Mobile phone addiction and academic burnout: the mediating role of technology conflict and the protective role of mindfulness.

With the rapid development of Internet technology, more and more college students are facing the threat of mobile phone addiction. However, the relationship and underlying mechanism between mobile phone addiction and academic burnout haven't been explored in depth. This study proves the mediating role of technology conflict and the moderating role of mindfulness in the relation between mobile phone addiction and academic burnout. 752 college students were recruited to complete the questionnaire of mobile phone addiction, technology conflict, mindfulness and academic burnout. Results showed that mobile phone addiction was significantly and positively associated with academic burnout, and this relationship could be mediated by technology conflict. Besides, the direct effect of mobile phone addiction on academic burnout and the indirect effect of technology conflict in this link were moderated by mindfulness. Both these two effects are stronger for college students with lower level of mindfulness. Our findings enrich our understanding of how and when mobile phone addiction was related to academic burnout. Educational professionals and parents should take timely measure to the academic burnout of college students suffering from mobile phone addiction, particularly for those with lower level of mindfulness.

Factorial structure and measurement invariance of the Chinese version of the Oral Health Impact Profile-14 among clinical populations and non-clinical populations: an evidence for public oral investigations.

OBJECTIVE: Oral health-related quality of life (OHRQoL) is a multidimensional concept that is commonly used to examine the impact of oral health status on quality of life. The purpose of this study was to examine the optimal factor model of the Chinese version of the Oral Health Impact Profile (OHIP-14) questionnaire in clinical populations, measurement invariance across clinical status and gender cohorts. This would ensure equal validity of the Chinese version of OHIP-14 in different populations and further support public oral investigations. METHODS: The Chinese version of OHIP-14 was used to investigate 490 dental patients and 919 college students. Confirmatory factor analysis (CFA), item analysis and reliability, measurement invariance, and the t-test were used for data analyses. RESULTS: We found that the 7-factor structure had the best-fit index in the sample (CFI = 0.970, TLI = 0.952; SRMR = 0.029, RMSEA = 0.052(0.040,0.063)). The reliability of the scales was satisfactory (Cronbach's α = 0.942). The error variance invariance fitted the data adequately in measurement invariance, indicating that measurement invariance is acceptable both across the clinical and non-clinical populations (∆CFI=-0.017, ∆RMSEA = 0.010) and across genders in the clinical population (∆CFI = 0.000, ∆RMSEA=-0.003). T-test for scores showed that the clinical populations scored significantly higher than the non-clinical populations, as did the overall score (t = 7.046, p < 0.001, d = 0.396), in terms of functional limitation (t = 2.178, p = 0.030, d = 0.125), physical pain (t = 7.880, p < 0.001,d = 0.436), psychological discomfort (t = 8.993, p < 0.001, d = 0.514), physical disability (t = 6.343, p < 0.001, d = 0.358), psychological disability (t = 5.592, p < 0.001, d = 0.315), social disability (t = 5.301, p < 0.001,d = 0.304), social handicap (t = 4.452, p < 0.001, d = 0.253), and that in the non-clinical populations, females scored significantly higher than males, as did in terms of physical pain (t = 3.055, p = 0.002, d = 0.280), psychological discomfort (t = 2.478, p = 0.014, d = 0.222), and psychological disability (t = 2.067, p = 0.039, d = 0.188). CONCLUSION: This study found that the Chinese version of OHIP-14 has measurement invariance between the clinical and non-clinical populations and across genders in the clinical populations, and can be widely used in OHRQoL assessment for public oral investigations.

Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

Lactobacillusjohnsonii L531 Protects against Salmonella Infantis-Induced Intestinal Damage by Regulating the NOD Activation, Endoplasmic Reticulum Stress, and Autophagy.

Salmonella enterica serovar Infantis (S. Infantis) is an intracellular bacterial pathogen. It is prevalent but resistant to antibiotics. Therefore, the therapeutic effect of antibiotics on Salmonella infection is limited. In this study, we used the piglet diarrhea model and the Caco2 cell model to explore the mechanism of probiotic Lactobacillus johnsonii L531 (L. johnsonii L531) against S. Infantis infection. L. johnsonii L531 attenuated S. Infantis-induced intestinal structural and cellular ultrastructural damage. The expression of NOD pathway-related proteins (NOD1/2, RIP2), autophagy-related key proteins (ATG16L1, IRGM), and endoplasmic reticulum (ER) stress markers (GRP78, IRE1) were increased after S. Infantis infection. Notably, L. johnsonii L531 pretreatment not only inhibited the activation of the above signaling pathways but also played an anti-S. Infantis infection role in accelerating autophagic degradation. However, RIP2 knockdown did not interfere with ER stress and the activation of autophagy induced by S. Infantis in Caco2 cells. Our data suggest that L. johnsonii L531 pretreatment alleviates the intestinal damage caused by S. Infantis by inhibiting NOD activation and regulating ER stress, as well as promoting autophagic degradation.

