Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson's disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.

Effects of Root Extracts from <i>Panax ginseng</i>C. A. Meyer (Araliaceae) of Different Ages on K562 Cells

It is well accepted in China that elder ginsengs have more bioactivity and value than younger ones. However, there is little research about the comparison of beneficial effects of ginsengs with different ages. In this study, ginseng root extracts (GRE) were extracted from ginsengs of 5, 8, 12, 14, and 16 years old, respectively, using 55% ethanol and their effects on human leukemic K562 cells within 48 hours were tested by using Cell Counting Kit-8. The results show that there are significant increases in the cell viability of all the GRE groups compared with Control group within 32 hours. Furthermore, the growth curves of GRE groups were obviously distinct from each other. The cell viability of 5-year-old and 8-year-old GRE groups kept a rapid increase while that of 16-year-old GRE group showed a strong fluctuation within 28 hours. Our results demonstrate that root extracts from ginsengs of different ages contain different bioactivity constituents and have different effects on cell.

The Effects of Total Saponins of Panax Ginseng on Hematopoietic Progenitor Cells in Healthy Humans and Aplastic Anemia Patients

Ginseng is said to have beneficial effects on anemia. The proliferation effects of totalsaponins of Panax ginseng (TSPG) on hematopoietic progenitor cell in healthy individuals and 29 patientswith aplastic anemia (AA) were observed through bone marrow cultures of burst forming unit-erythroid(BFU-E) , colony forming unit-erythroid (CFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) in vitrcacompared with methyltestosterone (MT). The results suggest TSPG might prompt the prolif-eration of normal progenitor cellS at a concentration of 20 g/ml. The numbers of BFU-E ,CFU-E and CFU-GM increased by 37. 8±2.9 % , 31. 4±2. 9 % and 33. 3± 4. 0 % respectively over the controls ; further-more TSPG was still useful to BFU-E,CFU-E growth without Epo in vitro, although the colony nurnberswere much lower. Otherwise MT was useless to CFUGM. Of the 29 patients with AA, 14 who respondedto MT showed sensitivity to TSPG in marrow culture (the rising rate of colony formation exceeded 30 % ) ,but immune-mediated AA (patient's peripheral blood mononucleated cell suppressed normalhematopoiesis) and stem cell decreased AA (few of colonies were formed) showed almost no expressionfor TSPG activity because of the immunological suppression system and the absence of progenitors.

Rubus Parvifolius L.Inhibited the Growth of Leukemia K562 Cells in Vitro and in Vivo

Objective：To determine the antiproliferative activity of Rubus parvifolius L.（RP）extract,its medicinal serum and RP total saponins（RPTS）against K562 cells in vitro and in vivo.Methods：Nude mice models bearing leukemia tumors were treated with different concentrations of RP extract.The size,weight and histopathological change of leukemic tumors were determined.Semi-solid agar culture and methylthiazolyl tetrazolium（MTT）assay were used to determine in vitro the inhibition of colony formation and proliferation of K562 cells respectively by different concentrations of RP medicinal serum and RPTS.Results：RP extract had a tumor inhibition rate of 84.8%when administered to mice at a dose of 1.0 g/day of crude RP root equivalent.Semi-solid agar culture of K562cells in the presence of 20%（v/v）of RP medicinal serum and 150 mg/L RPTS demonstrated a 50.8%and 100%inhibition of the colony forming unit（CFU）-K562,respectively.The same doses of RP medicinal serum and RPTS showed a proliferation inhibition of 31.4%and 86.3%,respectively against K562 cells in MTT assay.Conclusion：RP extract and RPTS show effective antiproliferative activity against myeloid leukemia cells in vitro and in vivo.

人参总皂苷诱导K562细胞凋亡的实验研究

目的 研究人参总皂苷诱导白血病细胞凋亡及其机制 ,为进一步开发人参总皂苷提供实验依据。方法 采用细胞体外培养、图象分析技术、流式细胞术、形态学观察与免疫细胞化学等方法 ,研究人参总皂苷诱导 K5 6 2细胞凋亡。结果 人参总皂苷对 K5 6 2细胞有增殖抑制作用 ,并可诱导 K5 6 2细胞凋亡明显增加 ,同时 K5 6 2细胞癌基因产物 C- MYC,BCL- 2表达明显降低。结论 人参总皂苷能诱导 K5 6 2细胞凋亡 ,其机制可能与调控癌基因产物的表达有关。

