Objective]To study the feasibility of Cartilage engineering using fibrin gel and chondrocyte cell sheets.[Methods]rabbit auricular chondrocytes were isolated and cultured to form cell sheets in flasks. The cell sheets were harvested using cell scrapers,and cut into fragments. The two precursor solutions of Fibrin gel were used to suspend the cell sheet fragments and isolated chondrocytes,and then added into the wells of a 48-well plate to form Gelatinous chondroid disc constructs. After in vitro culture, the constructs were implanted into nude mice. After 8 weeks,the constructs were harvested,and the specimens were evaluated using grossly observing, histological and immunohistochemical observation. [Results]Mature cartilage discs were obtained. The histomorphology of the explanted discs appeared non-uniform cartilaginous tissue comprise of regenerated cartilage islands with different size and irregular shape. Immunohistochemistry staining demonstrated that type II collagen highly expressed in the ECM of the cartilage islands. In 1 of the 8 discs,partial ossification was observed.[Conclusion]Fibrin gel is a favourable carrier. Artificial cartilage with stereochemical structure was constructed via combining the fibrin gel and chondrocyte cell sheets.

BACKGROUND:The cell-sheet technology, based on a temperature-responsive culture, has been drawing more and more attention;however, the temperature-responsive culture dish is quite expensive. Therefore, it is imperative to develop a substitutive technique.OBJECTIVE: To study the feasibility of cell-sheet ulturing using common culture dish, and investigate the chondrogenesis of the cell sheet. METHODS: A piece of nasal septal cartilage was adopted from a patient with deviation of nasal septum to extract primary chondrocytes that were then cultured and amplified. The passage 3 chondrocytes were used to construct ell sheets. Monolayer cell sheet was formed by intensive culturing and allowing the extracellular matrix secretion. Bilayer cell sheet was constructed by seeding passage 2 chondrocytes on the monolayer cell sheet. The cell sheets were harvested using cell scraper, their properties were investigated prior to plantation into nude mice to construct the tissue-engineered cartilage. RESULTS AND CONCLUSION: Both bilayer and monolayer cell sheets with soft tremellose structures showed no significant difference through naked eyes. The newly harvested cell sheets appeared to have good fluidity and gelation. Eight weeks after mplantation into the nude mice, mature cartilage blocks were obtained. Histologically, the cell sheets were thin films composed by layered chondrocytes and extracellular matrix. Glycosaminoglycan formation and type Ⅱ collagen expressions were observed in the cell sheets cultured in vitro. The explanted samples exhibited ature cartilaginous tissue at 8 weeks after implantation. Biochemical analysis showed that the DNA contents of the neocartilages were higher than those of native human costal cartilage, while the contents of glycosaminoglycan and hydroxyproline were similar to native human nasal septal cartilage. To conclude, the hondrocyte cell sheets are likely to be constructed and harvested successfully using common culture dish, and the cell sheets exhibit favourable chondrogenesis.

Objective To retrospectively analyze medical data of patients with colorectal cancer (CRC) so as to provide evidence for clinical use of opportunistic screening.Methods A total of 2450 CRC patients (male 1377,female 1073) who were treated at five hospitals in North China during October 2001 and September 2011 and had complete medical records and pathological results were recruited.The correlations of incidenceofCRCwithage,gender,tumorlocationandhistologicaltypeswere analyzed.Results Of all the CRC patients,those less than 50 years old accounted for 18.14％ ; and the incidence of CRC was substantially increased in those over 50 years old.Seventy-three percent of tumor occurred at the rectum and sigmoid colon,6％ at descending colon,7％ at transverse colon and 14％ at ascending colon.Moderately,well or poorly differentiated adenocarcinoma accounted for 50.33％,40.35％and 9.32％,respectively.Histological differentiation was not correlated with age and gender ( P ＞ 0.05 ).Conclusions Age and gender should not be considered a determination of opportunistic screening for CRC.Colonoscopy is recommended as an alternative CRC screening procedure.

