Objective To construct an immortalized rat astrocyte strain expressing enkephalin which can be regulated by doxycycline.Methods pRevTet-On and recombinant plasmid pRevTRE/hPPE were transfected into packaging cell PT67 respectively by standard lipofectamine methods.The supernatant containing RevTet-On virus was used to infect immortalized rat astrocyte strain(IAST)and RevTRE/hPPE virus was used to infect the IAST/Tet-On cell.Immortalized rat astrocyte strain that stably expressed Tet-regulated enkephalin was established. hPPE gene expression level of IAST/Tet-On/hPPE cell strain was detected by real time-PCR,immuno- cytochemistry and radio-immunoassay.Results Real time-PCR,immuno-cytochemistry and radio-immunoassay confirmed the level of enkephalin expression was regulated by doxycycline in a dose-dependent manner.Conclusion An immortalized rat astrocyte strain which secrets enkephalin as controlled by doxycycline has been constructed successfully.

?AlM: To observe the effect of Qingguang'an on elastic fiber, MMP-7, TlMP-1 in scarring area of filtration canal after glaucoma surgery through the four Qingguang'an effective groups and Qingguang'an granules, to discuss and compare their mechanism of action on scarring area of filtration canal.?METHODS:Four effective components of Qingguang'an were used in groups D, E, F, G and H after glaucoma surgery, compared with group A ( blank ) , group B (model) and group C ( MMC) to observe the effect of elastic fiber, MMP-7, TlMP-1 in scarring filtration canal.?RESULTS:Compared with the preoperative basic lOP and 2d , 1, 2, 4wk postoperative lOP of groups C, E and H, the lOP of three group rose up slower than other groups, and kept the lowest data at 28d. There was significant difference compared with the rest of A, B, D, F, G groups (P<0. 05). The area and density of elastic fiber in surgery group were significantly different with that of black control group ( P<0. 05 ), but there were no statistical differences between groups C and H, groups C and F, groups H and E (P>0. 05). The difference was statistically significant among other groups (P<0. 01).?CONCLUSlON:The scarring area of filtration canal after glaucoma surgery is the major reason which lead to the failure of surgery. Qingguang'an effective group 2, Qingguang'an granules and MMC could reduced the scar tissue by restrained the elastic fiber, TlMP - 1 and increased the MMP-7. By observing the experimental results that both Qingguang'an effective group 2 and Qingguang'an granules could restrained the scarring area of filtration canal, the effects were unbiased, Qingguang'an granules group is better than effective group 2.

The glycosylation heterogeneity of recombinant human pro-urokinase (pro-UK) was assessed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Firstly, the source of heterogeneity was determined by measuring the M_r of intact protein before and after N-deglycosylation. Glycosylation sites and the proportion of O-glycopeptides then were determined at the peptide level. Finally, the N-glycans were confirmed and quantified using the N-glycan profile. Results show that the structural heterogeneity of pro-UK is mainly caused by glycosylation. All T₁₈ were fucosylated, and 6.4% of S_138/139 was O-glycosylated with two kinds of oligosaccharides with a ratio of 6.0% and 0.4% respectively. All N₃₀₂ positions were N-glycosylated by more than ten types of glycans, among which A2F and A3F accounted for 80% of the total. The assessment of glycosylation heterogeneity of pro-UK will provide a reference for quality standardization.