人参总皂甙逆转K562/A02细胞对DOX耐药性的研究

目的 :研究人参总皂甙 (Totalsaponinspanaxginseng ,TSPG)对人红白血病耐药细胞系K5 6 2 /A0 2耐药性影响及作用机制。方法 :MTT法测定TSPG作用后K5 6 2 /A0 2细胞对阿霉素 (DOX)的药物敏感变化。免疫组化法检测TSPG作用后K5 6 2 /A0 2细胞mdr - 1基因产物P -gp及凋亡调控基因产物bcl- 2的表达。RT -PCR法检测TSPG作用后K5 6 2 /A0 2细胞mdr - 1mRNA表达。结果 :TSPG能增强DOX对K5 6 2 /A0 2细胞的毒性作用 ,逆转效率与浓度相关。TSPG作用后P - gp表达下降有显著差异 (P <0 .0 1) ,bcl- 2表达变化无显著差异 (P >0 .0 5 )。TSPG作用后K5 6 2 /A0 2细胞mdr - 1mRNA表达降低有显著差异 (P <0 .0 1)。结论 :TSPG能部分逆转K5 6 2 /A0 2细胞的耐药性 ,机制主要是下调mdr - 1mRNA表达 ,导致产物P - gp减少 ;与bcl- 2蛋白表达变化无显著相关。

人参总皂苷对K562细胞红细胞生成素受体表达的影响

目的:探讨人参总皂苷(TSPG)对人红白血病细胞株(K562细胞)红细胞生成素受体(EpoR)表达的影响。方法:采用流式细胞仪检测TSPG作用前后的K562细胞膜EpoR阳性率的变化;免疫细胞化学检测TSPG作用前后K562细胞EpoR表达的变化;以免疫印迹法检测TSPG处理前后K562细胞全细胞、细胞膜和细胞质中EpoR含量的变化。结果:K562细胞膜表达EpoR蛋白的阳性率随TSPG剂量的增加而显著下降;免疫细胞化学染色可见对照组K562细胞胞膜着色深,胞质浅淡,核/质比例大;经TSPG 200mg/L作用72h后,K562细胞胞膜着色明显变浅,胞质着色明显加深且丰富,核/质比例明显降低;不同剂量的TSPG作用K562细胞72h后,全细胞总蛋白中的EpoR的表达无明显差异,经200mg/L TSPG诱导后的K562细胞膜中EpoR蛋白表达下降,而TSPG(100、200、300、400mg/L)诱导K562细胞72h后,胞质EpoR蛋白表达增强,且随着剂量的加大更加明显。结论:TSPG能使K562细胞的EpoR位置重塑,为研究TSPG诱导K562细胞向红系细胞分化的作用机制提供了依据。

人参皂甙对K562细胞作用的基因表达谱研究

目的运用基因芯片技术研究人参总皂甙(TSPG)作用后K562细胞基因表达谱的变化。方法200 μg/mlTSPG作用于K562细胞3d后,提取总RNA,合成cRNA并分别用cy3和cy5标记,与Agilent Human 1B寡核苷酸基因芯片杂交,研究基因表达谱的变化。结果TSPG刺激K562细胞后共有362个基因表达发生变化,与对照组K562细胞比较,表达上调的基因有20个,主要有代谢相关基因,信号转导相关基因,细胞受体相关基因等;表达下调的有342个,包括免疫防御相关基因,DNA结合与转录因子,代谢相关基因,细胞周期相关基因等。RT-PCR技术验证了FOSL1、E2F2、CCNE2和ODZ1四个基因表达的变化。结论TSPG刺激K562细胞后,影响了细胞内一系列基因的表达。这些基因可能与TSPG的抗肿瘤机制有关。

人参总皂苷对人红白血病细胞株(K562)中STAT5表达的影响

旨在探讨人参总皂苷对K562细胞STAT5表达的影响。MTT法显示TSPG对K562细胞增殖抑制程度呈剂量与时间依赖性增加,且呈正相关关系。流式细胞术表明TSPG能阻止K562细胞从G0/G1期向S、G2/M期移行。激光共聚焦显微镜观察可见,TSPG 200 mg/L作用K562细胞24 h,胞浆中的STAT5荧光强度增加,而胞核内STAT5荧光强度减弱。Westernblotting结果显示,TSPG作用K562细胞6、24、48 h,胞核中STAT5表达较对照组减少,TSPG作用72 h胞核蛋白中STAT5表达增加;TSPG作用K562细胞6、12、24、48、72 h,胞浆内STAT5表达增加。TSPG能减少K562胞核中STAT5的表达,这可能是TSPG抑制K562细胞增殖的作用机制之一。

