Establishment and identification of pancreatic stem cell strain derived from islets of Kunming mice under feeder layer conditions / 中国组织工程研究

BACKGROUND:Until now, little has been reported on establishment of pancreatic stem cell strains and lines,and the purification of pancreatic stem cells is difficult since the cell line establish rate is low.OBJECTIVE:To explore a more rational and effective technique of in vitro separation and continuous passage of pancreatic stem cells, with the hope to establish cell strains and even cell lines and to lay the foundation for the follow-up study of pancreatic stem cells in the treatment of diabetes.METHODS:Firstly, Percoll discontinuous density gradient centrifugation method was applied to separate the mouse pancreatic endocrine portion from the exocrine portion, then to obtain cell strains with highly proliferative ability and low differentiation from pancreatic endocrine portion-the islet. We used mouse embryonic fibroblasts treated with mitomycin C as a feeder layer, for in vitro continuous culture of islet-derived pancreatic stem cells under feeder layer conditions until they were transferred to the 30th passage to establish cell lines. Then pancreatic stem cell line derived from pancreatic islet was detected and identified by a series of tests including growth characteristic test, morphological observation, related molecular marker identification and differentiation characteristic identification.RESULTS AND CONCLUSION:In the continuous process of passage, pancreatic stem cells showed active proliferative ability, and maintained the typical morphological characteristics of stem cells and expression of pancreatic stem cell marker-Nestin. After induction, pancreatic stem cells showed insulin gene expression,reflecting their differentiation potential. Therefore, under the condition of feeder layer, the pancreatic stem cell line derived from Kunming mice was successfully established and the related identification was completed,which lays the foundation for the following research.

Effects of Flow Components C6 and C7 in N-butyl Alcohol Extract from the Leaves of Cestrum nocturnum on the Proliferation and Apoptosis of Human Gastric Cancer Cell SGC7901 / 中国药房

OBJECTIVE:To study the effects of flow components C6 and C7 in n-butyl alcohol extract from the leaves of Ces-trum Nocturnum(CN)on the proliferation and apoptosis of human gastric cancer cell SGC7901. METHODS:C6 and C7 were ob-tained by using different ratio of chloroform and methanol(1:9,1:7)to the gradient elution of CN leaves. After cultured with 0 (blank control),5,10,20,40,80 μg/ml C6 and C7 for 72 h,inhibitory effect of C6 and C7 on the proliferation of SGC7901 was determined by MTT assay. Inhibitory rate and IC50 were calculated. After SGC7901 were cultured with 10 μg/ml C6 and C7 for 72 h,colony formation assay was utilized to detect the effects of C6 and C7 on the cell colony formation,and the rate of colony for-mation was calculated. In addition,Wright/Giemsa and Hoechst33258/PI staining assay were used to observe the change of cytomor-phology. RESULTS:MTT showed that C6 and C7 had inhibitory effect on the proliferation of SGC7901 to different extent;inhibi-tory rates were 22.1%-80.0% and 19.6%-79.7%,and IC50 were 16.4,18.05 μg/ml,respectively. Compared with blank control group,colony formation rate of C6 and C7 group decreased,with statistical significance(P<0.05). The number of apoptotic cells was more in treatment group than in other groups. CONCLUSIONS:Flow components C6 and C7 in n-butyl alcohol extract from the leaves of CN can inhibit the proliferation of SGC7901 cells and induce the apoptosis of them.

Current situation of the clinical competence training for clinical medicine postgraduates with professional degree and its countermeasures / 中华医学教育探索杂志

Extensive attention was paid on how to ensure the cultivation quality for postgraduates with professional degree under the background of the enrollment expansion.The problems in the cultivation of postgraduates with professional degree including declined quality among enrolled students,inefficient training program,unsound management system and little clinical operation chance were analyzed combined with the practice and explore in the clinical competence training for postgraduates with professional degree in Guangxi university of Traditional Chinese Medicine.Some countermeasures were put forward in improving clinical competence for postgraduates with professional degree,for instance the improvement of the management system,tutor team,quality supervision system,clinical skill training and the construction of training bases.

