The Effect of Sampling and Storage on the Fecal Microbiota Composition in Healthy and Diseased Subjects

Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80°C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p<0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80°C versus the other methods and -80°C samples (p<0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80°C, but was higher in fecal swabs (p<0.05). Storage up to 24 hours (at +4°C or room temperature) or freezing at -20°C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.     

Use of a uterine-cell monolayer culture system for micromanipulated bovine embryos

The objective of this study was to evaluate the growth of micromanipulated bovine embryos in two in vitro culture systems. Sixty ova (day 7 from estrus) were collected in Dulbecco's phosphate-buffered saline (PBS), with 2% fetal calf serum, and transferred to a PBS holding medium containing 10% fetal calf serum to prepare for micromanipulation. Forty embryos (morula to expanded blastocyst stages) were selected for embryo splitting using a modified microsurgery procedure. Thirty-nine of these embryos were successfully bisected into demi-embryos (DE) and the halves allotted by post-manipulation quality grades into one of two treatment groups (Trt). DE in Trt A were cultured in Ham's F-10 medium with 10% FCS (HF-10) while the remaining DE halves from each embryo were cocultured in HF-10 on a monolayer of endometrial fibroblasts (8 x 10(4) viable fibroblast cells plated three days prior to culture) in Trt B. Embryo development, recorded at 12-hour intervals, was evaluated by a split-plot analysis of variance. Results indicated that embryo viability decreased (P<0.001) over time in culture. Overall viability was greater (P<0.001) for DE in Trt B than in Trt A, with a significant (P<0.05) Trt x Time interaction, indicating that embryo viability decreased more rapidly across time in HF-10 than in the monolayer coculture system. The percentage of DE developing at 12, 24, 36, 48, 60 and 72 hours in culture was: 44%, 41%, 33%, 28%, 21% and 18% for Trt A and 69%, 69%, 69%, 67%, 62% and 62% for Trt B. Fourteen of the DE in Trt B attached to fibroblast monolayer and initiated trophoblastic outgrowth and four additional DE remained viable for up to 17.5 days in vitro as intact blastocysts. These findings are the first reported that demonstrate that the zona-free bovine DE will develop during in vitro culture. Also, the bovine endometrial fibroblast monolayer system proved to be excellent for both short term (相似文献   

Conditions of the primary culture for rat hepatocytes and plasminogen activator release

The conditions of primary culture for rat hepatocytes was investigated on the releasing effect of Plasminogen Activator (PA). The culture method using Collagen Coated Dish (CCD-method) which is currently available and the ordinary culture method using Plastic Culture Dish (PCD-method) were employed for that purpose in a comparative way. The effect of the addition of some supplements, that is FN, Aprotinin, EGF were also investigated. The following results were obtained. The dissociated rat hepatocytes formed a monolayer with pavementlike morphology at 24-48 hours after seeding. No difference was observed in the morphology of hepatocytes during the culture period between the two methods, although CCD-method allowed 120 hours culture, whereas PCD-method allowed 72 hours. The PA activity was demonstrated on the hepatocytes by either culture method according to the fibrinolysis autography. The cultured hepatocytes released PA into the medium continuously as long as the viability and morphology of the cells were maintained in good state. The PA activity reached the maximum after 96 hours culture in CCD-method, whereas it reached the maximum after 48 hours in PCD-method. The addition of Aprotinin to the culture medium was not necessary for PA release in CCD-method in contrast to PCD-method. When EGF was discontinued in the culture medium, the release of PA was reduced in association with the occurring of morphological disintegration of hepatocytes.     

