Aim To evaluate the effect of intermedin (IMD)on cell proliferation and regeneration in rat tu-bular epithelial cell line (NRK-52E)that was subjec-ted to hypoxia-reoxygenation (H/R)injury.Methods The NRK-52E cells were divided into control group and three model groups (H/R,H/R +primitive vec-tor,H/R +IMD vector).The content of LDH was de-tected to observe the influence of IMD on H/R injury. The cell proliferation was detected by MTT.The cell cycle was detected by flow cytometry.Real-time PCR and western blotting were used to determine mRNA and protein levels.Results ① In comparison to the con-trol,H/R treatment decreased the cell viability and in-creased LDH activity (P <0.01 );in contrast,com-pared to H/R,IMD treatment ameliorated cell viability (79.1 5 ±1 .421 % vs 61 .22 ±1 .63%,P <0.05)and decreased LDH activities by 33.85% (P <0.01 ).②The proliferation of NRK-52E cells was significantly in-hibited by H/R treatment.In comparison to the con-trol,H/R treatment of NRK-52E cells increased the proportion of cells in the G0 /G1 phase but decreased the proportion of cells in the S and G2 /M phases. Moreover,the over-expression of IMD resulted in S and G2 /Mphase redistribution and the accumulation of G2 /M-phase cells.The real-time PCR and western blotting results indicated that the mRNA and protein expression levels of cyclin D1 ,CDK4 and p57 were increased in H/R-treated cells.IMD further stimulated this up-reg-ulated expression of cyclin D1 ,CDK4 and decreased the expression of p57 in NRK-52E cells.④Cyclin D1 had a predominantly nuclear localization in NRK-52Ecells,although cytoplasmic localization was also ob-served.Conclusion The study shows that the over-expression of IMD may promote renal cell proliferation and regeneration after renal tubular cell H/R injury via the up-regulation of cyclin D1 ,CDK and the down-reg-ulation of p57.

BACKGROUND:Studies have indicated that the abnormal expression of tumor necrosis factorreceptor-associated protein 1 (TRAP1) is closely related to the occurrence and development of a variety of tumors. Therefore, targeted inhibition of TRAP1 expression has become an important target for the treatment or intervention of tumor growth. OBJECTIVE:To explore the effect of the TRAP1 gene silencing on the proliferation and apoptosis of human laryngeal squamous cel carcinoma stem cel s. METHODS:CD24-CD44-human laryngeal squamous cel carcinoma stem cel s were isolated by flow cytometry. Interfering RNA (siRNA) sequences for smal molecule TRAP1 gene was designed and transferred into human laryngeal cancer stem cel s by LipofectamineTM 2000. Flow cytometry, MTT assay, cel clone formation assay and TUNEL apoptosis assay were used to evaluate the effect of silencing TRAP1 gene on the proliferation and apoptosis of CD24-CD44+laryngeal cancer stem cel s. RESULTS AND CONCLUSION:Compared with CD24+CD44-cel s, CD24-CD44+cel s upregulated OCT4, SOX2, NANOG and TRAP1 expression levels (P<0.05). However, the expression of TRAP1 protein in human laryngeal squamous cel carcinoma was significantly decreased after RNA interference (P<0.05). The growth rate of TRAP1 gene silenced human laryngeal squamous cel carcinoma was significantly reduced (P<0.05), the cel arrest was in the G0/G1 phase, the number of cel s in the S phase was decreased (P<0.05), and there was no significant change in the M phase. TRAP1 gene silencing significantly inhibited the proliferation of human laryngeal squamous cel carcinoma stem cel s (P<0.05). Compared to the non-transfected cel s, the TRAP1 gene silencing significantly reduced the clone formation ability of transfected human laryngeal squamous cel carcinoma stem cel s (P<0.05), and TRAP1 gene silenced-human laryngeal squamous cel carcinoma stem cel s were more easy to trigger apoptosis by upregulating BAD and BAX expression levels (P<0.05). Overal , our experimental results indicate that the specific interference of TRAP1 gene expression could inhibit the proliferation and promote apoptosis of human laryngeal squamous cel carcinoma stem cel s.

