Objective To optimize the parameters of the low-frequency/low-energy ultrasound combined with micro-bubbles in inducing early apoptosis of DU145 cells (an androgen-independent prostatic cancer cells). Methods In our study, the impact of ultrasonic power, micro-bubbles/cell suspension volume rate and irradiation time were investigated. Three levels of each factor were deifned as ultrasonic power (60, 80, 100 mW), micro-bubbles/cell suspension volume rate (10%, 20%, 30%), irradiation time (30, 60, 90 s). According to the three-factor three-level orthogonal design, nine experiments were carried out. The early apoptosis was detected by lfow cytometry. A new experiment was designed with the optimized parameters. Another group without ultrasound irradiation was designed as the control group. Flow cytometry and transmission electron microscope (TEM) were used to detect the early apoptosis. Results In descending order, the inlfuence of these factors on the cell early apoptosis were:ultrasonic power>micro-bubbles/cell suspension volume rate>irradiation time. Moreover, the inlfuence of each factor level were:80 mW>60 mW>100 mW in ultrasonic power, 20%>30%>10%in micro-bubbles/cell suspension volume rate, 60 s>90 s >30 s in irradiation time. The early apoptosis rate of experiment group was 10.41%, while the control group was 0.94%. TEM showed apoptotic cells in the experiment group. Conclusions The optimized parameter of low-frequency/low-energy ultrasound with micro-bubbles in inducing early apoptosis of DU145 cells are ultrasonic power of 80 mW, micro-bubbles/cell suspension volume rate of 20%, and irradiation time of 60 s. With the optimized parameters, the early apoptosis rate of the experiment group has signiifcant higher than the control group.

Objective To investigate the feasibility of low-frequency ultrasound combined with microbubbles improving transfection of enhanced green lfuorescent protein plasmid (pEGFP) to subcutaneously transplanted prostate cancer in nude mice, and to optimize the parameters of intensity. Methods The model of nude mice of subcutaneously transplanted prostate cancer were built. Then they were divided into 2 groups including low-frequency ultrasound group and low-frequency ultrasound combined with microbubbles group, each group with 15 mice. Then 250μl of mixture of saline and pEGFP (50μg) were co-injected in low-frequency ultrasound group and 250μl of mixture of SonVue and pEGFP (50μg) were co-injected in low-frequency ultrasound combined with microbubbles group through tail vein. According to intensity, they were divided into 3 subgroups respectively, including group 1 W/cm2, group 2 W/cm2 and group 3 W/cm2, each group with 5 mice. Ultrasound was applied at 80 kHz input frequency with 50%duty cycle for 10 minutes after pEGFP injection. The tumors were collected on the third day after treatment. The expression of pEGFP in tumor was examined by laser scanning confocal microscope (ISCM) and the average lfuorescence intensity were estimated. At the same time, routine pathological examination was performed. The mean lfuorescence intensity of low frequency ultrasound group and low frequency ultrasound combined with microbubble group were compared with one-way ANOVA and those of any two groups were compared by SNQ-q test. Results TThe mean lfuorescent light in low-frequency ultrasound combined with microbubbles group were 23.75±2.54, 30.25±1.67 and 59.60±2.03, which were obviously stronger than those of low-frequency ultrasound treatment group (14.04±1.35, 14.66±0.98 and 14.32±1.20), the difference was statistically signiifcant (the value of q were 18.26, 14.12 and 13.88, P<0.05). The rate of gene transfection increased along with the enhancement of the ultrasound intensity (the value of q were 15.33, 17.81 and 13.79, P<0.05). Histology analyses performed by HE staining showed that there was no damage to the tumor tissues in all groups, tumor tissues were intact and without infection. Conclusions Low-frequency ultrasound combination with microbubbles can significantly enhance pEGFP transfecting into subcutaneously transplanted prostate cancer in nude mice. In certain range, improving the ultrasound intensity can increase the rate of gene transfection.

