To study the apoptotic regularity of peripheral lymphocytes in radiation compound wound healing and explore their relationship to radiation delayed wound healing, apoptosis and Bax and Bcl 2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical method of alkaline phosphatase, respectively. White blood cells in radiation compound wound(RW) group decreased and reached the lowest 3 days after wound, it was still lower in RW group than in simple wound(SW) group 5 days after wound; apoptosis of peripheral lymphocytes of both groups increased, however, the lymphocyte apoptosis in RW group was higher than that in SW from beginning to end in the whole process of wound healing; the expression of Bax protein of lymphocyte increased at different times after wound, and the expression in RW group was obviously higher than that in SW group, while Bcl 2 showed an opposite tendency. It is suggested that reduction of white blood cells and increment of lymphocyte apoptosis are the major causes of radiation delayed wound healing; Bax and Bcl 2 proteins take part in the regulation of lymphocyte apoptosis in wound healing.

Objective To study the ultrasonic extraction technology of tyrosol in Rhodiolae Crenulatae Radix et Rhizoma;To mathematically simulate the extraction process.Methods The content of tyrosol was set as index;HPLC was used;the ultrasonic extraction process of tyrosol in Rhodiolae Crenulatae Radix et Rhizoma was optimized by an orhogonal experiment and mathematic simulation.Results The optimum ultrasonic extraction conditions were as follows:80%ethanol was used as extraction solvent, the ratio of material to liquid was 1:35 (g:mL), with the extraction time of 30 min and ultrasonic power of 240 W. In these conditions, the extracting rate of tyrosol was 0.150 1%. Compared with the heating reflux method, the extraction time should be shortened by 66.7% and the extracting rate should be increased by 12%.Conclusion The extraction method is simple and the extraction rate of effective components is high. Mathematical simulation values based on ultrasonic extraction are consistent with experiment values.

By use of color Doppler ultrasonography (ATLHDI 5000,made in USA),a prospective study was made in 362 aged (greater than or equal to 60 years) patients (252 males and 110 females) for a period of 3 years.There were 176 patients (48.2%,130 males and 46 females) had degenerative vavular disease (DVD).The incidence increased with the aging of the population.There were no evident differences between males and females.Degenerative aortic valvular deformation was most frequently found (in 171 cases),followed by mitral (in 24 cases).The complications were hypertension,diabetes and coronary heart disease.It suggests that the DVD in the elderly mainly occurs in aortic valves,then in mitral value.Its incidence increases with aging.Complications such as hypertension,diabetes and coronary heart disease are important risk factors of DVD.

Objective:To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC).Methods:(1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD 133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD 63 and CD 9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD 133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results:(1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD 133 positive ( CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% ( t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD 63 and CD 9 were positive. (3) Three groups′ OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) μm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD 133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD 133 was higher than that in the control group( F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant ( F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant ( F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups ( F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased ( P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group ( t=23.202, P<0.05). Conclusions:Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.

BACKGROUND:The development of keloid is a progress of fibrosis in wound healing, and involves various fibrosis-related cytokines. Bone morphogenetic protein 7 (BMP7), Gremlin and high mobility group box-1 (HMGB1) play an important role in fibrosis of many organs, but their role in keloid tissue has rarely been reported.
OBJECTIVE:To investigate the role of BMP7, Gremlin, vascular endothelial growth factor (VEGF) and HMGB1 in the development of keloid.
METHODS:The protein levels and distribution of BMP7, Gremlin, VEGF and HMGB1 in 20 cases of keloid and 20 cases of normal skin were detected by immunohistochemistry and western blot analysis, respectively. And the correlations among expression levels of BMP7, Gremlin, VEGF and HMGB1 in keloid were analyzed.
RESULTS AND CONCLUSION:In keloid tissue, the expression levels of Gremlin, VEGF and HMGB1 were significantly higher than that in normal skin (P<0.01), while the expression levels of BMP7 were significantly lower (P<0.01). The levels of Gremlin were negatively correlated with the levels of BMP7 (r=-0.539, P<0.05). And the levels of VEGF were positively correlated with the levels of HMGB1 (r=0.56, P<0.05). The overexpression of Gremlin and decreased expression of BMP7, as wel as the increased expression of HMGB1 and VEGF, may contribute to the pathogenesis of fibrosis in the development of keloid.

