BACKGROUND: It was reported that the reason why the fracture healing become more and more difficult with the increasing age may be associated with the varied gene regulation of bone marrow mesenchymal stem cells differentiation into osteoblast and lipoblast. However, the exact mechanism under it remains poorly understood.OBJECTIVE: To explore the association of the age-related changes on expression of peroxisome proliferator-activated receptor γ (PPARγ) and core binding α1 (Cbfα1) with the age-relatad fracture healing impairment, we studied the expression changes of PPARγ and Cbfα1 during aging fracture healing.DESIGN, TIME AND SETTING: A randomized control animal trial was performed in the Third Xiangya Hospital of Central South University between October 2007 and Febuary 2008. MATERIALS: Six male SD rats of 3 months and the other six of 23 months, randomly-selected, were divided into two groups: the old experiment group (23 months) and the young control group (3 months). METHODS: Transverse osteotomies on the middle-upper parts of left tibiae were performed to the rats, with the self-made extemal mini-fixator implanted there simultaneously. Tractions were taken twice a day (0.2 mm/d) for 14 days from day 2 till day 15 post operation. On day 15 post operation, rats were sacrificed to harvest left tibea samples. MAIN OUTCOME MEASURES: Both imaging examination and histological observation were performed to the fractures of rats. The PPARγ and Cbfα1 expression were detected with reverse transcription-polymerase chain reaction (RT-PCR) technique. RESULTS: All samples were involved in the results analyses. The imaging examination showed that many osteotylus generated between the broken ends of fractures in the young control group; that the bone formation of the young control group was much better than that of the old experiment group. The histological observation showed that large amounts of osteotylus growth occurred to every animal in the young control group and their membrane bone formation was remarkable; comparatively, the osteotylus growth in the old group was weakened dramatically, and only fibrous joints were seen. The RT-PCR detection showed that differences of significance existed in both PPARγ and Cbfα1 mRNA expressions between the two groups, with the much higher Cbfol mRNA expression in the bone marrow of rats in the young group and the much higher PPARy mRNA expression in the bone marrow of rats in the old group.CONCLUSION: Rats of different ages show different fracture healing abilities, i.e. the higher ages, the lower fracture healing abilities. In addition, expressions of PPARγ and Cbfα1 in the bone marrow of rats alter with the increasing age, which indicates that there are certain associations between the changed expression level of the two factors and the fracture healing impartment.

Objective To investigate the optimized parameters of dye (SYBR Green Ⅰ) realtime fluorescent polymerase chain reaction (RF-PCR) for detecting αβT lymphocyte clones in the peripheral blood and its application in monitoring specific T cell clone in the peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis B (CHB). Methods The total RNA was extracted from the PBMC of six healthy donors, and was reversely transcripted into cDNA. Then the cDNA was amplified using RF-PCR with the primers specific for T cell receptor β viable region (TCRBV) gene families as upstream primers and the primer for T cell receptor (TCR) β constant region (TCRBC) as downstream primer. The annealing temperature,concentration of primers and the total number of cycles were comparatively analyzed. The optimized PCR was performed to investigate the 24 TCRBV gene families from 12 patients with CHB, and the PCR products were monitored by melting curve analysis, and the clone expansion of peripheral blood T cell was detected by peak-motif of melting curve analysis. Results The optimized annealing temperature, final premier concentration,the number of cycles were 60.6 ℃, 0.5 μmol/L and 40 cycles, respectively. The begin temperature for melting curve analysis was better as 80 ℃ compared to 75 ℃. There was mono-peak on melting peak chart for TCRBV gene families in PBMC from patients with CHB, and PCR products of the single peak were determined as monoclonal T cell by sequencing. Conclusions The optimized reaction parameters of RF-PCR for monitoring 24 TCRBV gene families are determined. The melting peak chart could be used to monitor the clone expansion of the peripheral lymphocytes and to detect the clone-specific T cells in the peripheral blood from patients with CHB.

