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1.
生物正交化学反应是一类可以在生理条件下发生的化学反应,具有简单、高效、高特异性的特点,在生物医学的研究中被广泛应用.基于生物体天然生命过程的代谢工程,可对生物分子进行无损、高效的生物代谢修饰,是一种理想的生物修饰技术.通过生物代谢途径可有效地将各种化学报告基团引入靶标物的生物分子中,有利于携带配对基团的标记物与其发生生物正交反应,从而在活体系统中实现生物分子的标记示踪和药物递送.这种基于代谢工程与生物正交化学的标记策略因为具有两者之间的优势,在生物医学工程中的标记、成像示踪、诊断等领域展现出巨大的研究价值与应用潜力.本文介绍了生物正交和代谢工程的原理与生物医学研究进展,阐述了生物正交化学在分子成像和药物传递等方面的研究与应用.  相似文献   

2.
Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34+ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood  相似文献   

3.
Palsson BO  Oh DJ  Koller MR 《Cytotechnology》1995,18(1-2):125-131
The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC8 (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC5 in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC8. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC5 expansion has now been extended to LTC-IC8 expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.  相似文献   

4.
The purpose of this study was to determine the effect of permeation enhancers on the transbuccal delivery of 5-fluorouracil (FU). The effect of permeation enhancers on in vitro buccal permeability was assessed using sodium deoxycholate (SDC), sodium dodecyl sulphate (SDS), sodium tauroglycocholate (STGC), and oleic acid and their concentrations for absorption enhancement were optimized. STGC appeared to be most effective for enhancing the buccal permeation of FU than the other enhancers. These enhancements by STGC were statistically significant (p < 0.05) compared to control. The order of permeation enhancement was STGC > SDS > SDC > oleic acid. Histological investigations were performed on buccal mucosa and indicated no major morphological changes. The enhancing effect of STGC on the buccal absorption of FU was evaluated from the mucoadhesive gels in rabbits. The absolute bioavailability of FU from mucoadhesive gels containing STGC increased 1.6-fold as compared to the gels containing no permeation enhancer. The mean residence time and mean absorption time considerably increased following administration of gel containing penetration enhancer compared with the gel without penetration enhancer.  相似文献   

5.
This study aimed at developing a topical formulation of lapachol, a compound isolated from various Bignoniaceae species and at evaluating its topical anti-inflammatory activity. The influence of the pharmaceutical form and different types of emulsifiers was evaluated by in-vitro release studies. The formulations showing the highest release rate were selected and assessed trough skin permeation and retention experiments. It was observed that the gel formulation provided significantly higher permeation and retained amount (3.9-fold) of lapachol as compared to the gel-cream formulation. Antinociceptive and antiedematogenic activities of the most promising formulation were also evaluated. Lapachol gel reduced the increase in hind-paw volume induced by carrageenan injection and reduced nociception produced by acetic acid (0.8% in water, i.p.) when used topically. These results suggest that topical delivery of lapachol from gel formulations may be an effective medication for both dermal and subdermal injuries.  相似文献   

6.
The study aim was concerned with formulation and evaluation of bioadhesive buccal drug delivery of tizanidine hydrochloride tablets, which is extensively metabolized by liver. The tablets were prepared by direct compression using bioadhesive polymers such as hydroxylpropyl methylcellulose K4M, sodium carboxymethyl cellulose alone, and a combination of these two polymers. In order to improve the permeation of drug, different permeation enhancers like beta-cyclodextrin (β-CD), hydroxylpropyl beta-cyclodextrin (HP-β-CD), and sodium deoxycholate (SDC) were added to the formulations. The β-CD and HP-β-CD were taken in 1:1 molar ratio to drug in formulations. Bioadhesion strength, ex vivo residence time, swelling, and in vitro dissolution studies and ex vivo permeation studies were performed. In vitro release of optimized bioadhesive buccal tablet was found to be non-Fickian. SDC was taken in 1%, 2%, and 3% w/w of the total tablet weight. Stability studies in natural saliva indicated that optimized formulation has good stability in human saliva. In vivo mucoadhesive behavior of optimized formulation was performed in five healthy male human volunteers and subjective parameters were evaluated.  相似文献   