Objective To investigate the effect of 20 kHz low-intensity ultrasound combined with microbubbles on autophagy of both PC3 cells and DU145 cells. Methods Ultrasound with a frequency of 20 kHz and intensity of 80 mW in continuous wave mode was used. Both PC3 cells and DU145 cells were divided into four groups, including control group (A), microbubbles group (B), ultrasound group (C) and ultrasound combined with microbubbles group (D). Twenty-four hours after treatment, the acid vesicular organelles were detected by acridine orange lfuorescence staining, and transmission electron microscopy (TEM) was used to observe autophagosomes. Results Acridine orange lfuorescence staining showed formation of acid vesicular organelles (AVOs) in the cytoplasm with normal nucleus in both PC3 cells and DU145 cells in group D, while in group A cells were basically normal. Lots of autophagosomes with double-membrane structure were detected by transmission electron microscope in group D. Conclusions Low-frequency and low-intensity ultrasound combined with microbubbles can induced autophagy in both PC3 cells and DU145 cells.

OBJECTIVE:To determinate vitamin B1 and vitamin B6 in Gengnianling capsules by HPLC simultaneously .METHODS: The separation was performed on Hypersil-ODS C18 column, methanol - sodium hexanesulfonate solution(20 : 80) was used as mobile phase with a flow rate of 0.8ml/ min and detection wavelength of 280nm.RESULTS: Linear correlations with peak area scores were achieved when the sample size of vitamin B1 and vitamin B6 were with a range of 0.884?g-2.652?g (r = 0.9 999) and 0.714?g-2.142?g(r = 0.9 999) .respectively, the average recovery of which were 95.87%(RSD = 0.82%) and 101.96% (RSD = 0.86%), respectively .CONCLUSION: The method is simple, accurate and it can be used for quality control of Gengnianling Capsule.

Objective To investigate the rationality and safety of dengzhanxixin injection used in elderly inpatients. Methods The clinical data of 986 inpatients including 620 males and 366 females were collected, and questionnaires containing age, sex, discharge diagnosis, symptoms, drug dosage, course of treatment, laboratory examination, adverse drug reaction and drug effect were analyzed. Results For the 986 cases, the average age was(74.3±7.5)years. The average dose of dengzhanxixin injection was (38.2±4.4) ml, once daily by intravenous drip, and the average period of treatment was (10.8±5.2) days. The adverse reaction rate was 0.81%. The levels of blood glucose and hemoglobin were decreased after treatment(t orμ=1226.5,2620.0, both P<0.05), but there were no statistical differences in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), blood urea nitrogen (BUN) and white blood cell count (WBC) before and after treatment (t or μ=122.5, 405.0, 513.5, 996.5, 956.5, all P>0.05). Conclusions It is safe to use dengzhanxixin injection according to the medication description for elderly inpatients.

Objective To construct and identify retroviral-mediated short hairpin RNA ( shRNA ) expression vectors of ERβ419, and explore ERβ419 unknown biological function in beagles in future.Methods To screen out the most effective gene silencing sequence of beagle ERβ419 mRNA using qRT-PCR and Western Blot assays, imitate beagle estrogen target cells.Results qRT-PCR results showed, ERβ419-shRNA1 ( P <0.01 ) and ERβ419-shRNA3 ( P <0.01)differed significantly, Western Blot result as same as qRT-PCR,ERβ419-shRNA3 is the best choice.Conclusion Beagles ERβ419-shRNA3 retrain most effectively target gene repression. It is applied to explore ERβ419 unknown biological function in beagles reproductive system, and to prevent and treat beagles reproductive function diseases.