Objective To approach the effects of full Marathon on striated muscle and renal function of Marathon amateurs without complaints. Methods A prospective self-paired design study was conducted. The amateurs without body discomfort, hematuria, brown urine, or persistent muscle pain within 1 week after the 2012 Xiamen International Marathon Race were enrolled voluntarily. The peripheral blood and random urine specimens of all subjects under static status 1 week before the race and after the race instantly (within 10 minutes after finishing the race) were collected to detect markers of renal function and striated muscle injury. Results Sixty-one subjects were included in the final analysis of the study with full Marathon of 42.195 km and mean race time of (297.05± 55.60) minutes. Compared with those under static status before the race, the markers of renal function including the levels of urinary N-acetyl-beta-D-glucusamidase [NAG (U/L): 64.00 (54.50, 85.50) vs. 9.50 (8.10, 11.50)], urinary β2-microspheres protein [β2-MG (μg/L): 261.00 (128.50, 1 608.00) vs. 66.60 (33.75, 123.00)], random urinary creatinine [UCr (μmol/L): 19 066.56±10 938.31 vs. 5 872.52±4 363.20] and serum creatinine [SCr (μmol/L): 129.97±25.84 vs. 97.39±14.51] immediately after the race were significantly increased (all P < 0.01); the markers of muscle injury including the levels of serum creatine kinase [CK (U/L): 864.00 (504.00, 1 644.00) vs. 164.00 (128.00, 256.00)], lactic dehydrogenase [LDH (U/L): 383.26±141.69 vs. 182.23±41.12], myoglobin [Mb (mg/L): 1 880.00 (1 080.00, 3 300.00) vs. 42.00 (36.00, 54.50)], alanine aminotransferase [ALT (U/L): 27.0 (19.5, 38.0) vs. 24.0 (15.0, 29.5)] and aspartate transaminase [AST (U/L): 52.07±25.13 vs. 28.28±11.86] were also significantly increased (all P < 0.01), and the increase in CK, Mb, and LDH were more significant. It was shown by correlation analysis that CK after race was negatively correlated with age (r = -0.352, P = 0.005) and body mass index (r = -0.271, P = 0.035), and it was positively correlated with racing time (r = 0.387, P = 0.002) and urinary β2-MG after the race instantly (r = 0.364, P = 0.004). Mb after race was negatively correlated with body mass index (r = -0.331, P = 0.009), and it was positively correlated with urinary β2-MG after the race instantly (r = 0.315, P = 0.013). LDH after race was negatively correlated with age (r = -0.275, P = 0.032) and body mass index (r = -0.377, P = 0.003), and it was positively correlated with urinary β2-MG after the race instantly (r = 0.424, P = 0.001). Conclusion Full Marathon could significantly impact striated muscle and renal function of Marathon amateurs without complaints.

BACKGROUND:Increasing attention has been paid on the role of advanced glycation end products in bone tissue. Glucose metabolic disorder is one of the main reasons for the increase of advanced glycation end products.
OBJECTIVE:To observe the change of advanced glycation end products expressed in type 2 diabetes rats, and to investigate the relationship between impaired fracture healing and change of advanced glycation end products expression in vivo.
METHODS:Thirty Sprague-Dawley rats were randomly and equal y divided into two groups:control group (normal feeding) and experimental group (high fat and sucrosum diet feeding to establish type 2 diabetes model). After diabetes models were established, the model of distraction osteogenesis in the left tibiae of al the rats was produced. Distraction was given 0.3 mm per day and continued for 14 days.
RESULTS AND CONCLUSION:After the traction was complete, cal us formation in distraction gap was obviously reduced in experimental group compared with control group by X-ray examination. The array of microcolumn formation was disordered and the area of primary matrix front was catachromasis by histology examination. The enzyme-linked immunosorbent assay results showed that, the level of advanced glycation end products was obviously elevated (P<0.01) while osteocalcin was obviously reduced (P<0.01) in experimental group in comparison with control group. The formation of distraction cal us was impaired in the process of fracture healing and blood of type 2 diabetes rats. The increase of advanced glycation end products may be one of the reasons that cause impaired fracture healing in diabetic rats.

