Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca²⁺ on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca²⁺ on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca²⁺ (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca²⁺ stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca²⁺ significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.     

Ghrelin is a Regulator of Cellular Apoptosis and Proliferation in the Rat Ovary

Apoptosis and proliferation are the common and essential events of reproductive function and development in the ovary, especially during follicular growth and atresia or luteal regression. Therefore, this study was set to investigate the influence of ghrelin treatment on apoptosis and proliferation specific indices in the rat ovary. Twenty-eight adult female Wistar rats were randomly allocated into control and treatment groups. Treatment group (n = 14) received 3 nmol of ghrelin as subcutaneous injection for 14 consecutive days or vehicle (normal saline) to the control rats. The animals from each group were equally sacrificed on days 9 and 14 after onset of ghrelin treatment and their ovaries were taken for immunohistochemical evaluation and caspase-3 assay. Accumulation of apoptosis-associated peptide Bax was significantly reduced following ghrelin treatment particularly in granulosa and luteal cells on day 14 (P < 0.01). In contrast, immunoreactivity against anti-apoptotic protein Bcl-2 was significantly elevated in ghrelin-exposed animals in granulosa, theca and luteal cells (P < 0.05). However, ghrelin administration was not able to change caspase-3 activity prominently, so that the means of enzyme activity were not statistically significant between groups (P > 0.05). Moreover, significant up-regulation of proliferation-associated peptide PCNA was also seen in the granulosa, theca and luteal cells of ghrelin-treated rats by day 14 (P < 0.05), but not on day 9. These findings indicate the first evidence of ghrelin involvement in the control of key gonadal functions, apoptosis and proliferation in the rat ovary, which is mainly mediated through decrease in Bax/Bcl-2 ratio consistent with upstream of PCNA level, however not depends on the reduction of caspase-3 activation. This may have potential implications that ghrelin can be considered as an apoptotic modulator of some ovarian disorders.     

Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle

Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P < 0.0001). After 28-day myogenic induction, higher expression levels of skeletal muscle-specific genes were observed in MDSCs than FMSCs (P < 0.01), and the lowest expression levels were demonstrated in ADSCs among three cells (P < 0.01). Besides, M-Cad and MyHC expressions in ADSCs were not detected by immunofluorescence or real-time quantitative PCR. Furthermore, after 14 days adipogenic induction, PPARγ2, LPL and aP2 mRNA expressions were higher in ADSCs vs. MDSCs (P < 0.01). Besides, MSCs from adult or fetal muscle expressed higher OCN and OPN than ADSCs after 28 days osteogenic induction (P < 0.01). Taken together, our results suggested that cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.     

Is high prevalence of vitamin D deficiency a correlate for attention deficit hyperactivity disorder?

The aim of the study was to determine the association between vitamin D and attention deficit hyperactivity disorder (ADHD), and difference in the level of vitamin D in ADHD children and control. This a case–control study carried out in school health and primary health care clinics. A total of 1,331 children and adolescents who were diagnosed with ADHD based on clinical criteria and standardized questionnaires were enrolled in this study and were matched with 1,331 controls, aged 5–18 years old. Data on body mass index (BMI), clinical biochemistry variables including serum 25-hydroxyvitamin D were collected. The study found significant association between ADHD and vitamin D deficiency after adjusting for BMI and sex (adj. OR 1.54; 95 % CI 1.32–1.81; P < 0.001). Majority of the ADHD children were in the age group 5–10 years (40.7 %), followed by 11–13 years (38.4 %). The proportion of BMI <85th percentile was significantly over represented in ADHD group as compared to healthy control (87.8 vs. 83 %; P < 0.001, respectively), while on the other hand, BMI >95th percentile was over represented in the control than ADHD group (7.6 vs. 4.6 %; P < 0.001, respectively). Mean values of vitamin D (ng/mL) were significantly lower in ADHD children (16.6 ± 7.8) than in healthy children (23.5 ± 9.0) (P < 0.001). There was significant correlation between vitamin D deficiency and age (r = ?0.191, P = 0.001); calcium (r = 0.272, P = 0.001); phosphorous (r = 0.284, P = 0.001); magnesium (r = 0.292, P = 0.001); and BMI (r = 0.498, P = 0.001) in ADHD children. The vitamin D deficiency was higher in ADHD children compared to healthy children.     

