BACKGROUND:Compared with traditional autogenous bone graft, composite, as the carrier of seed cells, possesses advantages of fewer traumas and no limitation of donor site in repairing bone defect.OBJECTIVE: To observe the ability of the composite of β-tricalcium phosphate artificial bone-hyaluronic acid-type I collagen (β-TCP/HA/COL-I), as induced bone marrow stromal cell (MSC) carrier, to repair rabbit radial defect, and the feasibility with the composite as bone substitute material.DESIGN: A randomized and controlled trial.SETTING: Department of Orthopaedics, Renmin HospitaL, Wuhan University.MATERIALS; The study was conducted in the Department of Orthopaedics Renmin Hospital, Wuhan University between September 2003 and July 2004. Thirty-one New Zealand big white rabbits, aged 6 months, with body mass of 1.5 to 2.0 kg were enrolled in this study. The rabbits were randomized into control group (n =4) and experimental group (n =27).METHODS : ①In vitro induction and culture of MSCs was performed on 31 white rabbits, and the alkaline phosphatase (ALP) positive ratio of induced MSCs was observed. The structure of β-TCP/HA/COL-I was observed under scanning electron microscope. ② A 2 cm radial defect was created through operation. Eight weeks later, composites of β-TCP/HA/COL-I/MSCs were implanted into one side of rabbits in the experimental group, and autogenous bone was implanted into the other side. Rabbits in the control group were untouched. ③All the animals in the experimental group were randomly sacrificed at postoperative 4,8 and 12 weeks, 6 rabbits at 4 and 8 weeks, 15 at 12 weeks; Animals in the control group were sacrificed at 12 weeks. Gross observation, X-ray photographing, haematoxylin-eosin (HE) dyeing, and assessment of inorganic ingredient were performed. Osteogenic area and biomechanical tests were performed at 12weeks. Repairing effects on bone defect in each group were compared.MAIN OUTCOME MEASURES: The ALP positive ratio of induced MSCs; The structure of composite ofβ-TCP/HA/COL-I;Gross observation; X-ray photographing; HE dyeing and assessment of inorganic ingredient; Osteogenic area and biomechanical tests.RESULTS: All the 31 rabbits entered the stage of result analysis. ① The ALP positive ratio of cells reached 75% after induction and culture. ② Scanning electron microscope observation showed that 3 kinds of materials with abundant cellular structure distributed evenly. ③ The osteogenic area at 12 weeks was (72.5±3.6)% and (76.7±6.2)% in the experimental group and autogenous group, respectively (P ＞ 0.05). ④The maximum bending moment was (521.0±61.1) and (554.3±53.3)N·mm in the experimental group and control group, respectively; The maximum displacement at point of application of force was (0.816±0.071)and (0.870±0.103)respectively, without significant difference (P ＞ 0.05). ⑤Inorganic ingredient in the composite was 75%, 57% and 42% at 4,8 and 12 weeks respectively, suggesting that the inorganic component in the material was gradually decomposed with the elongation of time. ⑥Results of gross observation,X-ray photographing, histopathological examination, biomechanical test showed that with the elongation of time, composite of β-TCP/HA/COL-I/MSCs could repair bone defect in the experimental group, while bone defect in the control group had not been repaired.CONCLUSTON: Composite of β-TCP/HA/COL-I /MSCs possesses the same effect on repairing bone defect as autogenous bone, so it may be used as autogenous bone graft substitute.

Aim To study the application status of odds ratio for medical animal experiments.Methods Odds ratio and seven kinds of animal models were used as retrieval strategy to search medical animal experiment related papers in several Chinese and English databases.Papers relating to each kind of animal model and using odds ratio in abstract and text were counted. Data from different databases were compared. Calculation of odds ratio was exemplified and the significance of different odds ratio values was illustrated in this paper.Results Few medical animal experiments cited odds ratio as statistics.Conclusions The importance of odds ratio has not been fully recognized in Chinese references.

OBJECTIVETo investigate the effects of Pyrroloquinoline quinine (PQQ) on hydrogen peroxide-induced apoptosis of Schwann cells (SCs) and its mechanism.

METHODSSCs were isolated and cultured in vitro, and identified by S-100 immunofluorescence staining. The cultured SCs were divided into control group, hydrogen peroxide-treated group, hydrogen peroxide and PQQ treated groups. The intracellular superoxide dismutase (SOD) and malondialdehyde (MDA) content was detected; the apoptotic rate of SCs induced by hydrogen peroxide was determined by flow cytometry assay. The Hoechst33342 staining was used to detect the nuclear fragmentation and apoptotic nuclear condensation of SCs; the Rhodamine123 staining was used to detect the changes of mitochondrial membrane potential in SCs, the Western blot analysis was used to detect the expression of Bcl-2 in hydrogen peroxide induced SCs.

