纤维素酶降解人参细胞胞壁产生的激发子诱导人参培养细胞的反应

用纤维素酶降解人参细胞壁制备的激发子刺激人参细胞后,电子显微镜的观察结果显示激发子处理诱导了细胞内淀粉粒的降解,出现了细胞内动员能量物质的应急反应;同时还出现了大量拟脂类圆球体。进一步的研究表明,涉及降解淀粉的淀粉酶活力是升高了,而降解脂类物质的酯酶活力是降低的;同时细胞中可溶性糖和总类脂含量是增加的。这些都说明了人参细胞经激发子处理后能量物质的动员以及脂类代谢途径的调整。另外,适当浓度激发子的处理会诱导人参细胞内人参皂苷含量和苯丙氨酸解氨酶活力的升高,而过氧化物酶和多酚氧化酶活力是下降的。

平阳霉素作用静脉畸形内皮细胞后VCAM-1、ICAM-1、ICAM-3表达

目的:研究平阳霉素(PYM)作用于人静脉畸形内皮细胞(HVMECs)后Ig粘附分子(VCAM-1、ICAM-1、ICAM-3)表达。方法:体外培养HVMECs,采用细胞ELISA和RT-PCR技术检测不同浓度PYM作用人HVMECs后Ig粘附分子表达。结果:PYM作用人HVMECs后粘附分子表达具有时间浓度效应。PYM能诱导VCAM-1在人HVMECs表达,2h后明显增高,8h后达到峰值,12h后下降,48h后呈阴性表达。PYM能促进ICAM-1在人HVMECs表达,12h后显著增高,18h最高,24h逐渐下降。PYM能促进ICAM-3表达,12h后逐渐增高,24h达到峰值,72h后1CAM-3仍高表达。0.01～1mg/L PYM能诱导或促进粘附分子表达,表达水平与药物浓度成正相关,1mg/L PYM作用人HVMECs后粘附分子表达较高,10mg/L PYM作用人HVMECs后,表达下降。RT-PCR检测PYM作用人HVMECs 2、6、12h能诱导或促进VCAM-1、ICAM-1和ICAM-3mRNA表达,8、12、18h后mRNA表达最高。1mg/L PYM作用于HVMECs后,粘附分子mRNA表达最高,mR-NA表达具有时间浓度效应。结论:低浓度PYM能诱导或促进人HVMECs粘附分子表达,具有时间浓度效应。

细胞培养/链特异性RT-PCR方法检测甲型肝炎减毒活疫苗病毒滴度

目的建立检测甲型肝炎减毒活疫苗病毒滴度的细胞培养/链特异性逆转录聚合酶链式反应(strand-specific reverse transcriptase-polymerase chain reaction,strand-specific RT-PCR)方法。方法根据甲型肝炎病毒(HAV)L-A-1株基因组序列,设计5条特异性引物。将甲型肝炎减毒活疫苗原液感染2BS细胞,收获增殖高峰期的HAV,提取RNA,进行2轮链特异性PCR扩增,检测HAV复制过程中的负链RNA,通过Reed-Muench公式计算HAV病毒滴度,并对建立的细胞培养/链特异性RT-PCR法进行特异性及敏感性验证,同时用该方法检测4批冻干甲型肝炎减毒活疫苗病毒滴度,并与《中国药典》三部(2010版)中甲型肝炎减毒活疫苗病毒滴度检测方法(细胞培养时间为28 d/ELISA法)进行比较。结果 HAV负链RNA经2轮扩增获得阳性扩增条带,大小与预期相符。该方法检测HAV复制过程中的负链RNA,特异性、敏感度均较好。两种方法检测的病毒滴度接近,差异无统计学意义(P>0.05)。结论建立的细胞培养/链特异性RT-PCR方法检测周期短,灵敏度、特异性好,可用于疫苗的滴度检测。

Biotransformation of Gastrodin by Mucor spinosus

Microbial transformation of gastrodin by Mucor spinosus strain 3.3450, resulted in a product with a transformation rate close to 100 per cent. This product was identified as p-hydroxy benzyl alcohol on the basis of its 1H, 13C NMR and EI-MS spectral data. It could be inferred that the enzyme responsible for the biotransforma-tion reaction was a kind of extracellular and constitutive enzyme since the transformation reaction of the substrate could be carried out in cell free extracts of the fermentation broth of the Mucor spinosus.

Comparative Study of PCR Kits with Cell Culture and LCR in the Detection of Chlamydia Trachomatis Infections

Objective: To determine the diagnostic performancesof six Chinese PCR kits for detection of Chlamydiatrachomatis in patients with sexually transmitteddiseases using cell culture and LCR as references.Methods: Endocervical or urethral swab specimenswere collected from 673 patients attending STDclinics in Beijing, Shanghai, Nanjing and Tianjin. C.trachomatis culture and PCR were performed onspecimens from all patients while LCR detection wasperformed only on specimens with discordant cultureand PCR results.Results: Of the 616 patients, 6.3% (39) wereculture-positive while 23.5% to 28.7% were positiveby PCR testing. Compared to cell culture, the sensi-tivity of all six PCR methods was 90% or higher. In200 cases with discrepant reports, LCR and PCRshowed excellent consistency (YI index: 0.523-0.881 ), the sensitivity and specificity of PCR methodswere 83.9%- 98.6% and 66.7%- 94.7% respectively,while PCR2 showed the highest YI index (0.881). Withthe reference standard defined as culture positive orLCR positive plus at least one PCR positive fordiscrepant results, we found that the specificity andsensitivity of all six Chinese PCR kits were higherthan 95% and 85%, respectively.Conclusions: Domestically-produced PCR kits forChlamydia trachomatis detection are highly sensi-tive and specific, however, quality control remainsimportant in their clinical application.