AIM:To examine the role and dynamic changes of mast cell(MC) and its subpopulations in simple and irradiated-wound of rats.METHODS:MC and its subpopulations were estimated using alcian blue-safranin (ABS)double staining RESULTS:(1)The total number of MC in two groups decreased coincidently on day 2 after wounding, and the total MC increased rapidly and reached maximal gradually on day 5 and 7 after wounding, the increment of MC remained consistently 28 day after wounding (2)Both mucosal MC( MMC) and Mix MC decreased obviously on day 2 after wounding, hereafter,they remained the low level all the time However, the CTMC kept in the high level after wounding (3)The Mix MC on day 5 and the total MC during day 5-15 after wounding were lower in irradiated group than in simple wound group CONCLUSION: MC and its subpopulations could delay the healing process of simple and irradiated wound

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-Patched1 (PTCH1) and Gli1 in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEP1), respectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines. Cyclopamine decreased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G(0)/G(1) phase, while Shh induced the reverse effects and could partially prevent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic approach to NB.

OBJECTIVE:To optimize the ultrasonic extraction technology of salidroside in Rhodiola rosea. METHODS:With the content of salidroside as the index,L16(45)orthogonal test was designed to observe the effects of 4 factors including the mass fraction of ethanol,solid-liquid ratio,extraction time and ultrasonic power on the content of salidroside in R. rosea extraction solu-tion. By using Matlab 6.5 software,mathematical simulation of ultrasonic extraction process of salidroside was made with the data of orthogonal test. Multiple regression analysis method was adopted to optimize the ultrasonic extraction technology of salidroside, and then the extraction result of optimal technology and that of traditional heating reflux technology were compared. RESULTS:The optimal extraction technology of salidroside was as follows as ethanol mass fraction of 77%,solid-liquid ratio of 1∶34 (g∶ml),extraction time of 34 min,ultrasonic power of 237 W(ultrasonic frequency of 40 kHz). Under the above-mentioned condi-tions,the content of salidroside was up to 0.982 4%,close to the predicted value of 0.989 4%. Compared to heating reflux meth-od,the ultrasonic method has similar content of salidroside but extraction time was shortened by 62.2%. CONCLUSIONS:The ul-trasonic extraction method for extracting the salidroside in R. rosea is simple,requires shorter time,and giving rise to higher extrac-tion rate.

Objective:To summarize the evidence for the link between rheumatoid arthritis and risk of vertebral fractures or vertebral deformities with a meta-analysis, so as to provide objective proof for early preventing and the development of vertebral deformity and fractures.Methods:Wanfang,CNKI,VIP,PUBMED,Springlink and Elsevier were retrieved for all publications relating to rheumatoid arthritis and vertebral fractures in women.According to inclusion and exclusion criteria,two investigators collected their data individually,then statistical analysis was performed using Stata 12.0 software.Results: Eight case-control studies were enrolled, including 86 741 participants,2 258 of them with RA.The results of Meta-analysis showed that a higher incidence of vertebral fractures in RA,and Odds ratio was 3.70 with a 95%confidence interval(2.47-5.55,P<0.000 1).The publication bias analysis did not reveal any evidence of obvious asymmetry, and the sensitivity analysis showed that omission of any individual study made no significant difference for all comparison models,suggesting that our results were statistically robust.Conclusion:RA may be one of the risk factors for the vertebral fractures.

The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-- Patchedl (PTCH1) and Glil in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Glil in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEPI), re-spectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concen-trations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expres-sion of PTCH1 and Gill was detected in both NB samples and S-type NB cell lines. Cyclopamine de-creased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopaminc could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase, while Shh induced the reverse effects and could partially pre-vent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies re-vealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic ap-proach to NB.