12种不同产地铁皮石斛指纹图谱研究及重金属元素含量测定

以12种不同产地铁皮石斛新鲜茎段为材料,以Ultimate XB C18(4.6mm×250mm,5μm)乙腈-0.1%磷酸溶液为流动相进行梯度洗脱,流速1.0mL·min-1,检测波长270nm,柱温30℃。通过中药色谱指纹图谱相似度评价系统(国家药典委员会)进行相似度分析,并建立12个不同产地铁皮石斛指纹图谱。经微波消解处理材料后,采用ICP-AES原子吸收光谱法测定不同产地铁皮石斛新鲜茎段中重金属铜、镉、铅、汞的含量,分析不同产地铁皮石斛质量,为建立铁皮石斛质量标准提供理论依据。结果表明:(1)不同产地铁皮石斛指纹图谱共有23个特征峰,相似度为0.918~0.987,其中产自安徽大别山、云南文山的铁皮石斛相似度分别为0.918、0.935,相比其他产地具有一定的差异性。(2)通过聚类分析将12种不同产地铁皮石斛分为4类:安徽霍山、江苏南京、云南石屏、云南玉溪、云南思茅为一类,云南文山、浙江仙居为一类,云南红河、广西天峨、安徽大别山、福建宁化为一类,浙江天台单独为一类。(3)产自云南玉溪的铁皮石斛茎段中铜含量超标26.25%,产自福建宁化和江苏南京的镉含量分别超标27.33%和122.30%,其它产地铜、镉、铅、汞含量在国家现行安全值以内。     

Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.     

Effect of influent COD/SO4(2-) ratios on mesophilic anaerobic reactor biomass populations: physico-chemical and microbiological properties

The competitive and syntrophic interactions between different anaerobic bacterial trophic groups in sulphate limited expanded granular sludge bed (EGSB) reactors was investigated. The outcome of competition between the sulphate-reducing, methanogenic and syntrophic populations after development in reactors at varying influent COD/SO4 (2-) ratios was examined in batch activity tests with the inclusion of specific sulphate reducing bacteria (SRB) and methane producing archaea (MPA) inhibitors. SRB species could not out-compete MPA species for acetate at influent COD/SO4 (2-) ratios as low as 2. The SRB were seen to play a more significant role in the conversion of hydrogen but did not become completely dominant. HMPA were responsible for hydrogen utilization at an influent COD/SO4 (2-) ratio of 16, and were still dominant when the ratio was reduced to 4. It was only when the COD/SO4 (2-) ratio was reduced to 2 that the HSRB assumed a more influential role. SRB species were significant in the degradation of propionate at all COD/SO4 (2-) ratios applied. Sludge samples were analysed by scanning electron microscopy (SEM), granule size distribution and fluorescent in situ hybridization (FISH), combined with confocal laser scanning microscopy (CLSM), to monitor any changes in granule morphology under the various COD/SO4 (2-) ratios imposed during the reactor trial. In situ hybridization with domain- and species-specific oligonucleotide probes demonstrated a layered architecture with an outer layer harboring mainly Eubacterial cells and an inner layer dominated by Archaeal species.     

大鼠骨骼肌卫星细胞诱导分化为胰岛素生成细胞的研究

目的探讨大鼠骨骼肌卫星细胞（MDSCs）定向诱导分化为胰岛素生成细胞（IPCs）,为1型糖尿病的干细胞治疗提供一种新的研究思路。 方法通过二次酶消化法和差速贴壁培养法分离、培养大鼠MDSCs,利用不同的诱导培养液使MDSCs定向分化为IPCs,并对诱导后细胞进行形态观察,通过双硫腙染色和免疫组化染色对MDSCs-IPCs形态进行鉴定,采用Q-PCR和Western Blot方法检测MDSCs-IPCs中C-peptide和Insulin的表达,通过胰岛素释放实验检测MDSCs-IPCs的生物学功能,β细胞和MDSCs-IPCs两组间比较采用t检验。 结果MDSCs在接种4 h后开始贴壁部分细胞伸出小的突起,48 h后绝大多数细胞贴壁呈梭形、胞浆丰富、折光度高。随着培养时间的延长,细胞的梭形形状更为明显且生长迅速。免疫组化结果显示细胞表达Desmin、α-Sarcomeric Actinin、MyoD1、Myf5和PAX7。成胰诱导后MDSCs形成胰岛样的圆形细胞团,双硫腙染色呈猩红色,Insulin免疫组化染色阳性。Q-PCR结果显示MDSCs-IPCs中C-peptide和Insulin mRNA表达量分别是β细胞的0.73倍（P > 0.05）和0.79倍（P > 0.05）。胰岛素释放实验显示,5.6 mmol/L和16.7 nmlol/L葡萄糖刺激培养2 h后,β细胞和MDSCs-IPCs分泌胰岛素量分别为[（20.3±4.2）mU/L]、[（16.1±3.7）mU/L]、[（60.5±9.3）mU/L]和[（40.9±7.3）mU/L],葡萄糖可调节MDSCs-IPCs胰岛素的分泌。 结论MDSCs易于分离培养、增殖能力强,体外可诱导分化为有功能的IPCs,适合作为再生医学的种子细胞。     