Aim To investigate the changes of cerebral blood flow in rats with diabetic cognitive impairment by two-channel laser Doppler flowmeter,and to explore the changes of cerebral blood flow in diabetic rats with cognitive impairment and to investigate the changes of cerebral blood flow lesions and the central nervous system function changes in the study to provide pre-foundation.Methods Thirty-four male Wistar rats were randomly divided into two groups:blank control group and diabetic model group.The rats in the model group were treated with streptozotocin (STZ) 60 mg · kg-1,and the glucose oxidase method was used to determine fasting blood glucose ≥ 16.7 mmol · L-1 as the standard of the model.The Water maze was used to observe the behavioral changes of diabetic rats.Dual-channel laser doppler flowmeter was used to measure cerebral blood flow in diabetic rats with cognitive impairment.Another 34 male Wistar rats were randomly divided into two groups:control group and diabetic model group.Dual-channel laser doppler flowmeter was used to dynamically monitor cerebral blood flow on 0 d,7 d,14 d,21 d,28 d,35 d,42 d,56 d and 75 d.ELISA was applied to detect the concentration of iNOS,eNOS and ET-1 in cerebrospinal fluid.Results Morris water maze test showed that the time of the platform (latency) was significantly longer than that of the blank control group(P ＜0.05).The cerebral blood flow/100 g of diabetic rats with cognitive impairment was significantly lower than that of the control group (P ＜ O.05),and the blood flow in the model group was significantly lower than that in the model group (P ＜ 0.05).Compared with control group,iNOS and eNOS concentrations were markedly elevated,while ET-1 concentration obviously decreased.Conclusions The decrease of blood flow in the frontal cortex of diabetic rats with cognitive impairment suggests that it may be one of the factors leading to cognitive impairment in diabetes mellitus.Cerebral blood flow reduction occurs in the early stages of diabetes,followed by no significant deterioration.Cerebral blood flow may not be related to the changes of NO and ET-1,but the trend of cerebral blood flow may be related to the change of the two.

A reliable method for simultaneous determinition of eleven representative components (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, shanzhiside, geniposidic acid, genipin-1-β-D-gentiobioside, geniposide and secoxyloganin) in combination of chromatographic fingerpint analysis for Reduning injection was developed by ultra high-performance liquid chromatography (UPLC). The method was performed on an Agilent ZORBAX SB-C18 anlytical column (3. 0 mm x 100 mm, 1. 8 µm) with a guard column of Agilent UPLC Guard ZORBAX SB-C18 (3.0 mm x 5 mm) at the column temperature of 30 °C. The gradient mobile phase consisted of acetonitrile (A)-0. 1% phosphoric acid (B) with a flow rate of 0. 4 mL . min-1. The injection volumn was 2 µL. The detection wavelengths were set at 324 nm and 238 nm for quantit tive analysis and 225 nm for fingerpint chromatography. Neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, shanzhiside, geniposidic acid, genipin-1-β-D-gentiobioside, geniposide and secoxyloganin were baseline seperated with good linearity relationships (r >0. 999) between concentration and peak areas over the linear ranges. The average recoverys of the investigated compounds were 103.5%, 100. 2%, 103. 3%, 102. 8%, 101. 3%, 102. 8%, 97. 36%, 99. 62%, 98. 16%, 102. 8%, 99. 27%, respectively. Reduning injection of forty-five batches was analyzed by UPLC finge print chromatography. Thirty batches were selected to generate the reference fringerprint chromatography with fourteen common peaks. The similarity values between the reference fringerprint chromatography and the remaining fifteen batches were higher than 0. 99. The developed method was fast, accurate and sensitive. It could be used as a reference for the quality control of multiple components determination and fingerprint chromatography for Reduning injection in future.
Chlorogenic Acid ; chemistry ; isolation & purification ; standards ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; standards ; Glucosides ; chemistry ; isolation & purification ; standards ; Iridoids ; chemistry ; isolation & purification ; standards ; Quality Control ; Reference Standards ; Reproducibility of Results ; Sensitivity and Specificity ; Time Factors