Klf4 methylated by Prmt1 restrains the commitment of primitive endoderm.

The second cell fate decision in the early stage of mammalian embryonic development is pivotal; however, the underlying molecular mechanism is largely unexplored. Here, we report that Prmt1 acts as an important regulator in primitive endoderm (PrE) formation. First, Prmt1 depletion promotes PrE gene expression in mouse embryonic stem cells (ESCs). Single-cell RNA sequencing and flow cytometry assays demonstrated that Prmt1 depletion in mESCs contributes to an emerging cluster, where PrE genes are upregulated significantly. Furthermore, the efficiency of extraembryonic endoderm stem cell induction increased in Prmt1-depleted ESCs. Second, the pluripotency factor Klf4 methylated at Arg396 by Prmt1 is required for recruitment of the repressive mSin3a/HDAC complex to silence PrE genes. Most importantly, an embryonic chimeric assay showed that Prmt1 inhibition and mutated Klf4 at Arg 396 induce the integration of mouse ESCs into the PrE lineage. Therefore, we reveal a regulatory mechanism for cell fate decisions centered on Prmt1-mediated Klf4 methylation.

Melatonin as Immune Potentiator for Enhancing Subunit Vaccine Efficacy against Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) is a pathogen associated with substantial economic losses in the dairy cattle industry. Currently, there are no effective vaccines against BVDV. Melatonin (MT) has been shown to have anti-inflammatory and anti-viral properties, and the use of MF59 in vaccines significantly enhances vaccine efficiency. Here, MT and MF59 were added into the Erns-LTB vaccine. Subsequently, their inhibitory activity on the NF-κB signaling pathway in Mardin-Darby Bovine Kidney cells and the hippocampus was assessed using western blot and quantitative reverse transcription PCR. The findings revealed that MT in the Erns-LTB vaccine decreases the phosphorylation of p65 proteins caused by BVDV infection. In addition, MT decreased the mRNA levels of IL-1ß and IL-6 in vitro, but increased the production of IFN-α, IFN-ß, Mx1 in vitro, brain-derived neurotrophic factor, cyclic amp response element-binding protein, and the stem cell factor in vivo. Furthermore, treatment with Erns-LTB + MF59 + MT stimulated the production of T lymphocytes, alleviated pathological damage, decreased expressions of BVDV antigen, and tight junction proteins in mice. These findings imply that MT has potential for use in the Erns-LTB vaccine to inhibit BVDV infection and regulate the immune responses of T-cells by inhibiting the NF-κB signaling pathway.

Epidemiology of Takayasu arteritis in Shanghai: A hospital-based study and systematic review.

BACKGROUND: Takayasu arteritis (TAK) is a rare large vessel vasculitis, and epidemiological data on TAK are lacking in China. Thus, we designed this study to estimate the TAK prevalence and incidence in residential Shanghai, China. METHODS: Data on diagnosed TAK cases aged over 16 years were retrieved from 22 tertiary hospitals in Shanghai through hospital electronic medical record systems between January 1, 2015 and December 31, 2017 to estimate the prevalence and incidence. A systematic literature review based on searches in PubMed, Ovid-Medline, Excerpta Medica Database (EMBASE), Web of Science, and China National Knowledge Infrastructure (CNKI) was performed to summarize TAK distribution across the world. RESULTS: In total 102 TAK patients, with 64% female, were identified. The point prevalence (2015-2017) was 7.01 (95% CI 5.65-8.37) cases per million, and the mean annual incidence was 2.33 (1.97-3.21) cases per million. The average age of TAK patients was 44 ± 16 years, with the highest prevalence (11.59 [9.23-19.50] cases per million) and incidence (3.55 [0.72 3.74] cases per million) in the 16 to 34 years population. Seventeen reports were included in the system review, showing that the epidemiology of TAK varied greatly across the world. The incidence and prevalence were both relatively higher in Asian countries, with the prevalence ranging 3.3-40 cases per million and annual incidence ranging 0.34-2.4 cases per million. CONCLUSIONS: The prevalence and incidence of TAK in Shanghai was at moderate to high levels among the previous reports. The disease burden varied globally among racial populations.

