Leucine Supplementation in a Chronically Protein-Restricted Diet Enhances Muscle Weight and Postprandial Protein Synthesis of Skeletal Muscle by Promoting the mTOR Pathway in Adult Rats

Low protein intake causes a decrease in protein deposition in most animal tissues. The purpose of this study was to investigate whether leucine supplementation would increase the synthesis rate of protein and muscle weight in adult rats, which chronically consume only 58.8% of their protein requirements. Thirty-six male Sprague-Dawley rats were assigned to one of three dietary treatments including a 20% casein diet （CON）, a 10% casein ＋ 0.44% alanine diet （R）, and a 10% casein ＋ 0.87% leucine diet （RL）. After a 10 d dietary treatment, plasma amino acid levels were measured after feeding, the gastrocnemius muscles and soleus muscles were harvested and weighed, and the fractional synthesis rate （FSR） and mammalian target of rapamycin （mTOR） signaling proteins in skeletal muscle were measured. Regarding the plasma amino acid level, the RL group had the highest concentration of leucine （P 〈 0.05） and the lowest concentration of isoleucine （P 〈 0.05） among the three groups, and the CON group had a lower concentration of valine （P 〈 0.05） than the R and RL groups. Compared with the R and RE groups, the CON group diet significantly increased （P 〈 0.05） feed intake, protein synthesis rate, and the phosphorylation of eukaryutic initiation factor 4E binding protein 1 （4E-BP1）, and decreased the weight of abdominal adipose. Compared with the R group, the RL group significantly increased in gastrocnemius muscle weight, protein synthesis rate, and phosphorylation of both ribosomal protein $6 kinase 1 （56K1） and 4E-BP1. In conclusion, when protein is chronically restricted in adult rat diets, leucine supplementation moderately improves body weight gain and increases muscle protein synthesis through mTOR activation,

A comprehensive study of long-term skeletal changes after spinal cord injury in adult rats

Spinal cord injury（SCI）-induced bone loss represents the most severe osteoporosis with no effective treatment.Past animal studies have focused primarily on long bones at the acute stage using adolescent rodents. To mimic chronic SCI in human patients, we performed a comprehensive analysis of long-term structural and mechanical changes in axial and appendicular bones in adult rats after SCI. In this experiment, 4-month-old Fischer 344 male rats received a clinically relevant T13 contusion injury. Sixteen weeks later, sublesional femurs, tibiae,and L4 vertebrae, supralesional humeri, and blood were collected from these rats and additional non-surgery rats for micro-computed tomography（m CT）, micro-finite element, histology, and serum biochemical analyses.At trabecular sites, extreme losses of bone structure and mechanical competence were detected in the metaphysis of sublesional long bones after SCI, while the subchondral part of the same bones showed much milder damage. Marked reductions in bone mass and strength were also observed in sublesional L4 vertebrae but not in supralesional humeri. At cortical sites, SCI induced structural and strength damage in both sub- and supralesional long bones. These changes were accompanied by diminished osteoblast number and activity and increased osteoclast number and activity. Taken together, our study revealed site-specific effects of SCI on bone and demonstrated sustained inhibition of bone formation and elevation of bone resorption at the chronic stage of SCI.

Application of Luxol Fast Blue staining in locating the corticospinal tract in adult rats

BACKGROUND: There are many methods for myelin staining, mordant, or oil-soluble dye or the special reaction of osmic acid with lipoid is used according to different principles. The commonly used methods are classic Well staining, classic lithium carbonate-haematine staining, fast green staining, silver staining, etc. Luxol Fast Blue can brightly stain myelin sheath, and has certain specificity. The background can be very clean if there is proper differentiation, whereas Luxol Fast Blue is cheap and convenient to operate, thus it is an ideal staining reagent for routine myelin sheath. OBJECTIVE: To show the corticospinal tract of normal adult rats with Luxol Fast Blue staining method. DESIGN: A repetitive measurement design. SETTINGS: Institute of Nuerobiology, Nantong University; Department of Rehabilitation Medicine, Affiliated Hospital of Nantong University. MATERIALS: Six healthy adult male SD rats of clean degree, weighing averagely 300 g, were provided by the experimental animal center of Nantong University. 1 g/L Luxol Fast Blue solution was provided by Sigma Company; Leica CM1900 cryostat microtome by Leica Company; Leica DMR microscope by Leica Company. METHODS: The experiment was carried out in the Staff Room of Human Anatomy, Nantong University in May 2005. The rats were given intraperitoneal injection of combined anesthetic (2 mL/kg), then the chest was open for perfusing saline and phosphate buffer containing formamint via heart. Brain and spinal cord were removed after 1 hour then fixed, then changed to phosphate buffer (pH 7.4) containing 300 g/L saccharu at 4 ℃, and stayed overnight, tissue blocks at pyramid, decussation of pyramid and cervical, thoracic, lumbar and sacral segments of spinal cord were removed to prepare continuous horizontal frozen sections (30 μm) after sedimentation, the sections were dried at room temperature. The corticospinal tract of normal adult rats were shown with Luxol Fast Blue staining method, and observed under Leica DMR microscope. MAIN OUTCOME MEASURES: Positive fibers in Luxol Fast Blue staining. RESULTS: After the Luxol Fast Blue staining, the labeled myelinated nerve fibers were bright blue. They located in the pyramid, decussation of pyramid and the ventral part of posterior funiculus in cervical, thoracic, lumbar and sacral segments of spinal cord. CONCLUSION: Luxol Fast Blue staining method may manifest the distribution of corticospinal tract with clear distinct in adult rats.