RESULTSThe SOD activity was significantly decreased and MDA level was increased in H2O2 induced SCs (P < 0.05), after addition of PQQ, the SOD content increased and MDA content decreased (P < 0.05). Flow cytometry results showed that the early apoptotic rate was 58.8% in H2O2 induced SCs, which has significant difference compared with the control group (P < 0.05), after addition of 10, 50, 100 nmol/L PQQ, the apoptotic rates were reduced to 33.7%, 18.7%, 3.9% respectively, showing significantly different with injured group (P < 0.05). Hoechst 33342 staining showed that H2O2 induced SCs had typical morphological characteristics, such as uptake of nuclear chromatin, nuclear shrinkage, nuclear fragmentation phenomenon. The proportion of apoptotic cells after PQQ treatment reduced. Rhodamine staining results showed that the H2O2 induced mitochondrial membrane potential reduction in SCs, which was reversed by addition of PQQ. Western blot analysis showed that the expression of Bcl-2 was decreased in H2O2 induced SCs, while it increased significantly after addition of PQQ (P < 0.05).

CONCLUSIONPQQ has a protective effect on oxidative stress-induced apoptosis of SCs.

Apoptosis ; drug effects ; Benzimidazoles ; Cell Nucleus ; drug effects ; DNA Fragmentation ; Fluorescent Dyes ; Humans ; Hydrogen Peroxide ; pharmacology ; Malondialdehyde ; metabolism ; Oxidants ; pharmacology ; Oxidative Stress ; Pyrroles ; pharmacology ; Quinine ; pharmacology ; Quinolines ; pharmacology ; Schwann Cells ; cytology ; drug effects ; Superoxide Dismutase ; metabolism

BACKGROUND:It has been confirmed that carboxymethylated chitosan has an promoting effect on Schwann cel proliferation and secretion, but its impact on the cyclic adenosine monophosphate-mediated protein kinase A signaling pathway in schwann cel stil needs further study. OBJECTIVE:To investigate the effect of carboxymethylated chitosan on cyclic adenosine monophosphate/ protein kinase A signaling pathway in rat schwann cels. METHODS:The Schwann cels of the second generation neonatal rats were obtained and seeded in 6-wel plate at a concentration of 1×109/L. These Schwann cels were cultured and divided into four groups. The Schwann cels in the control group were cultured by adding PBS. The Schwann cels in the experimental groups were cultured by adding 50, 100 and 200 mg/L of carboxymethyl chitosan solution, respectively. After 24 hours, the concentration of cyclic adenosine monophosphate, protein kinase A activity and cyclic adenosine monophosphate response element binding protein mRNA expression were detected. RESULTS AND CONCLUSION:Compared with the control group, carboxymethyl chitosan increased cyclic adenosine monophosphate concentrations, the activity of protein kinase A and cyclic adenosine monophosphate response element binding protein mRNA expression within the Schwann cels in a dose-dependent manner (P < 0.05). These results demonstrate that carboxymethyl chitosan can increase the concentration of cyclic adenosine monophosphate within the Schwann cels and promote protein kinase A activity, thereby activating cyclic adenosine monophosphate/protein kinase A signaling pathway.

BACKGROUND:Carboxymethylated chitosan is shown to promote some kinds of cells proliferation, but its effects on proliferation of Schwann cells need further studies.
OBJECTIVE:To investigate the effects of carboxymethylated chitosan on proliferation of Schwann cells and expression of nuclear factor-κB in cultured Schwann cells.
METHODS:Schwann cells from Sprague-Dawley rats at logarithmic growth phase were seeded in 96-wel plates, and cultured respectively with PBS, 0, 10, 50, 100, 200, 500, 1 000 mg/L carboxymethyl chitosan for 24 hours. cellproliferation was detected using the cellcounting kit-8 assay. After trypsin digestion, Schwann cells from Sprague-Dawley rats at logarithmic growth phase were used to prepare cellsuspensions, which were seeded in 6-wel cellculture plates and cultured respectively with 50, 100 and 200 mg/L carboxymethyl chitosan and PBS for 24 hours. Then, 5-bromo-2-deoxyuridine, real-time PCR and western blot assay were performed.
RESULTS AND CONCLUSION:cellcounting kit-8 and 5-bromo-2-deoxyuridine detection results showed that carboxymethyl chitosan at 50-1000 mg/L, especial y at 200-500 mg/L, could promote Schwann cellproliferation. Real-time PCR and western blot results showed 50-200 mg/L carboxymethyl chitosan could promote nuclear factorκB mRNA and protein expression in Schwann cells in a dose-dependent manner, suggesting carboxymethyl chitosan can promote Schwann cellproliferation and expression of nuclear factor-κB in Schwann cells cultured in vitro.

