Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN: A comparative experiment. SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03). METHODS: ① Preparation of cerebral cortical neurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37 ℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours. ④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES: ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons; ② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining; ③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④ DNA agarose gel electrophoresis ladder-like strap appeared or not; ⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS: ① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons (P > 0.05). ② The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [(13.00±4.52)%,(12.72±2.15)%,(11.80±1.18)%,(38.03±1.05)%, P < 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture (36.77±1.45)%, (36.60±1.61)%, (36.37±2.02)%, (38.03±1.05)%, P > 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L (P < 0.01). Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation (P < 0.01). ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia; ⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased (P < 0.01), and those of Bax protein and Caspase-3 protein were reduced (P < 0.01), and the ratio of Bcl-2/Bax was increased (P < 0.01). CONCLUSION: Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Quantitative detection and comparison of sulfate glycosaminoglycans content in extracellular matrix of in vitro cultured epiphyseal, articular and rib chondrocytes

Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached ( 70. 12 ± 7. 72 )μg/cm2, articular chondrocytes (92.00 ± 10.15) μg/cm2 and rib chondrocytes (80.61 ± 11. 40) μg/cm2, respectively. On the third pasage chondrocytes, epiphyceal chondrocytes decreased to (53.27 ± 9. 50 ) μg/cm2, articular chondrocytes to (63.88 ± 11.92) μg/cm2 and rib chondrocytes to (58.94 ±8.21) μg/cm2, respectively. The change of TB in every passage

Cerebrospinal fluid used as culture medium prior to autologous olfactory ensheathing cell transplantation

BACKGROUND: Cerebrospinal fluid can be an inducer for neural stem cells in vitro, but few studies employ cerebrospinal fluid to culture olfactory ensheathing cells. OBJECTIVE: To investigate the growth of nasal mucosa olfactory ensheathing cells in normal cerebrospinal fluid, and to analyze the feasibility of cerebrospinal fluid for culturing olfactory ensheathing cells used for transplantation. DESIGN, TIME AND SETTING: A completely randomized, block design study was performed at the Cell Laboratory, Wuxi Third People's Hospital, and Jiangsu Institute of Parasitic Diseases, China, in August 2008. MATERIALS: Dulbecco's modified Eagle's medium/F12 (DMEM/F12) and fetal bovine serum (Gibco BRL, USA), mouse anti-rat P75 monoclonal antibody and rabbit anti-glial fibrillary acidic protein polyclonal anti body (Santa Cruz Biotechnology, USA), mouse anti-rat myelin basic protein monoclonal antibody (Cymbus, UK), mouse anti-rat microtubule-associated protein-2 monoclonal antibody (Transduction Laboratories, USA), FITC conjugated rabbit anti-mouse monoclonal antibody (Boster, China), TRITC conjugated goat anti-rabbit monoclonal antibody (Sigma, USA) were used. METHODS: Nasal mucosa olfactory ensheathing cells were separately incubated in DMEM/F12, cerebrospinal fluid, and changing DMEM/F12 into cerebrospinal fluid. Adult female Sprague Dawley rat models of spinal hemisection were established. Nerve injury was repaired by transplantation of nasal mucosa olfactory ensheathing cells cultured in cerebrospinal fluid or DMEM/F12. MAIN OUTCOME MEASURES: The proliferative ability of olfactory ensheathing cells cultured in cerebrospinal fluid was determined by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The morphology and purity of olfactory ensheathing cells were detected using immunohistochemistry. Animal behavior was evaluated by the Basso, Beattie and Bresnahan locomotor rating scale. Morphological repair was assessed by a horseradish peroxidase-tetramethylbenzidine retrograde tracer technique and immunohistochemistry. RESULTS: Changing from DMEM/F12 to cerebrospinal fluid did not change overall culture morphology and purity on day 14. These cells also contributed to myelinization and the conduction velocity of regenerated axons, and improved motor abilities of denervated muscle fibers in rats with spinal cord injury. The recovery of behavioral function and neuronal regeneration was similar in the two groups. CONCLUSION: Cerebrospinal fluid culture prior to autologous olfactory ensheathing cell transplantation is feasible for clinical use.

