BACKGROUND:The reported time of bone marrow mesenchymal stem cel s induced to differentiate into chondrocytes is different. Few studies have observed and compared the cel s’ dynamic transformation during the induction process.
OBJECTIVE:To observe the dynamic differentiation and the mature time of rabbit bone marrow mesenchymal stem cel s which were directional y induced to chondroblasts for 8, 11, 14, 17, 20 days.
METHODS:Bone marrow was aspirated from the femur of New Zeal rabbits, and bone marrow mesenchymal stem cel s were isolated by gradient centrifugation. After cultivation and amplification, bone marrow mesenchymal stem cel s at passage 3 were directional y induced to chondrocytes by the serum-free medium containing transforming growth factor beta-1. The experiments were divided into five groups according to different induction time points:8 days, 11 days, 14 days, 17 days, 20 days. Then cel ular morphology, toluidine blue staining, typeⅡ col agen immunohistochemistry, aggrecan content in induction medium, and chondrogenic differentiation in each group were observed and compared.
RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cel s had apparently transformed in morphology at 8 days of induction, and presented obvious chondrocytes’ morphology at 14 days. The aggrecan in induction medium could be detected at a low level at 4 days, significantly increased at 8 days, and maintained slow increasing at 20 days. At 14 days, the metachromatic particles could be found by toluidine blue staining, and the col agen type Ⅱimmunohistochemistry was significantly positive in cel climbing slice. Experimental findings indicate that, bone marrow mesenchymal stem cel s that are monolayer cultured in a high density can be induced into chondroblasts at the effect of transforming growth factor beta-1 and other factors. There are a few chondroblasts in the early induction process, then cel s begin to have chondrocytes morphology and function after induced for 8 days, and may differentiate to mature chondrocytes at 14 days. In addition, they can keep a high biological activity in the induction process.

Objective To evaluate the category of the variations on bilateral renal artery and clinical significance with 64-slice spiral CT angiography. Methods CT angiography of 250 patients were retrospectively analyzed. The variations on bilateral renal artery were observed and classified. Results According to the number of the main renal artery, the anatomy of renal artery was classified and typed in details. The incidence of the variations on renal artery in the patients and kidneys was 39.20% (98/250) and 22.80% (114/500), respectively. The incidence of early branch of the main renal artery and accessory renal artery was 9.60% (48/500) and 11.80% (59/500), respectively. No statistical difference of variation incidence was found between male and female, nor between left and right kidneys. The incidence of unilateral and bilateral variations on renal artery was 32.80% (82/250) and 6.40% (16/250), respectively, and the combinations of the latter were multiform. Conclusion Anatomical variations on renal artery can be showed clearly with 64-slice spiral CT angiography. Variations on bilateral renal artery are important to select the donor in case of renal transplantation.