In vitro survival of fresh and frozen/thawed bovine demi-embryos

Three experiments were conducted to investigate the effects of type of culture medium in freshly bisected bovine embryos and the effects of agar embedding and of 1.2 propanediol (PROH) as the cryoprotectant in frozen/thawed bisected bovine embryos on development in vitro. A total of 265 bovine embryos were used as controls or were microsurgically bisected and were cultured in vitro for 48 hours and development was determined 24 and 48 hours after the onset of culture. Whitten's medium supported more (P<0.05) intact and demi-embryos to grow to expanded blastocysts (92.9 and 73.1%, respectively) compared with Ham's F10 (43.8 and 26.3%, respectively) and PBS (53.8 and 12.5%, respectively). Embedding in agar and culture in Whitten's medium resulted in a higher (P<0.05) percentage of in vitro development of frozen/thawed demi-embryos after 24 hours than the freezing of nonembedded demi-embryos (44.1 versus 19.6%, respectively). This difference disappeared, however, after a 48 hours culture period (17.6 versus 11.8%, respectively). Following freezing in PROH, survival rates of 40 and 28%, respectively after 24 hours of culture were obtained for intact and demi-embryos. The respective percentages after 48 hours were 8.6 and 16%. Since neither embedding in agar nor the use of PROH as the cryoprotectant resulted in high survival rates of frozen/thawed demi-embryos in vitro, new freezing procedures are needed to overcome the sensitivity of demi-embryos to freezing and thawing.     

The effect of human serum albumin on the extended storage of human oral keratinocyte viability under mild hypothermia

Isolated oral keratinocytes in suspension provide a number of advantages for use in maxillofacial surgery, however, the poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. The purpose of the present study was to evaluate whether human serum albumin (HSA) could serve as an effective constituent of a storage medium to enhance human oral keratinocyte (HOK) viability under conditions of mild hypothermia. Primary human oral keratinocytes were isolated from small pieces of the non-inflamed gingival tissues obtained during the extraction of the third molars of patients. HOK were cultured on collagen type I-coated culture dishes in keratinocyte growth medium (KGM). After the trypsinization of a culture dish (passage 2 or 3), freshly isolated HOK were stored for 24, 48, and 72 h at 4 degrees C or at room temperature in KGM, saline, Dulbecco's modified Eagle's medium (DMEM), saline supplemented with 10% HSA or DMEM supplemented with 10% (v/v) HSA under one atmosphere pressure. After storage, HOK cell survival was determined by dye exclusion using trypan blue and colony-forming assay and cell cycle change was obtained by flow cytometry. Highest cell viability was obtained in saline supplemented with 10% HSA and DMEM supplemented with 10% (v/v) HSA at 4 degrees C and at room temperature. Under these conditions no significant decline in keratinocyte viability was observed for at least 48 h. The cell cycle profiles of these cells were also maintained for at least 48 h at room temperature. These observations demonstrate that HSA might be better at preserving the viability of HOK stored under hypothermic and mild hypothermic conditions up to 48 h.     

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.     

Efficiency of a Transport Medium for the Recovery of Aerobic and Anaerobic Bacteria from Applicator Swabs

The survival of four aerobic and four anaerobic pathogens was evaluated quantitatively on cotton swabs and calcium alginate swabs stored in dry tubes as compared with swabs stored in Amies Transport Medium without charcoal. Survival of the pathogens was markedly improved when stored in Amies Transport Medium, although there was considerable loss of viability after a few hours of storage.     

Production of chlamydospores by Candida albicans cultivated on dilute oxgall agar

Summary A simple easily prepared clear medium consisting of 2% oxgall in 1.8% agar is described. This has proved to be excellent for the rapid identification ofCandida albicans in swabs and cultures, provided that the inoculated area is covered by a coverslip and the incubation temperature period is maintained at 28°C for 24 to 48 hours. No special technical skill is required to prepare this medium. The results presented show thatC. albicans will always produce chlamydospores on this medium if the recommended procedure is followed.Head, Medical Mycology Section.Mycologist, Medical Mycology Section.     