Effect of Panax notoginseng saponins on expression of alpha-secretase mRNA in brains of senescence-accelerated SAMP8 mice / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of Panax notoginseng saponins (PNS) on expression of alpha-aecretase mRNA in the brains of senescence-accelerated SAMP8 mice.</p><p><b>METHOD</b>SAMP8 mice were randomly divided into the control group, the PNS high-dosage group (200 mg x kg(-1)), the PNS low-dosage group (100 mg x kg(-1)) and the huperzine A group (0.3 mg x kg(-1)), with eight mice in each group. The control group and each administration group were orally administered with the same volume of double distilled water once for consecutively two months. The expression of alpha-secretase (ADAM 9, ADAM10, ADAM17) mRNA was assayed by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR).</p><p><b>RESULT</b>The expression of ADAM9 mRNA in PNS high-dosage group and huperzine A group were significantly higher than that of the control group (P < 0.05). The expression of ADAM10 in the PNS high-dosage group, the PNS low-dosage group and the huperzine A group showed no significant difference from the control group.</p><p><b>CONCLUSION</b>PNS can up-regulate expressions of ADAM9 mRNA and down-regulate expressions of ADAM10 mRNA in the brains of SAMP8 mice.</p>

Effect of Panax notoginseng saponins on syp and tau gene expression in brain of senescence accelerated mouse / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of Panax notoginseng saponins (PNS) on (synaptophysin, syp) and tau gene expression in the brain tissue in senescence accelerated mouse prone 8 (SAMP 8).</p><p><b>METHOD</b>SAMP8 were randomly divided into 4 groups: PNS 23.38, 93.50 mg x kg(-1) group, huperzin A 0.038 6 mg x kg(-1) x d(-1) group and blank control group; the drug groups were treated with the designed drugs respectively per day by intragastric administration for 4 consecutive weeks, and double distilled water was given to blank control group. After treatment, the mRNA content of tau and syp were assayed by reverse transcription (RT) and real-time polymerase chain reaction (real-time PCR).</p><p><b>RESULT</b>Compared with blank control group, the syp mRNA contents were increased in PNS groups (P < 0.05 or P < 0.01), and the tau mRNA content were not significant difference in all groups.</p><p><b>CONCLUSION</b>This study suggests that PNS can up-regulate syp gene expression at transcriptional level in the brain of SAMP 8.</p>

Protective effects of panax notoginseng saponins on cholinergic neurons in rats with Alzheimer disease / 中国组织工程研究

BACKGROUND: There are no effective methods to cure Alzheimer disease (AD). Now, researches have shown that panax notoginseng saponins (PNS) play an important role in improving AD, but its mechanism is unclear.OBJECTIVE: To observe the protective effect of PNS characterized by removing blood stasis to stop bleeding and promoting blood circulation to relieve pain on pathological lesion of cholinergic neuron in rat with AD.DESIGN: Completely randomized grouping design and controlled study.SETTING: Neuroscience Institute of Guangxi Traditional Chinese Medical University.MATERIALS: This experiment was completed in the Chinese Herb Pharmacodynamic Laboratory of Guangxi Traditional Chinese Medical University between June 2003 and April 2005. A total of 90 health Wistar rats of clean grade and half gender were selected in this study. Among them, there were 75 old rats with 15 months old and 15 young rats with 3 months old. METHODS: This experiment was completed in the Chinese herb Pharmacodynamic Laboratory (Key Laboratory) of Guangxi Traditional Chinese Medical University between June 2003 and April 2005. ① A total of 90 healthy Wistar rats of clean grade and half gender were selected in this study. Among them, there were 75 old rats with 15 months old and 15 young rats with 3 months old. Fifteen young rats with 3 months old were regarded as young control group, and other 15 selected from 75 rats with 15 months old were regarded as old control group. The rest 60 rats were modeled on the basis of subacute injury induced by intravenous injection of D-galactose and bilateral cerebral Meynert basal nuclei injured by ibotenic acid. Parallel control was performed with saline on rats in young control group and old control group under the same condition. ② Two weeks later,survival modeling rats were divided randomly into 4 groups: model group,high-dosage PNS group, low-dosage PNS group and huperzine A group with 12 in each group. Rats in high-and low-dosage PNS groups were perfused with 200 and 100 mg/kg PNS (provided by Yunnan Yuxi Weihe Pharmaceutical Factory), respectively, once a day; rats in huperzine A group were perfused with 0.3 mg/kg huperzine A once a day for 4 weeks; rats in model group, young control group and old control group were perfused with the same volume of saline for 4 weeks. ③ After administration, pathological sections of brain tissue were cut, and immunologic-reaction activity of choline acetyltransferase (ChAT), morphological changes and numbers of positive neuron in cerebral sections were determined by immunohistochemistry analysis. ChAT immuno-positive neurons were analyzed with IBAS imaging analysis system to assay average area of section and average absorbance (A), and amount of ChAT immuno-positive neurons was calculated with microscope micrometer. ④ Measurement data were compared with single-factor analysis of variance.MAIN OUTCOME MEASURES: Effect of PNS on distribution of cholinergic neuron and ChAT content in cerebral tissue of AD rat models.RESULTS: A total of 75 old rats and 15 young rats entered the final analysis. ① Amount of ChAT immuno-positive neurons was the most, and the color was the deepest in young control group; amount of ChAT immuno-positive neurons was higher in high-dosage PNS group than that in huperzine A group and model group; ChAT immuno-positive neurons were smaller in model group than those in other goups, and the amount was decreased obviously. Axis-cylinder and dendrite of soma were shortened remarkably. ② Amounts of ChAT immuno-positive neurons in basal forebrain were less in model group than those in other groups (P ＜ 0.05), less in lowdosage PNS group, huperzine A group and model group than those in old control group (P ＜ 0.05), less in huperzine A group and model group than those in high- and low-dosage PNS group (P ＜ 0.05), and less in young control group than those in other groups (P ＜ 0.05). The mean A value of ChAT immuno-positive neurons in basal forebrain was similar to amounts in each group. Average area of section of ChAT immuno-positive neurons in basal forebrain was smaller in low-dosage PNS group and model group than that in young control group (P ＜ 0.05), and differences in other groups were not significant (P ＞ 0.05).CONCLUSION: PNS plays a protective role in pathological lesion of cholinergic neuron in AD rat models. PNS can also increase survival amount and quality of cell and increase content and activity of ChAT so as to protect and improve central cholinergic system, and inhibit aging and dementia through improving and repairing injured cholinergic neurons.