Background and purpose:It has confirmed that docetaxel, in combination with cisplatin or oxaliplatin have good effect in the treatment of advanced gastric cancer. This study in order to observe the clinical efficacy and adverse reaction of weekly docetaxel combined with cisplatin (DP) versus weekly docetaxel combined with oxaliplatin (DO) as the firrst-line treatment of advanced gastric cancer.Methods:A total number of 76 cases of advanced gastric cancer were randomly assigned 2 arms, 38 per arm. DP regimen group (docetaxel 35 mg/m2,ivgtt,dl,d8 combined with cisplatin 60 mg/m2, ivgtt,dl,repeated every 3 weeks) and DO regimen group(docetaxel 35 mg/m2,ivgtt,dl,d8 combined with oxaliplatin 120 mg/m2 ivgtt,dl,repeated every 3 weeks). Results:No significant difference was found between DP regimen group and DO regimen group on the objective RR(37%vs 41%),PFS (4.9vs 4.4 months) and OS (9.7vs 12.3 months). The main grade 3 or 4 toxicity in the DP and DO groups was neutropenia, DO was less associated with nausea and vomiting, but more associated with peripheral neuropathy than DP group. No signiifcant difference was found between DP regimen group and DO regimen group on the anemia, thrombocytopenia, diarrhea, alopecia (P>0.05).Conclusion:Weekly docetaxel combined with cisplatin (DP) shows similar efifcacy and toxicity compared with weekly docetaxel combined with oxaliplatin (DO) as the ifrst-line treatment of advanced gastric cancer and worthy of further study.

Objective To study the effects of carboxymethylated chitosan (CMCS) to nitric oxide (NO)-induced apoptosis on rat chondrocytes,and explore p38MAPK signal transduction pathway in the process and its mechanism.Methods The rat articular cartilage cells were cultured in vitro,collagen type-2 (collagen-2) immunohistochemical staining was used to identify the cartilage cells.The model of chondrocyte apoptosis was built by different concentrations of sodium nitroprusside (SNP) induction.The cells were divided into the control group,the SNP treated group SNP+CMCS treated group,and the SNP+p38 MAPK inhibitor SB203580 treated group.The apoptotic rate of chondrocytes was calculated by FCM,apoptotic nuclei was identified by Hoechst33342 stain,the mitochondrial membrane potential changes was detected by Rhodamine123 (Rho123) stain,the expression of p38 and p-p38 were detected by Western blotting analysis.Results 1-3 mmol/L SNP could induce chondrocyte apoptosis,the apoptotic rate was increased with the SNP increasing,the most obvious apoptosis was occurred in 3 mmol/L SNP treated chondrocytes,which was 69.8％ (P＜0.05).SNP could increase the nuclear fragmentation of chondrocytes,the cells with nuclear fragmentation was significantly higher than that in the control group.SNP could reduce mitochondrial membrane potential in chondrocytes,which decreased significantly compared with the control group.SNP could increase the p-p38 expression in chondrocytes,which was 4.3 times compared to the control group.CMCS of different concentrations could reduce the apoptotic rate of SNP-induced chondrocytes,which was 51.0％,29.9％ and 15.2％,which was decreased significantly (P＜0.05) when compared with 3 mmol/L SNP induced group,CMCS decreased the cells number of SNP-induced nuclear fragmentation.CMCS increased the mitochondrial membrane potential in SNP-induced chondrocytes.CMCS reduced the expression levels of p-p38 in SNP-induced chondrocytes.Conclusion CMCS has protective effect on SNP-induced apoptosis of chondrocytes.This process is completed by inhibiting the activity of p38 MAPK signal pathway.