Relationships Between Single Nucleotide Polymorphisms of the H-FABP Gene and Slaughter and Meat Quality Traits in Chicken

Using PCR-SSCP with five primer pairs, we detected six single nucleotide polymorphisms of the H-FABP gene: 332G → A, 534G → A, 783C → T, 835C → T, 1198T → C, and 2329C → T. Chi-square results showed significant differences (P < 0.05) in genotype frequency among breeds in Fragment 1 and extremely significant differences (P < 0.01) in Fragments 2–4. We found a significant association between Fragment 2 genotype and muscle fiber number, Arg and Thr (P < 0.05); between Fragment 3 genotype and living weight, carcass weight, breast muscle weight, abdominal fat weight, and abdominal fat percentage (P < 0.05); between Fragment 4 genotype and Thr, Phe, and inosinic acid (P < 0.05). It was concluded that H-FABP was the major gene influencing slaughter performance and meat quality or was linked with the major gene in these strains and that the C783T mutation could be used as a candidate molecular genetic marker for breeding selection. The combination M1C2–B2B2–D1D1 is an ideal model for breeding in these strains because it can improve slaughter and meat quality traits.     

Enterocin M and its Beneficial Effects in Horses—a Pilot Experiment

Probiotic bacteria or their antimicrobial proteinaceous substances called bacteriocins (enterocins) hold promising prophylactic potential for animal breeding. This study present the results achieved after application of Enterocin M in horses. Enterocin M has never been applied to horses before. Clinically healthy horses (10) were involved in this pilot experiment. They were placed in the stables of the University of Veterinary Medicine and Pharmacy, Ko?ice, Slovakia, with the approval of the University Ethics Committee. The animals were fed twice a day with hay and oats, or alternatively grazed with access to water ad libitum. The experiment lasted 6 weeks. Sampling was performed at the start of the experiment, at day 0–1, at day 21 (3 weeks of Enterocin M application), and at day 42 (3 weeks of cessation). Feces were sampled directly from the rectum and blood from the vena jugularis; the samples were immediately treated and/or stored for analyses. Each horse itself represented a control animal (compared to its status at the start of the experiment, day 0–1). After initial sampling, the horses were administered 100 μl of Ent M (precipitate, 12,800 AU/ml) in a small feed bolus to ensure it was consumed; Ent M was applied for 3 weeks (21 days). Fecal samples were treated using the standard microbial dilution method; phagocytic activity was assessed with standard and flow cytometry; biochemistry and metabolic profiles were tested using commercial kits and standard methods. Administration of Ent M led to mathematical reduction of coliforms, campylobacters (^abP?<?0.05), and significant reduction of Clostridium spp. (^abP?<?0.001, ^bcP?<?0.001); increase of PA values was noted (P?<?0.05, P?<?0.0001); no negative influence on hydrolytic enzyme profile or biochemical blood parameters was noted.     

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P < 0.01) change in expression levels were screened according to the screening standard (signal log ratio ≥1 or ≤?1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335 ± 0.0116 72 h after transfection. This value was significantly (P < 0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P < 0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.     

