Stimulatory effect of green tea extract on the growth of neurites in the rat spinal ganglion culture

Addition of green tea extract in concentrations of 0.004–0.006% to the nutrient medium markedly stimulated the growth of spinal ganglion neurites of 1–2-day-old rats. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 9, pp. 296–298, September, 2004     

Stimulatory effect of green tea extract on the growth of neurites in the rat spinal ganglion culture

Addition of green tea extract in concentrations of 0.004–0.006% to the nutrient medium markedly stimulated the growth of spinal ganglion neurites of 1–2-day-old rats.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 9, pp. 296–298, September, 2004     

Low dose of morphine microinjected in the ventral periaqueductal gray matter of the rat depresses responses of nociceptive ventrobasal thalamic neurons

The effect of low doses of morphine (2 micrograms in 0.2 microliter) microinjected in the periaqueductal gray matter (PAG) was studied on responses of 11 nociceptive (N) and 3 non-nociceptive (Nn) ventrobasal (VB) thalamic neurons in lightly anesthetized rats. Responses of Nn neurons were unmodified but their receptive field was consistently enlarged during 50-60 min. Responses of N neurons to calibrated pinches were strongly depressed when the microinjection site was located in the PAG or in the dorsal raphe (NDR) (7 cases), and not significantly changed when morphine was applied in the dorsal PAG. For the 7 depressed neurons, the mean value of the responses expressed as a percentage of the control was 20.12 +/- 5.17, 15 min after morphine application. This depressive effect was variably reversed by naloxone (0.1-0.5 mg/kg).     

无血清原代培养人前脂肪细胞并诱导分化

目的无血清原代培养人前脂肪细胞并诱导其分化为成熟脂肪细胞。方法采用胶原酶消化法分离并原代培养人前脂肪细胞,通过对细胞形态学的观察、油红O染色,以及脂肪细胞标志性酶G-3-PDH活性的测定对细胞进行鉴定。结果摸索出无血清培养并诱导人前脂肪细胞分化为成熟脂肪细胞的条件。在无血清的基础培养基中前脂肪细胞能够维持增殖。无血清分化培养基培养4d后细胞形态逐渐变圆,并出现球性脂滴,脂滴的数量逐渐增多至分化培养的第21天到达顶峰。结论在无血清培养的状态下成功诱导前脂肪细胞向脂肪细胞的分化,以作为激素或细胞因子对前脂肪细胞增殖或分化影响的研究基础。     

人胚胎干细胞无血清无饲养层培养体系的建立

目的 拟建立人胚胎干细胞无血清无饲养层的培养体系.方法 将干细胞分别置于Knockout培养基(第1组)、自制无血清培养基(第2组)和添加bFGF及肝素的改良无血清培养基(第3组)中培养25代后观察比较3组干细胞形态学变化、克隆数目及大小.并借助细胞免疫组化、流式细胞术和体外拟胚体形成等方法检测干细胞的未分化状态和全能性.结果 传代25次后,第2组克隆逐渐分化.第1组和第3组的干细胞均呈典型的人胚胎干细胞形态特征;细胞表达Nanog,Oct-4,Tra-1-60,SSEA-4,但不表达SSEA-1;流式检测也显示SSEA-4高表达,SSEA-1低表达;体外分化能形成拟胚体.通过单位视野下克隆个数及大小的比较,结果显示第2组与第1组和第3组间的差异显著.结论 本研究通过改良本实验室已获得国家专利的无血清培养基,初步建立了人胚胎干细胞无血清无饲养层培养体系,培养效果与公认培养体系无明显差异.     

