TGF-ß1 gene-engineered mesenchymal stem cells induce rat cartilage regeneration using nonviral gene vector.

This study evaluated the potential of utilizing transfected pTGFß-1 gene-engineered rat mesenchymal stem cells (MSCs) using nonviral vector to promote cartilage regeneration. Pullulan-spermine was used as the nonviral gene vector and gelatin sponge was used as the scaffold. MSCs were engineered with TGF-ß1 gene with either the three-dimensional (3D) reverse transfection system or the two-dimensional (2D) conventional transfection system. For the 3D reverse transfection system, pullulan-spermine/pTGF-ß1 gene complexes were immobilized to the gelatin sponge, followed by the seeding of MSCs. Pullulan-spermine/pTGF-ß1 gene complexes were delivered to MSCs cultured in the plate to perform the 2D conventional transfection system, and then MSCs were seeded to the gelatin sponge. Then, TGF-ß1 gene-transfected MSC seeded gelatin sponge was implanted to the full-thickness cartilage defect. Compared with the control group, both groups of TGF-ß1 gene-engineered MSCs improved cartilage regeneration through optical observation and histology staining. So, with pullulan-spermine as the nonviral vector, TGF-ß1-gene engineered MSCs can induce cartilage regeneration in vivo.

Impregnation of plasmid DNA into three-dimensional PLGA scaffold enhances DNA expression of mesenchymal stem cells in vitro.

Current efforts had been made to undertake a three-dimensional (3-D) reverse transfection of bone marrow-derived mesenchymal stem cells (BM-MSCs) in PLGA scaffolds. As a kind of multipotent stem cells, BM-MSCs show great potential and tremendous capacity in the gene transfection field and PLGA 3-D scaffold has been shown to be a biomaterial that provides structural support to cells proliferation and tissue engineering. The objective of this study was to assess the transfection efficiency of BM-MSCs with a 3-D reverse transfection method by using PLGA scaffold and observe the SEM photographs of BM-MSCs cultured on PLGA scaffold. BM-MSCs were cultured in 3-D PLGA scaffold which was incorporated with pullulan-spermine/pGL3. It was shown that the gene expression duration of BM-MSCs transfected using 3D reverse method with pullulan-spermine/DNA in the presence of serum maintained 12 days at high levels as compared with the plasmid DNA in medium, and scanning electronic microscopy (SEM) photographs of BM-MSCs cultured on PLGA scaffold exhibited robust cell attachment and viability when cultured in PLGA scaffold in vitro. This study demonstrates that the 3-D reverse transfection method of BM-MSCs using PLGA scaffold could achieve long gene expression in a relatively high level, therefore this transfection system is promising in gene transfection and tissue engineering.

Effective gene delivery to mesenchymal stem cells based on the reverse transfection and three-dimensional cell culture system.

PURPOSE: To enhance the level and prolong the duration of gene expression for gene-engineered rat mesenchymal stem cells (MSCs) using non-viral vector. METHODS: A novel transfection system based on reverse transfection method and three-dimensional (3D) scaffold was developed. The reverse gene transfection system was evaluated for transfection efficiency compared to conventional methods. Collagen sponge and polyethylene terephthalate non-woven fabric were introduced as scaffolds to perform 3D culture with reverse transfection. pDNA coding TGFß-1 was delivered to MSCs to assess its ability in inducing chondrogenesis with the 3D non-viral reverse transfection system. RESULTS: The reverse transfection method induced higher transgene levels than the conventional transfection in the presence of serum. The electric charge of the anionic gelatin plays an important role in this system by affecting the release pattern of the gene complexes and through the adsorption of serum protein to the substrate. During a long-time in vitro culture, MSCs cultured on 3D scaffolds exhibited a higher transgene expression level and more sustained transgene expression than those cultured and transfected on the two-dimensional substrate. CONCLUSIONS: The combination of reverse transfection system with 3D cell culture scaffold benefits the cell proliferation and long-time gene transfection of MSCs.

[Effect of Chidamide on the Killing Acitivity of NK Cells Targeting K562 Cells and Its Related Mechanism In Vitro].

