仙茅叶片的组织培养及其细胞学观察

目的通过对仙茅叶片组织培养的研究与细胞学观察,为仙茅的快速繁殖提供技术依据。方法将仙茅幼叶培养在MS培养基,通过设计不同的光照条件,附加不同的激素、水解酪蛋白和活性炭成分,调查出愈率和成苗率。细胞学观察采用石蜡切片法。结果对仙茅叶片的愈伤诱导,黑暗的效果好于光照;在所试验的培养基成分中,适宜的激素配比是2.0mg/L2,4-D或6-BA1.5mg/L 2,4-D2.5mg/L,并且附加300mg/L水解酪蛋白和0.2%活性炭,对于仙茅叶片的离体成苗较好;培养后,愈伤组织主要由叶片的中脉产生,位于中脉上表皮内侧的薄壁细胞首先启动分裂,随后维管束鞘薄壁细胞及其外侧的叶肉细胞也启动分裂,参与愈伤组织的形成。愈伤分化时,芽发生于愈伤组织的表面,根发生于愈伤组织的内部。结论可以通过仙茅幼叶的组织培养进行仙茅的快速繁殖。     

Complete replication of human cytomegalovirus in explants of first trimester human placenta

Tissue integrity and viability of first trimester placenta explants were obtained in culture for 3 weeks. Explants were infected with human cytomegalovirus (HCMV), several cycles of HCMV replication were obtained and the progression of the infection was observed within a tissue that maintains its normal cellular organization. In agreement with recent clinical data, 3 weeks were necessary for the virus to colonize the placenta fully. Complete HCMV replication was observed in trophoblasts, followed by subsequent transmission of the infection to the stromal fibroblasts and fetal endothelial capillary cells. Viral DNA replication was monitored and the production of infectious viral progeny documented.     

Anti-DNA antibodies cross-reacting with laminin inhibit trophoblast attachment and migration: implications for recurrent pregnancy loss in SLE patients

PROBLEM: Systemic lupus erythematosus (SLE), an autoimmune disease, is associated with reduced fetal survival, recurrent abortions, and other pregnancy complications. Some of the autoantibodies found in SLE bind to laminins (LNs), which play an important role in the implantation of the fertilized ovum in humans. METHOD OF STUDY: To elucidate the role of these specific autoantibodies, chorionic villous explants from 6 7-week-old human placentas were established as organ cultures on laminin-1 (LN-1), collagen IV (CN-IV) or uncoated culture dishes. The cultures were then exposed to a mouse monoclonal anti-DNA/anti-LN-1 antibody, to human polyclonal lupus antibodies cross-reacting with LN-1, a function-blocking polyclonal antibody to LN-1, polyclonal antibodies to CN-IV, or IgG control. RESULTS: The explants attached to LN-1 and CN-IV, but not to uncoated culture dishes. LN-1 promoted migration of trophoblast, whereas CN-IV promoted migration of fibroblast-like cells. Trophoblast attachment and migration were abolished in a dose-dependent manner by all three antibodies to LN-1, but not by antibodies to CN-IV or IgG control. Furthermore, the effect of anti-LN antibodies was abolished by preincubating them with LN-1. CONCLUSIONS: These studies suggest that anti-DNA antibodies cross-reacting with LNs may play a role in early pregnancy failure in SLE patients by interfering with placental implantation.     

Cell vitality in cartilage tissue culture following excimer laser radiation: An in vitro examination

Excimer laser is used for cartilage debridement, although the resulting cell damage is yet unclear. For examination of cartilage survival after treatment, we used short-term tissue cultures of human joint cartilage. Specimens were treated with a XeCl-Excimer laser using different laser parameters, pulse energies, and repetition rates. Following treatment, discs were cultured for 8 days prior to examination. In contrast to the 20 μm damage zone as instant visible effect in histomorphologic examinations, we found a 0.3 mm zone in which ～ 50% of cartilage cells had morphological signs of damage on light microscopic examinations. Autoradiography revealed that cartilage cells in an 0.5–0.7 mm area surrounding the laser craters had no collagen synthesis. This examination indicates that cell damage of excimer laser is higher than expected from prior studies. © 1993 Wiley-Liss, Inc.     