Immunization with recombinant Erns-LTB fusion protein elicits protective immune responses against bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV), a major infectious pathogen and is associated with major economic losses and significant impact on animal welfare worldwide. Here, recombinant Erns-LTB protein vaccine containing MF59 adjuvant was prepared and assessed using a mouse model. The recombinant plasmid (pET32a-Erns-LTB) was constructed and transformed into BL21 (DE3) cells to produce Erns-LTB protein. The Erns-LTB protein was formulated with MF59 adjuvant, when delivered intraperitoneally in mice, exhibited higher immunogenic and induced superior levels of anti-BVDV IgG compared with the MF59 adjuvanted Erns protein. Importantly, after challenged with different BVDV BJ175170 and BJ1305 isolate strains, mice inoculated with Erns-LTB protein displayed alleviated pathological damage and decreased plasma virus shedding compared with mice inoculated with Erns protein. The enhanced protection from Erns-LTB protein is mediated by T cell immunity and primarily based on CD4+ T helper (Th) and CD8+ cytotoxic T lymphocyte (CTL), these results suggest that Erns-LTB protein has potential to protect against a broad range of BVDV strains thereby providing a novel direction for developing broadly protective vaccines.

Exploring the interactional details between aldose reductase (AKR1B1) and 3-Mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid through molecular dynamics simulations.

Aldose reductase (AKR1B1) has been considered as a significant target for designing drugs to counteract the development of diabetic complications. In the present study, molecular dynamics (MD) simulations and molecular mechanics generalized Born surface area (MM-GB/SA) calculations were performed to make sure which tautomer is the preferred one among three tautomeric forms (Mtia1, Mtia2, and Mtia3) of 3-Mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (Mtia) for binding to AKR1B1. The overall structural features and the results of calculated binding free energies indicate that Mtia1 and Mtia2 have more superiority than Mtia3 in terms of binding to AKR1B1. Furtherly, the local active site conformational characteristics and non-covalent interaction analysis were identified. The results indicate that the combination of Mtia2 and AKR1B1 is more stable than that of Mtia1. Furthermore, two extra hydrogen bonds between AKR1B1 and Mtia2 are found with respect to Mtia1. In addition, Mtia2 makes slightly stronger electrostatic interaction with the positively charged nicotinamide group of NADP+ than Mtia1. Based on the results above, Mtia2 is the preferred tautomeric form among the three tautomers. Our study can provide an insight into the details of the interaction between AKR1B1 and Mtia at the atomic level, and will be helpful for the further design of AKR1B1 inhibitors.

Identify signature regulatory network for glioblastoma prognosis by integrative mRNA and miRNA co-expression analysis.

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor in adults. Patients with this disease have a poor prognosis. The objective of this study is to identify survival-related individual genes (or miRNAs) and miRNA -mRNA pairs in GBM using a multi-step approach. First, the weighted gene co-expression network analysis and survival analysis are applied to identify survival-related modules from mRNA and miRNA expression profiles, respectively. Subsequently, the role of individual genes (or miRNAs) within these modules in GBM prognosis are highlighted using survival analysis. Finally, the integration analysis of miRNA and mRNA expression as well as miRNA target prediction is used to identify survival-related miRNA -mRNA regulatory network. In this study, five genes and two miRNA modules that significantly correlated to patient's survival. In addition, many individual genes (or miRNAs) assigned to these modules were found to be closely linked with survival. For instance, increased expression of neuropilin-1 gene (a member of module turquoise) indicated poor prognosis for patients and a group of miRNA -mRNA regulatory networks that comprised 38 survival-related miRNA -mRNA pairs. These findings provide a new insight into the underlying molecular regulatory mechanisms of GBM.

Transport of Corilagin, Gallic Acid, and Ellagic Acid from Fructus Phyllanthi Tannin Fraction in Caco-2 Cell Monolayers.