OBJECTIVE: To investigate the effect of chidamide on the killing activity of NK (Natural killer cell, NK) cells targeting K562 cells and its related mechanism. METHODS: K562 cells were pretreated with chidamide at different concentrations and cocultured with NK cells at different effect-target ratios. The killing effect of chidamide on K562 cells by NK cells, the expression of natural killer group 2 member D (NKG2D) ligands and apoptosis rate of K562 cells were detected by flow cytometry. RESULTS: The killing sensitivity of NK cells to K562 cells could be enhanced by chidamide. The expression of ULBP2 on K562 cell surface could be up-regulate, however, the expression of ULBP1 and MICA/MICB showed no statistically difference as compared with control group. Chidamide showed no obvious cytotoxicity to K562 cells. CONCLUSION: Chidamide can significantly improve killing efficiency of NK cells on K562 cells, which may be related to the up-regulation of ULBP2 expression.

[Effects of fasudil on the expression of Rho kinase-1 and airway inflammation in a mouse model of asthma.].

OBJECTIVE: To explore the role of Rho kinase-1 (ROCK-1) in airway inflammation of asthma by observing the effects of fasudil, a specific inhibitor of ROCK-1, on the expression of Rho kinase-1 and airway inflammation in a mouse model of asthma. METHODS: Twenty-four female BALB/c mice were randomly divided into 3 groups (n = 8 each): a control group, an asthmatic group and a treatment group. Mice in the asthmatic and the treatment groups were sensitized by intraperitoneal injection of OVA (25 microg) precipitated with 1 mg of alum in 200 microl of saline on days 1 and 15, and subsequently challenged by nebulization of 2% OVA on days 22-26. Mice in the control group were sensitized with Al(OH)3 saline and challenged with saline instead of OVA. Mice of the treatment group were injected intraperitoneally with fasudil (10 mg/kg) 1 h before each OVA challenge. All the mice were killed 24 h after the final challenge, and bronchoalveolar lavage fluid (BALF) was collected for counting total inflammatory cells and eosinophils (EOS). Cytokines and chemokines in BALF were measured by ELISA. The lung tissue slides were examined histologically. The protein and mRNA expression of ROCK-1 were measured by immunohistochemistry and RT-PCR respectively. RESULTS: (1) OVA challenge in mice of the asthmatic group caused a marked increase in the number of the total cells and eosinophils in BALF (q = 25.909, 35.002, respectively, all P < 0.01). When fasudil was applied, both the total cell counts and the eosinophil numbers were significantly decreased. The total cell number was decreased from (1.45 +/- 0.12) x 10(9)/L to (0.89 +/- 0.09) x 10(9)/L (q = 16.676, P < 0.01), and the number of eosinophils was decreased from (0.52 +/- 0.06) x 10(9)/L to (0.20 +/- 0.04) x 10(9)/L (q = 21.537, P < 0.01). (2) Compared with the control group, OVA challenge in mice of the asthmatic group induced eotaxin, IL-5 and IL-13 release into BALF (q = 18.246, 23.009, 25.826, respectively, all P < 0.01). The eotaxin, IL-5 and IL-13 levels in BALF after OVA challenge were (45 +/- 8) ng/L, (157 +/- 23) ng/L and (429 +/- 46) ng/L, respectively. Application of fasudil resulted in inhibition of the augmented levels of eotaxin, IL-5 and IL-13 in BALF, decreased to (20 +/- 5) ng/L, (57 +/- 14) ng/L and (254 +/- 28) ng/L, respectively (q = 13.119, 17.503, 8.449, respectively, all P < 0.01). (3) Mice in the control group showed no detectable inflammatory response in the lung, whereas OVA-challenged mice induced infiltration of inflammatory cells around airways and blood vessels. The majority of the infiltrated inflammatory cells were eosinophils. Application of fasudil significantly reduced the infiltration of inflammatory cells in the peribronchial areas compared with the asthmatic mice. (4) The expression levels of ROCK-1 mRNA and protein in mice of the asthmatic group (0.67 +/- 0.05 and 1.09 +/- 0.06) were much higher than those of the control group (0.26 +/- 0.05 and 0.87 +/- 0.09) (q = 25.614, 8.156, all P < 0.01). When fasudil was administered, the expression levels of ROCK-1 mRNA and protein were significantly attenuated to 0.35 +/- 0.04 and 0.98 +/- 0.08, compared with those of the asthmatic group (q = 20.379, 4.135, all P < 0.01). (5) The expression level of ROCK-1 mRNA was positively correlated with the number of eosinophils and the levels of eotaxin, IL-5 and IL-13 in BALF (r = 0.709, 0.600, 0.613, 0.650, all P < 0.01). CONCLUSION: Airway inflammation of bronchial asthma was improved by inhibiting expression and activity of ROCK-1 by fasudil, suggesting that ROCK-1 may be involved in asthmatic airway inflammation induced by OVA challenge.