7.
A survivorship model was developed for UV-B irradiated Catla catla (17 days) larvae with the help of Kaplan and Meier Product-Limit (PL) method. Larvae were exposed to UV-B radiation (145 μW cm−2) for three different exposure times: 5, 10 and 15 min on every other day. The mean survival time of fish was calculated for each treatment using uncensored and censored survival data during 74 days study period. The mean uncensored and censored survival data for the 5-min exposed fish were 7 and 43, respectively. In 10-min exposure period, the uncensored and censored survival data were 19 and 31, respectively. During maximum exposure of 15 min, the uncensored survival data was 20 and censored data was 30. The mean survival time of fish calculated using PL estimate in 5, 10 and 15-min exposure treatments were 69.61 ± 0.50, 65.25 ± 0.96 and 60.60 ± 1.55 days, respectively. The mean survival time showed a decreasing trend with the increase of exposure period. The survival time was significantly (P < 0.001) higher in 5-min exposure treatment than others. This is clear from the present study that the exposure of UV-B radiation affects the survival rate of surface feeder catla larvae.  相似文献   

8.
A novel three-dimensional culture system for the ex vivo expansion of human umbilical cord blood (CB) hematopietic progenitor cells (HPCs) was developed by growing CB mononuclear cells on highly porous CultiSpher G microspheres coated with human bone marrow stromal cells in stirred flasks in the presence of supplemented cytokines. After 12 days, the number of total viable cells, colony-forming units in culture (CFU-C) and CD34+ cells present in the cultures reflected average increases of 7.7, 23.3 and 9.6-fold, respectively, and marked hematopoietic islands were formed on the surface of CultiSpher G.  相似文献   

9.
We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)8-(,-Lys)4-(,-Lys)2-(,-Lys)-Ala-NH2 (D1) and ((Lys)8-(,-Lys)4-(,-Lys)2-,-Lys)-Ala-[Lys(Plm)]2-Ala-NH2 (D2), as well as the starburst polymeric derivatives of D1, (pVIm) 8 -D1 and (pLys) n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.  相似文献   

10.
Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.  相似文献   

11.
In situ gel-forming systems have drawn much attention of current researchers to overcome the poor bioavailability from the conventional eye drops. The present work described formulation and pharmacoscintigraphic evaluation of timolol-maleate-loaded chitosan/hydroxy propyl methyl cellulose (HPMC)-based polymer matrix for enhanced ocular retention. Chitosan and HPMC ratio was optimized and formulation was characterized for various in vitro parameters. The ocular retention was studied on New Zealand rabbits by gamma scintigraphy, which is a very simple and noninvasive technique. For scintigraphy study, the drug timolol maleate was radiolabeled 99mTc by direct labeling method using SnCl2·2H2O as reducing agent. The labeling procedure was optimized to get maximum labeling efficiency (>98%). In vitro stability of the radiolabeled drug (99mTc-timolol maleate complex) was checked and it was found to be stable for up to 24 h. Plain drug eliminates rapidly as significant activity was recorded in kidney and bladder after 2 h of ocular administration. It was evident from the scintigraphic images and the time–activity curve plotted from the data that the plain drug solution cleared very rapidly from the corneal region and reached into systemic circulation via nasolachrymal drainage system, as significant activity was recorded in kidney and bladder after 2 h of ocular administration. Developed formulation cleared at a slow rate and remained at corneal surface for longer time duration. No radioactivity was observed in systemic circulation after 2 h. Ocular irritation of the developed formulation was also checked by hen’s egg chorioallantoic membrane test and formulation was found to be practically nonirritant. The study signified the potential of gamma scintigraphy in evaluation of novel drug delivery systems in a noninvasive manner.  相似文献   

12.
Li Q  Cai H  Liu Q  Tan WS 《Biotechnology letters》2006,28(6):389-394
Ex vivo expanded CD34+ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34+ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34+ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing. Revisions requested 27 September 2005; Revisions received 14 December 2005  相似文献   

13.
Brucella species are gram-negative bacteria which belong to alpha-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified.  相似文献   

14.
To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold, total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34+ cells by 15.7-fold. Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines.  相似文献   