人参总皂苷对K562细胞的增殖抑制及诱导分化作用

目的:研究人参总皂苷TSPG对人慢性粒细胞白血病细胞株(K562细胞)增殖及分化的影响,探讨TSPG抗肿瘤作用。方法:取对数生长期K562细胞,分为阴性对照组、不同浓度TSPG组及阳性对照组进行体外培养,以噻唑蓝(MTT)比色法、台盼蓝活细胞计数法观察TSPG对K562细胞增殖的影响;用联苯胺染色及血红蛋白测定检测其诱导K562细胞向红系细胞分化情况;采用流式细胞术测定细胞凋亡。结果:MTT和台盼蓝计数显示TSPG浓度在200 mg/L-800mg/L范围内对K562细胞增殖有抑制作用,并呈浓度和时间依赖性。联苯胺染色提示不同浓度TSPG作用K562细胞的阳性率与对照组比较无明显差异;血红蛋白测定结果显示不同浓度TSPG作用K562细胞的血红蛋白含量与对照组相比有显著差异(P<0.05),但不同浓度TSPG组间并无差异。流式细胞术检测结果显示TSPG400 mg/L组细胞凋亡率与对照组比较差异有统计学意义(P<0.05)。结论:TSPG不仅能抑制K562细胞增殖,还可以诱导其分化,TSPG增殖抑制作用可能与诱导K562细胞凋亡有关。

人参总皂苷对K562细胞STAT5表达的影响

目的探讨人参总皂苷(TSPG)对人红白血病细胞株(K562细胞)信号转导与转录因子5(STAT5)表达的影响。方法紫外-可见分光光度法测定血红蛋白含量及Wright′s染色法检测TSPG诱导K562细胞向红系细胞分化的变化;免疫细胞化学观察TSPG作用前后K562细胞STAT5表达的变化;以ELISA和Western blotting法检测TSPG处理前后K562细胞的细胞质和细胞核中STAT5含量的变化。结果TSPG(200mg/L)诱导K562细胞6h、12h、24h后,随着TSPG作用K562细胞时间的延长,细胞中血红蛋白的含量逐渐增加;而加入AG490(40μmol/L)部分抑制了K562细胞中血红蛋白含量的增加。免疫细胞化学染色可见对照组K562细胞的细胞质和细胞核着色浅淡;经TSPG 200mg/L作用12h后,K562细胞的细胞质和细胞核着色明显加深,表达增强,与对照组相比差异有统计学意义(P<0.05);TSPG(200mg/L)诱导K562细胞6h、12h、24h后,随着TSPG作用K562细胞时间的延长,细胞质和细胞核中STAT5的表达逐渐增强。结论TSPG能增强K562细胞STAT5的表达,这可能是TSPG诱导K562细胞向红系细胞分化的作用机制之一。

微生物对人参果总皂苷中人参皂苷化合物K的转化作用

目的利用微生物转化法对人参果总皂苷(SFPG)进行生物转化,制备人参皂苷化合物K(C-K)。方法从种植人参的土壤中筛选、分离4种微生物m14、m3、m8、m9对SFPG进行转化,筛选最佳活性菌株;以C-K的量为指标,采用TLC和HPLC法检测微生物的转化结果。结果真菌m14为转化C-K的最佳活性菌株,最佳转化条件为转化时间6d,转化温度30℃,摇床转数160r/min,转化初始pH值为自然pH5.5,转化底物质量浓度为120mg/mL。转化后的C-K的量是转化前的41.65倍。结论真菌m14转化SFPG中C-K的专属性强、转化效率高,为C-K的工业化生产提供了一条新的途径。