Effect of bushen yizhi formula on neurotransmitter release in rat models with Alzheimer disease / 中国组织工程研究

BACKGROUND: The decrease of activity level of choline acetyltransferase (ChAT) and reduction of membrane protein of synaptic vesicles are the main biochemical indexes in Alzheimer disease. Traditional Chinese bushen yizhi formula is of the effects of strengthening the kidney, replenishing qi, generating marrow and nourishing the brain, its medicine-containing serum can effectively protect these changes resulting from amyloid protein.OBJECTIVE: To investigate whether the medicine-containing serum of bushen yizhi formula can improve the pathological injury of neurons induced by amyloid protein metabolites in Alzheimer disease, thus to estimate the therapeutic effect of bushen yizhi formula on Alzheimer disease.DESIGN:A randomized and controlled trial. SETTING:Institute of Neuroscience, Second Affiliated Hospital, Guangxi College of Traditional Chinese Medicine, Nanning 530011, Guangxi Zhuang Nationality Autonomous Region, China; Encephalopathic Institute of Guangzhou University of Traditional Chinese Medicine.MATERIALS:The experiment was conducted from September 2003 to March 2004 at the Research and Development Center of New Drugs of Guangxi College of TCM. Totally 60 healthy Wistar rats of clean grade,three months, were at random divided as blank control and bushen yizhi groups, with 30 in each group. The bushen yizhi formula consisted of Fructus Lycii, Fructus Cnidii, Radix Gingeng, Radix Polygoni Multiflori,Cortex Moutan Radicis, Borneolum, etc. The medicine was prepared before use as 0.176 g/Ml liquid(including 1.13 g/Ml rude drugs).METHODS:① Each of the rats in bushen yizhi group was by garage given 1.0-1.5 Ml prepared liquid per day for consecutive 30 days. Within 4hours after the last administration the serum was isolated at 5 000 r/min for 5 minutes, filtered aseptically, the compliment was inactivated at 56 ℃,then the medicine-containing serum was obtained. The rats in blank control group were given saline of the same volume, and the serum was prepared with the same method. ② For NG108-15 cells, the concentration of amyloid protein 25-35 segments in the culture liquid was 5 μ mol/L, or 1-42 segments, 200 nmol/L, was suitable. ③ NG108-15 cells was first in cubated for a day in CO2 incubator with Dulbecco-improved Eagle medium of 0.1 molarity at 37 ℃, then 5 μmol/L amyloid protein 25-35 segment or 200 nmol/L segment 1-42 was added to continuously incubate for a day, after that the cell models of Alzheimer disease was ready. Then 50 g/L medicine-containing serum or Dulbecco-improved Eagle medium of blank control group serum was taken to replace the original culture liquid, and the amyloid protein segments of the above-mentioned concentration was still added for maintaining the cell models of Alzheimer disease, after 5 days'differentiation incubation, the indexes were detected with those in non-amyloid protein as control. ④ Western blotting, radioimmunoassay and electrophysiological examination were used to investigate the ChAT activity,synapsin protein level and rate of functional synapse formation of NG108-15cells in Alzheimer disease after treatment with the medicine-containing serum of bushen yizhi formula.MAIN OUTCOME MEASURES :The effects of the medicine-containing serum of bushen yizhi formula on ChAT activity, synapsin protein level,rate of functional synapse formation of NG108-15 cells and frequency of miniature plate potential in NG108-15 cells.RESULTS:Totally 60 rats involved all entered the final result analysis.① The effect of the medicine-containing serum of bushen yizhi formula on ChAT activity: In the conditions when there was no amyloid protein or the final concentration of amyloid protein 1- 42 was 200 nmol/L, the ChAT activity in bushen yizhi group was obviously higher than that in blank control group [(1651.2±134.5), (1336.1±268.2), ( 586.1±223.4),(1290.7±381.5)μmol/(g·h); P ＜ 0.05] . ② The effect of the medicinecontaining serum of bushen yizhi formula on synapsin protein level: The absorbance values of synapsin Ⅰa and Ⅰb, in the conditions when there was no amyloid protein, amyloid protein 25-35 and 1-42, were respectively 5.02, 1.36 and 2.46 in bushen yizhi group; and 3.18, 0.57 and 0.71 in blank control group. The values in bushen yizhi group were respectively 1.6,2.4 and 3.5times those in blank control group. ③ The effect of the medicine-containing serum of bushen yizhi formula on functional synapse formation of NG108-15 cells: In the conditions when there was no amyloid protein or amyloid protein 1-42, the levels of functional synapse formation in bushen yizhi group wereall higher than those in blank control group [(90.6±6.0)%, (63.2±17.0)%, (58.0±13.1)%, (34.2±13.0)% ;P＜0.05].④ The effect of the medicine-containing serum of bushen yizhi formula on frequency of miniature plate potential in NG108-15 cells: In the conditions when there was no amyloid protein or amyloid protein 1-42, the frequencies of miniature plate potential in bushen yizhi group were all higher than those in blank control group [(9.28±4.1), (5.48 ±5.14), (5.55±5.85),(3.05±4.46)/min; P ＜ 0.01, P＜ 0.05].CONCLUSION:In the conditions when there existed no amyloid protein or amyloid protein, the medicine-containing serum of bushen yizhi formula can raise ChAT activity, not only correct the decrease of cellular synapsin caused by amyloid protein, but also raise the expression of synapsin protein, the rate of functional synapse formation and neurotransmitter release under the condition of no amyloid protein. It was shown that the prescription can antagonize the pathological development of Alzheimer disease, and play the therapeutic effect through enhancing the release power of neurotransmitter.