Preliminary studies on bovine embryo survival following short-term storage at 4 degrees C

A total of 113 non-surgically collected bovine embryos, 5-8 days of age, were stored for 48 hours at 4 degrees C in a modified phosphate-buffered saline solution (PBS). Following storage, embryos were cultured for 8-12 hours at 37 degrees C, and those which were morphologically normal were transferred to synchronized recipients by several methods designed to achieve twin pregnancies. Embryos which were collected and transferred on the same day served as controls. Of 113 embryos stored, 47 (42%) appeared to be transferable after the brief culture period. There was a marked breed effect on viability after refrigeration, with Hereford embryos surviving significantly better than Angus embryos (71% vs. 12%, respectively, p < .001). Post-transfer embryo survival of stored and control embryos, based on actual calvings, was 34 and 48 percent, respectively, a difference which was not significant (p=0.3). A marked difference in pregnancy rate following non-surgical transfer by 2 different technicians was noted (50% vs. 21.7%, respectively).     

Effects of frozen and liquid hypothermic storage and extender type on calcium homeostasis in relation to viability and ATP content in striped bass (Morone saxatilis) sperm

The effect of hypothermic storage on striped bass sperm calcium homeostasis was determined by Fluo-3 flow cytometry. Calcium homeostasis was defined as the ability of cells to maintain a low concentration of intracellular free calcium as measured by Fluo-3 fluorescence. Sperm were stored frozen in striped bass extender (SBE) and Tris–NaCl medium (T350) modified with 50 mM glycine and 7.5% dimethylsulfoxide and in nonfrozen form diluted 1:3 (vol/vol) in SBE and T350 for 1, 24, and 48 hours at 4 °C in an oxygen atmosphere. Fluo-3 fluorescence was detected in less than 5% of fresh viable sperm cells indicating maintenance of calcium homeostasis. In contrast to sperm in fresh semen, frozen-thawed and nonfrozen sperm cells lost to a considerable extent the ability to maintain low intracellular free calcium even in the absence of exogenous calcium; positive Fluo-3 fluorescence was found in 26% and 39% of thawed sperm frozen in SBE- and T350-based freezing diluents, respectively, and increased (P < 0.05) to 67% during nonfrozen storage in SBE and T350 at 24 and 48 hours. Sperm viability measured by exclusion of propidium iodide by flow cytometry was 99% in fresh milt and maintained at 86% (P > 0.05) in SBE after 48 hours of nonfrozen storage but decreased (P < 0.05) to 55.7% after 48 hours in T350. Energy status in terms of ATP content, determined by luciferin–luciferase bioluminescence assay, was higher (P < 0.05) in sperm frozen in SBE than in T350 during the first 5 minutes post-thaw and decreased to essentially zero by 15 minutes post-thaw and did not differ among nonfrozen storage treatments. In conclusion, sperm cells impervious to propidium iodide after frozen or nonfrozen storage were unable to maintain low intracellular calcium content. SBE is a better medium than T350 for frozen or nonfrozen storage of striped bass sperm. The inability to regulate intracellular calcium in striped bass sperm may be associated with poor activation of motility after 4 °C storage and cryopreservation.     

Effect of vitrification on viability and chromosome abnormalities in 8-cell mouse embryos at various storage durations

This study was designed to investigate the effect of vitrification and post-thaw survival and chromosomal aberrations caused by vitrification of vitrified 8-cell mouse embryos in comparison with a control group. To this purpose the survival rate and the frequency of chromosomal aberrations were assessed in frozen-thawed 8-cell mouse embryos after various storage durations in the presence of ethylene glycol as cryoprotectant. eight-cell mouse embryos were obtained from NMRI mice 3 days after mating. Retrieved embryos were transferred to vitrification solution containing ethylene glycol as cryoprotectant, then transferred into a vitrification straw using standard technique, and vitrified in liquid nitrogen. Six groups of embryos according to storage duration (24 hours, 1 and 2 weeks, 1-6 months) were frozen. After appropriate storage periods embryos were thawed and studied for their viability 4-6 hours after thawing and intact embryos were transferred to fresh medium containing colcemid. After 48 hours, the embryos were fixed and studied for their chromosome abnormalities using Tarkowsky's drying technique. Results indicate that freezing affects the viability and chromosome structure of embryos when compared with the control group. Furthermore increasing the storage duration reduces the viability and increases the chromosome aberrations of embryos (such as aneuploidy and polyploidy). This result might indicate that the effects of vitrification on the cytoskeleton or other cellular organelle might produce chromosomal alterations leading to cell death.     