Synergistic effects of brain-derived neurotrophic factor and retinoic acid on inducing the differentiation of bone marrow stromal cells into neuron-like cells in adult rats in vitro

BACKGROUND： Under induction of retinoic acid （RA）, bone marrow stromal cells （BMSCs） can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor （BDNF） can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE： To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN： Randomized controlled animal study SETTING： Department of Neurology, Affiliated Hospital of Xuzhou Medical College MATERIALS： The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification： SYXK （su） 2002-0038]. Materials and reagents： low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein （GFAP） antibody, SABC kit, and diaminobenzidine （DAB） color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS： Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group （primary culture）, BDNF group （20 μg/L BDNF） and BDNF＋RA group （20 μg/L BDNF plus 20 μg/L RA）. On the 3^rd and the 7^th days after induction, BMSCs were stained immunocytochemically with nestin （sign of nerve stem cells）, neuron-specific enolase （NSE, sign of diagnosing neurons） and GFAP （diagnosing astrocyte）, and evaluated cellular property. MAIN OUTCOME MEASURES ： Induction and differentiation in vitro of BMSCs in 3 groups RESULTS： （1） Induction and differentiation of BMSCs： Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. （2） Results of immunocytochemical detection： Three days after induction, rate of positive cells in BDNF＋RA group was higher than that in BDNF group and control group [（86.15±4.58）%, （65.43±4.23）%, （4.18±1.09）%, P 〈 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF＋RA group than that in both groups at 3 days after induction [（31.12±3.18）%, （29.35±2.69）%, P 〈 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction （P 〈 0.01）; meanwhile, the amount in BDNF＋RA group was remarkably higher than that in BDNF group （P 〈 0.01）. CONCLUSION： Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.

Allograft of developing metanephroi into omenta of cyclosporine-treated adult rats after preservation

To evaluate the animal models and the effect on organogenesis and function of embryonic metanephroi allografted into cyclosporine-treated aduld rats after their preservation in histidine-tryptophan-ketoglutarate (HTK) solution.Methods Whole metanephroi preserved in HTK for 3 days from embryonic day 16 and 17 (E16,E17) of Sprague-Dawley (SD) rats were grouped and allografted into the omenta of cyclosporine-treated SD adult rats which had one kidney resected.E16 metanephroi implanted directly into omenta were used as controls.3 to 4 weeks after implantation,metanephroi allografted in host rats were removed for histopathological and electronic microscopy (EM) examination or anastomosed for renal function measurement 8 weeks later.Results 3 to 4 weeks post-implantation,E16 and E17 metanephroi had formed mature nephrons and collecting ducts with few lympocytic infiltration.EM examination showed they had cellular ultrastructure of normal nephrons and collecting ducts.E16 and E17 metanephroi allografted after preservation for 3 days had no significantly different values of wet weight (P=0.346,P=0.705),urine volume (P=0.396,P=0.687),and creatinine clearances (P=0.469,P=0.543) from those of E16 metanephroi implanted directly.Conclusion E16 and E17 metanephroi allografted into cyclosporine-treated adult rats undergo growth and differentiation and exhibit excretory function after preservation in HTK solution for 3 days.9 refs,1 tabs.

Beta-amyloid precursor protein cleavage enzyme-1 expression in adult rat retinal neurons in the early period after lead exposure

Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 （BACE-1） in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer＇s disease.Adult rats were intraocularly injected with different doses of lead acetate （10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L）.The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer＇s disease.