Component Analysis of Essential Oil Extracted with Supercritical CO_2 from Eucalyptus tereticornis by GC-MS / 中国药房

OBJECTIVE:To analyze the chemical components extracted with supercritical CO2(SFE-CO2)from Eucalyptus tereticornis.METHODS: Volatile oil was extracted from E.tereticornis with SFE-CO2.The chemical component analyzed by GC-MS and its proportion was determined by normalization method.RESULTS: 28 compounds which account for 88.13% of the total peak area were separated and identified.The main components of volatile oil were eucalyptol (33.99%),borneol(8.88%),?-pinene (5.39%),caryophyllene (4.51%),(+)-4-carene (4.19%).CONCLUSION:This study can be served as a scientific basis for the further exploitation and utilization of E.tereticornis.

Experimental Studies on Anti-aging Actions of Total Saponins of Radix Notoginseng / 广州中医药大学学报

[Objective] To observe the anti-aging actions of total saponins of Radix Notoginseng, i.e., Panax Notoginseng saponins (PNS) on rats with Alzheimer's Disease (AD). [Methods] Among 90 Wistar rats, 15 rats aged 3 months were allocated to the youth group, 15 aged 15 months to the aged group, and other 60 aged 15 months were given with intraperitoneal injection of D-galactose and injection of ibotenic acid into bilateral Meynert nucleus basalis to establish the models of AD. After then, the surviving model rats were randomized into four groups: model control, high-dose PNS (200mg?kg-1?d-1), low-dose PNS (100 mg?kg-1?d-1) and huperzine A (0.3 mg?kg-1?d-1). Except the model group, the youth group and the aged group were given the same amount of normal saline by gavage, the other groups were treated with the designed drugs respectively for 4 weeks. After treatment, serum levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), triglyceride (TG) and cholesterol were detected. [Results] High- and low-dose PNS increased the serum levels of SOD, GSH and CAT, the differences being significance as compared with the model group (P