Thermoprotection of preimplantation bovine embryos from heat shock by glutathione and taurine.

To determine if deleterious effects of heat shock on embryos could be reduced in vitro by glutathione or taurine, morulae from superovulated cows were placed in modified Hams-F10 medium supplemented with 50 nM glutathione (GSH), 50 mM taurine or neither. Morulae were incubated for 2 hours at 38.5 degrees C, then at 42.0 degrees C (heat shock) or 38.5 degrees C for 2 hours and followed by incubation at 38.5 degrees C for 20 hours. Neither GSH nor taurine enhanced viability or blastocyst development at 38.5 degrees C. At 42.0 degrees C, however, GSH and taurine increased (P less than 0.02) viability (73%, 41% and 26% live for GSH, taurine and control); GSH increased (P less than 0.05) blastocyst development (55% for GSH vs. 30% for control). In conclusion, partial thermoprotection of bovine embryos from heat shock can be achieved in vitro by administration of GSH. Taurine is only slightly effective.     

Shelf-Life Evaluation of Bilayered Human Skin Equivalent, MyDerm™

Skin plays an important role in defense against infection and other harmful biological agents. Due to its fragile structure, skin can be easily damaged by heat, chemicals, traumatic injuries and diseases. An autologous bilayered human skin equivalent, MyDerm™, was engineered to provide a living skin substitute to treat critical skin loss. However, one of the disadvantages of living skin substitute is its short shelf-life, hence limiting its distribution worldwide. The aim of this study was to evaluate the shelf-life of MyDerm™ through assessment of cell morphology, cell viability, population doubling time and functional gene expression levels before transplantation. Skin samples were digested with 0.6% Collagenase Type I followed by epithelial cells dissociation with TrypLE Select. Dermal fibroblasts and keratinocytes were culture-expanded to obtain sufficient cells for MyDerm™ construction. MyDerm™ was constructed with plasma-fibrin as temporary biomaterial and evaluated at 0, 24, 48 and 72 hours after storage at 4°C for its shelf-life determination. The morphology of skin cells derived from MyDerm™ remained unchanged across storage times. Cells harvested from MyDerm™ after storage appeared in good viability (90.5%±2.7% to 94.9%±1.6%) and had short population doubling time (58.4±8.7 to 76.9±19 hours). The modest drop in cell viability and increased in population doubling time at longer storage duration did not demonstrate a significant difference. Gene expression for CK10, CK14 and COL III were also comparable between different storage times. In conclusion, MyDerm™ can be stored in basal medium at 4°C for at least 72 hours before transplantation without compromising its functionality.     

Assessment of canine oocyte viability after transportation and storage under different conditions

To improve assisted reproductive technologies in the domestic dog, different transport treatments were evaluated for their ability to maintain viability of canine oocytes, as assessed by esterase activity 8h after storage or after 48 h of in vitro maturation (IVM) culture. In Experiment 1, ovaries were transported within reproductive tracts or were excised and stored at either 20 or 37 degrees C in phosphate buffered saline. Oocytes collected from reproductive tracts transported at 37 degrees C had the greatest viability after storage (P<0.05). However, after IVM there were no significant differences among any of the four storage conditions in oocyte viability or meiotic resumption (P=0.05). In Experiment 2, isolated oocytes were transported in either TCM-199 with Hank's salts and Hepes buffer or in TL-Hepes at either 20 or 37 degrees C, or in maturation medium equilibrated with 5% CO(2) at 37 degrees C. In Experiment 2, oocytes transported in Hepes buffered media at 37 degrees C had greater viability rates after storage than did those transported in these same media at 20 degrees C or in sodium bicarbonate buffered medium at 37 degrees C (P<0.001). After IVM, oocytes transported in the 37 degrees C treatment groups had greater viability rates than did those transported at 20 degrees C (P<0.01). Overall, isolated oocytes transported at 37 degrees C had greater rates of meiotic resumption than did those transported at 20 degrees C (P<0.05). Taken together, these data indicate that canine oocytes exhibited sensitivity to lesser temperatures and maintained greater rates of viability during transport at 37 degrees C. Isolated oocytes maintained greater viability than oocytes transported in situ. Hepes buffered media increased viability rates for isolated oocytes transported at 37 degrees C compared to a similar medium buffered with sodium bicarbonate.     