Objective To detect the expressions of CD70 mRNA and protein and to determine the methylation status of CD70 gene promoter in T cells from patients with systemic lupus erythematosus (SLE).Methods Peripheral blood CD4+ and CD8+ T cells were isolated from 15 patients with active SLE,15 patients with inactive SLE and 15 healthy control subjects.Real-time quantitative reverse transcription-PCR was carried out to quantify the mRNA expression of CD70,flow cytometry to determine the frequency of CD4+CD70+ and CD8+ CD70+ T cells,and bisulfite sequencing to evaluate the methylation status of CD70 gene promoter in CD4+ and CD8+ T cells.Differences in these parameters among these groups were analyzed by one-factor analysis of variance and SNK-q test.Results Compared with the healthy controls,the patients with active SLE and inactive SLE showed a significant increase in CD70 mRNA expression in CD4+ T cells (0.82 ± 0.12 and 0.73 ± 0.11 vs.0.45 ±0.09,F =53.017,P ＜ 0.01) and in the frequency of CD70+CD4+ T cells (80.30％ ± 11.04％ and 66.80％ ± 3.98％ vs.12.48％ ± 3.45％,F =311.517,P ＜ 0.01).Also,the expression of CD70 mRNA in CD4+ T cells and the frequency of CD70+CD4+ T cells were significantly higher in patients with active SLE than in patients with inactive SLE (both P ＜ 0.05).There was a positive correlation between the frequency of peripheral CD70+CD4+ T cells and disease activity in SLE in these patients (r =0.792,P ＜ 0.01).The average methylation index of the region between-600 bp and-300 bp of CD70 gene promoter in CD4+ T cells was 0.32 ± 0.05 and 0.36 ± 0.05 respectively in the patients with active and inactive SLE,significantly lower than that in the healthy controls (0.62 ± 0.05,F =152.64,P ＜ 0.01),and the patients with active SLE showed a significantly lower methylation index than those with inactive SLE (P ＜ 0.05).Conclusions The CD70 gene promoter in CD4+ T cells is significantly hypomethylated in patients with SLE,which may directly lead to the overexpression of CD70.

BACKGROUND:To seek for ideal scaffold materials is still an important task for cartilage tissue engineering.OBJECTIVE:To investigate the application of the AviteneTM microfibrillar collagen hemostat sponge in cartilage tissue engineering.METHODS:Rabbit auricular cartilage was harvested via surgical operation,and primary chondrocytes were isolated and amplified.Microfibrillar collagen hemostat sponge was cut into small bricks.The passage 2 chondrocytes were suspended and seeded onto the spongy bricks.After 1 week of in vitro culture,the constructs were then implanted into nude mice.After 8 weeks,the specimens were collected and evaluated using gross,histological and immunohistochamical observation.RESULTS AND CONCLUSION:During the cell seeding,the scaffold maintained its dimensions.No shrinkage was observed when the cell suspension was added.There was no considerable change in dimensions during the 1-week in vitro culture and at 8 weeks after implantation in nude mice.At 8 weeks post-implantation,mature cartilage blocks were harvested,which were white,translucent,and flexible.Histologically,the constructs appeared to have typical mature cartilaginous tissues,with robust extracellular matrix secretion,in which the microfibrillar collagen was incompletely degraded.We conclude that the microfibrillar collagen is a favorable scaffold material for cartilage tissue engineering.

Objective To observe the effect of ulinastatin in the treatment of children with severe pneumonia. Methods 86 children with severe pneumonia were divided into two groups according to the hospital card number:treatment group(n=45) and control group(n=41).All of them were given routine treatment,while the patients of the treatment group were given ulinastatin(5 000 U· kg-1 · time-1 ,1-2times/d,7d) in addition.The time of tem-perature drop to normal,the time of dyspnea improve,the time of dyspnea disappear,the time of lung rale disappear and length of hospital stay were observed.Then,the therapeutic effect was evaluated after treatment for 7 days.Results The treatment group had 21 cases excellence,21 cases improvement and 3 cases failure, the total effective rate was 93.33%,the control group had 17 cases excellence,15 cases improvement and 9 cases failure,the total effective rate was 78.04%,there was significant difference between the two groups(χ2 =4.17,P<0.05).The time of temperature drop to normal,the time of dyspnea improve,the time of dyspnea disappear,the time of lung rale disappear and length of hospital stay in the treatment group were (3.37 ±1.51)d,(3.12 ±1.72)d,(7.15 ±2.45)d,(10.75 ±2.47)d, respectively,which in the control group were (5.02 ±1.78)d,(4.82 ±1.51)d,(9.08 ±2.85)d,(13.22 ±2.85)d, there were significant differences between the two groups(t=4.71,4.91,3.42,4.41,all P<0.01).Conclusion Ulinastatin has good effect on children with severe pneumonia.It has value in application.