人参总皂甙诱导造血生长因子生成的实验研究

为探讨人参“补气生血”的现代生物学机理，采用造血祖细胞体外培养，造血生长因子生物活性检测等技术，研究人参总皂甙（ＴＳＰＧ）对造血生长因子的诱导生成作用。结果表明，经ＴＳＰＧ诱导分别制备的脾细胞、巨噬细胞、成纤维细胞和骨髓基质细胞的培养上清对髓系造血祖细胞（ＢＦＵ－Ｅ、ＣＦＵ－ＧＭ、ＣＦＵ－ＭＫ）的体外集落增殖均有显著刺激作用。ＴＳＰＧ也能促进脾细胞、成纤维细胞和巨噬细胞分泌ＩＬ－６；刺激脾细胞产生ＩＬ－２。这提示，ＴＳＰＧ可通过诱导造血基质细胞与脾细胞等产生造血生长因子样物质（如ＣＳＦ，ＩＬ，Ｅｐｏ等），进而促进血细胞生成，这或许是人参“补气生血”的生物学机理之一。

人参总皂甙协同造血生长因子体外诱导CD34^+造血干/祖细胞扩增与分化的作用

为探讨人参总皂甙(totalsaponins of panaxginseng,TSPG)协同造血生长因子体外诱导CD34+造血干/祖细胞(HSC/HPC)扩增与分化的作用,收集人脐血、骨髓细胞并采用StemsepTM干细胞分选系统分离纯化CD34+HSC/HPC,用不同剂量TSPG加入不同组合的造血生长因子进行培养,检测细胞总数、CD34+细胞和CD33+细胞比例及集落形成细胞总数(CFC)、粒系祖细胞(CFU-GM)数量变化。结果显示:10-70μg/mlTSPG均可不同程度地提高脐血细胞总数、CFC数和CD34+细胞数,50μg/ml是最佳刺激浓度,可使细胞总数、CFC数和CD34+细胞数分别增至(2470.5±79.96)×103、(53.96±4.29)×100%和(21.86±3.09)×100%;20μg/ml是液体培养诱导骨髓CD34+细胞向粒系分化的最佳浓度,可使细胞总数、CFU-GM和CD33+细胞分别增至(133.2±9.03)×103、(26.78±1.91)×100%和(16.98±1.73)×100%;甲基纤维素半固体培养检测显示,TSPG(10-50μg/ml)均能提高CD34+细胞形成CFU-GM的集落产率,以TSPG20μg/ml效果最为明显。结论:合适剂量的TSPG能够促进CD34+造血干/祖细胞体外扩增与定向诱导分化。

人参总皂苷增强成骨分化的间充质干细胞促造血作用研究

目的观察人参总皂苷(TSPG)诱导间充质干细胞向成骨细胞分化的作用,探讨TSPG增强成骨分化的间充质干细胞促造血的可能机制。方法贴壁法培养人源骨髓间充质干细胞(h MSCs),成骨细胞分化和免疫表型进行鉴定。不同剂量的TSPG联合成骨细胞条件诱导液培养h MSCs,MTT法检测细胞增殖活性,对-硝基苯磷酸盐(p NPP)法检测培养上清碱性磷酸酶含量,茜素红染色法观察钙结节数量,Western blot法检测成骨细胞分化转录因子-核心结合蛋白因子2(RUNX2)蛋白表达水平,Elisa法检测培养上清造血相关因子含量,造血祖细胞集落生成实验检测培养上清造血支持能力。结果 MTT和p NPP结果均显示随着TSPG剂量增加,吸光值逐渐增加,呈剂量依赖性。钙结节数量和RUNX2蛋白表达水平明显高于对照组。造血相关生长因子和造血祖细胞集落数明显高于对照组。结论TSPG通过上调RUNX2蛋白表达诱导h MSCs向成骨细胞分化,同时增强成骨分化的MSCs支持造血。

人参总皂苷对人胚胎神经干细胞增殖及定向诱导为多巴胺能神经元的影响

目的：探讨人参总皂苷（TSPG）对人胚胎神经干细胞（NSC）增殖和分化为多巴胺（DA）能神经元的影响。方法：从7～12周人工流产胚胎脑组织中分离培养人胚胎NSC,免疫细胞化学鉴定NSC特异性nestin抗原表达,在NSC的培养体系中加入TSPG,采用流式细胞术和MTT法检测不同浓度TSPG以及TSPG与EGF,bFGF联合应用对人胚胎NSC增殖的影响;经免疫细胞化学检测培养细胞酪氨酸羟化酶（TH）表达,分析不同浓度TSPG以及TSPG与IL-1联合应用对NSC定向诱导为DA能神经元的影响。结果：TSPG对NSC的增殖有促进作用,TSPG与EGF、bFGF联合应用的促增殖作用是EGF和bFGF联合应用时的2倍;TSPG能促进神经干细胞定向分化为DA能神经元,TSPG与IL-1联合诱导的效果是单用IL-1的5倍。结论：TSPG能促进人胚胎NSC的增殖,并能诱导NSC向DA能神经元分化。