Detection of Grass Carp Hemorrhage Virus (GCHV) from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as "Red Spot Disease", is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and comparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplification. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain induces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the 6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines, including CAB, FHM, CIK, EPC, CCO and GCO, infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHV strains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and the two strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-V viruses have been replicated and amplified despite there being no cytopathogenic effects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published recently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp fragment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is identical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously, the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product, and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.

In vitro Induction of primary antibody responses to particulate and soluble protein antigens in T cell-replaced murine spleen cell cultures

Specific antibody responses could be induced in serumfree condition.Specific anti-SRBC or anti-SRBC ghost antibody were induced from anti-Thy treated (T-depleted) murine spleen cells in serum-free culture in the presence of Con A conditioned medium.This induction system may facilitate the study of lymphokine functions on antigen triggered B cells. In T cell-replaced cultures,the antibody responses of B cells could be successfully induced when soluble SRBC membrane proteins were used as antigens.It thus indicates that antigen together with lymphokines are sufficient to drive B cells to become antibody secreting cells in the absence of T cells.The T cell-replaced system provides a more stable way for in vitro immunization and may be applied to monoclonal antibody production when in vivo immunization is difficult to be carried out.

Application of photobioreactors to cultivation of microalgae

An overview of photobioreactors now in use for production of microalgae world wide is presented, and the application of photobioreactors to the cultivation of microalgae is discussed in detail. It is pointed out that high cell density and industrial production of microalgae can be achieved using many kinds of closed photobioreactors including fermentor, tubular and flat plate photobioreactors, and the cultivation of Spirulina, Chlorella, Dunaliella tertiolecta and Porphyridium cruentrim by photobioreactors can achieve higher and steadier productivity than the cultivation of microalgae by an open air system. More and more researches indicate that tubular and flat plate photobioreactors are the development trend for photobioreactors with bubbles and air lift stirrers, and high bright light emitting diodes are the most economic light source with great potential for future development of photobioreactors. Photobioreactors can also be used for the production of high value metabolite (EPA or DHA) using some microalgae species for energy development and environment protection.

THE PREPARATION OF FRUCTOSE-MODIFIED CHITOSAN MICROCARRIERS AND CULTURE OF PRIMARY RAT HEPATOCYTES

The fructose modified chitosan microcarries (CMs) were prepared by the reaction of glutaraldehyde with fructose-modified chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Morphology of rat hepatocytes cultured on CMs was observed using phase contrast microscope and scanning electron microscope, and the metabolic activities were measured. Rat hepatocytes cultured on CMs retained the spherical shape as they have in vivo and had high metabolic activities. Fructose can enhance the metabolic activity of hepatocytes and the modified CMs are promising scaffold for hepatocytes attachment.

The Use of Bone Morphogenetic Protein-7 and Resveratrol in Collagen Type II of Articular Cartilage

This study aimed to investigate the effects of resveratrol and bone morphogenetic protein 7 on type II collagen from superficial and middle zone of porcine articular chondrocytes. Articular cartilage was isolated from dissected porcine knee joint n = 12. Isolated cells were plated as monolayers at a density of 1 × 105 cells/well in 12-well culture plates and incubated at 37℃ in a humid atmosphere of 5% carbon dioxide and 95% air. Cell cultures were treated for four days with various concentrations of bone morphogenetic protein-7 and resveratroL Cells were then collected and analysed for collagen type II expression by real time polymerase chain reaction and protein level quantification by enzyme-linked immunosorbent assay. Cartilage tissue sections were localised for collagen type II by immunohistochemistry. Moreover, resveratrol and bone morphogenetic protein-7 effects on cartilage matrix contents were analysed by histology. Resveratrol and bone morphogenetic protein-7 stimulates expression of collagen type II mRNA and protein level accumulation in the surface zone and middle zone at 50μM ＋ 300 ng/ml （RSV ＋ BMP-7）. Immunohistochemistry results confirmed the presence of collagen type II on articular cartilage. Histological tissue sections confirmed that chondrocytes were obtained from different zones of articular cartilage. The study suggests that a combination of bone morphogenetic protein-7 and resveratrol up-regulate the expression and synthesis of collagen type II.

Biomaterial Scaffolds with Biomimetic Fluidic Channels for Hepatocyte Culture

Biomaterial scaffolds play an important role in maintaining the viability and biological functions of highly metabolic hepatocytes in liver tissue engineering. One of the major challenges involves building a complex microchannel network inside three-dimensional （3D） scaffolds for efficient mass transportation. Here we presented a biomimetic strategy to generate a mi- crochannel network within porous biomaterial scaffolds by mimicking the vascular tree of rat liver. The typical parameters of the blood vessels were incorporated into the biomimetic design of the microchannel network such as branching angle and diameter. Silk fibroin-gelatin scaffolds with biomimetic vascular tree were fabricated by combining micromolding, freeze drying and 3D rolling techniques. The relationship between the micro-channeled design and flow pattern was revealed by a flow experiment, which indicated that the scaffolds with biomimetic vascular tree exhibited unique capability in improving mass transportation inside the 3D scaffold. The 3D scaffolds, preseeded with primary hepatocytes, were dynamically cultured in a bioreactor system. The results confirmed that the pre-designed biomimetic microchannel network facilitated the generation and expansion of hepatocytes.