Ethanol induced apoptosis in human HL-60 cells

In this study flow cytometric and morphologic methods of apoptosis detection in human promyelocytic leukemia cell line HL-60 were compared. HL-60 cells were harvested at 4, 7, 16, 24 a 48 hours after induction of apoptosis by 3 % ethanol. Little changes were observed both by flow cytometry (decrease of forward scatter, increase of unprocessed cells staining with APO2.7 antibody) and viability determination by Trypan-blue staining until after 7 hours. However, after 4 hours morphologic changes were observed in the nuclear and cytoplasmic structures using Diff-Quik stained cytospin preparations and standard light microscopic techniques (50% apoptotic cells). The same results were obtained by flow cytometric measurement of sub-diploid DNA content (sub-G1 cells), and an increase of staining with APO2.7 antibody in cells permeabilised by digitonin prior to staining. After 7 hours almost all cells exhibited apoptotic morphology. After 16 hours the cell size (forward scatter) decreased significantly, and 54% of unprocessed cells were APO2.7 positive. After 24 hours only 6% of cells were alive (high forward scatter) and these cells were APO2.7 negative. The HL-60 cells did not proliferate during the cultivation in 3% ethanol, and after 48 hours all stained by Trypan blue. HL-60 leukemic cells were CD34-/AC133-, CD33+/CD15+, and only 2% of the cells were CD95+. Induction of apoptosis by ethanol did not enhance CD95 antigen expression.     

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H₂O₂ exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H₂O₂ to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 M) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.     

Spirulina enhances the viability of Lactobacillus rhamnosus E/N after freeze-drying in a protective medium of sucrose and lactulose

Aims: Response surface methodology (RSM) was used to optimize a protective medium for enhancing the viability of Lactobacillus rhamnosus E/N cells during lyophilization. Methods and Results: Spirulina, sucrose and lactulose were selected, on the basis of a Plackett‐Burman factorial design, as important protectants having the following protective effects on cell viability: 102·025, 36·885 and ?34·42, respectively. A full‐factorial central composite design was applied to determine optimal levels of three used agents. Conclusion: The optimal protective medium composition was determined to be: Spirulina 1·304% (w/v), lactulose 5·48% (w/v), and sucrose 13·04% (w/v) (Polish Patent P‐393189). The predictive value of cell viability in this medium was 89·619%, and experimental viability obtained during freeze‐drying was 87·5%. Significance and Impact of the Study: In this study, Spirulina was used for the first time as the protective agent in freeze‐drying medium, significantly increasing lactobacilli viability and giving synbiotic character of the final product.     

Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares

The endometria of 77 barren mares was swabbed simultaneously using a swab guarded with a single cannula and distal, gelatin capsule (completely guarded swab - CGS) and a partially guarded swab (PGS) with an open cannula. Sheep blood (5%) agar plates were inoculated with each swab, while MacConkey's agar plates were inoculated with the swabs from 44 mares. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. Organisms present were identified, counted, and categorized as saprophytic or pathogenic flora. The endometria of all mares were biopsied immediately following swabbing. Histologic evidence of inflammation in biopsy specimens was classified as (1) none, (2) slight, discrete, focal, and (3) slight or moderate, diffuse, widespread infiltration of inflammatory cells. The number of inflammatory cells migrating through the luminal epithelium was counted and averaged. There were significantly fewer CGS than PGS cultures that yielded growth at 24 and 48 hours of incubation after being streaked on blood agar and MacConkey's agar plates. There were fewer pathogenic bacterial or fungal colonies present at 48 hours of incubation on blood agar plates after being streaked with CGS as compared to PGS. There were no differences in the number of pathogenic bacterial or fungal colonies present at 24 hours of incubation on blood agar or at 24 and 48 hours of incubation on MacConkey's agar plates. There was no correlation between CGS or PGS culture of pathogens and severity of histologic inflammation. There was a positive correlation between culture of pathogens and number of inflammatory cells migrating through the luminal epithelium.     