Oxidative stress response of rat testis to model prooxidants in vitro and its modulation

Understanding the effects of prooxidants on mammalian testis either in vitro or in vivo is important, since recent evidence shows that oxidative stress can play a vital role in the etiology of male infertility. In this investigation, we have examined the oxidative stress response of adult rat testis in vitro as induced by model prooxidants (tert-butyl hydroperoxide (t-bHP) and cumene hydroperoxide (cHP) deploying two models—testicular cell suspensions (TCS) and testicular explants (TE). Significant induction of oxidative stress was observed in both models as evidenced by increased thiobarbituric acid reactive substances (TBARS) levels on incubation with hydroperoxides. The response was both concentration and time dependent. At the highest concentration (200 μ ), both hydroperoxides induced a 100% increase in the TE model, compared with a dramatic (380–560%) increase in the TCS model during a 30-min incubation. Further evidence of oxidative stress such as reduction in the GSH levels and alterations in the activity of antioxidant enzymes (catalase and glutathione peroxidase) were also obtained in the TE model. In the TE model, radical scavengers, namely thiourea, urea and mannitol, as well as antioxidants such as glutathione and catalase inhibited the t-bHP-induced lipid peroxidation response to varying degree. A similar degree of protection was also evident with known antioxidants such as ascorbic acid, butylated hydroxyanisole and butylated hydroxytoluene in the TE model. Further co-incubation of TE either with mercaptosuccinate (a potent glutathione peroxidase inhibitor) or 3-aminotriazole (an irreversible catalase inhibitor) resulted in a marked increase in t-bHP-induced lipid peroxidation, clearly suggesting the importance of both of these enzymic antioxidants in rat testis in vitro. These data suggest that the TE model may be further utilized to screen antioxidants in vitro and also investigate the prooxidant potency of xenobiotics in testicular cells.     

Reversal by retinoids of keratinization induced by benzo[a]pyrene in normal hamster tracheal explants: Comparison with the assay involving organ culture of tracheas from vitamin A-deficient hamsters

In a defined culture system for hamster tracheal explants, the activity of 12 different retinoids was evaluated for reversal of keratinization induced by exposure to the carcinogen, benzo[a]pyrene (BP-HTOC assay). The effects of retinoids in this system were compared to those in a defined culture system for tracheal explants from vitamin A-deficient hamsters (standard-HTOC assay). In both assays, all-trans-retinoic acid (RA) and 13-cis-RA were the most active retinoids. For RA and 13-cis-RA, the values of ED₅₀ determined in the BP-HTOC bioassay were 4 × 10⁻¹² and 1 × 10⁻¹¹ M, respectively, whereas the corresponding values in the standard HTOC assay were 2 × 10⁻¹¹ and 3.3 × 10⁻¹⁰ M. For all 12 retinoids, the ED₅₀ values from the BP-HTOC were lower than those from the standard-HTOC assay, and there was also a statistically significant correlation between the rank-ordering of ED₅₀ values from the 2 assays. Among 3 N-(retinoyl)amino acids examined in both assays, N-(retinoyl)leucine was the most active, N-(retinoyl)phenylalanine the least active, and N-(retinoyl)alanine intermediate. Among a novel series of bifunctional retinoic acid analogues, the dicarboxyl derivative was the most active. On the basis of these results, the BP-HTOC assay appears to be one of the most sensitive assays for retinoids yet developed. This assay is an appropriate model for evaluating the chemopreventive potential of new retinoids in vitro.     