Objective. To investigate the absorption property of the representative hydrolyzable tannin, namely corilagin, and its hydrolysates gallic acid (GA) and ellagic acid (EA) from the Fructus Phyllanthi tannin fraction (PTF) in vitro. Methods. Caco-2 cells monolayer model was established. Influences of PTF on Caco-2 cells viability were detected with MTT assay. The transport across monolayers was examined for different time points, concentrations, and secretory directions. The inhibitors of P-glycoprotein (P-gp), multidrug resistance proteins (MRPs), organic anion transporting polypeptide (OATP) and sodium/glucose cotransporter 1 (SGLT1), and tight junction modulators were used to study the transport mechanism. LC-MS method was employed to quantify the absorption concentration. Results. The apparent permeability coefficient (Papp) values of the three compounds were below 1.0 × 10-6 cm/s. The absorption of corilagin and GA were much lower than their efflux, and the uptake of both compounds was increased in the presence of inhibitors of P-gp and MRPs. The absorption of EA was decreased in the company of OATP and SGLT1 inhibitors. Moreover, the transport of corilagin, GA, and EA was enhanced by tight junction modulators. Conclusion. These observations indicated that the three compounds in PTF were transported via passive diffusion combined with protein mediated transport. P-gp and MRPs might get involved in the transport of corilagin and GA. The absorption of EA could be attributed to OATP and SGLT1 protein.

The Genus Phyllanthus: An Ethnopharmacological, Phytochemical, and Pharmacological Review.

The plants of the genus Phyllanthus (Euphorbiaceae) have been used as traditional medicinal materials for a long time in China, India, Brazil, and the Southeast Asian countries. They can be used for the treatment of digestive disease, jaundice, and renal calculus. This review discusses the ethnopharmacological, phytochemical, and pharmacological studies of Phyllanthus over the past few decades. More than 510 compounds have been isolated, the majority of which are lignins, triterpenoids, flavonoids, and tannins. The researches of their remarkable antiviral, antioxidant, antidiabetic, and anticancer activities have become hot topics. More pharmacological screenings and phytochemical investigations are required to support the traditional uses and develop leading compounds.

[Research progress on salvianolic acids of Salvia miltiorrhiza].

Salvia miltiorrhiza is one of the most common traditional Chinese medicines. It has rich resources in China. According to modern studies, phenolic acids are the main effective components in S. miltiorrhiza. These components have cardiovascular and cerebrovascular protective effect, and anti-tumor, antioxidant, anti-inflammatory, and antifibrotic activities, etc. It has been widely used for the treatment of cardiovascular and cerebrovascular diseases and others. In this paper, the chemicals and pharmacological effects of phenolic acids from S. miltiorrhiza were summarized in the last decade. Its researches and development prospects were also analyzed for further studying and comprehensive utilization of these phenolic acids.

Positive therapy of andrographolide in vocal fold leukoplakia.

PURPOSE: Vocal fold leukoplakia is a premalignant precursor of squamous cell carcinoma. Although many efforts have been contributed to therapy of this disease, none exhibits a satisfactory result. The aims of this study were to investigate the effectiveness and feasibility of andrographolide therapy in vocal fold leukoplakia and to explore the preliminary mechanism underlying. MATERIALS AND METHODS: Forty-one eligible patients were enrolled in the study. The patients were treated for 10-minute exposures of 5 ml (25mg/ml) andrographolide injection aerosols twice a day, and 2 weeks was considered as one treatment course. Electronic laryngoscope was used to observe the condition of vocal fold leukoplakia during the treatment. Every patient received one or two treatment courses, and the follow-up was carried out for 12 months. Toxic reactions of treatments were evaluated on the basis of the standards of the United States MD Anderson Cancer Center. Moreover, laryngeal carcinoma cell line Hep2 was applied to explore the mechanism of effect of andrographolide. Anti-proliferative effect on Hep2, cell nuclear morphology, express of mitogen-activated protein kinases (MAPK) and pro-apoptotic protein were detected after andrographolide treatment. RESULTS: We found that andrographolide exhibited significant curative effects on treatments, which were accompanied by thinning of the lesion of leukoplakia, reduction in the whitish surface area, and return of pink or red epithelium. A complete response up to 85% was observed, and no toxic side effect events occurred during the study. No patient with a complete response had a recurrence in the follow-up. Moreover, cellular experiments in Hep2 indicated that andrographolide activated MAPK pathway and caspase cascade, and finally induced apoptosis in laryngeal carcinoma cell. CONCLUSIONS: The advantages of andrographolide are connected with minimally invasive and localized character of the treatment and no damage of collagenous tissue structures, which are more convenient and less painful for patients. These results suggest that andrographolide treatment is a viable strategy for curing vocal fold leukoplakia.