Objective To investigate the effects of hypoxia-inducible factor (HIF)-1α on glycolysis of human esophageal squamous carcinoma cells and the possible mechanism.Methods TE13 and Eca109 cells were cultured under normal oxygen (20％O2) and hypoxia (1％O2) conditions.The hypoxia was duration 6 hours,12 hours,24 hours and 48 hours.HIF-1α gene was stable silented by RNA interference method and TE13/small interfering HIF cells and Eca109/siHIF cells were obtained.The cell culture condition and time was same as TE13 and Eca109 cells.The changes of HIF-1α expression were detected by Western-blot.The changes of lactic acid concentration in cell culture supernatant were determined by Spectrophotometry.The changes of glucose transporter-1 (GLUT-1) and lactic dehydrogenase A (LDHA) expression at mRNA level were examined by realtime polymerase chain reaction.The changes of GLUT-1 and LDHA expression at protein level were tested by Western blot.Using t or t' test to analyze the effects of hypoxia duration on HIF-1αexpression at protein level.One-way ANOVA was applied for the difference analysis between the groups.Results In TE13 and Eca109 cells,the HIF-1α expression significantly increased under hypoxia condition and reached the peak at 12 hour (t=6.11,8.31; both P＜0.05).The lactic acid secretion of TE13/siHIF cells and Eca109/siHIF cells was (1.24±0.33) and (1.28±0.37) mmol/L,which significantly decreased when compared with TE13 and Eca109 cells [(3.25±1.34) and (4.91±1.69) mmol/L,t=2.53,3.59,both P＜0.05].The lactic acid secretion of TE13 and Eca109 cells significantly increased after hypoxia [(6.48±1.73) and (8.02± 1.95) mmol/L,t=2.715,2.050,both P＜0.05].There was no significant lactic acid secretion in TE13/siHIF cells and Eca109/siHIF cells after hypoxia (P ＞ 0.05).The expressions of GLUT-1 and LDHA at mRNA level were significantly suppressed in TE13/siHIF cells and Eca109/siHIF cells (normal oxygen:t=6.98,3.92,7.25,3.67,all P＜0.05).The expression of GLUT-1 at protein level remarkably weaked (normal oxygen:t=4.57、16.56,hypoxia:t=6.19、6.09,all P＜0.05),while the expression of LDHA at protein level slightly decreased (P＞0.05).Conclusions The level of glycolysis can be lowered by suppression HIF-1α expression in human esophageal squamous carcinoma cells.The pathway may be involved in the suppression of GLUT-1 and LDHA expression.Except for HIF-1α,there may be other regulating factors in LDHA protein expression at same time.

Objective To assess the feasibility of applying intensity-modulated radiotherapy(IMRT)simultaneous integrated boost(SIB)to replace conventional radiotherapy(CR)plus brachytherapy of whole pelvis in locally advanced cervical eaneer(LACC).Methods Five LACC patients based difference position of uterus were chosen and worked out CR and IMRT SIB plans respectively.Dose distributions were compared between IMRT SIB and CR.Results When uterus was in ante-,neutral-,retro-pnsition and deviation respectively,IMRT SIB could provide enough and homogeneous dose distribution for target volume and reduce irradiated volumes and doses for organs at risk(recta,bladder and small intestine)than CR.The doses of the A,B,and fundus of uterus were higher in IMRT SIB than CR.However,in ease of small intestine was close to or encircled the uterus,the targets volume dose would be inadequacy.Conclusions LACC IMRT SIB's dose distribution is better than CR(except excess ante-position)and may help to treat those patients who couldn't be suitable with brachytherapy.

Objective To analyze the differentially expressed genes in esophageal squamons cell carcinoma (ESCC), para-cancerous tissue (PCT) and normal esophagus tissue (NET) using oligomicroarray and to identify the target genes related to the development and progression of esophageal carcinoma. Methods The total RNAs isolated from ESCC, PCT or NET using one step Trizol method were purified and reversely transcribed into cRNAs. The cRNAs were then fluorescence labeled and hybridized with Agilent oligomicroarray (21 074 probes). The fluorescence intensity features were detected by Agilent scanner and quantified by feature extraction software. The selected candidate genes were confirmed by real time real time fluorescent quantitative RT-PCR immunohistochemistry andWestern blotting.Results ① The oligomicroarray demonstrated that there were 38 up-regulated genes and 61 down-regulated genes. ② The real time fluorescent quantitative RT-PCR revealed that five genes (CTHRC1, INHBA, SPP1 ,LUM, HRK)were more differentially expressed in up-regulated genes. Of which, CTHRC1 displayed more disparity.③ Immunohistochemistry examination showed that the higher expression of CTHRC1 (56.5 %, 26/46) was observed in ESCC. There was significantly difference in expression of CTHRC1 between patients with or without lymph node metastasis (P<0.05). ④ CTHRC1 protein was expressed in both TE-13 and Eca-109 cell lines. Conclusion CTHRC1 is probably one of the most significant biomolecules in ESCC.