Objective To investigate the effect of exogenous hydrogen sulfide (H2S) on intestinal mucosal barrier after cardiopulmonary resuscitation (CPR) in cardiac arrest (CA) rabbits. Methods Forty-four male New Zealand rabbits were divided into sham operation group (Sham group, n = 12), post-cardiac arrest syndrome (PCAS) group (n = 16) and H2S intervention group (PCAS+NaHS, n = 16) according to random number table method. The rabbit model of PCAS was established by tracheal clamping and suffocation, and CPR was started at 5 minutes after CA. However, Sham group did not clamp the tracheal intubation after anesthesia, and the other operations were the same as those in PCAS group. In the PCAS+NaHS group, a bolus of NaHS (0.5 mg/kg), a H2S donor, was injected via era vein 1 minute before the start of CPR, followed by a continuous injection of NaHS (1.5 mg·kg-1·h-1) for 3 hours, while the rabbits in other group were intravenously injected with the same volume of normal saline (NaCl 0.9%). Intestinal and portal vein blood samples were collected 24 hours after return of spontaneous circulation (ROSC). The level of serum fluorescein isothiocyanate-dextran (FD-4) was detected by fluorescein isothiocyanate (FITC) labeling method to reflect intestinal mucosal permeability. After hematoxylin-eosin (HE) staining of small intestine tissues, the morphological changes of mucosa were observed under light microscope, and the intestinal mucosa injury score was calculated. The expression of tight junction protein ZO-1 in intestinal mucosa was detected by immunohistochemistry. The content of malondialdehyde (MDA) in small intestinal tissue was determined by thiobarbituric acid chromogenic method, the activity of superoxide dismutase (SOD) was determined by xanthine oxidation method, and the level of myeloperoxidase (MPO) was determined by double antibody sandwich enzyme linked immunosorbent assay (ELISA) to reflect the oxidative stress and inflammatory reaction in small intestinal tissue. The expression of apoptosis protein (caspase-3) and autophagy related protein (Beclin-1, LC3) in small intestine tissue was detected by Western Blot. Results 12, 13 and 14 animals were successfully resuscitated in Sham group, PCAS group and PCAS+NaHS group respectively, while 12 animals in each group survived to the end of experiment. Compared with Sham group, the level of FD-4 in portal vein serum was significantly increased in PCAS group (mg/L: 11.95±0.59 vs. 1.43±0.48, P < 0.05), the pathological injury and inflammation infiltration were obviously aggravated under light microscope, the score of small intestine injury was significantly increased (4.21±0.37 vs. 0.36±0.18, P < 0.05), the expression of tight junction protein ZO-1 in the intestine was visibly down-regulated detected by immunohistochemistry, MDA content and MPO activity were significantly increased [MDA (nmol/mg): 3.65±0.32 vs. 1.54±0.24, MPO (U/g): 362±35 vs. 134±18, both P < 0.05], while SOD activity was significantly decreased (U/mg:78.84±7.49 vs. 115.48±8.48, P < 0.05), the expression levels of cleaved capase-3, Beclin-1 and LC3 proteins in the intestine were significantly increased (caspase-3/β-actin: 1.11±0.08 vs. 0.21±0.02, Beclin-1/β-actin: 2.08±0.11 vs. 0.42±0.03, LC3/β-actin: 1.05±0.07 vs. 0.37±0.05, LC3-Ⅱ/ LC3-Ⅰ: 1.28±0.14 vs. 0.17±0.02, all P < 0.05). Compared with PCAS group, the portal vein serum FD-4 level in PCAS+NAHS group was significantly decreased (mg/L:5.59±0.48 vs. 11.95±0.59, P < 0.05), the intestinal mucosal pathological injury and inflammatory cell infiltration were significantly decreased, the score of small intestine injury was significantly decreased (2.18±0.47 vs. 4.21±0.37, P <0.05), the expression of ZO-1 in intestine was significantly increased, MDA content and MPO activity in intestine were significantly decreased [MDA (nmol/mg): 2.65±0.31 vs. 3.65±0.32, MPO (U/g): 251±21 vs. 362±35, both P < 0.05], while SOD activity was significantly increased (U/mg: 96.86±7.52 vs. 78.84±7.49, P < 0.05), while the expression of activated caspase-3, Beclin-1 and LC3 proteins was significantly decreased (caspase-3/β-actin: 0.72±0.06 vs. 1.11±0.08, Beclin-1/β-actin: 0.96±0.08 vs. 2.08±0.11, LC3/β-actin: 0.72±0.06 vs. 1.05±0.07, LC3-Ⅱ/ LC3-Ⅰ:0.83±0.09 vs. 1.28±0.14, all P < 0.05). Conclusion H2S has a protective effect on intestinal mucosal injury induced by CA/CPR, which may be related to tight junction protein ZO-1 up-regulation, oxidative stress alleviation, inflammation reduction, apoptosis and autophagy inhibition.