Theoretical mechanistic study of the reaction of the methylidyne radical with methylacetylene.

A detailed doublet potential energy surface for the reaction of CH with CH(3)CCH is investigated at the B3LYP/6-311G(d,p) and G3B3 (single-point) levels. Various possible reaction pathways are probed. It is shown that the reaction is initiated by the addition of CH to the terminal C atom of CH(3)CCH, forming CH(3)CCHCH 1 (1a,1b). Starting from 1 (1a,1b), the most feasible pathway is the ring closure of 1a to CH(3)-cCCHCH 2 followed by dissociation to P ( 3 )(CH(3)-cCCCH+H), or a 2,3 H shift in 1a to form CH(3)CHCCH 3 followed by C-H bond cleavage to form P ( 5 )(CH(2)CHCCH+H), or a 1,2 H-shift in 1 (1a, 1b) to form CH(3)CCCH(2) 4 followed by C-H bond fission to form P ( 6 )(CH(2)CCCH(2)+H). Much less competitively, 1 (1a,1b) can undergo 3,4 H shift to form CH(2)CHCHCH 5. Subsequently, 5 can undergo either C-H bond cleavage to form P ( 5 ) (CH(2)CHCCH+H) or C-C bond cleavage to generate P ( 7 ) (C(2)H(2)+C(2)H(3)). Our calculated results may represent the first mechanistic study of the CH + CH(3)CCH reaction, and may thus lead to a deeper understanding of the title reaction.

A modified technique of bone grafting pedicled with femoral quadratus for alcohol-induced osteonecrosis of the femoral head.

BACKGROUND: Quadratus femoris pedicled bone grafting has yielded satisfactory long-term clinical outcome for osteonecrosis of the femoral head (ONFH) in pre-collapse ONFH without extensive lesion. However, for pre-collapse ONFH with extensive necrotic area, it is still challenging to preserve the femoral head. The current study aimed to introduce a new technique of deliquesce strut with titanium mesh containing bone grafting pedicled with the femoral quadratus and to evaluate its short-term outcomes. METHODS: From January 2008 to December 2008, 10 ONFH patients (12 hips) underwent operations by a new technique of deliquesce strut with titanium mesh containing bone grafting pedicled with the femoral quadratus (group A). According to the ARCO classification system, there were two hips in stage II B and 10 hips in stage II C. Also in the same period, 12 ONFH patients (16 hips) underwent operations by the conventional procedure of quadratus femoris pedicled bone grafting (group B). There were 6 hips in stage II B and 10 hips in stage II C. All patients were males and suffered from alcohol induced ONFH. For the new technique, the necrotic area was evaluated, and a titanium mesh piece of the same size (range from 2.5 cm × 2.8 cm to 2.8 cm × 3.4 cm) was obtained and shaped to match the contour of the head. The cancellous bone was first placed underneath the subchondral bone and was densely impacted (about 1 to 2 mm thick). Then the titanium mesh piece was inserted. The length of the decompressive trough was measured. A titanium cylinder mesh cage with a diameter of 1.6 cm of the same length was obtained, with a "U" shaped window in the wall being created to make room for the muscle pedicle. The muscle pedicle bone was inserted into the titanium mesh cage to form a bone graft-titanium cage complex and, then the complex was inserted. The hundred percent score method was used for outcome evaluation. Clinical and radiographic outcomes were compared between group A and group B. RESULTS: The average operative time was 150 minutes (130 to 185 minutes) in group A, with an average of 130 minutes (120 to 180 minutes) in group B. The mean blood loss was 400 ml (300 to 500 ml) in group A and 350 ml (250 to 500 ml) in group B. Group A patients were followed up for an average of 19.2 months (14 to 24 months), with an average of 18.5 months (12 to 24 months) for Group B. Full weight bearing was allowed 5 to 7 months postoperatively. Pain and function were obviously improved. For group A, pain score improved from a mean of 9.8 points preoperatively to an average of 24.6 points postoperatively, and function score improved from a mean of 9.0 points preoperatively to an average of 17.4 points postoperatively. In group B, pain score improved from a mean of 9.5 points preoperatively to an average of 24.2 points postoperatively and function score improved from a mean of 9.2 points preoperatively to an average of 17.2 points postoperatively. The range of motion changed the least, with score improvement from a preoperative mean of 13.9 points to postoperative 16.8 points for group A and from a preoperative mean of 13.7 points to postoperative 16.5 points for group B. Radiographic score improved from preoperative 31 points to postoperative 38 points for group A, in comparison with an improvement from preoperative 31 points to postoperative 37 points for group B. At the latest follow up, 11 hips were rated as excellent and 1 hip was better for group A, with 14 hips being rated as excellent and 2 hips being better in group B. There was no statistically significant difference between groups A and B in clinical and radiographic outcomes. CONCLUSION: For ONFH in stage ARCO IIC, satisfactory clinical outcome can be achieved by the new technique in the short-term period while the long-term clinical outcome has yet to be determined.