人参总皂甙体外诱导CD34^+造血干/祖细胞增殖的作用

目的：探讨人参总皂甙(TSPG)协同造血生长因子体外诱导CD34^+造血干/祖细胞(HSC/HPC)增殖的作用。方法：收集人脐血细胞,采用Stemsep TM干细胞分选系统分离纯化CD34^+HSC/HPC,经不同剂量TSPG加入不同造血生长因子组合进行培养,检测细胞总数、CD34^+细胞及集落形成细胞总数(CFCs)的变化。结果：TSPG 10、20、50和70μg/ml均可不同程度地提高细胞总数、CFCs和CD34^+细胞扩增倍数,TSPG 50μg/ml是最佳刺激浓度,可使细胞总数、CFCs和CD34^+细胞分别增至(2470．50±79．96)倍、(53．96±4．29)倍和(21．86士3．09)倍。结论：合适剂量的TSPG能够促进CD34^+造血干/祖细胞体外增殖。

人参总皂苷体外诱导大鼠骨髓间充质干细胞分化为神经元样细胞

目的：探讨人参总皂苷体外诱导青年SD大鼠骨髓间质干细胞（MSCs）分化为神经元样细胞的作用。方法：贴壁筛选法分离纯化MSCs。10μg·L^-1 bFGF预诱导第5代的MSCs24h，换200μg·mL^-1人参总皂苷的无血清培养液诱导诱导5～6h，观察细胞形态变化，免疫组织化学法检测神经元特异性烯醇化酶（NSE）、微管联合蛋白-2（MAP-2）、胶质纤维酸性蛋白（GFAP）的表达。结果：第5代间质干细胞形态达到均一，呈梭形。用人参总皂苷诱导30min到3h后，MSCs胞体逐渐增大，并伸出细长突起形似神经元样细胞。诱导5～6h后，NSE、MAP-2和GFAP阳性细胞数分别为（76±4．7）％、（61±5．2）％、（10±2．4）％。对照组上述染色均呈阴性。结论：人参总皂苷在体外能定向诱导MSCs分化为神经元样细胞。

三七悬浮细胞高密度培养生产人参皂甙和多糖

研究了在摇瓶培养中接种量和各种营养成分对三七悬浮细胞高密度培养的影响。结果表明，磷起始浓度为３．７５ｍｍｏｌ／Ｌ对其细胞培养较佳。接种量对细胞量和产物形成量有一定程度影响，而对细胞倍增数影响更大。在摇瓶培养中采用１２层纱布较棉花塞有利于氧供应。进一步考察了培养过程中糖和其它营养成分的补料策略，发现仅添加合适浓度的糖可使干细胞达到３５ｇ／Ｌ，人参皂甙和多糖分别可高达１．５７ｇ／Ｌ和５．２２ｇ／Ｌ；其它营养物质的添加（如微量成分、氮源、激素等）则对生长不利。人参多糖的产量为目前报道的最高值。

人参总皂甙诱导人造血基质细胞表达IL-3的实验研究

目的 研究人参总皂甙 (TSPG)对人早期血细胞发生的影响及其机理 ,进一步探讨人参“补气生血”的现代分子生物学机理。 方法 采用造血祖细胞体外培养、造血生长因子 (HGF)生物活性检测、免疫细胞化学、核酸分子原位杂交技术 ,研究TSPG对造血基质细胞表达白细胞介素 3(IL 3)的影响及其机理。 结果 TSPG体外作用能明显促进人早期髓系多向性造血祖细胞 (CFU Mix)的集落形成 ;经TSPG诱导制备的人骨髓基质细胞 (BMSC)、脐静脉内皮细胞株 (EcV30 4 )、单核细胞株 (THP)条件培养液对CFU Mix的增殖分化有明显促进作用 ;经TSPG诱导后BMSC、EcV30 4、THP细胞内IL 3的蛋白及mRNA表达显著提高。 结论 TSPG能促进人早期血细胞的增殖分化 ,其机理可能与TSPG诱导人造血基质细胞表达IL 3有密切关系。