15.
The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.  相似文献   

16.
大鼠海马场电位记录是研究学习记忆的一个重要手段,尤以在体记录更具生理学意义.为克服目前在体海马场电位记录的弊端和诸多不便,提高实验效率,设计并完善了一套简便易行,融刺激、记录、给药于一体的大鼠海马在体CA1区场电位实验技术.将雄性SD大鼠麻醉后固定于脑立体定位仪上,利用自制的刺激/记录/给药联合装置,引导海马CA1区场兴奋性突触后电位(fEPSP).结果表明,使用刺激/记录/给药联合装置能长时间稳定记录由测试刺激诱发的海马CA1区fEPSP.高频刺激条件下,能成功诱导早期时相长时程增强(E-LTP)和晚期时相型长时程增强(L-LTP).海马内注射AMPA受体阻断剂CNQX(100μmol/L,1μl)可迅速抑制fEPSP,注射NMDA受体拮抗剂AP-5(100μmol/L,1μl)可明显压抑LTP,药物发挥作用的时间较侧脑室给药明显缩短,剂量减少.采用此联合装置还成功实现了PPF的稳定记录.总之,采用刺激/记录/给药一体化技术进行在体海马CA1区场电位记录的特点是简单、可靠、高效,可以为开展脑认知功能活动的电生理学研究奠定良好的基础.  相似文献   

17.
A specific method was developed for monitoring the concentration of cyanobacteria (blue-green algae) before waterblooms, based on their characteristics ofin vivo fluorescence. The excitation and emission spectra of cyanobacteria are very different from those of eukaryotic algae, due to the importance of phycocyanin, rather than chlorophylla, in determining the fluorescence characteristics. Our results, based on four cyanobacteria:Microcystis aeruginosa, Anabaena cylindrica, Phormidium tenue andSpirulina platensis, indicate that excitation at 620 nm and its emission at 645 nm is a sensitive and specific method for their detection. Furthermore, the addition of 10 M photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) induced only 3% increase in phycocyanin fluorescence, suggesting that this measurement is almost independent of the ongoing rate of photosynthesis.Author for correspondence  相似文献   

18.
Summary The production of whole plants from explants of protein pea (Pisum sativum L.) using an efficient, reliable and rapid strategy, while maintaining trueness to type, will be required before regeneration can be exploited for genetic transformation. Seeds of the pea genotypes Terese, Solara, Frisson and P64 (a hypernodulating mutant line of Frisson) were surface-sterilized and imbibed overnight, whereafter embryo axes were dissected and germinated on hormone-free medium for 7–10 d. Hypocotyl sections lacking pre-existing meristems were harvested and cultured on a range of media with various concentrations and combinations of growth regulators in order to induce either caulogenesis or somatic embryogenesis. Differences in responsiveness were apparent between genotypes, but regeneration via caulogenesis was consistently more reliable than via the induction of somatic embryos. Few explants underwent somatic embryo production and their conversion into plants has remained elusive so far, irrespective of the genotype studied. Conversely, large numbers of buds were produced within 10 d by organogenesis, and healthy, rootable shoots were obtained. A clear relationship was observed between the growth regulators employed for bud regeneration and shoot rooting phases and the subsequent competence of the regenerated plants for flowering, pod formation and viable seed production.  相似文献   

19.
20.
The ability of self-emulsifying drug delivery systems (SEDDS) to improve solubility, dissolution rate and bioavailability of a poorly water-soluble calcium channel blocker, nimodipine (NM) was evaluated in the present investigation. Solubility of NM in various oils, surfactants and cosurfactants was determined. The influence of the ratio of oil to surfactant + cosurfactant, pH of aqueous phase on mean globule size of resulting emulsions was studied by means of photon correlation spectroscopy. The NM loaded SEDDS selected for the in vitro and in vivo studies exhibited globule size less than 180 nm. In vitro dissolution studies indicated that NM loaded SEDDS could release complete amount of NM irrespective of the pH of the dissolution media. Pharmacokinetics of NM suspension, NM oily solution, NM micellar solution and NM SEDDS were evaluated and compared in rabbits. Relative bioavailability of NM in SEDDS was significantly higher than all the other formulations. NM loaded SEDDS were subjected to various conditions of storage as per ICH guidelines for 3 months. NM SEDDS successfully withstood the stability testing.  相似文献   

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