Objective To explore the effects of gossypol acetate on the morphological features and the gene expression in the femoral head of Sprague-Dawley rat in vivo after treated with dexamethasone .Methods Dexamethasone(Dex) was injected into the abdominal cavity of SD rats at an dose of 10 mg/kg ,twice a week ,and feed gossypol acetate 5 mg · kg -1 · d-1 .The controls re-ceived saline 2 mL injection .The treatment lasted for 12 and 20 weeks .The slices of the femoral head were made for HE and immu-nohistochemical study .The total mRNA was extracted for RT-PCR assessment .Results The cancellous bone trabecular became sparse ,trabecular bone area ratio decreased ,bone marrow fat tissue increased .These changes were fitted for pathological character of bone necrosis .The gossypol acetate could not affect the pathological changes .The proportion of the positive stained osteoblasts increased ,adipocytes decreased .PPARγ,C/EBPα,11β-HSD1 expression enhanced ,Runx2 down regulated in the treatment groups and GAA group .Conclusion Dex can induce evident pathological changes conform to the characters of femoral head necrosis .They may have closed correlation between 11β-HSD1 and the gene expression .But GAA could not affected the pathological changes and abnormality of the gene expression .

Objective To explore the clinical effect of treatment on facial soft tissue depression by autologous fat granule mixed with adipose-derived stem ceils.Methods A lof of 36 patients with facial soft tissue depression from September 2011 to March 2013 in this clinic were as the research objects.The autologous fat granules were harvested from patients' lateral thigh.The autologous fat granules mixed with adipose-derived stem cells (ADSCs) which were harvested by separating and cleaning were injected into facial soft tissue depression.The standard was based on the subjective satisfaction of patients and the physicians' objective assessment;there were two unrelated doctors to reach an objective assessment of the clinical efficacy based on the comparison of preoperative and postoperative photographs.Results A lof of 36 patients were followed up for 5 16 months,low absorption rate of injec ted fat and significant improvement in shape filling areas were observed,and skin texture,pores,spots and wrinkles were significantly improved.Conclusions Autologous fat granule mixed with AD SCs in the treatment of facial soft tissue depression is a minimally injuried,easily-handled,cheap and safe procedure.It is an ideal treatment of facial soft tissue depression.All the patients have no complications such as infection,induration and fat liquefaction.

Objective To investigate the changes of hypoxia-inducible factor(HIF)-1α and hexokinase-Ⅱ(HK-Ⅱ)expression in human esophageal squamous cell carcinoma and its effect in glycolysis.Methods TE13 cells and Eca109 cells were cultured under hypoxic condition(1 ％O2)for different hypoxic time(6,12,24 and 48 hours).Cells cultured under normal oxygen condition(20％O2)were set as control.The changes of HIF-1α and HK-Ⅱ expressions at protein level were detected by Western blot.HIF-1α genes were specifically silenced with RNA interference technology(RNAi),and then the changes of HIF-1α and HK-Ⅱ expression were determined by realtime PCR and Western blot.Under normal oxygen and hypoxic condition,the changes of lactic acid concentration in cell culture medium were detected by spectrophotometric method.Results Under hypoxic condition,the expression of HIF-1α and HK-Ⅱ gradually increased as hypoxic time extended(P＜0.05),reached a peak at 12h and then gradually decreased as time extended.Compared with that under normal oxygen condition,the expression of HK-Ⅱ in TE13 cells and Eca109 cells significantly increased under hypoxic condition(P＜0.05),which was more significant after 12 hours hypoxia.The result of realtime PCR indicated that under normal oxygen condition the expression of HIF-1α at RNA level in TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference(P＜0.05).The expression of HK-Ⅱ at RNA level was consistent with the result of HIF-1α.Under normal and hypoxia condition,the expression of HK-Ⅱ at protein level in TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference,and the difference was statistic significant(P＜0.05).The lactic acid secretion of TE13 cells and Eca109 cells under hypoxia condition(14.707 ± 3.594 and 15.062 ±3.901)was higher than that under normal oxygen condition(6.070±1.839 and 6.891±1.592,P＜0.05).The lactic acid secretion of TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference,and the difference was statistic significant(P＜0.05).Conclusion The expressions of HIF-1α and HK-Ⅱ in human esophageal squamous cell carcinoma significantly increased under hypoxia conditions.The expression of HK-Ⅱ is closely correlated with lactic acid concentration and HIF-1α expression.HIF-1α may affect cell glycolysis through HK-Ⅱ.

