Subculture, cryopreservation and recovery of osteoclasts / 中国骨伤

<p><b>OBJECTIVE</b>To explore the feasibility of passage, cryopreservation, and recovery of osteoclasts in order to develop new techniques facilitating osteoclast research.</p><p><b>METHODS</b>Passage of osteoclasts: adult male SD rat(SPF grade, weight of 250 g) was sacrificed and the abdominal aorta was exposed for blood draw. Monocytes isolated from peripheral circulation was treated with RANKL and M-CSF for 2 weeks. After formation of osteoclasts, they were trypsinized with pipetting, centrifuged, re-suspended with α-MEM containing RANKL and M-CSF, and cultured in 6 well-plates and 35 mm culture dishes. Freezing of osteoclasts: trypsinized osteoclasts were centrifuged and resuspended with DMSO, FBS, α-MEM (1:2:7), and were stored in liquid nitrogen(-196 °C). Recovery of osteoclasts: frozen osteoclasts were taken out of liquid nitrogen tank and thawed quickly at 37 °C in water bath. After wash with PBS, the cells were resuspended with α-MEM containing RANKL and M-CSF, and were cultured in 6 well dishes and 35 mm culture dishes. Meanwhile, cells were checked with inverted phase contrast microscope and observed in the live cell station for real time imaging. TRAP staining was performed 3 days after plating.</p><p><b>RESULTS</b>Trypsinization together with pipetting and shaking can detach the adherent osteoclasts, and the resuspended cells can be used for passage and storage in liquid nitrogen. The passaged cells became fully attached to the culture dishes in 2 hours, and the multinucleated feature could be clearly seen. The osteoclasts recovered from liquid nitrogen could completely spread out for 2 to 3 hours so that the multinucleated cells were clearly seen. These cells were still TRAP positive.</p><p><b>CONCLUSIONS</b>Although osteoclasts strongly adhere to the bottom of culture dishes, a large majority of the osteoclasts can be detached after appropriate digestion with trypsin, pipetting and shaking. These cells can be used for passage and cryopreservation. After recovering from liquid nitrogen, these cells still preserve the viability and the feature of osteoclasts. The results provide a new and powerful tool for future study of osteoclast biology.</p>

Tonglian Decoction () arrests the cell cycle in S-phase by targeting the nuclear factor-kappa B signal pathway in esophageal carcinoma Eca109 cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction (, TLD) on esophageal carcinoma Eca109 cells.</p><p><b>METHODS</b>Eca109 cells were treated with TLD and its separated formulae, including the clearing-heat and detoxification formula (Q), activating-blood and promoting-qi formula (H) and nourishing-yin and blood formula (Z). Cell proliferation was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, cell morphology was observed using a microscope, the cell cycle was measured using flow cytometry and the activity of the nuclear factor-kappa B (NF-κB) signal pathway was detected by Western blot.</p><p><b>RESULTS</b>The half maximal inhibitory concentrations of TLD, Q and H were 386, 771 and 729 mg/L, respectively. TLD, Q and H significantly inhibited cell proliferation, with 69.43%, 60.84% and 61.90% of treated cells in the G phase of the cell cycle. The percentage of cells in S phase increased significantly after treatment with TLD, Q, and H compared with the control group (P<0.05), and TLD showed the strongest effect. Z had no influence on the cell cycle compared with the control group (P>0.05). Western blot detection indicated slight differences in the inhibition of the NF-κB pathway by the different formulae. TLD formula strongly inhibited IKKβ, NF-κB, interleukin-6 and tumor necrosis factor-α expression compared with the control group.</p><p><b>CONCLUSIONS</b>TLD inhibited Eca109 cell proliferation by arresting cells in S phase. The possible mechanism might be related to inhibiting the NF-κB transduction cascade. The combination of the herbs found in the three separate formulae, H, Q and Z, work synergistically in TLD to produce the inhibitory effects of TLD treatment on Eca109 proliferation.</p>