Relative expressions of miR-205-5p, miR-205-3p, and miR-21 in tissues and serum of non-small cell lung cancer patients

Objective was to assess and compare the relative expressions of miR-205-5p, miR-205-3p, and miR-21-3p in tissues and serum among non-small cell lung carcinoma (NSCLC) patients, benign pulmonary conditions patients, and healthy volunteers. Serum samples were obtained between October 2011 and September 2012 from 20 NSCLC patients undergoing surgical treatment, 20 patients diagnosed with a benign lung disease (pulmonary tuberculosis, pneumonia, chronic obstructive pulmonary disease, or interstitial pneumonia) (lesion group), and 20 healthy volunteers (control group). NSCLC patients provided cancer tissues and cancer-adjacent normal tissues during surgery (paired specimens). Quantitative RT-PCR was used to assess miR-205-5p, miR-205-3p, and miR-21-3p expressions in serum and tissue samples. The relative expressions of miR-205-5p and miR-205-3p were significantly higher in NSCLC tissues compared with cancer-adjacent paired specimens (both P < 0.001). In the serum, significantly higher miR-205-5p, miR-205-3p, and miR-21-3p relative expressions were observed in the NSCLC group compared with the two other groups (all P < 0.001). The relative expressions of miR-205-5p and miR-21-3p in NSCLC tissues and serum were significantly correlated (r = 0.553, P = 0.011; and r = ?0.541, P = 0.014, respectively), while no significant correlation was observed for miR-205-3P (P = 0.120). Expressions of miR-205-5p and miR-205-3P in squamous cell carcinoma specimens were significantly higher than in lung adenocarcinoma specimens (both P = 0.001). Similarly, higher serum miR-205-5p and miR-205-3p levels were observed in squamous cell carcinoma patients (P = 0.033 and P = 0.002, respectively). In this preliminary and novel study, miR-205-5p was more useful as a marker for NSCLC than miR-205-3p or miR-21, indicating a potential for future applications in NSCLC diagnosis and prognosis.     

Neuroprotective Effect of Atorvastatin Involves Suppression of TNF-α and Upregulation of IL-10 in a Rat Model of Intracerebral Hemorrhage

We evaluated the neuroprotective effects of atorvastatin (2, 5, and 10 mg/kg) on experimentally induced intracerebral hemorrhage (ICH) in adult rats; controls were administered PBS. Plasma TNF-α and IL-10 levels before and after ICH were analyzed at various time points by enzyme-linked immunosorbent assay (ELISA) and neurological behavior of rats was assessed by climbing scores. At 3-days postoperatively, brain water contents and TNF-α/IL-10 expression in brain tissue were determined. Histopathological changes and microglial cells in the brain tissue were evaluated by light-microscopy. Post-ICH neurological deficits differed significantly between sham-operated group A and experimental-ICH group B (P < 0.05). Brain water contents were significantly less in group A than in group B (P < 0.05). Significant differences (P < 0.05) between two groups were observed regarding activated microglia, TNF-α and IL-10 levels. Compared with group B, neurological deficits, brain water contents, pathological changes, and activated microglia were reduced (P < 0.05) in groups C (Experimental-ICH + atorvastatin 2 mg/kg), D (Experimental-ICH + atorvastatin 5 mg/kg) and E (Experimental-ICH + atorvastatin 10 mg/kg). Atorvastatin-induced a dose-dependent reduction of TNF-α and increase of IL-10 levels (P < 0.05). Therefore, it was concluded that atorvastatin improved neurofunctional rehabilitation in rats through the suppression of cytokines-mediated inflammatory response and attenuation of brain damage following intracerebral hemorrhage.     

Differential effects of mesenchymal stem cells on a heterogeneous cell population within lung cancer cell lines

Although mesenchymal stem cells (MSCs) promote lung cancer growth in vivo, in vitro studies indicate that they inhibit the proliferation of lung cancer cells. Because malignant tumors contain a heterogeneous cell population with variable capacity for self-renewal, the aim of this study was to determine whether the inconsistencies between in vitro and in vivo studies are a result of differential effects of MSCs on the heterogeneous cell population within lung cancer cell lines. Human MSCs were isolated from the bone marrow, and their cell surface antigen expression and multi-lineage differentiation capacity was examined at passage 10. CD133+ cells were isolated from A549 and H446 cell lines using immunomagnetic separation. The effects of MSCs on the growth and microsphere formation of heterogeneous cell populations within two lung cancer cell lines (A549 and H446) were compared. MSCs inhibited the in vitro proliferation of both cell lines, but significantly accelerated tumor formation and stimulated tumor growth in vivo (P < 0.05). In CD133+ cells isolated from both A549 and H446 cells, co-culture with MSCs for 1–3 days significantly increased their proliferation (P < 0.05). MSCs also significantly increased microsphere formation in both cell lines (P < 0.05). Selective stimulation of CD133+ cell growth may account for the discrepant effects of MSCs on lung cancer progression.     