Objective The purpose of this study is to study the clinical and pathological features of neurilemoma .Methods We observed the clinicopathologic features and immunohistochemical staining from eight patients with orbital neurilemoma between 2010.1~2012.12.Results Eight patients with classic neurilemoma were included in the study ,in which there were five males and three females ,aged between 21 and 63,mean age 35.The main symptom of the patients was exophthalmos ,including five cases of right eyes and three left eyes;2 cases of orbital floor and six above orbit ,lasting for one to ten years .The tumor diameter ranged between 1cm and 5 cm,an average of 3 cm,being pale and light yellow color .There were five cases of type Antoni A and one case of type was Antoni B among the six classic type neurilemoma .Two cases of ancient were neurilemoma ,in which one case was the histological structure of the classic type neurilemoma ,but there were more hypertrophy tumor cells , chromatin was coarse block atypia cells .The other one case with cells arranged disorderly ,which was mainly fine striated cells with scattered deeply stained atypia cells ,stromal transparent degeneration ,cystic degeneration .Im-munohistochemistry results showed that S -100(+),vimentin(+),ki67(-).Conclusion Antoni type B and ancient schwannoma are rare ,with complicated histologic characteristics .Combined with clinical features and im-munohistochemistry staining ,it can be diagnosed .

BACKGROUND: Up to now, there are few reports addressing the biological properties and differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). OBJECTIVE: To observe the biological characteristics, osteogenic and adipogenic differentiation potential of UCB-MSCs. METHODS: MSCs were harvested and cultured from UCB at various gestational ages (GA). Harvested UCB-MSCs were cultured primarily and subcultured, and then induced to differentiate into osteoblasts and adipocytes. RESULTS AND CONCLUSION: Under the inverted phase contrast microscope, UCB-MSCs adhered to the wall, showing fibroblast like morphology and whirlpool like growth alignment. Observation of the ultramicrostructure under transmission electron microscope showed that UCB-MSCs had a big cellnucleus, fewer cellular organelles and big karyoplasmic ratio.Allofthe growth curves of primary and passaged UCB-MSCs presented S-shaped. The 3rd and 5th passages of MSCs showed the strongest proliferation activity. The count of colony forming unit fibroblasts varied with GA, significant difference was found among the three GA groups (P < 0.05), and the lower GA group had a higher count of colony forming unit fibroblasts than that in the older GA group. Flow cytometry showed that these cells expressed CD29, CD44 and CD90 positively, but they failed to express hematopoiesis related molecules such as CD34 and CD45. When the MSCs were induced to osteogenic and adipogenic differentiation for 3 weeks, strong expression of alkaline phosphatase was found and the formation of a mineral extracellular matrix was detected by alizarin red staining were detected; and neutral lipid vacuoles were detected by oil red O staining. UCB-MSCs have similarmorphologicaland biological characteristics and cell surface molecule markers with MSCs derived from bone marrow, both of which have great capability of proliferation and regeneration. UCB-MSCs can be induced to osteoblasts and adipocytes in a suitable condition in vitro.