Objective To investigate the inhibitory effect of blocking PI3K/AKT pathway by wortmannin on hypoxia-inducible factor 1α (HIF-1α) and the effect on the expression of glycolysis associated genes in human esophageal carcinoma cell lines TE1 and TE13,and to analyze the relation between PI3K/AKT-HIF-1α pathway and glycolysis in esophageal carcinoma cells. Methods Esophageal carcinoma cell lines TE1 and TE13 pretreated with wortmannin (2 μmol/L) were incubated under normoxic and hypoxic conditions.And each cell line was divided into four groups.The expression of HIF-1α and glycolysis associated genes GLUT-1,LDHA and HK-Ⅱ at protein level were measured by.Western blot.The expression of HIF-1α,GLUT-1,LDHA and HK-Ⅱ at mRNA level was determined by real-time PCR. The activities of LDH and HK-Ⅱ and lactic acid (LA)concentration in the culture supernatant were tested with spectrophotometer method.Results Under normoxic condition,HIF-1α was expressed in TE1 cells and the expression of HIF-1α was inhibited by wortmannin (2 μmol/L),the most significant inhibitory effect was at 12 hours,therefore 12 hours was selected for the subsequent hypoxia experiment.Compared with untreated cells,the expression of HIF-1α、HK-Ⅱ 、GLUT-1、LDH-A at protein level significantly decreased in TE1 and TE13 cells after pretreated with wortmannin (P ＜ 0.05),and the expression of HIF-1α、HK-Ⅱ at mRNA level significantly decreased (P＜ 0.05).Under normoxic and hypoxic conditions,the HK-Ⅱ and LDH activities in TE1 and Te13 esophageal carcinoma cells significantly decreased after treated with wortmannin compared with untreated cells (P＜0.05).Under hypoxia condition,the enzyme activity increased in untreated cells (P＜ 0.05). Under normoxic and hyp0xic conditions,the lactic acid concentration in the culture supernatant obviously decreased in cells treated with wortmannin compared with untreated cells (P＜ 0.05). Under hypoxia condition,lactic acid concentration increased in wortmannin treated cells (P ＜ 0.05). Conclusions Under normoxic and hypoxic conditions,wortmannin decrease lactic acid concentration through inhibiting the expression of HIF-1α and glycolysis associated genes, which indicate PI3K/AKT-HIF-1α pathway was closely related to glycolysis in esophageal carcinoma cells.

Objective To evaluate the value of multislice spiral CT in preoperative staging of bladder carcinoma.Methods Eighty-two patients(78 males and 4 females)with pathologically proved bladder carcinoma and the radiology materials were retrospectively analyzed.All the diagnosis results were compared with the results of surgical pathology.Results The location accuracy and qualitation accuracy in the diagnosis of bladder carcinoma with spiral CT WaS 78.0%(64/82)and 93.9%(77/82)respectively.Compared with the results of surgical pathology,the accuracy of judgement in bladder surrounding involvement,lymph node metastasis and surrounding organ involvement with CT was 90.2%(74/82),96.3%(79/82)and 89.0%(73/82),respectively.The CT staging of bladder carcinoma was positively correlated to that of surgical pathology.Conclusions Spiral CT has high value in the diagnosis of bladder carcinoma.It could be adopted as a conventional and main examination of bladder carcinoma before operation.