Improved development of DNA-injected bovine embryos co-cultured with mouse embryonic fibroblast cells

The in vitro development of DNA-injected bovine zygotes, produced in vitro, was compared when cultured with or without mouse embryonic fibroblasts (MEF). The in vivo viability of the embryos produced in these in vitro culture systems was assessed by single or double transfer to recipients taken to term. For these experiments, in vitro fertilized oocytes were not injected (Experiment 1) or were injected with pBL1 gene (Experiment 2) and then cultured for 2 days in CR1aa medium supplemented with 3 mg/ml BSA at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air. Embryos that developed to the 4- to 8-cell stage at the end of this period were randomly assigned to the two cultured systems and cultured for a further 5 days in groups of 10 to 15 embryos in 0.75 ml medium. These two culture systems were CR1aa medium alone or co-culture with MEF in CR1aa medium supplemented with 10% fetal bovine serum (FBS). Every 48 h, 0.5 ml of the medium was replaced with fresh CR1aa medium and at Day 5 of culture, both media were supplemented by the addition of 5.56 mM glucose and 1x GMS-X supplement solutions. Results were assessed as morphological development of the embryos and data were analyzed by Chi-square test or Student's t-test.The development rate of in vitro fertilization (IVF)-derived embryos co-cultured with MEF (24.4%, 49/201) was significantly higher than those cultured alone (14.4%, 28/194; P<0.05) in Experiment 1. There was a similar difference between the treatments in the proportions of embryos which reached the hatching stage or hatched (10.9%, 22/201 vs. 4.1%, 8/194, respectively; P<0.05). DNA-injected embryos co-cultured with MEF (13.7%, 28/205) showed a higher developmental rate than that of the embryos cultured without MEF (6.7%, 13/193; P<0.05) in Experiment 2. Following the transfer to recipients of one or two DNA-injected blastocysts, the pregnancy rates for two culture systems were similar (MEF co-culture 27.4%, 23/84; CR1aa culture 24. 2%, 16/66). However, the numbers of calves born alive from these pregnancies were higher on the MEF co-culture group (82.6%, 19/23) than the CR1aa culture group (56.2%, 9/16). It was concluded that in vitro embryo development to the blastocyst stage and subsequent in vivo development to term of DNA-injected bovine embryos was improved in comparison to culture in CR1aa alone when the last 5 days of in vitro culture were in a MEF co-culture system.     

5-脂氧合酶激活蛋白小干扰RNA诱导人乳腺癌细胞凋亡的初步研究

目的：研究5-脂氧合酶激活蛋白（FLAP）的表达抑制对乳腺癌细胞凋亡的诱导作用。方法：通过小干扰RNA（siRNA）抑制乳腺癌细胞MDA-MB-231中FLAP的表达,用流式细胞仪检测膜联蛋白（annexin）-V标记的早期凋亡细胞,用Western印迹检测细胞凋亡相关蛋白的水平。结果：转染了FLAP siRNA的乳腺癌细胞,24h后FLAP的表达被抑制,17％的细胞出现早期凋亡;48h时早期凋亡细胞增加到32．1％;72h时早期凋亡细胞下降到13．8％,而死亡或凋亡晚期细胞占到61．3％。在细胞凋亡过程中,Bcl-2水平下降,而细胞色素c、胱冬蛋白酶（caspase）-3的水平逐渐增高。结论：FLAP的表达抑制可以诱导乳腺癌细胞通过Bcl-2和胱冬蛋白酶-3途径发生凋亡。