基因重组碱性成纤维细胞生长因子对大鼠视网膜神经节细胞的影响

目的:研究基因重组碱性成纤维细胞生长因子(rbFGF)对大鼠视网膜神经节细胞(RGCs)的影响。方法:大鼠在视神经部分损伤后,球后注射生理盐水、维生素B₁₂、rbFGF,伤后4周进行轴突定量、视网膜神经节细胞定量以及RGCs凋亡的检测,观察视神经损伤修复情况。结果:伤后4周时,生理盐水和维生素B₁₂对RGCs无挽救作用,800U、1600U和2400U的rbFGF对RGCs挽救率分别为24.5%、27.3%、28.5%,800UrbFGF组、1600UrbFGF组和2400UrbFGF组未发生溃变的轴突数分别是损伤未治疗组的2.03、2.43、2.31倍。流式细胞仪检测结果显示:rbFGF治疗7d后,RGCs凋亡率显著减少。结论:rbFGF可提高视网膜神经节细胞的存活率,减少轴突溃变,有抗凋亡作用,对视神经损伤有显著的促功能修复作用。     

Variations in the number and position of human lumbar sympathetic ganglia and rami communicantes

The purpose of this study is to provide precise anatomical and statistical information about the number and location of lumbar sympathetic ganglia and the number and length of the related rami communicantes, and to consider the neurological pathway for nociception from the low back. Three-hundred ninety-three ganglia and 719 rami communicantes from 50 human cadavers were identified by gross dissection. The number of ganglia in a single lumbar chain ranged from 2 to 6, the mean being 3.9. The mean lengths of rami connected to the 1st and 5th lumbar spinal nerves, respectively, were significantly longer and shorter than those connected to the 2nd, 3rd, and 4th nerves. The lumbar sympathetic ganglia and rami communicantes were not distributed segmentally. The present results may assist in understanding the nociceptive pathway from the low back.     

Differences in sensitivity to nerve growth factor of axon formation and tyrosine hydroxylase induction in cultured sympathetic neurons

Superior cervical ganglia from 2-day-old and 3-week-old rats were maintained in vitro for up to 2 weeks in the presence of a range of concentrations of nerve growth factor up to 100 micrograms/ml. Nerve fibre length and density were measured and tyrosine hydroxylase activity of these cultures assayed after various times. Ganglia were also examined for catecholamines and neuronal numbers using fluorescence histochemistry and histology respectively. In cultures maintained without nerve growth factor, or in those containing low concentrations of nerve growth factor (3 ng/ml), tyrosine hydroxylase decreased to 5-10% of the initial levels by 14 days in vitro. The presence of the high concentration of 1 microgram/ml nerve growth factor in the culture medium or the addition of such a concentration during the culture period did not prevent an initial decrease in tyrosine hydroxylase but subsequently increased the enzyme activity. The maximal effect of nerve growth factor on nerve fibre density was at low concentrations whereas its maximal effect on neuronal survival, tyrosine hydroxylase activity or nerve fibre elongation was at high concentrations. After 2 days in culture, maximum neurite production occurred in cultures containing 10 ng/ml, while maximum nerve fibre elongation and tyrosine hydroxylase activity occurred in cultures containing 100 micrograms/ml nerve growth factor. We conclude that low concentrations of nerve growth factor, as occur in plasma, cause maximum axon formation while high concentrations of nerve growth factor, as occur in effector organs, induce maximum tyrosine hydroxylase activity and cell survival. The former process may be mediated via cell surface receptors and the latter via retrograde axonal transport of nerve growth factor to the cell body, following uptake by the terminal regions of the axons.     

Phenotypical changes of embryonic chick adrenal medullary cells in vitro induced by nerve growth factor and ciliary neuronotrophic factor

This study investigates the survival properties and changes in the morphological phenotype of adrenal medullary (chromaffin and neuronal) cells cultured from embryonic chicks at different developmental ages (embryonic days E8 to E16) in response to nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). The 4-day survival of medullary cells from all embryonic ages except E8 was about 80% of the seeded cells and was only slightly enhanced by the addition of saturating doses of CNTF (10 ng/ml). With no factors, after 4 days 10-30% of the surviving medullary cells extended neurites. NGF (100 ng/ml) and, even more, CNTF (10 ng/ml) and their combination substantially increased the proportions of neurite-bearing cells (up to 70%). The effect of the factors were maximal at E10 and E12 and declined at older developmental ages. Neurite growth was virtually unaffected by NGF and CNTF at E8. These results show that in vitro survival and neurite growth of chick adrenal medullary cells in response to trophic factors is developmentally regulated.     