Survival,development, and electrical activity of central nervous system myelinated axons exposed to tumor necrosis factor in vitro

Spinal cord explants from CD-1 mouse embryos were cultured in Maximow slide assemblies to promote myelin development. At about 20 days in vitro, recombinant human or mouse tumor necrosis factor alpha (TNFa) was added. Observed 3–8 days later, myelin was largely intact. The myelin blistering and oligodendrocyte damage seen in other strains were generally absent. Axonal conduction was measured optically through the use of a voltage-sensitive dye. Action potential shape, conduction velocity, and refractory period were all unchanged by exposure to TNFa. Two series of explants were grown with TNFa present continuously throughout the culture period. Observed with light and electron microscopy, myelin developed in at least 50% of the explants treated with recombinant mouse TNFα and 80% of those exposed to recombinant human TNFα. Optically recorded action potentials were of normal shape and refractory period. Conduction velocities were slightly lower than controls. CD-1 mouse central nervous system contains TNFα receptors and yet was resistant to myelin damage. The apparent strain specificity of TNFα disruption of myelin may result from more indirect modes of action, including interaction with other cytokines produced by glial cells. Survival of axonal conduction suggest that Na⁺ channel function remains intact following TNFα exposure. © 1995 Wiley-Liss, Inc.     

The effect of in vitro tracheal occlusion on branching morphogenesis in fetal lung explants from the rat nitrofen model of congenital diaphragmatic hernia

Background/Purpose

Fetal tracheal occlusion (TO) has been investigated as a treatment option for lung hypoplasia secondary to congenital diaphragmatic hernia. Tracheal occlusion has been shown to accelerate lung growth, but its effect on bronchial branching is unknown. In this study, we characterize the effects of in vitro TO on bronchial branch development in fetal lung explants derived from the nitrofen rat model of congenital diaphragmatic hernia.

Methods

Rat dams were gavaged nitrofen on gestational day 9.5, and fetal lungs were harvested for explant culture on gestational day 14 (term, 22 days). Four experimental groups were investigated, with TO performed ex vivo using cautery: control, control + TO, nitrofen, and nitrofen + TO. Explants were incubated for 72 hours. Representative photographs were taken at 0, 24, 48, and 72 hours from the time of culture, and the number of distal branches was counted for each explant. The Student t test was used to compare distal branch measurements.

Results

A minimum of 12 fetal lung explants were cultured for each group. By 24 hours, all explants undergoing TO had more branch iterations than explants that did not. Moreover, TO in nitrofen-exposed explants increased bronchial branching to control levels by 24 hours in culture.

Conclusion

Our results suggest that TO at day 14 increases branching in normal and nitrofen-exposed lung explants. In addition, TO increases airway branching in nitrofen-exposed explants to control levels suggesting that early TO reverses the lung hypoplasia seen in this model.     

Secretory Factors From Rat Adipose Tissue Explants Promote Adipogenesis and Angiogenesis

Adipogenic differentiation of adipose‐derived stem cells (ASCs) is known to be affected by many promoting and inhibiting factors, and correlated with surrounding cells and extracellular matrix. However, few studies have evaluated the effect of secreted biological molecules from adipose tissue explants on the growth of ASCs. In this study, ASCs were isolated and the secretory factors from adipose tissue explants (SFAE) were prepared from adipose tissue explants. The multilineage differentiation potential of ASCs was determined using different inductive media. The influence of SFAE on the colony formation and proliferation of ASCs was determined using colony‐forming efficiency assay and Cell Counting Kit‐8 assay, respectively. Real‐time polymerase chain reaction and Western blot were performed to analyze the beneficial effect of SFAE on the adipogenesis and angiogenesis of ASCs. Utilizing gelatin scaffold, the influence of SFAE on ASCs was investigated in vivo. Subsequently, cell/gelatin constructs were cultured in a subcutaneous pocket on a rat dorsum for 2 and 9 weeks, and the resultant samples were histologically evaluated. Results showed that ASCs derived from adipose explants can differentiate along multiple lineages in vitro. Utilizing SFAE, the proliferation and colony‐forming efficiency of ASCs were inhibited, while the expression of adipogenesis markers such as C/EBPβ, PPARγ2, and LPL, as well as angiogenesis factor VEGF‐A were promoted. Moreover, the beneficial effect of SFAE on adipogenesis was revealed in vivo. In conclusion, our results suggested that SFAE has beneficial influence on adipogenesis and angiogenesis both in vitro and in vivo.