BACKGROUND: Mesenchymal stem cells (MSCs) exist in umbilical cord blood (UCB), currently, there is not a method to in vitro separate, culture and amplificate human UCB-MSCs effectively. OBJECTIVE: To explore factors that influence yields of UCB-MSCs. METHODS: The relationship between the success rate of yielding UCB-MSCs and several factors, such as gestational ages (≥40 weeks, 37 weeks and ≤32 weeks), the number of mononuclear cells (MNCs) in UCB (≥2.5×109/L, <2.5×109/L), the inoculum density of MNCs (1×107, 1×109, 1×1011/L), the concentration of fetal bovine serum (FBS, 5%, 10%, 15%, 20%) in culture medium, and whether the culture flask being coated with FBS or not beforehand, as well as relationships among these factors were investigated. RESULTS AND CONCLUSION: The success rate of yielding UCB-MSCs was up to 58.3%. The success rate decreased as the gestational ages increasing (P < 0.01). The success rate could be enhanced to 76.9% when the MNCs count was more than 2.5×109/L, and there was significant difference when comparing to that of the group (36.4%) with MNCs count less than 2.5×109/L (x2=8.07, P=0.005). There was a negative correlation between the MNCs count and the gestational ages in the specimens with the same volume of UCB (r=-0.95, P < 0.01). In the group with the cell inoculum density of 1×1011/L, the growth and proliferation of primary and subculturing MSCs were better than that of the groups with the cell inoculum density lower than 1×1011/L. The adherence of MSCs in the group with the culture medium containing 5% FBS happened much later than other 3 groups, while the purity of MSCs in this group was much higher. When comparing the passage rate, there was no significant difference among the 4 groups with different concentration of FBS. In the group of culture flask being coated with FBS beforehand, the purity and proliferation ability of MSCs was higher than that in the groups with culture flask not being coated. It is suggested that culture of UCB-MSCs was influenced by several factors. The success rate could be increased by choosing the fetus with relative lower gestational ages, collecting enough volume of UCB, inoculating cells with a higher density, choosing the medium with lower concentration of FBS, and coating the culture flask with FBS beforehand.

Objective To compare the clinical outcomes of irradiated versus non-irradiated deepfrozen bone-patellar tendon-bone (B-PF-B) ullograft in anterior cruciate ligament (ACL) reconstruction. Methods A total of 66 patients undergoing arthroecopic ACL reconstruction were prospectively random-ized consecutively into two groups, ie, Group A ( irradiated deep-frozen allograft, n = 34) and Group B ( non-irradiated deep-frozen allograft, n = 32). All ACL reconstructions were done by the same senior surgeon with the same arthroscopic technique. Before and after surgery, the clinical results were compared in aspects of general conditions, range of motion ( ROM), Pivot shift test, Lachman and Anterior Drawer Test (ADT), Daniel's one-leg hop and Harner's vertical jump tests, overall international knee docu-mentation committee (IKDC) rating and KT-2000 arthrometer testing. Results Of all, 63 patients in-eluding 32 patients in Group A and 31 in Group B were available for a follow-up of average 38 months ( mean 38.3 months in Group A and mean 37.7 months in Group B) and three lost follow-up. There was one patient with late septic infection. While there was no statistical difference in aspects of general condi-tions including hospital stay, duration of fever and complications ( P > 0. 05 ), but there was a trend that the patients in Group A had a longer postoperative duration of fever ( mean 8.9 days ) than Group B (mean 7.8 days). Physical examinations showed no statistical difference upon ROM in both groups ( P > 0. 05), while there was statistical difference between Lachman test and ADT ( P < 0.05 ). The positive Pivot shift test was found in 12 patients in Group A and 3 in Group B, with statistical difference ( P < 0. 05 ). KT-2000 testing showed a side-to-side difference of less than 3 mm in 26 patients in Group B and only 8 in Group A, and a side-to-side difference of more than 5 mm in two patients in Group B and 12 in Group A, with statistical difference (P < 0.05 ). The anterior and rotational stability was decreased sig-nificantly in Group A. No statistical difference was found between two groups in overall IKDC rating, Daniel' s one-leg hop and Harner' s vertical jump tests ( P > 0.05 ). However, the function and IKDC score tended to decrease in Group A. Conclusion The short term clinical outcomes of the ACL reconstruction with irradiated B-PT-B allograft are adversely affected and unsatisfactory, indicating a cautious use.