BACKGROUND:Recently, non-fusion technology representing as artificial cervical disc replacement continues to improve. On the basis of reconstruction of disc structure and function of involved segments, cervical spine structure of surgery area segment is significantly close to dynamic and static load stress distribution required by natural physiological systems. It effects are apparent in protecting intervertebral facet joints of degenerated segment and structure and function of the cervical spine of adjacent segments and in maintaining cervical dynamic stability, which presented obvious methodological strengths compared with segmental fusion technology.
OBJECTIVE:To evaluate the clinical outcomes of anterior cervical discectomy and fusion and Bryan artificial cervical disc replacement in the treatment of single-level cervical spondylotic myelopathy or radiculopathy.
METHODS:A total of 43 middle and old age patients with single-level cervical spondylotic myelopathy or radiculopathy, who were treated from March 2010 to March 2012, were enrol ed in this study. They were randomly assigned to anterior cervical discectomy and fusion group (fusion group) and Bryan artificial cervical disc replacement group. Range-of-motion of cervical overal and adjacent intervertebral area near the intervertebral space was observed with radiography. During fol ow-up, postoperative recovery of neurological function was evaluated using Japanese Orthopaedic Association scale, visual analog scale and neck disability index.
RESULTS AND CONCLUSION:None patients experienced complications of neurovascular injury during and after the surgery. Range-of-motion of postoperative overal cervical vertebra and adjacent joint was improved in the Bryan artificial cervical disc replacement group compared with the fusion group. Neurological function was apparently improved after surgery in each group. At 3 months after surgery, scores of Japanese Orthopaedic Association, visual analog scale and neck disability index were significantly improved in the Bryan artificial cervical disc replacement group compared with the fusion group (P<0.05). During final fol ow-up, there were significant differences in visual analog scale scores between the two groups. Japanese Orthopaedic Association scale score and neck disability index score were similar between the two groups. During fol ow-up, no prosthesis sinking, displacement or heterotopic ossification were detected. These data indicated that artificial cervical disc replacement could effectively keep the range of motion of cervical segments and protect disc degeneration of adjacent segment. Mid-term fol ow up obtained similar improvement of neurological function of fusion surgery. The moderate-term and short-term efficacies of non-fusion technology were better than fusion technology in the treatment of single-level cervical spondylopathy.

Objective:To obtain purified recombinant Litopenaeus vannamei allergen protein Lit v 1.2.Methods: The target gene of Lit v 1.2 was inserted into clone vector pGEM-T and then ligated to the expression vector pET 44a.The pET44a-Liv 1.2 was transformed into Rosetta and screened by ampicillin resistance .The recombinant protein was expressed by IPTG induction .The protein was purified by 6-His tag affinity chromatography and the purification was analyzed by SDS-PAGE gel electrophoresis .Results:The ex-pression plasmid pET44a-Lit v 1.2 was constructed.SDS-PAGE showed that expressed Lit v 1.2 was efficient and soluble in E.coli Rosetta.The protein molecular weight was consistent with the theoretical value .The highly purified target protein was obtained.Conclusion:In this study ,we successfully gained highly purified recombinant allergen protein Lit v 1.2 which was expressed in prokaryotic system and purified by affinity chromatography column .The purified Lit v 1.2 protein will facilitate us to further study its role in immunological responses .

Objective To investigate the effect of analgesia with dezocine on CD4+ and CD8+ T lymphocytes in patients after pulmonary lobectomy.Methods Seveuty patients with early non-small-cell lung cancer were selected,and they were scheduled the pulmonary lobectomy.The patients were divided into control group and dezocine group by random digits table method with 35 cases each.The patients in dezocine group were given 0.5 mg/ml dezocine by patient-controlled analgesia (PCA) pump,and the patients in control group were given 0.9％ sodium chloride by PCA pump.The peripheral blood lymphocytes count and percentages of CD4+ T lymphocytes,CD8+ T lymphocytes,natural killer cell (NK cell) before anesthesia induction and 4,24,48 h after operation were detected.Results In control group,the peripheral blood lymphocytes count and NK cell 24 h after operation were (1.08 ± 0.21) × 109/L and 0.141 4 ± 0.021 8,which before anesthesia induction were (1.71 ± 0.33) × 109/L and 0.190 9 ± 0.022 8,and there were statistical differences (P ＜ 0.05).In dezocine group,the peripheral blood lymphocytes count and NK cell 24 h after operation were (1.14 ±0.28) × 109/L and 0.124 9 ± 0.027 6,which before anesthesia induction were (1.69 ± 0.28) × 109/L and 0.198 6 ± 0.027 5,and there were statistical differences (P ＜ 0.05).The CD4+ T lymphocytes 24 h after operation in dezocine group was significantly higher than that in control group (0.355 6 ±0.031 1 vs.0.273 5 ±0.029 4),and there was statistical difference (P ＜0.05).There was no statistical difference in CD8+ T lymphocytes between the 2 groups (P ＞ 0.05).Conclusion Analgesia with dezocine can notably improve immunosuppression in patients after pulmonary lobectomy.