Association of Insulin Resistance and β Cell Function with Lipid Metabolism in Middle-aged and Elderly Hui and Han Populations / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the association of insulin resistance and β cell function with lipid metabolism in middle-aged and elderly Hui and Han populations.</p><p><b>METHODS</b>A total of 1000 subjects age over 40 years were recruited from five urban communities in Yinchuan and Wuzhong cities of Ningxia. The composition ratio between Hui and Han nationality was 1:2. A questionnaire-based survey was performed. Physical examinations were carried out to measure the height, body mass, waistline, and hipline. The levels of triglyceride (TG), total cholesterol (TC), blood uric acid (BUA), fasting blood glucose and insulin were measured. The boby mass index (BMI), waist-hip ratio (WHR), and secretion related index including insulin resistance index (IR), insulin sensitivity index (IAI), and beta cell function index (HBCI) were calculated.</p><p><b>RESULTS</b>The BMI, WHR, IAI, HBCI, and the prevalence rate of diabetes in Hui nationality were significantly higher than those in Han nationality (P<0.01). The levels of BUA, fasting blood glucose, TC, and IR in Han nationality were significantly lower than those in Hui nationality (P<0.01). In Hui populations, TG, BMI, WHR, and BUA were positively correlated with IR (r=0.234, r=0.193, r=0.143, and r=0.129, respectively; P<0.01) and were negatively correlated with IAI (r=-0.234, r=-0.193, r=-0.143, r=-0.129, respectively; P<0.01), whereas TC was negatively correlated with HBCI (r=-0.169, P<0.01). In Han populations, TC, TG, BMI, WHR, and BUA were positively correlated with IR (r=0.140, r=0.257, r=0.288, r=0.163, r=0.104, P<0.01) and negatively correlated with IAI (r=-0.140, r=-0.257, r=-0.288, r=-0.163, and r=-0.104, P<0.01), whereas BMI was negatively correlated with HBCI (r=-0.111, P<0.01). After the influential factors such as gender, nationality, and age were adjusted, the TC, TG, BMI, WHR, BUA levels were positively correlated with IR (r=0.109, r=0.256, r=0.253, r=0.139, and r=0.142, P<0.01) and negatively correlated with IAI (r=-0.109, r=-0.256, r=-0.253, r=-0.139, and r=-0.142, P<0.01). TC and BMI were negatively correlated with HBCI (r=-0.113, r=-0.086, P<0.01). TG and BMI were independently associated with IR and IAI (r=0.218, r=0.182, r=-0.218, r=-0.182), while TC and BMI were independently associated with HBCI (r=-0.113, r=-0.086).</p><p><b>CONCLUSIONS</b>The distributions of TC, TG, BMI, WHR, BUA, IR, IAI, and HBCI differ between Han and Hui populations. The development of insulin resistance is closely related with the increased levels of TC, TG, BMI, WHR, and BUA. However, the HBCI increases with the increased level of TC and BMI. TG and BMI may be related with insulin resistance. Also, TC and BMI may affect the secretion function of β cells.</p>

Influenza early warning model based on Yunqi theory / 中国结合医学杂志

<p><b>OBJECTIVE</b>To establish an early warning model to simulate the outbreak of influenza based on weather conditions and Yunqi theory, an ancient calendar theory of Chinese medicine (CM).</p><p><b>METHODS</b>Tianjin, a northeastern city in China, was chosen as the region of research and applied the influenza-like illness attack rate (ILI)% as the baseline and warning line to determine the severity of influenza epidemic. Then, an influenza early warning model was constructed based on the theory of rough set and support vector machines (RS-SVM), and the relationship between influenza and meteorology was explored through analyzing the monitoring data.</p><p><b>RESULTS</b>The predictive performance of the model was good, which had achieved 81.8% accuracy when grouping the obtained data into three levels that represent no danger, danger of a light epidemic, and danger of a severe epidemic. The test results showed that if the host qi and guest qi were not balanced, this kind of situation was more likely to cause influenza outbreaks.</p><p><b>CONCLUSIONS</b>The outbreak of influenza closely relates to temperature, humidity, visibility, and wind speed and is consistent with some part of CM doctrine. The result also indicates that there is some reasonable evidence in the Yunqi theory.</p>