OBJECTIVE: Using the method of lactate dehydrogenase (LDH) assay, to observe the activities of rat cerebral microvascular endothelial cells (CMECs) intervened by Tongluo Jiunao Injection (TLJNI), a traditional Chinese compound drug removing toxin to dredge brain collaterals, and then further study the effects of different kinds of conditioned mediums (CMECs-CM) of cerebral microvascular endothelial cells on ischemia and ischemia/reperfusion cerebral cortex cells, and to probe into the drug pharmacological mechanisms of CMECs in modulating the neurons. METHODS: Three kinds of CMECs (normal, ischemic and ischemic/reperfusional) were all treated by TLJNI previously, and then the three pairs of CMECs-CM without serum were collected respectively for LDH assay. Rat cerebral cortex neurons were also primarily cultured and then divided into similar three groups (normal, ischemic and ischemic/reperfusional). The neuron responses caused by CMECs-CM at different concentrations were observed by using LDH transudation rate assay. RESULTS: The LDH release values of ischemic and ischemic/reperfusional CMECs with TLJNI treatment were obviously reduced (P<0.01) compared with the same kinds of CMECs untreated. For ischemic neurons, both conditioned medium of ischemic CMECs (Is-CM) and conditioned medium of ischemic CMECs with drug treatment (IsT-CM) in high concentration of 100% increased the LDH transudation rate (P<0.01), while in low concentration of 10%, IsT-CM reduced the transudation rate (P<0.05). For ischemia/reperfusion neurons, all kinds of CMECs-CM reduced the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, 10% or 50% showed relatively stronger effects, and both conditioned medium of normal CMECs (N-CM) group and conditioned medium of ischemic/reperfusional CMECs (Rp-CM) group had statistical significance (P<0.05 or P<0.01). For normal neurons, all kinds of CMECs-CM increased the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, only conditioned medium of normal CMECs (N-CM) had statistical significance (P<0.05 or P<0.01). CONCLUSION: The study shows that TLJNI is capable of preventing the damage of CMECs from both ischemia and ischemia/reperfusion states. Chinese drug can restrain the brain ischemia and ischemia/reperfusion damage by the media that CMECs modulate the neurons, demonstrating the pharmacological mechanisms of TLJNI. This work also indicates that there exist some active substances against ischemia/reperfusion injury secreted from CMECs-CM with TLJNI treatment.

0.05).However,phosphor-ERK and phosphor-STAT3,NF-?B were higher in CVB_3 group than those in control groups(P

Objective To investigate protective activity against Aβ25-35-induced cytotoxicity in PC12 cells of different extracts and ursolic acid, which were isolated from pyrola decorata. Methods Aβ25-35-induced cytotoxicity in PC12 cells was established as the model in vitro. The cultured PC12 cells were divided into blank control group, DMSO control group, model control group, different extract groups of pyrola decorate and ursolic acid(UA) group. The different extract groups included ether extract (PE), acetidin extract (AE), n-butanol extract (BE), the water extract (WE), 50% ethanol extract (HEE). MTT assay was used to test the optimum concentration, and the number of viable cells in culture medium was measured by ELISA at 490 nm wavelength. Results The cell viabilities in different extracts groups(PE, AE, BE, WE, HEE) were respectively 89.3%, 77.2%, 79. 2%, 75. 1% ,74. 0% at the concentration of 5. 0 mg ? mL-1 . Moreover, ursolic acid showed the best neuroprotective activity (88.9%) at the concentration of 500 μg?L-1 . Compared with model control group, the survival rate of each group was remarkably increased, and the protective activities of PE and UA were more significant among them. Conclusion Different polar extracts of pyrola decorata and isolated ursolic acid have neuroprotective effects on Aβ25-35-induced cytotoxicity in PC12 cells in certain degrees.

It designs a way that can easily screen high-pyruvate-producing strain.It is a intelligently selected method which can highly improve the efficiency of strain screening.The principle can be described as the following:On the CaCO-3 medium,a transparent ring can be exhibited based on the reaction of PYR produced by the strain and CaCO-3 in the medium for pyruvate-calcium is a kind of soluble substance,it is obviously that the high-pyruvate-producing strain has a bigger dimension of the transparent ring.On the other hand,color reaction between TTC and ADH indicate the enzyme activities which have a proportional relation with color,our object strain is a weak-ADH-enzyme-activities type with a weak metabolic flux from PYR to alcohol.So the white color strain may be the right choice.