GABAA) receptor subunits α1,β1, γ2in different parts of the brain of rats with hepaticencephalopathy.METHODS: Twelve adult male Sprague-Dawley rats were randomly divided into two groups: (1) hepatic encephalopathy model group (n = 6), which was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg/kg) for threeconsecutive days; (2) control group (n = 6), in which the rats were treated with same dose of normal saline solution. After the freeze slice of cerebrum was made,in situ hybridization was used to detect the mRNA of GABAA receptor subunits α1, β1, and γ2 in rat cerebral cortex, basal nuclei, substantia nigra and hippocampi. Image data were collected and analyzed quantitatively by QWin550CWmodel image signal gather and analysis system. RESULTS: In rats with hepatic encephalopathy, mRNA expression levels of GABAA receptor subunits α1, β1 increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis and hippocampi (144.7±15.67/184.14±4.41, 60.61±33.66/113.07±32.44,87.71± 21.25/128.40±18.85, 122.34±5.56/161.60±4.56,123.29±5.21/140.65±4.15, 123.40±4.42/140.09±4.52,124.76±4.18/140.09±4.12, 141.62±15.09/182.80 ±5.20,69.13±30.74/134.21±43.76, 87.87±25.16/151.01±19.49,122.14±6.30/162.33±3.92, 122.81±5.09/137.19±7.12,123.00±4.63/138.11±5.92, 125.75 ±2.43/138.81±6.10,P＜0.01), but did not change in the cerebral cortex compared to the control group. Similar changes were found in the mRNA expression levels of GABAA receptor subunit γ2,which increased significantly in basal nuclei, substantia nigra pars compacta, substantia nigra pars reticularis (136.81±26.41/167.97±16.23, 51.00±36.14/113.18±36.52, 86.35±20.30/126.90±19.74, P＜0.01), CA1 of hippocampal (162.15±9.05/178.62±6.45, P＜0.05), and no changes were found in the cerebral cortex and CA2, CA3, CA4 of hippocampi.CONCLUSION: In rats with hepatic encephalopathy, mRNA expression levels of GABAA receptor subunits α1,β1, γ2 increase significantly in basal nuclei, substantia nigra and hippocampi, suggesting that the changes of mRNA expression levels in GABAA receptor subunits may contribute to the pathogenesis of hepatic encephalopathy.

Effect of peripheral nerve on the neurite growth from retinal explants in culture

The effect of peripheral nerve (PN) on neurite outgrowth from retinal explants of adult hamsters was examined. Cultures of retinal explants, and co-cultures of retinal explants and PN were performed using chick retinal basement membrane (BM) as substrate. The presence of PN increases the number and length of neurite outgrowth. In addition, a high proportion of neurites situated close to PN tend to grow towards it. Since there was no contact between retinal ex-plants and PN, we suggest that PN might secrete diffusible substances to attract the neurites to grow towards it.

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified oytochrome oxidase and horseradish peroxidase histochemieal methods. The results show that a population of large retinal ganglion cells was consistently observed with the eyto3hrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birrth. These oytochrome oxidase rich ganglion cells appeared to have large somata, 3-6 primary dendrites and extensive dendritic arbors, and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). However, the morphological details of some of the cells revealed by the cytoahrome oxidase staining method are frequently better than those shown by the HRP histochemieal method. These results suggest that the mit03hondrial enzyme oytoohrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal ganglion cells with high metabolie rate in the rat.

Ultracytochemical localization of H+-adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

H+-adenosine triphosphatase (H+-ATPase) activity was demonstrated oytoohemioally in autophagio vaouoles (AVs) of rat hepatooytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase (p-NPPase) activity [1]. When an inhibitor of H+-ATPase, N-ethylmaleimide (NEM) or 4,4'-diisothiooyanostilbene-2,2'disalfonio aold, di-sodium salt (BIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H+-ATPase. Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate (VBL). The number of AVs increased remarkably in hepatooytes from 40 min after VBL treatment. H+-ATPase activity was observed mainly on the membranes of lysosomes and AVs. However, early forms of AVs containing only incompletely digested material showed no H+-ATPase activity. Most AVs revealing a positive reaction seemed to be in advanced stages of development. Acid phosphatase aotioity was demonstrable in mature but not in early forms of AVs. The present investigation showed that membranes of advanced stage A Vs possess an H+-ATPase which may be derived from lysosomal membranes.

Opisthorchiasis-associated biliary stones:Light and scanning electron microscopic study

AIM:Biliary stones are frequently encountered in areas endemic for opisthorchiasis in Thailand.The present study was to describe the prevalence and pathogenesis of these stones. METHODS:Gallstones and/or common bile duct stones and bile specimens from 113 consecutive cholecystectomies were included.Bile samples,including sludge and/or microcalculi,were examined for Opisthorchis viverrinieggs, calcium and bilirubin.The stones were also processed for scanning electron microscopic(SEM)study. RESULTS:Of the 113 cases,82 had pigment stones,while one had cholesterol stones.The other 30 cases had no stones.Most of the stone cases(76%,63/83)had multiple stones,while the remainder had a single stone.Stones were more frequently observed in females.Bile examination was positive for O.viverrini eggs in 50% of the cases studied.Aggregates of calcium bilirubinate precipitates were observed in all cases with sludge.Deposition of calcium bilirubinate on the eggshell was visualized by special staining.A SEM study demonstrated the presence of the parasite eggs in the stones.Numerous crystals, morphologically consistent with calcium derivatives and cholesterol precipitates,were seen. CONCLUSION:Northeast Thailand has a high prevalence of pigment stones,as observed at the cholecystectomy,and liver fluke infestation seems involved in the pathogenesis of stone formation.

Expression changes in tau and microtubule-associated proteins in rat testicular interstitium

Objective: To examine the expression of the tau protein and mi-crotubule-associated proteins (MAP) in the testicular interstitium of aged and young rats. Methods: Sprague-Dawley male rats were divided into a young group (6 months) and an aged group (28 months) of 10 animals each. The two-step immunohistochemistry method with the antibody against tau protein and MAPa was performed with the testis tissues. Results: The immunoreactive cells of the testicular interstilial tau protein were significantly increased (P<0.01) and those of the MAP significantly decreased (P<0.01) in the aged than in the young rats. Conclusion: The changes in the expression of the tau protein and MAP may be related to the aging process of the testis.

Studies on the role of microtubules in myofibrillogenesis

Co-localization of mierotubule (MT) and muscle myosin (MHO) myofibril immunofluoresoence in developing myotubes of chicken' skeletal muscle cultures was observed by using double staining of tubulin and MHO indirect immunofluorescence. 12-o-tetradeeanoyl-phorbol-13-acetate (TPA) selectively and reversibly blocks mycfibrillogenesis and alters the morphology of myotubes into myosacs where MTs are present in radiating pattern,when the arrested myogenic cells recover and start myofibrillogenesis after released from TPA, prior to the emergence of myofibrils, the pre-existing MTs become bipolarly aligned ooincidently with the tubular restoration of cell shape. Single nascent myofibrils overlapping with MTs extend into the base of growth tips where MTs go farther to the end of the tips. That MT might act as scaffold in guiding the bipolar elongation of the growing myofibrils was suggested. Taxol and ooloemid disturbed MT polymerization and disposition, and interfered with the normal spatial assembly of myofibrils in developing myotubes.

Substance P－and Choline Acetyltransferase－like Immunoreactivity in the Motoneuron Areas of the Cat Ventral Spinal Cord

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Transforming Growth Factor-β_2 Gene Cloning and Protein Expression in Human Trabecular Meshwork Cells

Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.