The newly established human liver cell line: a potential cell source for the bioartificial liver in the future

The clinical use of a bioartificial liver (BAL) device strongly depends on the development of human liver cell lines. The aim of this study was to establish and assess the potential use of the stable HepG2 cell line expressing human augmenter of liver regeneration (hALR). The cDNA encoding hALR protein was inserted into pcDNA3.1 to generate pcDNA3.1/hALR, following which pcDNA3.1/hALR was transfected to HepG2 to establish a cell line that stably expressed hALR (HepG2 hALR). A total of 800 million HepG2 hALR cells were loaded into laboratory-scale BAL bioreactors and cultured for 4 days, during which time the parameters of hepatocyte-specific function and general metabolism were determined. The cell line that stably expressed human ALR was successfully established. The expression of recombinant hALR was higher in the HepG2 hALR cell line than in the HepG2 cell line based on immunofluorescence and immunoblot assays. In samples removed from the BAL bioreactor on day 4, compared to HepG2 cells, HepG2 hALR cells produced significantly more alpha-fetoprotein (127.3 %; P < 0.05) and urea (128.8 %; P < 0.05) and eliminated more glucose (135.7 %; P < 0.05); the level of human albumin was also higher (117 %) in HepG2 hALR cells, but the difference was not significant (P > 0.05). After 24 h of culture, the mean lidocaine removal rate was 65.1 and 57.3 % in culture supernatants of HepG2 hALR and HepG2 cell lines, respectively (P < 0.01). Based on these results, we conclude that HepG2 hALR cells showed liver-specific functionality when cultured inside the bioreactor and would therefore be a potential cell source for BAL.     

Cu(Nor)2·5H2O, a complex of Cu(II) with Norfloxacin: theoretic approach and biological studies. Cytotoxicity and genotoxicity in cell cultures

Norfloxacin is a fluoroquinolone antibiotic used in the treatment of bacterial infections. In this article, we studied the potential antitumoral action of a complex of Norfloxacin with Cu(II), Cu(Nor)₂·5H₂O on osteosarcoma cells (UMR106) and calvaria-derived cells (MC3T3-E1), evaluating its cytotoxicity and genitoxicity. We have also elucidated the more stable conformation of this complex under physiologic conditions by Molecular Dynamic simulations based on the model of the canonical ensemble and PM6 force field. When solvent effect was taken into account, the complex conformation with both carbonyl groups in opposite sides displayed lower energy. Cu(Nor)₂·5H₂O caused an inhibitory effect on the proliferation on both cell lines from 300 μM (P < 0.01). Nevertheless, the decline on cell proliferation of UMR106 cells was more pronounced (45 % vs basal) than in MC3T3-E1 cells (20 % vs basal) at 300 μM (P < 0.01). Cu(Nor)₂·5H₂O altered lysosomal metabolism (Neutral Red assay) in a dose-dependent manner from 300 μM (P < 0.001). Morphological studies showed important transformations that correlated with a decrease in the number of cells in a dose-dependent manner. Moreover, Cu(Nor)₂·5H₂O caused statistically significant genotoxic effects on both osteoblast cell lines in a lower range of concentrations (Micronucleus assay) (P < 0.05 at 10 μM, P < 0.001 from 25 to 50 μM). UMR106 cells displayed a dose-related genotoxic effect between 5 and 25 μM while the MC3T3-E1 cells showed a narrower concentration dependent range. Altogether, these results suggest that Cu(Nor)₂·5H₂O is a good candidate to be further evaluated for alternative therapeutics in cancer treatment.     