[Cartilage-derived morphogenetic protein 1 initiates chondrogenic differentiation of human dermal fibroblasts in vitro].

OBJECTIVE: To explore the factors influencing the differentiation of fibroblasts into chondrocyte phenotype induced by a growth factor, cartilage-derived morphogenetic protein 1 (CDMP1). METHODS: Fibroblasts isolated from foreskin obtained during circumcision were cultured in the forms of micromass and monolayer culture. The culture fluid of the fibroblasts at the passage 2, 7, and 10 was added with CDMP1 of the concentrations at the concentrations of 10, 30, 100, and 300 ng/ml respectively and co-cultured for 7 days. RT-PCR was used to detect the expression of bone morphogenetic protein receptor (BMPR), activin receptor-like kinase (ALK) receptor, collagen types II, IV, and X before and after CDMP1 induction. Western blotting was used to detect the protein expression of collagen types II, a transcriptional factor Sox9, and aggrcan before and after the induction. Flow cytometry was used to detect the superficial markers CD29, CD105, CD106, and CD166, and the expression of collagen types I and II. RESULTS: Western blotting showed that the collagen type II positive cell rate in the passage 5 cells was 74.3% +/- 0.4%, not significantly different from that of the passage 2 cells (73.4% + 0.5%). When the concentrations of CDMP1 were 10 and 30 ng/ml no expression of aggrecan and collagen type II was detected, When the concentrations of CDMP1 was 100 and 300 ng/ml, the expression of aggrecan and collagen type II could be detected and without significant differences between these 2 concentrations. The expression of aggrcan and collagen type II mRNA disappeared in the monolayer cultured P2 and P5 fibroblasts induced by CDMP1 for 14 days, However, RT-PCR and Western blotting showed expression of collagen type II, aggrcan and SOX9 in the micromass cultured fibroblasts. RT-PCR showed that all fibroblasts cultured in vitro expressed ActR-I/ALK-2, BMPR-IA/ALK-3, and BMPR-IB/ALK-6 genes, and the expression of these genes significantly increased after CDMP1 for 7 days. CONCLUSION: CDMP1 stimulates the human dermal fibroblasts expanded in vitro to differentiate into chondrogenic phenotype in a dose dependent manner. Three-dimensional culture environments accelerate the chondrogenic differentiation. The expression of ALK receptors may involve the CDMP1 stimulated differentiation of fibroblasts.

[Construction and clinical application of tissue engineered epidermal membrane].

OBJECTIVE: To Construct tissue engineered epidermal membrane for promoting healing of clinical skin graft donor site wound. METHODS: Epidermal cells /Chitosan-Gelatin (CG) membrane was constructed with cultured human epidermal cells(EC) and CG membrane, at the donor site of split skin graft, the wound was divided into three groups: the control group was covered with CG membrane without KC, KC/CG membrane was grafted on the treatment part of the wound area, and blank group was covered with traditional vaseline gauze. The engineered epidermal membrane and its effect on wound were evaluated with gross observation, HE, immunohistochemistry, collagen type I/III ratio by picrosirius polarization method and RT - PCR test at various time points before and after operation. RESULTS: The result showed that human tissue engineered epidermis could be constructed with cultured human EC and CG membrane, and this artificial epidermal membrane could be used for promoting the healing of skin graft donor site wound successfully (16 cases with 3 months' oberservation). The average healing time is (16.2 +/- 3.8) days for control group, (8.1 +/- 1.3) days for experimental group and (23.0 +/- 5.7) days for blank group. The artificial epidermis was well survived with normal structure. In addition, less hypertrophic scar development was observed in treated wound at 90 days (3 in 16 cases, 20.0%) than in the blank sites (11 in 16 cases, 74.4%). The difference is statistically significant (chi2 = 8.127, P < 0.01). CONCLUSIONS: The constructed EC-CG membrane can be constructed in vitro, survived in vivo and has good clinical application in promoting healing of skin graft donor site wound and inhibiting hypertrophic scar formation.