In Vitro-Propagation of Agave tequilana Weber cv.azul in a Temporary Immersion System

In Mexico,there is a need to produce large quantities of plantlets for the establishment and replanting of blue(cv.azul)agave production areas.Most of these plots are within the origin denomination area(DOT,Spanish acronym)of the distilled product of this plant,known as tequila.The objective of this study was to develop an in vitropropagation protocol for Agave tequilana Weber cv.azul using segmented stems in both:solid and liquid media.A disinfection and in vitro technique were developed to obtain shoots,through plantlets collected in commercial plots,which attained 100%surface-disinfection and budding rate.At the multiplication stage,the effects of 6-Benzylaminopurine(BA)(0.0,4.4 and 13.2μM)and kinetin(0.0,9.4,18.8 and 37.6μM)were evaluated on lateralshoot production of segmented sagittal stems.These were cultivated on Murashige&Skoog(MS)medium,with the addition of 3.0%sucrose and 8 g L−1 agar.It was observed that BA and kinetin increased the number of shoots per explant,obtaining up to 18 and 26,respectively.Furthermore,it was found that just the sagittal segmentation of explants increased axillary budding.On the other hand,segmented-stem bases were grown in MS liquid medium with 3.0%sucrose,inside a RITA
system,programmed by a 5 min immersion step with a frequency of every 4 h.The effect of Indole−3-Acetic acid(IAA)(0.57,2.9,5.7μM)was evaluated,while maintaining a concentration of BA(13.2μM).It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant.These results offer a new methodology to increase the efficiency of A.tequilana Weber cv.azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.

In Vitro Propagation of Grapevine Cultivars with Potential for South of Brazil

The micropropation is an important biotechnological tool for obtaining and maintaining mother vine plants with high quality plant health. The objective was to evaluate the establishment and multiplication in vitro and ex vitro acclimatization of grape genotypes with potential for Southern Brazil. Vine nodal segments were cultured in five culture medium formulations without adding growth regulators. It was evaluated the number of leaves and roots, length of roots and shoots, replication rate, relative chlorophyll index, percentage of regenerated and rooted plants, dry biomass of shoot, root and total plants grown in vitro and after acclimatization. In vitro propagation of IAC 571-6 rootstock and cv. Poloskei Muskotaly through nodal segments provided high rates of regeneration and rooting. High survival rates were obtained in the acclimatization of IAC 571-6 and P?l?skei Muskotaly. Considering all the variables, the culture medium Roubelakis showed the best growth rates and development for shoots and roots, and in vitro multiplication rate for IAC 571-6 and Poloskei Muskotaly grape varieties.

Study on Virus-free and Rapid Propagation Technology of Artemisia selengensis sp.in Yangxin County

[Objectives]This study was conducted to culture virus-free tissue culture plantlets of mid-maturing green-stalk Artemisia selengensis sp.varieties in Yangxin County.[Methods]With bud tips of A.selengensis in Yangxin as explants and MS as the basal medium,screening and proportioning of plant growth regulators were performed to establish a virus-free and rapid propagation system for mid-maturing green-stalk varieties of A.selengensis in Yangxin.[Results]The optimal callus induction medium,adventitious shoot differentiation medium,adventitious shoot elongation medium and rooting medium for explants were MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+sucrose 25 g/L+agar 7 g/L,MS+6-BA 0.1 mg/L+NAA 0.1 mg/L+sucrose 25 g/L+agar 7 g/L,MS+6-BA 0.02 mg/L+NAA 0.02 mg/L+sucrose 25 g/L+agar 7 g/L and 1/2 MS+NAA 0.05 mg/L+sucrose 25 g/L+agar 7 g/L,respectively,and the seedling rate reached more than 95%.The culture conditions were as follows:temperature(25±2)℃,relative humidity 85%,illumination intensity 3000 lx,and illumination time 14 h/d.[Conclusions]This study has important practical significance for the purification and rejuvenation and large-scale industrial breeding of Yangxin A.selengensis seedlings.

Unveiling how vitrification affects the porcine blastocyst: clues from a transcriptomic study

Background:Currently,there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes.To date,vitrification(VIT)is the most efficient method for pig embryo cryopreservation.Despite a high number of embryos survives in vitro after vitrification/warming procedures,the in vivo embryo survival rates after embryo transfer are variable among laboratories.So far,most studies have focused on cryoprotective agents and devices,while the VIT effects on porcine embryonic gene expression remained unclear.The few studies performed were based on vitrified/warmed embryos that were cultured in vitro(IVC)to allow them to re–expand.Thus,the specific alterations of VIT,IVC,and the cumulative effect of both remained unknown.To unveil the VIT-specific embryonic alterations,gene expression in VIT versus(vs.)IVC embryos was analyzed.Additionally,changes derived from both VIT and IVC vs.control embryos(CO)were analyzed to confirm the VIT embryonic alterations.Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA–sequencing:(1)VIT embryos(vitrified/warmed and cultured in vitro),(2)IVC embryos and(3)CO embryos.Results:RNA–sequencing revealed three clearly different mRNA profiles for VIT,IVC and CO embryos.Comparative analysis of mRNA profiles between VIT and IVC identified 321,differentially expressed genes(DEG)(FDR<0.006).In VIT vs.CO and IVC vs.CO,1901 and 1519 DEG were found,respectively,with an overlap of 1045 genes.VIT-specific functional alterations were associated to response to osmotic stress,response to hormones,and developmental growth.While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects.Conclusions:Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs.IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos.The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts.Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.