Induction of MHC class I-related chain B (MICB) by 5-aza-2'-deoxycytidine.

5-Aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, exerts antitumor activity through induction of cell cycle arrest, apoptosis and DNA damage. In this study, we showed that MHC class I-related chain B (MICB), a ligand of the NKG2D receptor expressed by natural killer cells and activated CD8(+) T cells, was upregulated following 5-aza-dC treatment. The upregulation of MICB was accompanied by promoter DNA demethylation and DNA damage. Furthermore, the upregulation of MICB was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase. Our results suggest that promoter DNA demethylation, in combination with DNA damage, contribute to the upregulation of MICB induced by 5-aza-dC.

[Effect of MiR-99a-5p on the Differential Ability of Human Bone Marrow Mesenchymal Stem Cells].

OBJECTIVE: To investigate the effect of microRNA-99a-5p (miR-99a-5p) on differentiation ability of human bone marrow mesenchymal stem cells (BM-MSC). METHODS: BM-MSC was cultured and then transfected with miR-99a-5p mimics or inhibitors. The transfection efficiency was detected by real-time quantitative PCR. The effects of miR-99a-5p on the adipogenic and osteogenic differentiation ability of BM-MSC were detected by differentiation experiment. RESULTS: As compared with the negative control group, the expression of miR-99a-5p was significantly up-regulated after transfection with miR-99a-5p mimics(P<0.001), the expression of miR-99a-5p was down-regulated after transfection with miR-99a-5p inhibitor (P<0.001). In osteogenic differentiation experiments, the miR-99a-5p overexpression could promote the osteogenic differentiation, while the downregulation of miR-99a-5p expression inhibited the osteogenic differentiation. The same results were obtained by semi-quantitative detection through spectrophotometry. In the adipogenic differentiation test, transfection of miR-99a-5p mimics or inhibitors had no significant effect on the adipogenic differentiation of BM-MSC. CONCLUSION: Overexpression of miR-99a-5p can promote the osteogenic differentiation of BM-MSC, but no significant effects are observed in the adipogenic differentiation.

Dynamics of a nonlocal discrete Gross-Pitaevskii equation with defects.

We study the dynamics of dipolar gas in deep lattices described by a nonlocal nonlinear discrete Gross-Pitaevskii equation. The stabilities and the propagation properties of traveling plane waves in the system with defects are discussed in detail. For a clean lattice, both energetic and dynamical stabilities of the traveling plane waves are studied. It is shown that the system with attractive local interaction can preserve the stabilities, i.e., the dipoles can stabilize the gas because of repulsive nonlocal dipole-dipole interactions. For a lattice with defects, within a two-mode approximation, the propagation properties of traveling plane waves in the system map onto a nonrigid pendulum Hamiltonian with quasimomentum-dependent nonlinearity (induced by the nonlocal interactions). Competition between defects, quasimomentum of the gas, and nonlocal interactions determines the propagation properties of the traveling plane waves. Critical conditions for crossing from a superfluid regime with propagation preserved to a normal regime with defect-induced damping are obtained analytically and confirmed numerically. In particular, the critical conditions for supporting the superfluidity strongly depend on the defect type and the quasimomentum of the plane waves. The nonlocal interaction can significantly enhance the superfluidity of the system.

Non-viral gene delivery carrier and its three-dimensional transfection system.

An increasing number of non-viral vectors are being developed for the use of gene delivery nowadays, among which cationic polymers and lipoplexes receive most attention. Most of these researches are focused on how to increase the transfection efficiency of non-viral vectors as well as the reduction of toxicity. In this review, we go over new strategies to reduce the toxicity of cationic polyplexes such as poly(ethylene-imine) and the construction of highly effective gene transfer vector lipoplexes. In addition, since transformation of gene expression system from two-dimensional (2D) substrate to 3D scaffold triggers far better transfection efficiency, the non-viral vectors applied in 3D transfection system have also been reviewed.