Objective:To investigate the effect of hypoxia-inducible factor 1α (HIF-1α)/Bcl-2 and adenovirus E1B19kDa-interacting protein 3 (BNIP3)-mediated mitophagy on neuronal apoptosis after traumatic brain injury (TBI).Methods:HT22 cells (mouse hippocampal neurons) were chosen; oxygen-glucose deprivation/re-oxygenation (OGD/R) was used to establish in vitro TBI models. Expressions of HIF-1α and BNIP3 were regulated by HIF-1α, BNIP3-specific small interfering RNA (siRNA) or plasmid vector transfection. Experiment 1 was performed to investigate the effect of BNIP3-mediated mitophagy on neuronal apoptosis after TBI; HT22 cells were divided into 4 groups ( n=3): siRNA control group (normal culture after negative siRNA transfection), siRNA+TBI group (OGD/R after negative siRNA transfection), BNIP3-siRNA group (normal culture after BNIP3-siRNA transfection), and BNIP3-siRNA+TBI group (OGD/R after BNIP3-siRNA transfection); the expressions of mitochondrial autophagy related proteins such as HIF-1α, BNIP3, LC3-II, and P62 were detected by Western blotting, mitochondrial autophagosomes were observed by transmission electron microscopy, apoptosis was detected by TUNEL, and lactate dehydrogenase (LDH) activity in cell supernatant was determined by LDH kit. Experiment 2 was performed to investigate the effect of HIF-1α on BNIP3-mediated mitophagy and neuronal apoptosis after TBI; HT22 cells were divided into 8 groups ( n=3): siRNA control group (normal culture after negative siRNA transfection), siRNA+TBI group (OGD/R after negative siRNA transfection), HIF-1α-siRNA group (normal culture after HIF-1α-siRNA transfection), HIF-1α-siRNA+TBI group (OGD/R after HIF-1α-siRNA transfection), empty plasmid group (normal culture after pcDNA3.1[+] transfection), HIF-1α overexpression group (normal culture after HIF-1α plasmid transfection), empty plasmid+TBI group (OGD/R after empty plasmid transfection), HIF-1α overexpression+TBI group (OGD/R after HIF-1α plasmid transfection); the expressions of HIF-1α, BNIP3, LC3-II, P62, TOMM20 and COX IV, apoptosis rate and LDH activity in neurons of each group were determined. Results:(1) In Experiment 1, compared with siRNA control group, siRNA+TBI group had significantly increased BNIP3 expression and LC3-II/I ratio, significantly decreased P62, TOMM20 and COX IV expressions, and statistically increased apoptosis and LDH activity ( P<0.05); compared with siRNA+TBI group, BNIP3-siRNA+TBI group had significantly decreased BNIP3 expression and LC3-II/I ratio, significantly increased P62, TOMM20, and COX IV expressions, and significantly increased apoptosis and LDH activity ( P<0.05); under projective electron microscope, siRNA+TBI group had increased autophagosomes compared with siRNA control group, while BNIP3-siRNA+TBI group had decreased autophagosomes compared with siRNA+TBI group. (2) In Experiment 2, compared with siRNA+TBI group, HIF-1α-siRNA+TBI group had significantly decreased expressions of HIF-1α and BNIP3, and LC3-II/I ratio, significantly increased expressions of P62, TOMM20 and COX IV, and significantly increased apoptosis and LDH activity ( P<0.05); compared with empty plasmid+TBI group, HIF-1α overexpression+TBI group had statistically higher expressions of HIF-1α and BNIP3, and LC3-II/I ratio, significantly decreased expressions of P62, TOMM20 and COX IV, and significantly decreased apoptosis and LDH activity ( P<0.05). Conclusion:HIF-1α can mitigate TBI-induced neuronal apoptosis via promoting BNIP3-mediated mitophagy.