AIM:To clarify the mechanism of treating autoimmune cardiomyopathy at different stages with anti-L3T4 monoclonal antibody.METHODS:Mice immunized with human mitochondria ADP/ATP peptides were used as the cardiomyopathy(DCM) group,and the sham-immunized mice were regarded as the controls.Mice receiving early treatment were immunized with the same peptides,followed by the injection of 400 ?g of anti-L3T4 on day 0,1 and 2 post-immunization.Mice in the late treatment group were immunized as of the early treatment group but anti-L3T4 was administered 3 months post-immunization.The cytokine expression was measured with three-color flow cytometry to quantitate the splenic Th1/Th2 cell subsets in the different groups of mice.In addition,serum and myocardial cytokines were measured by enzyme-linked immunosorbent assay and real-time PCR.RESULTS:Th1 and Th2 subsets in the early treatment group were similar to those in control group,but were drastically lower than those in DCM group.Mice in the late treatment group showed an increased level of Th1-related cytokines,while the Th2 level was between the DCM and early treatment group.IFN-? and IL-6 levels in early treatment group were similar to those in control group.In the early treatment group,IL-4 level was higher than that in control and lower than that in DCM group,whereas IL-2 and TNF-? contents were lower than those in control and DCM group.In the late treatment group,IFN-? and IL-2 levels were higher than those in DCM group and lower than those in the early treatment group,while IL-6 and IL-4 levels were lower than those in DCM group.CONCLUSION:These results suggest that the cytokine production in cardiomyopathic mice may be repressed by treatment with anti-L3T4 at different stages.Early treatment with anti-L3T4 has better inhibitory function than treatment in late stage of autoimmune cardiomyopathy.

AIM: To investigate the changes of the L-type calcium channel subunit expression and calcium currents (I_~Ca -L) in cultured rat ventricular myocytes infected by coxsackie virus B_3 (CVB_3). METHODS: Primary cultured neonatal rat ventricular myocytes were infected with CVB_3. The changes of L-type calcium channel subunits mRNA in normal and infected myocytes were measured by semi-quantitative reverse transcription-polymerase chain reaction. I_~Ca -L was recorded in two groups respectively using whole cell patch-clamp techniques. RESULTS: The expression of ?_1 and ? subunits of L-type calcium channel mRNA increased in the infected group compared with the normal one (4.00?0.07 vs 2.21?0.41, P0.05). The average current density of I_~Ca -L significantly increased by CVB_3 infection [(-8.66?0.99) pA/pF vs (-6.97?1.75) pA/pF, P

BACKGROUND: Tumor necrosis factor-associated protein 1 (TRAP1) is a heat-shock protein 90-related mitochondrial chaperone. Accumulative evidence has demonstrated that TRAP1 overexpression is closely related to carcinogenesis. However, the exact function and mechanism of TRAP1 in the occurrence of laryngeal carcinoma remains unclear. OBJECTIVE: To investigate whether RNA interference can inhibit TRAP1 overexpression and to explore its effects on growth and apoptosis of CD133+CD44+ laryngeal carcinoma stem cells. METHODS: CD133+CD44+ laryngeal carcinoma stem cells were sorted from human laryngeal carcinoma Hep-2 cellsusing immunomagnetic beads. The shRNA sequence of TRAP1 was designed and synthesized and CD133+CD44+ laryngeal carcinoma stem cells were transfected with LipofectamineTM 2000. Cell counting kit-8 assay, colony formation assay and flow cytometry were used to investigate the effects of interference of TRAP1 expression on growth and apoptosis of CD133+CD44+ laryngeal carcinoma stem cells. Spectrophotometric method was used to detect the activity of caspase-3, -8 and -9. RESULTS AND CONCLUSION: TRAP1 mRNA and protein expression levels were significantly decreased in TRAP1 shRNA-transfected CD133+CD44+ laryngeal carcinoma stem cells (P < 0.01). Compared with the blank control and negative control groups, the growth and colony formation of CD133+CD44+ laryngeal carcinoma stem cells were significantly inhibited in the TRAP1 shRNA-transfected group (P < 0.05). Apoptosis of CD133+CD44+ laryngeal carcinoma stem cells was significantly inhibited in the TRAP1 shRNA-transfected group as compared with the blank control and negative control groups (P < 0.05). TRAP1 shRNA-mediated cell apoptosis was associated with the activation of caspase-3, -8 and -9. These results suggest that RNA interference targeting inhibition of TRAP1 suppresses cell growth but promotes apoptosis in CD133+CD44+ aryngeal carcinoma stem cells. TRAP1 is likely to be a gene target for treatment of laryngeal carcinoma.