Microemulsions as drug delivery systems to improve the solubility and the bioavailability of poorly water-soluble drugs.

IMPORTANCE OF THE FIELD: Microemulsions have been studied extensively as potential drug delivery vehicles for poorly water-soluble drugs. An understanding of the physicochemical and biopharmaceutical characteristics of the microemulsions according to administration routes will provide guidance for designing the formulations of microemulsions. AREAS COVERED IN THIS REVIEW: In this paper, the use and the characteristics of microemulsions as drug delivery vehicles are reviewed. As the formulations of the microemulsion always include a great amount of surfactant and co-surfactant, which may cause hemolysis or histopathological alterations of the tissue, the potential toxicity or the irritancy of microemulsions is also discussed in this paper. WHAT THE READER WILL GAIN: Developments of microemulsions for poorly water-soluble drugs in recent years are included in this review. Several factors limiting the commercial or clinical use of microemulsions are also discussed. TAKE HOME MESSAGE: Considering the potential in enhanced drug uptake/permeation and facing the limitations, their unique properties make microemulsions a promising vehicle for poorly water-soluble drugs.

Preparation, characterisation and anti-tumour activity of Ganoderma lucidum polysaccharide nanoparticles.

OBJECTIVES: The aim was to prepare novel Ganoderma lucidum polysaccharide nanoparticles and to evaluate the physicochemical properties and anti-tumour activity in in-vitro cytotoxicity studies using HepG2, HeLa and A549 cancer cell lines, and growth promotion effects on mouse spleen cells. METHODS: Chitosan nanoparticles loaded with G. lucidum polysaccharide were prepared using the ion-revulsion method. The diameter distribution of the particles and the surface charge were measured using a zetasizer analyser. The entrapment efficiency and drug loading capacity were examined by the diethylaminoethanol weak anion exchange method. The cytotoxic effects of nanoparticles on tumour cells and the growth promotion effects on mouse spleen cells were tested using the MTT assay. KEY FINDINGS: Nanoparticles loaded with G. lucidum polysaccharide at 6 microg/ml and chitosan/sodium tripolyphosphate (mass) ratio of 5.5 had significantly greater cytotoxic effects on tumour cells and growth promotion effects on mouse spleen cells than empty nanoparticles. CONCLUSIONS: G. lucidum polysaccharide nanoparticles showed significant anti-tumour efficacy, having both cytotoxic effects on tumour cells and growth promotion effects on spleen cells, making it a promising candidate in the clinical setting.

N/P ratio significantly influences the transfection efficiency and cytotoxicity of a polyethylenimine/chitosan/DNA complex.

For designing a complex vector that has the advantages of both polyethylenimine (PEI) and chitosan for gene delivery, a PEI/chitosan/DNA complex was constructed at various N/P ratios (the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of DNA) and both the cytotoxicity and the transfection efficiency of the vector were evaluated. The results demonstrated that the chitosan/DNA binding degree was depended on the N/P ratio. The mean size of the complex vector was between 100 nm and 150 nm. Compared with PEI/DNA, the complex vector (PEI/chitosan/DNA with chitosan/DNA N/P=4, PEI/DNA N/P=10) appeared to have low cytotoxicity, which maintained the cell survival rate at greater than 80%, and showed higher transfection efficiency of nearly 1000 fold compared with that using chitosan/DNA alone. Furthermore, the expression efficiency of the complex vector carrying enhanced green fluorescent protein was not inhibited in the presence of serum in both HeLa cells and A549 cells. The PEI/chitosan complex may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.

A novel camptothecin derivative incorporated in nano-carrier induced distinguished improvement in solubility, stability and anti-tumor activity both in vitro and in vivo.