Effects of streptozotocin-induced long-term diabetes on parietal cell function and morphology in rats

Gastric pathology is a common complication in diabetes mellitus. The aim of the study was to evaluate the functions and morphological changes of the parietal cells of the rat stomach after streptozotocin-induced diabetes. Diabetes mellitus was induced in Wistar rats by a single intraperitoneal injection of streptozotocin (60 mg/kg body weight). The rats were weighed weekly and sacrificed after 6 months. The glandular portion of the stomach was removed and processed for H⁺-K⁺-ATPase immunohistochemistry and light and electron microscopy studies. Acid secretion was measured in vivo. After 6 months of diabetes, the mean weight of the rats was significantly lower (P < 0.001) compared to control. The mean weight of the stomach to body weight percentage increased significantly (P < 0.001) compared to control. The blood glucose level in diabetic rats was significantly higher (P < 0.001) than in normal control. Diabetic rats showed significant (P < 0.001) decrease in basal and stimulated acid secretion when compared to control. Electron micrographs of the parietal cells of glandular stomach of diabetic rats revealed significant (P < 0.0002) reduction in the number of mitochondria and a small though not significant increase in the number of canaliculi in the parietal cells compared with normal. Immunohistochemistry showed reduced H⁺-K⁺-ATPase (P < 0.00001) compared to control. Long-term diabetes induces morphological as well as functional changes in gastric parietal cells. The decrease in the number of mitochondria accompanied by reduced in H⁺-K⁺-ATPase in parietal cells may explain the reduced acid secretion observed in diabetics.     

Efficacy of the Combination of Voriconazole and Caspofungin in Experimental Pulmonary Aspergillosis by Different Aspergillus Species

Objectives

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus, Aspergillus flavus, or Aspergillus niger is associated with high mortality. We evaluated the efficacy and compared the therapeutic effect differences of voriconazole (VRC) in combination with caspofungin (CAS) in transiently neutropenic rats infected by A. fumigatus, A. flavus, or A. niger.

Methods

Treatment groups consisted of VRC (10 mg/kg q12 h) monotherapy, CAS (1 mg/kg/day) monotherapy, combination of VRC (10 mg/kg q12 h) + CAS (1 mg/kg/day), and no drug for 10 consecutive days. The efficacy and the difference in the treatments were evaluated through prolongation of survival, reduction in serum galactomannan levels and residual fungal burden, and histological studies.

Results

For all the strains, the combination of VRC and CAS led to significant prolongation in survival (P < 0.05) and reduction in residual fungal burden (P < 0.05) compared with CAS alone, and decrease in serum galactomannan levels (P < 0.05) compared with either agent alone. The survival in the combined therapy groups was significantly improved compared to VRC monotherapy for the strains of A. flavus and A. niger (P < 0.05), but no significant difference for the strains of A. fumigatus (P > 0.05).

Conclusions

Combination of VRC and CAS was synergistic in IPA by A. flavus and A. niger, but small efficacy benefits in IPA by A. fumigatus.     

Inhibitory Effect of Gallic Acid on CCl4-Mediated Liver Fibrosis in Mice

The aim of this study was to investigate the effect of gallic acid (GA) on liver fibrosis induced by carbon tetrachloride (CCl₄). Male BALB/c mice were randomly divided into four groups: normal control group (group A), CCl₄-induced liver injury control group (group B), and CCl₄ induction with GA of low dose (5 mg/kg) and high dose (15 mg/kg) treatment group (group C and group D). GA was intra-gastric given for mice once a day after 2 weeks of CCl₄ induction. Animals were killed at the eighth week. Degrees of fibrosis and collagen percentage were measured. Hyaluronic acid (HA), type IV collagen (cIV), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γ-GT) were determined. Expression of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) mRNA levels were examined by RT-PCR. Western blotting was carried out to evaluate the changes of MMP-2 protein. HE and VG stainings showed GA in a dose-dependent manner improved significantly the fibrosis condition in CCl₄-injured mice (P < 0.05 or P < 0.01). Also, the concentrations of HA, cIV, and MDA, as well as the serum levels of ALT, AST, and γ-GT were markedly reduced by GA (P < 0.05 or P < 0.01), and decreases in MMP-2, TIMP-1 mRNA, and MMP-2 protein were observed as well (P < 0.05 or P < 0.01). GA could exert protective effect on liver injury and reduce liver fibrosis induced by CCl₄ in mice, which might be through the inhibition of hepatic stellate cell activity.     

Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods

Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16–20 h is an effective and simple modified enzyme digestion method.     

Establishment of a Spinal Cord Injury Model in Adult Rats by an Electrocircuit-Controlled Impacting Device and its Pathological Observations

One of the crucial challenges in medicine is the treatment and rehabilitation of spinal cord injury (SCI). In this study, we established a stable and reproducible acute spinal cord injury model in adult rats. The SCI was inflicted by our self-innovated spinal cord impact device controlled by electrical circuit. The Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB) score, electrophysiology, histological, and immunohistochemical changes after SCI were observed. The BBB score of the injured rats began to increase from the 3rd day of SCI and reached at the score 7.2 ± 1.3 at the 28th day. The latency of cortical somatosensory evoked potentials (CSEP) was not observed 2 and 6 h after injury, but appeared 24 h after injury which was significantly prolonged. It recovered from day 3 gradually to 27.3 ± 2.7 ms on day 28. H&E staining showed that the structure of gray and white matter was disrupted after the SCI. The result also showed dramatic neuron degenerations, cellular swelling, and the proliferation of glial cells. The immunohistochemical analysis showed that the expression of neuron specific enolase (NSE) and neurofilament 200 (NF200) started lowering at 2 h and dropped to the bottom at 24 h. Their expression rebound from day 3 and yet to the original level at day 28 (P < 0.05). The number of cells expressing glial fibrillary acidic protein (GFAP) hiked from day 3, peaked at day 14, and began recovering from day 28 (P < 0.05). The changes of NSE, NF200, GFAP, and CSEP were significantly associated with the BBB score (P < 0.05). In conclusion, our self-innovated device can reproduce the injury model stably. The changes of NSE, NF, and GFAP after spinal cord injury reflect the characteristics of pathological change, which are closely associated with the functional recovery from the spinal cord injury.     

Effect of ARHI on lung cancer cell proliferation, apoptosis and invasion in vitro

The purposes of this study were to elucidate the effects of ARHI (aplysia ras homolog I) on several biological features of lung cancer cells, including growth, proliferation and invasion, to collect experimental evidence for the future biological treatment of human lung cancer. The eukaryotic expression vector, pcDNA3.1–ARHI, was constructed and transfected into the human lung cancer cell line SK-MES-1. The biological properties of the resulting ARHI-expressing lung cancer cell line were evaluated using methyl thiazolyl tetrazolium assay, flow cytometry, and a Transwell invasion assay. Additionally, the influence of ARHI on the gene expression levels of cyclin D1, p27^KIP1, death-associated protein kinase 1 (DAPK1), and matrix metalloproteinases1/2 (MMP-1/2) was determined. Compared to the non-transfected SK-MES-1 cells and the cells transfected with the empty pcDNA3.1 plasmid, the ARHI-transfected cells displayed significantly reduced growth rates and decreased viability (P < 0.05). The ARHI-transfected cells also displayed a significantly higher percentage of cells in G1 phase (P < 0.05) and a lower percentage of cells in S phase (P < 0.05); a higher percentage of apoptosis (P < 0.05); and finally, a notable reduction in the basement membrane-penetration rate in the Transwell invasion assay (P < 0.05). Furthermore, it was determined that ARHI is capable of inhibiting the expression of cyclin D1, MMP-1, and MMP-2; however, ARHI promotes the expression of both p27^KIP1 and DAPK1 in SK-MES-1 cells. In conclusion, overexpression of ARHI gene might be associated with the inhibition of lung cancer cell growth, proliferation and invasion, and the promotion of apoptosis.