ObjectiveTo explore the potential mechanism of acupuncture with the method of soothing the liver and regulating the mind in improving depressive disorder. MethodsEighteen C57BL/6J mice were randomly divided into blank group， model group， and acupuncture group， with 6 mice in each group. The model group and the acupuncture group were subjected to depression induction by intraperitoneal injection of lipopolysaccharide （LPS）， while the blank group received an equal volume of normal saline once daily for seven consecutive days. Concurrently， the acupuncture group received "soothing the liver and regulating the mind" acupuncture intervention starting from the first day of modeling， once daily for 14 days； whereas the blank group and the model group were only restrained without acupuncture. The sucrose preference test was used to assess sucrose preference rate， the open-field test to measure center stay time and total travel distance， and the forced swim test to evaluate immobility time. Hematoxylin-eosin （HE） staining was performed to observe hippocampal morphological changes. Enzyme-linked immunosorbent assay （ELISA） was used to detect levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） in hippocampal tissue. Western blot analysis was conducted to examine the protein expression levels of Toll-like receptor 4 （TLR4） and nuclear factor-κB （NF-κB） in the hippocampus. ResultsCompared to the blank group， the model group showed a significant reduction in sucrose preference rate， center stay time， and total travel distance， along with a significant increase in immobility time in the forced swim test， hippocampal IL-1β， IL-6， and TNF-α levels， as well as TLR4 and NF-κB protein expression （P＜0.01）， and the histological examination revealed blurred hippocampal neuronal boundaries， loose arrangement， and some neurons exhibiting nuclear pyknosis and deep staining. Compared to the model group， the acupuncture group demonstrated a significant increase in sucrose preference rate， center stay time， and total travel distance， along with a significant reduction in immobility time in the forced swim test， hippocampal IL-1β， IL-6， and TNF-α levels， and TLR4 and NF-κB protein expression （P＜0.01）， and the histological analysis showed that hippocampal neurons in the acupuncture group were more tightly arranged， with reduced nuclear pyknosis and deep staining. ConclusionAcupuncture with the "soothing the liver and regulating the mind" method can significantly improve depression-like behavior， potentially by inhibiting the hippocampal TLR4/NF-κB signaling pathway and alleviating inflammatory responses.

Objective: To explore the utility of pharyngeal pH monitoring which positive standard is Ryan index in diagnosis of laryngopharyngeal reflux disease. Methods: In a retrospective study, clinical data of 590 patients who had symptoms laryngopharyngeal reflux disease from February 2016 to March 2017 were analyzed. All patients were received electronic laryngoscopy, assessment of reflux symptom index(RSI) and reflux finding score(RFS), and pharyngeal pH monitoring. SPSS 19.0 software was used to analyze the date. Results: There were 94 patients whose Ryan index were positive(15.93%). Among the 94 patients, 70 were positive during upright, 12 during supine and 12 during both upright and supine. There were 40 patients(6.78%)with pH decline events related to symptoms, while those Ryan index were normal. There were 536(90.85%), 417(70.68%), 233(39.49%) and 117(19.83%) patients with pH<6.5, pH<6.0, pH<5.5 and pH<5.0 events respectively. The positive rate of RSI, RFS, RSI and RFS, RSI or RFS were 44.24%, 16.78%, 7.12%, 53.90% respectively. The RFS score in Ryan index positive group was higher than that in Ryan index negative group[(8.2±2.4) vs (4.0±2.9), u=5.424, P<0.05], while the RSI score in Ryan index positive group was not statistically different from that in Ryan index negative group[(11.3±6.2) vs (12.7±5.8), t=1.247, P=0.167]. Conclusions Pharyngeal pH monitoring is an objective and non-invasive method which can reflect laryngopharyngeal reflux directly. However, with the Ryan index as a criterion for the diagnosis of laryngopharyngeal reflux disease, partial patients may be missed. Further studies are needed to obtain more accurate and objective laryngopharyngeal pH statistical index for diagnosis of laryngopharyngeal reflux disease.