Use of serum-free hormonally defined media to evaluate the effects of growth factors and inhibitors on proliferation of estrogen-responsive mammary and pituitary tumor cells in culture

Summary Serum-free defined media have been developed for assay of the mitogenic effects of growth factors on human MCF-7, human T-47D, and mouse COMMA-D mammary cells as well as for identification of mitogens and inhibitors of GH₄C₁ rat pituitary tumor cell growth. These lines were shown to grow in vivo in response to a variety of hormones including estrogens and thyroid hormones. With mammary cells, complete hormonally and nutritionally defined media were established that supported continuous passage at 50 to 90% of the serum stimulated rate. The strategy used to measure mitogens for mammary cells was to identify nutritional conditions where the growth rate was reduced greatly without impairing the response to picomolar to nanomolar concentration of growth factors. The effects of polypeptide growth factors and tissue extracts were estimated by their addition to basal medium and measuring cell number increases or labeled thymidine incorporation into DNA. In a variation of this methodology, the MTW9/PL2 rat mammary cells were used to identify secreted autocrine growth factors; nutritionally defined conditions were sought for growth of these rat cells in the complete absence of exogenous growth factors. The factors secreted into the medium were detected by bioassays with COMMA-D or MCF-7 mammary cell lines. The effects of growth factors-inhibitors on pituitary cells were measured by a related method; the GH₄C₁ cells were grown at less than optimal rates in a defined medium designated TRM-1. Addition of mitogens to TRM-1 stimulated pituitary cell growth whereas addition of inhibitors caused reduced levels of growth. The methods described in this report offer new means of assaying growth factors-inhibitors for a range of mammary and pituitary tumor cells.     

Platelet-derived growth factor promotes survival of rat and human mesencephalic dopaminergic neurons in culture

Summary The effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.     

促红细胞生成素/神经生长因子对大鼠压迫性损伤后背根神经节细胞的影响

目的:观察促红细胞生成素(EPO)/神经生长因子(NGF)对慢性压迫中的大鼠背根神经节(DRG)细胞的影响。方法:暴露大鼠多节段DRG,行慢性压迫造模型,并分离DRG,模型组分离的DRG细胞给予外源EPO或NGF,台盼蓝染色检测细胞存活率,TUNEL法检测细胞凋亡,免疫荧光法检测Bcl-2和Bax表达,荧光探针法检测活性氧(ROS)活性,ELISA法检测炎症因子IL-1β、TNF-α、IFN-γ的水平。结果:EPO或NGF可减轻DRG细胞的损伤和凋亡,下调促凋亡因子Bax的表达,并上调抗凋亡因子Bcl-2的表达,降低ROS活性。结论:EPO和NGF有利于受损的DRG细胞抗炎和抗氧化作用。     

Cultivation,frozen storage,and clonal growth of normal human epidermal keratinocytes in serum-free media

Summary Methods are described for serum-free culture of human epidermal keratinocytes derived from neonatal foreskin tissue. Cultures are initiated, stored frozen, and returned to active growth, all with bovine pituitary extract as the only undefined supplement. Clonal growth assays are then performed in a biochemically defined medium. The degree of stratification and differentiation in the defined medium (and also with pituitary extract) is controlled by the extracellular calcium ion concentration.     

Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.     

Differing effects on the pre- and paravertebral sympathetic ganglia of adult mice produced by antisera to nerve growth factors from mice and snakes

Antiserum to nerve growth factor isolated from mouse salivary glands causes, in adult mice, a reduction of approximately 50% in weight of the superior cervical ganglia. Prevertebral ganglia are less sensitive than paravertebral ganglia. The reduction in weight of the superior cervical ganglia results from a reduction in size of the principal sympathetic neurones (mean diameter falling by approximately 30%) rather than from a reduction in both cell size and cell numbers. If, as seems to be the case in neonatal mice, the antiserum acts by neutralizing endogenous nerve growth factor, no effect on adult neurone numbers would be expected. The origin of the reduction in neurone size is not understood.Antisera to nerve growth factor isolated from snake venoms do not affect the superior cervical ganglia of adult mice. It is suggested that chemical differences between the snake venom and mouse salivary gland nerve growth factors might lead to differences in the antisera produced from these respective antigens.