EXPERIMENTAL STUDY IN DIFFERENT EXPRESSION AND ORGANIZATION OF COLLAGEN IN SKIN WOUNDS OF ADULT AND FETAL RATS

Objective To observe the spatial and temporal distribution of collagen in fetal and adult rats wounds. Methods The organization of collagen deposition in fetal and adult rats skin wounds were observed by using van Gieson stain. The methods of immunohistochemistry and in situ hybridization were applied to examine collagen type Ⅰ and Ⅲ peptide and mRNA localization at serial time point during wound healing. Results Collagen type Ⅰ and Ⅲ were present in wounds of both fetal and adult rats, but the timing and pattern of collagen deposition varied. In the fetus, collagen wes detected by 48h postwounding (PW), but uns not present in the adult wounds until 5d PW. N in situ hybridization, signals in the area of the fetal wound were clearly greater and with increased number of cells as compered to that in the adjacent unwounded tissue. Adult rat wounds had evidence in increased signals of procollagen type Ⅰ and Ⅲ production by wound fibroblasts on day 5. Collagen deposited and wes arranged in reticular pattern as that of the nounal in fetal wounds. While in the adult wound, collagen deposited in the fashion of course bundles. bundles Conclusion Fetal rat wounds appeared to produce collagen mainly by an increased number offibroblasts in the area of the wound. In contrast, adult rat wounds underwent fibroblast migration and induction of procollagen mRNA synthesis. Our results Suggest that the deposition of collagen type Ⅰ and Ⅲ is regulated by their gene expression. Chllagen type Ⅲ plays an important role in the arrangement of collagen depoition.

Migration and differentiation of bone marrow-derived multipotent adult progenitor cells through tail vein injection in a rat model of cerebral ischemia

BACKGROUND： Multipotent adult progenitor cells （MAPCs） from the bone marrow have been shown to differentiate into neurons. OBJECTIVE： To observe migration, survival, and neuronal-like differentiation of MAPCs by tail vein injection. DESIGN, TIME AND SETTING： Randomized, controlled experiment of neural tissue engineering was performed at the Laboratory for Cardio-Cerebrovascular Disease, Hospital of Integrated Traditional and Western Medicine, Tongji Medical College of Huazhong University of Science and Technology between September 2006 and August 2007. MATERIALS： Eighty Sprague Dawley rats, 3-6 months old, underwent cerebral ischemia/reperfusion by thread technique, and were randomly divided into model and MAPCs groups （n = 40）. METHODS： Mononuclear cells were harvested from bone marrow using the FicolI-Paque density gradient centrifugation method. After removing CD45 and glycophorin A-positive cells （GLYA＋） with immunomagnetic beads, CD45 GLYA adult progenitor cells were labeled with bromodeoxyuridine （5-bromo-2-deoxyuridine, BrdU）. A total of 1 mL cell suspension, containing 5 × 10^6 MAPCs, was injected into the MAPCs group through the tail vein. A total of 1 mL normal saline was injected into the model rats. MAIN OUTCOME MEASURES： After 60 days, BrdU and neuron-specific enolase double-positive cells were observed using immunofluorescence. Cell morphology was observed under electron microscopy, and nerve growth factor mRNA was measured through RT-PCR. In addition, rat neurological functions were measured with behavioral tests. RESULTS： Immunofluorescence revealed that MAPCs positive for BrdU and neuron specific enolase were found surrounding the ischemic focus in the MAPCs group. Microscopic observation suggested that MAPCs-derived neuronal-like cells connected with other nerve cells to form synapses. Compared with the model animals, the level of nerve growth factor mRNA was significantly upregulated in rats injected with MAPCs （P 〈 0.05）. In addition, rats in the MAPCs group performed better in behavioral tests than the model group on days 28 and 60 （P 〈 0.05）. CONCLUSION： Transplanted MAPCs migrated to the ischemic region, survived, and differentiated into neuronal-like cells, resulting in stimulation of nerve growth factor mRNA and improved neurological function in ischemic rats.