PURPOSE: An oil/water nanoemulsion was developed in the present study to enhance the solubility, stability and anti-tumor activity of a novel 10-methoxy-9-nitrocamptothecin (MONCPT). MATERIALS AND METHODS: MONCPT nanoemulsion was prepared using Lipoid E80 and cremophor EL as main emulsifiers by microfluidization. The droplet size of the nanoemulsion was measured by dynamic light scattering. In vitro drug release was monitored by membrane dialysis. Kinetics of MONCPT transformed into carboxylic salt was performed in phosphate buffer at different pH. Hemolysis of MONCPT nanoemulsion was conducted in rabbit erythrocytes. Solubilization character of MONCPT in nanoemulsion was experimented using Nile red as a solvatochromic probe. In vitro cytotoxicity of the nanoemulsion was measured in A549 and S180 cells using Sulforhodamine B protein stain method, and suppression rate of tumor growth was investigated in S180-bearing mice. The cell cycle effects of MONCPT nanoemulsion on S180 cells were analyzed by flow cytometry. Distribution of the nanoemulsion in A549 cells and S180-bearing mice were also investigated by fluorescence image. RESULTS: MONCPT is incorporated in the nanoemulsion in form of lactone with concentration of 489 microg/ml, more than 200 folds higher than that in water. Experiments using Nile red as a solvatochromic probe indicated that more MONCPT might be located in the interfacial surfactant layer of the nanoemulsion than that in discrete oil droplet or continuous aqueous phase. Nanoemulsion could release MONCPT in a sustained way, and it was further shown to notably postpone the hydrolysis of MONCPT with longer hydrolysis half-life time (11.38 h) in nanoemulsion at pH 7.4 than that of MONCPT solution (4.03 h). No obvious hemolysis was caused by MOCPT nanoemulsion in rabbit erythrocytes. MONCPT nanoemulsion showed a marked increase in cytotoxic activity, 23.6 folds and 28.6 folds in S180 cells and A549 cells respectively via arresting the cell at G2 phase, compared to that induced by MONCPT injection. It correlated well to the in vivo anti-tumor activity of MONCPT nanoemulsion with suppression rate of 93.6%, while that of MONCPT injection was only 24.2% at the same dosage. Moreover, nanoemulsion exhibited enhanced capability of delivering drug into malignant cell's nucleus in vitro and induced drug accumulation in tumor in S180-bearing mice using in vivo imaging. CONCLUSION: The nanoemulsion prepared exhibited an improved MONCPT solubility, stability and anti-tumor activity, providing a promising carrier for cancer chemotherapy using MONCPT.

Decreased Dicer expression elicits DNA damage and up-regulation of MICA and MICB.

RNA interference (RNAi) acts constitutively to silence the innate immune response, and innate immunity genes are misregulated in Dicer-deficient Caenorhabditis elegans. Here, we show that inhibition of Dicer expression by RNAi in human cells up-regulates major histocompatibility complex class I-related molecules A and B (MICA and MICB). MICA and MICB are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. We reveal that knockdown of Dicer elicits DNA damage. Up-regulation of MICA and MICB by Dicer knockdown is prevented by pharmacologic or genetic inhibition of DNA damage pathway components, including ataxia telangiectasia mutated (ATM) kinase, ATM- and Rad3-related kinase, or checkpoint kinase 1. Therefore we conclude that up-regulation of MICA and MICB is the result of DNA damage response activation caused by Dicer knockdown. Our results suggest that RNAi is indirectly linked to the human innate immune system via the DNA damage pathway.

Constituents from the stems of Hibiscus taiwanensis.

Five new compounds, hibicuslide A (1), hibicuslide B (2), hibicuslide C (3), hibicutaiwanin (4), hibicusin (5), and fifty-one known compounds have been isolated from the stems of Hibiscus taiwanensis. The structures of these compounds were determined by spectroscopic and chemical transformation methods. Among them, mansonone H (19) and uncarinic acid A (30) inhibited HIV replication in H9 lymphocyte cells. The 9,9'-O-feruloyl-(-)-secoisolaricinresinol (12), myriceric acid C (29), and uncarinic acid A (30) showed cytotoxic activity against human lung carcinoma and breast carcinoma.

Cytotoxicity of phenylpropanoid esters from the stems of Hibiscus taiwanensis.

The separation of an extract prepared from the stems of the previously uninvestigated Hibiscus taiwanensis led to the isolation of three new phenylpropanoid esters, (7S,8S)-demethylcarolignan E (1), hibiscuwanin A (2), hibiscuwanin B (3), in addition to eight known ones. The structures of these compounds were elucidated by spectroscopic and chemical transformation studies. In cytotoxicity evaluation of the isolates, 9,9'-O-feruloyl-(-)-secoisolaricinresinol (8) showed strong cytotoxic activity against human lung carcinoma and breast carcinoma cell lines in an in vitro cytotoxicity assay with EC(50) values of 1.8 and 3.9 microg/mL, respectively.