提高表柔比星质量与生产水平的研究

柔红霉素产生菌S．Coeruleorubidus经N^ 、Co^60理化诱变剂选育后,获得的高产菌株经发酵罐应用后,其发酵产率分别提高25．8％、17．6％,累计净增利润超亿元,经分离收集的活性生物精品制成表柔比星,纯度提高4％,杂质比例下降2倍以上,收率提高50％,符合最新国际权威药典标准,产品畅销世界。     

Recombinant thermophilic enzymes for trehalose and trehalosyl dextrins production

Two thermophilic and thermostable enzymes, trehalosyl dextrins forming enzyme (TDFE) and trehalose forming enzyme (TFE), able to convert starch and dextrins to ,-trehalose were recently purified and characterized from Sulfolobales [I. Di Lernia, A. Morana, A. Ottombrino, S. Fusco, M. Rossi, M. De Rosa, Extremophiles, 2 (1998) 409; T. Nakada, S. Ikegami, H. Chaen, M. Kubota, S. Fukuda, T. Sugimoto, M. Kurimoto, Y. Tsujisaka, Biosci., Biotechnol., Biochem., 60 (1996) 267; T. Nakada, S. Ikegami, H. Chaen, M. Kubota, S. Fukuda, T. Sugimoto, M. Kurimoto, Y. Tsujisaka, Biosci., Biotechnol., Biochem., 60 (1996) 263; M. Kato, Y. Miura, M. Kettoku, K. Shindo, A. Iwamatsu, K. Kobayashi, Biosci., Biotechnol., Biochem., 60 (1996) 921; M. Kato, Y. Miura, M. Kettoku, K. Shindo, A. Iwamatsu, K. Kobayashi, Biosci., Biotechnol., Biochem., 60 (1996) 925]. The first enzyme transforms starch and dextrins to the corresponding trehalosyl derivatives, with an intramolecular transglycosylation process, which converts the glucosidic linkage at the reducing end from -1,4 to -1,1. The second, hydrolyzes the -1,4 linkage adjacent to the -1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. Herein, we report the cloning and high level expression of the two enzymes of Sulfolobus solfataricus strain MT4 in Escherichia coli using pTrc expression vector. The yield of TDFE and TFE obtained in this expression system was of 180 U/l and of 3630 U/l of medium, respectively.     

合成生物学与代谢工程

随着DNA重组技术的日趋成熟,代谢工程的理论和应用已经得到了迅速发展。合成生物学是近年来蓬勃发展的一门新兴学科,在许多领域都具有重要的应用。以下从改造细胞代谢的关键因子、代谢途径的调节和宿主细胞与代谢途径构建的关系等方面详细讨论了合成生物学的最新进展和合成生物学在代谢工程领域的应用。     

江米酒中凝乳酶产生菌的分离及产酶条件的优化

江米酒是我国南方的一种传统食品, 江米酒中微生物所产凝乳酶具有凝乳作用。从江米酒中分离纯化得到4株菌, 经菌落分离计数和酪蛋白平板实验研究确定产凝乳酶的优势菌为其中的霉菌, 并对该霉菌产凝乳酶条件进行了初步优化, 结果表明: 2倍浓度的PDA培养基中添加5%葡萄糖而不添加氮源是霉菌产凝乳酶的最佳培养基, 在此条件下其所产凝乳酶活力可比优化前提高144%。     

产脂酵母产脂培养条件研究及脂肪检测方法初探

目的：研究产脂酵母产脂条件及脂肪提取方法。方法：探讨了单因素如：pH、碳源、氮源、葡萄糖浓度、金属微量元素等对其产脂能力的影响,并进行了正交试验;脂肪测定方法研究,用对比分析法,并对其中的方法加以改进,采用光谱扫描以确定酵母脂肪最大吸收波长。结果：试验范围所得酵母产脂最佳条件是：接种量3%,MnCl_2 0．05%、FesO_4 0．04%、(NH_4)_2SO_4 0．3%、葡萄糖2%、pH 6．5,培养5d,45ml的发酵液其油脂的吸光度(252nm)可达2．4082;酵母脂肪最大吸收波长为252nm,在此波长下测定,获得了快速、准确的结果。结论：所得产脂条件可为进一步的开发研究打下基础。研究出的酵母脂肪测定方法,是一种快速、简便的方法,可满足产脂酵母菌种筛选和产脂条件研究的需要。