Chronic Administration of Sildenafil Citrate (Viagra) on the Frontal Cortex of Adult Male Rats: An Ultrastructural Study

Objectives: Sildenafil citrate (Viagra) is widely used as an aphrodisiac drug and for the treatment of erectile dysfunction. The present study was planned to study the morphological changes that might occur in the frontal cortex of the adult rat in response to chronic intake of Viagra and discover whether these changes are reversible or irreversible. Material and Methods: Forty adult male rats were used where they were classified into three groups: A control group (10 rats), B experimental treated group (20 rats), and C recovery group (10 rats). The treated and recovery groups received therapeutic dose of Viagra for 30 days. The control and the treated group were sacrificed at the first day after the designated period (30 days), the recovery group was sacrificed two weeks after the end of experiment. Results: The pyramidal cell neurons are little in number;some of them are atrophic, degenerated. Their cytoplasm showed varied degree of cellular degenerative changes with vaculation of their myelinated axons. Pyramidal neurons of recovery animals showed nearly complete recovery. Conclusion: Chronic intake of Viagra produced reversible morphological changes in pyramidal neurons of the frontal cortex of adult male rats.

Effects of intra-gastric beta-casomorphin-7 on somatostatin and gastrin gene expression in rat gastric mucosa

AIM： To investigate the in vivo effect of beta-casomorphin-7on the regulation of gastric somatostatin and gastrin messenger RNA in rat gastric mucosa.METHODS： Somatostatin and gastrin mRNA were quantified by RT-PCR and in situ hybridization （ISH）in 24 rats. The rats were divided into three treatment groups： basal diet ＋ physiological saline （n = 8）, basal diet ＋ beta-casomorphin-7 （7.5 × 10^-7 mol） （n = 8）,and basal diet ＋ poly-Gly-7 （containing equal mol of N with 7.5 × 10^-7 mol beta-casomorphin-7） （n = 8）.After oral administration for 30 days, rats were killed by exsanguinations.RESULTS： After intra-gastric administration of betacasomorphin-7 for 30 d, gastrin mRNA increased by 52.8% （P 〈 0.05, n = 8）, and somatostatin mRNA levels decreased by 30.7% compared with the controls （P 〈0.01, n = 8）. No significant differences in the expression of the two genes were observed in the poly-Gly-treated group, although gastrin mRNA expression was elevated by 35.6% as against the control group （P = 0.15, n =8）. The long-term oral administration of a casomorphin solution significantly decreased the even gray of D-cells,but did not lower the number of D-cells both in the antrum and fundus. Interestingly, the number of G-cells increased in the antrum and fundus, but its average density was augmented only in the antrum.CONCLUSION： Beta-casomorphin-7 is capable of modulating gene expression of the regulatory peptides from G and D cells. Data from in situ hybridization studies indicate that beta-casomorphin-7 affects gastrin gene expression indirectly by means of the paracrine action of somatostatin, and depends on its intrinsic molecular function.

Effects of NGF and TrkA on GAP-43^+ nerve regeneration in rat autotransplanted splenic tissue

Objective:To study the time-course of the regeneration of GAP-43+ nerve, and the effects of NGF and TrkA on this process. Methods: Adult Wistar rats underwent splenectomy and splenic autotransplantation, or sham-operation. On day 7, 14, 30, 60, 90, 120, and 180 after surgery, the density of GAP-43+ nerve fibers in spleen tissues were measured with the immunohistochemistry followed by computer image analysis. The expressions of GAP-43, NGF and TrkA were determined with in situ hybrdization, and their mRNA levels were detected with RT-PCR and image analysis qualification. Results: (1) The GAP-43+ nerve fibers began their regeneration on 30 d after operation and extended from greater omentum into splenic autotransplants. Density of the nerve fibers gradually became greater and almost normal 180 d after operation. (2) In splenic autografts, the mRNA expression of GAP-43, NGF and TrkA appeared on day 30 after the operation, gradually reached the peak on day 90. Conclusion: The renascent GAP-43+ nerve fibers may come from the greater omentum packaging the splenic autografts and NGF and TrkA can promote the nerval regeneration in the autotransplant spleen tissues.

Evaluation of the Haematinic Activities of Extracts of <i>Justicia secunda</i>Vahl Leaves in Red Blood Cells of Laboratory Rats

The use of plant parts (leaves, flowers, stems, barks, roots etc.) in traditional medicine is increasingly gaining ground in modern medicine, as plant sources have long been recognized as sources of secondary metabolites which can be used to treat a wide range of diseases. The effect of extracts of Justicia secunda leaves on red blood cells (RBC) count and haemoglobin (Hb) concentration was investigated in adult Sprague-Dawley rats to establish haematinic activity. Phenylhydrazine (PHZ)-induced anaemic rats were treated with water, methanol or ethyl acetate extracts at 200 mg/kg body weight. RBC counts and Hb concentration were analysed using a haematology analyser at 3-day intervals for 21 days. The extracts were compared with rats administered the haematinic tonic Feroglobin&reg;?and vehicle-treated (normal saline). Rats administered the water extract exhibited the most significant increase (P ) in the number of RBCs and Hb concentration compared with the vehicle-treated PHZ-induced anaemic rats. Rats administered the methanol extract followed with significant increase (P ) in RBC counts and Hb concentration (J. secunda leaves has excellent haematinic properties and this provides the pharmacological basis of its use in Ghanaian traditional medicine for the treatment of anaemia.