[Cartilage-derived morphogenetic protein 1 initiates chondrogenic differentiation of human dermal fibroblasts in vitro].

OBJECTIVE: To investigate the feasibility of human dermal fibroblasts in vitro differentiation into chondrogenic phenotype with induction of cartilage-derived morphogenetic protein (CDMP) growth factor. METHODS: Human dermal fibroblasts were isolated from foreskin and cultured in monolayer ex vivo. Dermal fibroblasts of passage2 was plated at density of 1 x 10(4) cells/cm(2) and induced with CDMP1 (100 ng/ml) in medium of F12 + 10% FBS. After 7 days of induction, morphology of cells was observed under phase-contrast microscopy and the length:width ratio of cells was calculated by Image Plus software analysis. Expression of type I, II, III collagen was detected by immunofluorescence and observed with confocal microscopy. The method of Western-Blot was applied to detect secretion of collagen type II. mRNA expression of chondrogenic related Sox9, Aggrecan as well as collagen type II, IX was detected by RT-PCR. The osteogenic related expression of collagen type X, Alkaline Phosphatase (AKP) was also detected by RT-PCR. Pellet cultured dermal fibroblasts at a density of 2 x 10(7) cells/ml was observed respectively for proteoglycan and collagen type II expression with Alcian blue and immunohistochemistry staining. RESULTS: With the induction of CDMP1, the morphology of cells changed from spindle fibroblastic appearance to that of typical chondrocyte-like polygon shape. By Image Plus software analysis, it was found that the length/width ratio changed significantly from 7.40 +/- 1.30 of preinduction to 1.40 +/- 0.15 of post-induction (P < 0.05). No significant difference was found between the postinduction and normal chondrocyte (1.29 +/- 0.24). By confocal microscope observation, expression of collagen type II was found intracellularly in CDMP1 treated fibroblasts. Western-Blot detection confirmed collagen type II expression by 7 days induction. RT-PCR gene expression analysis of characteristic chondrogenic related genes, such as Sox9, Aggrecan as well as collagen type II, IX, revealed induction of chondrocytic phenotype in monolayered culture upon stimulation with CDMP1 for 7 days. While osteogenic related gene expression of collagen type X, AKP was not detected by RT-PCR, which indicates that osteogenic differentiation was not initiated by CDMP1 in 7 days culture. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining cartilage specific type II collagen revealed deposition of typical cartilage extracellular matrix deposition in pellet cultured fibroblasts. CONCLUSION: These results suggests human dermal derived fibroblast could be differentiated into chondrogenic phenotype with CDMP1 induction in vitro.

[Experimental study on biological character changes of human epidermal cells during proliferation culture in vitro].

UNLABELLED: [Abstract] OBJECTIVE: The study was (1) to investigate the biological character changes of human epidermal cells during proliferation culture in vitro and (2) to provide data for construction of engineered skin. METHODS: The foreskin was collected from 20 healthy children. The epidermal cells were isolated with digestion of the foreskin and cultured in vitro. Growth curve was obtained from the data of cell counting. Cell growth kinetics was observed. Meanwhile, clonal analysis and cell size measurement was performed. The rate of keratin 19 (K19) and involucrin expression-positive cells was counted by flow cytometer. Expression of K19 and involucrin mRNA was detected by RT-PCR. RESULTS: When passaged to P6, the keratinocytes from child foreskin could be expanded by (700 +/- 37) times. Flow cytometer results showed that the percentage of K19 expression-positive cells decreased from (66.97 +/- 3.14)% to (4.65 +/- 1.38)% while the percentage of involucrin expression-positive cells increased from (11.65 +/- 1.62)% to (97.03 +/- 2.66)% at P0 and P6 respectively. RT-PCR results showed that expression of K19 mRNA decreased from P0 to P6 while involucrin mRNA kept stable with passage in vitro. CONCLUSIONS: Human epidermal cells of passage 5 maintain proliferation phenotype, which are suitable for skin tissue engineering. Decrease of proliferation phenotype content is partially responsible for the proliferation capacity loss of in vitro cultured epidermal cells.