Micropropagation of Curcuma Sp., a Threatened Medicinal Plant

A rapid and improved micropropagation protocol was developed for Curcuma sp., a threatened and high value medicinal plant, using main bud sprout and top of rhizome sprout as explants. Stepwise optimization of different plant is to change the growth regulator, reduce the level of macro-nutrition and add humate. The present study has created multiple shoot and root induction and plantlet in vitro culture, transfers the plantlet to ex vitro. The M2 medium (MS’s macronutrition 1/4, MS’s micronutrition + Morel’s vitamine + coconut milk 10% + sucrose 25 g/l + humate 1.0 ml/l + agar 7.5 g/l + 2,4D 0.5 mg/l + BAP1.0 mg/l + TDZ 1.0 mg/l) is the highest ratio of callus induction. The TA3 medium (MS’s macronutrition 1/4 + MS’s micronutrition + Morel’s vitamin+ coconut milk 10% + sucrose 25g/l + humate 2.0 ml/l + 2,4 D 0.5 mg/l + Kinetin 2.0 mg/l + TDZ 1.0 mg/l + BAP 1.0 mg/l2 + NAA 2.0 mg/l + activated carbon 2.0 g/l) is able to create buds and regeneration multiple bud for Curcuma sp. TA3 medium adding IAA 2 mg/l and IBA 0.5 mg/l has resulted in the highest indices of quantity, healthy shoot and large diameter of roots. A large number of healthy plantlets are induced by the medium of MS’s macronutrients 1/2, MS’s micronutrients full, Morel’s vitamin, humate 3 g/l, coconut milk 150 ml/l, activated carbon 3 g/l, composition phytohormone: IAA 2 mg/l + BAP 2 mg/l + TDZ 0.5 mg/l. Further studies should focus on optimizing the humate concentration for these species of Zingiberaceae. The duration of the research is from 5/2015 to 4/2016 at Faculty of Agriculture and Forestry, Tay Nguyen University, Vietnam.

Effect of Proline Pretreatment on Grapevine Shoot-Tip Response to a Droplet-Vitrification Protocol

Proline has been shown to accumulate in plants in response to biotic and abiotic stresses. Exogenous proline has thus been used for improving some plant cryopreservation protocols. Further enhancement of cryopreservation efficiency for in vitro grapevines could be expected if stresses linked to cryopreservation procedures could be reduced. We therefore studied the possible beneficial effect of proline in grapevine cryopreservation. Single-node explants from in vitro grown grapevine plantlets (Vitis vinifera L. cv Portan) were cultured on shooting media (half-strength MS + 1 μM BAP) containing no proline (control) or 50, 500, or 2000 μM filter-sterilized L-proline. Shoot tips excised from these microshoots were subjected to a PVS2-based droplet-vitrification procedure. Control and rewarmed explants were grown on a recovery medium containing 1 μM BAP. Shoot development on control medium and lower proline concentrations did not notably differ whereas the highest concentration of proline inhibited shoot development. Carry-over effects were observed since lower survival and regrowth were obtained both for non-frozen or LN-treated explants excised from micro-shoots obtained on the 2000 μM proline medium. No significant differences in survival and regrowth were observed for non-frozen explants subjected to pretreatment without LN exposure. A slightly enhancing effect (although non-significant) on post-cryopreservation survival was observed for explants derived from shoots developed on 50 or 500 μM proline, but no significant improvement of regrowth percentage was observed for these two conditions. Although a slight increase in survival could be observed, no significant beneficial effect of proline pretreatment on post-cryoconservation regrowth could be evidenced in our conditions. However, the 2-week period before explant excision could have allowed at least partial metabolism and catabolism of exogenous proline;the results observed could thus have been the consequence of complex interactions. Shorter proline treatments applied closer to the actual LN exposure step might produce different results and allow for clearer interpretation.

Propagation Techniques of Ewes at Early Age

The integration of hormone induction of young ewes,live oocyte collection,in-vitro fertilization and embryo transfer( JIVET),as a major breakthrough in livestock breeding technology,not only solves the problem of low oocytes obtained from adult ewes by hormone induction in production practice,but also significantly improves production efficiency. This technology can reduce the generation interval of cattle and sheep and speed up the acquisition of genes in genetic breeding. The application of this technology can help accelerate the cultivation process,shorten the generation interval,promote the industrialization and improve the economic benefits of beef cattle breed. This paper provides a reference for the research and application of new breeding and propagation techniques of beef cattle.

关于举办“第三届国际骨质疏松研讨会”准备工作意见的通知

关于举办“第三届国际骨质疏松研讨会”准备工作意见的通知各省、市、自治区老年学学会骨质疏松委员会、筹备组：中国老年学学会骨质疏松委员会二届委员、中国骨质疏松杂志编委：为了加强国际学术交流与合作，促进我国骨质疏松的防治和研究，在中国老年学学会、中国老龄协...