The Role of Oxygen Radicals in Rat Acute Lung Injury Induced by Phorbol Myristate Acetate

We tried to clarify the role of oxygen radicals released from granulocytes stimulated byphorbol myristate acetate(PMA) in rat acute lung injury. It was found that DNA strand-breakdamage(DSBD) in peripheral white blood cells (WBC) was significantly increased 40 min after injec-tion of PMA. DSBD in lung tissue of rats treated with PMA was also markedly increased comparedwith the controls. The PMA-treated rats showed significantly higher lipid-peroxide (LPO) level inplasma and lung tissue hemogenate than the controls did. These results suggest that determination ofDSBD, a simple and sensitive indicator for oxygen radical damaging, might be useful in thediagnosis of adult respiratory distress syndrome (ARDS), when it is used together with themeasurement of plasma LPO.

血清同型半胱氨酸水平升高促进大鼠颅内动脉瘤的形成

目的探讨颅内动脉瘤大鼠模型的构建方法及高同型半胱氨酸血症对大鼠颅内动脉瘤形成的影响。方法将36只SD雌性成年大鼠随机等分为3组,即传统模型组、新模型组和对照组,每组12只。传统模型组切除双侧卵巢,结扎双侧肾动脉后支和左侧颈总动脉,造模后1周喂以含8%氯化钠饲料;新模型组按同样方法造模后1周喂以含8%氯化钠和3%的L-甲硫氨酸的饲料;对照组仅暴露左侧颈总动脉和双肾,饲喂普通饲料。造模前及造模后1、6个月,测量血压1次;造模后6个月,检测血清雌二醇和同型半胱氨酸(Hcy),以及外周血基质金属蛋白酶-9(MMP-9)、诱导型一氧化氮合酶(iNOS)、单核细胞趋化蛋白-1(MCP-1)和超氧化物歧化酶(SOD)mRNA水平;扫描电镜观察颅内动脉瘤形成情况。结果三组大鼠初始血压无统计学差异(P>0.05);造模1个月,传统模型组和新模型组大鼠血压均显著升高(P<0.05);造模后6个月,新模型组大鼠血压显著高于传统模型组(P<0.05)。造模6个月,新模型组大鼠血清Hcy水平[(46.87±9.09)mol/L]明显高于传统模型组[(14.32±5.61)mol/L;P<0.05]和对照组[(15.29±4.90)mol/L;P<0.05]。造模6个月,新模型组[(103.16±7.20)pmol/L]和传统模型组[(95.43±8.28)pmol/L]大鼠血清雌二醇水平明显低于对照组[(176.72±11.41)pmol/L;P<0.05]。传统模型组和新模型组大鼠外周血MMP-9、iNOS、SODmRNA水平较对照组均明显增高(P<0.05),而且新模型组明显高于传统模型组(P<0.05)。新模型组颅内动脉瘤形成率(91.67%)明显高于传统模型组(41.67%;P<0.05)。结论L-甲硫氨酸喂养大鼠可诱导形成高同型半胱氨酸血症,血清同型半胱氨酸水平升高促进大鼠颅内动脉瘤的形成。

巴马火麻油对大鼠抗氧化功能的影响

为研究巴马火麻油对大鼠抗氧化功能的影响,依据《关于印发抗氧化功能评价方法等9个保健功能评价方法的通知》(国食药监保化[2012]107号)附件1抗氧化功能评价方法,采用巴马火麻油对成年大鼠进行实验,设溶媒对照组、空白对照组及火麻油高、中、低3个剂量组,连续灌胃30 d。结果表明,受试样品组肝组织匀浆和血清中的丙二醛低于溶媒对照组,差异显著(P<0.05),肝组织匀浆中的超氧化物歧化酶活性极显著高于溶媒对照组(P<0.01);受试样品组肝组织匀浆和血清中的还原型谷胱甘肽含量高于溶媒对照组,差异极显著(P<0.01)。这表明在本实验条件下,动物实验的脂质过氧化产物、抗氧化酶、抗氧化物质3项指标阳性,表明火麻油具有抗氧化功能。