OBJECTIVE: To observe the diagnostic capacity and its influencing factors in the toxicologic pathology diagnostic laboratories in China. METHODS: Inter-laboratory comparison of toxicologic pathology diagnosis was cosponsored by Chinese Society of Toxicology-Toxicologic Pathology Specialty Section and Guangdong Province Hospital for Occupational Disease Prevention and Treatment. Nine digital slices of digestive system lesions were screened as comparison samples,and the reference institution with toxicologic pathology diagnostic laboratories completed the diagnosis within the prescribed time. According to the three grades of “excellent”,“satisfaction”and “dissatisfaction”,the evaluation was carried out.RESULTS: A total of 74 reference institution participated in this comparison,which distributed in 20 provinces and 4 municipalities and 156 pathologists. The reference institutions were mainly distributed in North China,Southern China and East China. There was an average of 2 pathologists per laboratory,and in the quantity of academic title,the junior,intermediate,and senior was 15,70 and 46 persons respectively. Parasitic hepatocyte cysts( 97. 3%),adenocarcinoma of small intestine( 95. 9%) and polyarteritis nodosa of the pancreas( 89. 2%) had the highest rate of “excellent”grade,while the duodenal gland inflammation( 67. 6%),foam cell aggregation in colonic propria( 40. 5%) and hepatoma adenoma( 32. 4%) had the highest rate of dissatisfaction grade in the evaluation of single case. In the overall evaluation,reference laboratories reached the “excellent”grade and the “satisfaction”grade were 78. 4% and 21. 6% respectively.The number of pathologists provided by each reference laboratory had impacts on the overall evaluation level and the single case evaluation( neoplastic lesions) in the evaluation of single case( P < 0. 05). The types of the reference laboratory,the regional distribution and the grade of the academic title had no effect on the diagnostic ability( P > 0. 05). CONCLUSION: The reference laboratory is superior in diagnosing the digestive system lesions in the inter-laboratory comparison activity.The number of pathologists in the reference laboratory is one of the influencing factors of its diagnostic ability.

OBJECTIVE: To explore the non-neoplastic hepatic lesions in SD rats at different ages. METHODS: The specificpathogen free SD rats were collected from the blank control groups used in subacute toxicity tests,subchronic toxicity tests and chronic toxicity combined with carcinogenic tests for safety evaluation. At the end of each experiment,i. e. week 10,19,56 and 108(assigned into four groups: 10,19,56 and 108 weeks,each contained 100 rats with each sex),rats were executed. The liver organ coefficient was calculated,the pathological examination was performed,and the non-tumorous lesions in the liver were analyzed. RESULTS: The liver organ coefficients at the age of 19,56,108 weeks were lower than that of 10 weeks(P < 0. 05); those at the age of 56 and 108 weeks were lower than that of 19 weeks(P < 0. 05),and that of 108 weeks was greater than of 56 weeks(P < 0. 05). Among the 10-week-old,19-week-old,56-week-old and 108-week-old groups,the types of non-neoplastic hepatic lesions detected in the female rats were 6,6,13 and 15 respectively,meanwhile those in the male rats were 6,6,13 and 15 respectively. Both male and female rats,the incidences of hepatocyte fatty degeneration,edema and hepatic infiltration of inflammatory cells were significantly increased with the increase of age in each group(P < 0. 05). The incidences of intrahepatic bile duct proliferation and intrahepatic bile duct fibrosis in rats at the age of 56 and 108 weeks were higher than those at the age of 10 and 19 weeks(P < 0. 008).Moreover,the frequency of hepatic sinus expansion lesions in rats at the age of 108 weeks was higher than those of 19 weeks(P < 0. 008). CONCLUSION: Spontaneous non-neoplastic lesions in the liver of SD rats were common,primarily demonstrated as hepatocyte fatty degeneration,edema and infiltration of inflammatory cells. The incidences of lesions increased with the increase of age.

OBJECTIVE: To explore the spontaneous non-tumor lesion of kidney and its correlation with different age and sex in SD rats. METHODS: Eight hundred specific pathogen free SD rats were collected from the blank control groups used in subacute toxicity tests,subchronic toxicity tests and 1 or 2 years of chronic toxicity combined with carcinogenic tests. Rats were randomly divided into 4 groups with 10,19,56 or 108 weeks of experimental periods. Each group consisted of 100 female and 100 male rats. The renal tissues were collected at the end of each experiment,and the renal organ coefficients were calculated. The pathological non-tumor changes of the kidneys were analyzed. RESULTS: The renal organ coefficients in female rats at the age of 56 and 108 weeks were both lower than that of 10 and 19 weeks( P < 0. 008). The renal organ coefficient of male rats at the age of 56 weeks was lower than that of 10 and 19 weeks( P < 0. 008). The renal organ coefficient of male rats at the age of 108 weeks was higher than that of 56 weeks( P < 0. 008). The renal organ coefficient of male rats at the age of 108 weeks was higher than that of female rats of 108 weeks( P < 0. 008). The incidence of renal tubular calcium salt deposition,interstitial inflammatory cell infiltration and renal tubular dilatation in the female rats at the age of 108 weeks were higher than those in the male rats at the age of 108 weeks( P < 0. 05). The chronic progressive nephropathy incidence of female rats at the age of 108 weeks was lower than that of male rats aged 108 weeks( P < 0. 01).The renal tubular calcium salt deposition incidence of female rats aged 56 weeks was higher than that of male rats aged 56weeks( P < 0. 01). CONCLUSION: The spontaneous non-tumor lesions in the kidney of SD rats were common. The incidence of some lesions was different in the same age group with different sex.