银杏内酯B对成年大鼠神经干细胞向神经元分化的促进作用

目的 探讨银杏内酯B对神经干细胞分化为神经元的促进作用。 方法 采用无血清培养和单克隆实验技术在体外分离培养出大量来源于同一细胞的单细胞克隆球 ,并将其均匀种植于 2 4孔培养板中 ,分 3组分别加入含银杏内酯B、BDNF和不加任何细胞因子的完全培养液。培养 7d和 14d后终止 ,用神经元特异性标记物微管相关蛋白 2 (MAP 2 )的单克隆抗体进行免疫荧光标记 ,荧光显微镜下观察和计数MAP 2标记的阳性细胞并对细胞面积和周长进行图像处理。 结果 两个时期银杏内酯组MAP 2阳性细胞数均明显多于单纯对照组而少于BDNF组 ;分化 7d和 14d时银杏内酯组MAP 2阳性细胞的面积和周长明显大于单纯对照组 ,而略小于BDNF组。结论 银杏内酯B亦具有类似BDNF促进神经干细胞分化为神经元的作用。

成年糖尿病大鼠重要器官组织SOD、MDA含量变化

目的研究成年糖尿病大鼠重要器官组织超氧化物歧化酶(SOD)、丙二醛(MDA)含量变化。方法测定对照组(C)、胰岛素治疗糖尿病组(ID)、糖尿病组(D)大鼠重要器官组织SOD活力、MDA含量。结果大鼠心、肝、肺、脾、胰组织D组与C组比较,SOD活力下降(P<0.05、P<0.01),肝、脾、胰组织ID组与C组比较,SOD活力下降(P<0.05),肺D组与ID组比较,ID组SOD活力提高(P<0.05),肾组织SOD活力无变化;D组心、肺、胰、脾组织中MDA含量升高,与C组比较,有显著差异(P<0.05,P<0.01);ID组心、胰、脾组织MDA含量与C组比较有显著差异(P<0.05,P<0.01),心、脾组织MDA含量ID组与D组比较,有显著差异(P<0.05,P<0.01),肝、肾组织MDA含量无变化。结论糖尿病大鼠重要器官组织抗氧化酶水平低下,经胰岛素治疗后,有提高趋势。

提高成年大鼠神经干细胞单克隆形成率的方法

目的 探索提高神经干细胞单克隆形成率的方法并证实所分离培养的神经干细胞仍具有多分化潜能。 方法 对神经干细胞单克隆方法进行改良 ,即在单克隆培养液中加入 1 2原代克隆培养液。将所得到的单细胞克隆球消化、分离、增殖成大量的神经干细胞球 ,用含血清的DMEM分化培养液促其分化。 14d后 ,分别用神经元和胶质细胞的特异性标记物MAP 2标记神经元、GFAP标记星形胶质细胞和CNP标记少突胶质细胞。 结果平均每块含 1 2原代克隆培养液的 96孔培养板中有 2～ 3只孔可形成克隆球 ,而含纯新鲜培养液的培养板中仅 0 5～ 1 0只孔可形成克隆球。这些单细胞克隆球增殖后得到的大量亚细胞系克隆球分化后分别呈MAP 2、GFAP和CNP免疫荧光阳性。 结论 单细胞克隆实验中加入 1 2原代克隆培养液可提高神经干细胞的单克隆形成率 ,单克隆球增殖后得到的大量亚细胞系克隆球亦具有多分化潜能。

成年大鼠嗅鞘细胞的原代培养与鉴定

目的探索一种简便、经济实用的获得成年大鼠嗅鞘细胞的方法。方法无菌条件下取2.5月龄的SD大鼠嗅球最外二层,经分散后将细胞种植于含20%胎牛血清的DMEM/F12培养基中。采用差速贴壁和阿糖胞苷抑制相结合的方法培养。定期进行形态学观察并摄片。在培养第14d行胶质原纤维酸性蛋白(GFAP)和神经生长因子受体(NGFRp75)的免疫细胞化学染色鉴定,并计算嗅鞘细胞的纯度。结果培养的嗅鞘细胞呈双极、多突起形、扁圆形三种,其中扁圆形较少,嗅鞘细胞的纯度在90%以上。结论差速贴壁和阿糖胞苷化学抑制相结合进行成年大鼠嗅鞘细胞原代培养是一种简便经济实用的方法,可获得高纯度的嗅鞘细胞。