In order to establish a rapid method for identifying six constituents in Guizhi Fuling capsule, Q-TOF with DART ion source was used to perform the direct analysis of compounds in Guizhi Fuling capsule. The DART sampler delivery rate was 0.2 mm s(-1). The temperature of helium gas of DART was 450 degrees C. The capillary voltage was kept at 1 000 V. The temperature of the drying gas of Agilent 6538 Q-TOF MS was set at 350 degrees C. The flow rate of the drying gas of MS was set at 3.5 L x min(-1). The MS scan range was m/z 50-1 000. Based on accurate mass measurements and the elemental compositions of the product ions and fragmentation patterns of reference conpounds, six components, amygdalin, paeonol, paeoniflorin, cinnamic acids, gallic acid, benzoic acid were identified rapidly. The method can rapidly identify six chemical constituents in three batch of Guizhi Fuling capsule. The DART-Q-TOF-MS method is simple, rapid and specific and it can be used for rapid identification and characterization of compounds in traditional Chinese medicines.
Capsules ; Drugs, Chinese Herbal ; analysis ; Mass Spectrometry ; methods

OBJECTIVETo provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline.

METHODSRetrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/ hPPE was detected by RT-PCR, indirect immunofluorescence staining and radioimmunoassay.

RESULTSIAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline.

CONCLUSIONAn immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.

Animals ; Anti-Bacterial Agents ; pharmacology ; Astrocytes ; Cell Line, Transformed ; Chronic Disease ; Doxycycline ; pharmacology ; Enkephalins ; genetics ; metabolism ; Genetic Therapy ; methods ; Genetic Vectors ; Neurotransmitter Agents ; genetics ; metabolism ; Pain Management ; Protein Precursors ; genetics ; metabolism ; Rats ; Retroviridae ; genetics

OBJECTIVEThe study was to describe the breastfeeding status of children under the age of three in counties of western China and to provide evidence to the government for decision-making on intervention.

METHODSA cross-sectional study with probability-proportional-to-size (PPS) sampling method was used. The information on breastfeeding was obtained through memory of the mothers. Fourteen thousand and seventy-seven children were studied. Data on breastfed status in counties of western China was compared with those of the children from the survey of the counties of western China in 2001.

RESULTSThe breastfeeding rate of children under 3 years old in western China was 96.5%. However, the overall breastfed rate of children under 6 months were only 33.4%, with rates of 11.4% and 22.0% on exclusively and predominantly breastfed groups respectively. Timely first-suckling rate was 43.5% with the continued breastfeeding rate (1 year) as 64.9%, but the continued breastfeeding rate (2 year) was only 9.7%. Reasons causing mothers to wean would include according to her own intention and to be able to attend the physical labor while exclusive breastfeeding under 6 months was for the growth and development of children, which might reduce the two-week prevalence of diarrhea. Major risks of exclusive breastfeeding of children under 6 months were seen as: level of education of the mothers, economic depression of the counties and mother's nationality (if as minority).

CONCLUSIONMost of the children were ever or being breastfed at the time of interview with timely first-suckling took place earlier than in 2001. However continued breastfeeding did not last long. During these five years, the exclusive breastfeeding rate had been at low level, especially at the economic depression and the minority area.

Breast Feeding ; epidemiology ; Child, Preschool ; China ; epidemiology ; ethnology ; Humans ; Infant ; Infant, Newborn ; Weaning

OBJECTIVETo observe the effect and mechanism of zoledronate on prevention of collapse in an animal model of osteonecrosis.

METHODSIschemic osteonecrosis was surgically induced in 16 SD rats (which were further divided into zoledronate group and placebo group); another 8 rats were used as sham surgery group (n=8). The animals were killed 5 weeks after surgery. Radiographic, Micro-CT, histological, and immunohistochemical assessments were performed.

RESULTSRadiographic assessment showed better preservation of the femoral head shape in the zoledronate group than in the placebo group but not significantly different from the sham surgery group. Micro-CT assessment showed higher total volume, bone volume, and total mineralized content in the zoledronate group(all P0.05). Compared with the placebo group, the zoledronate group had reduced osteoclast and osteoblast activity, as confirmed by histological examinations.

CONCLUSIONZoledronate can decrease the femoral head deformity by reducing the osteoclast activity while suppressing new bone and vessels formation in a rat model of traumatic osteonecrosis, and therefore may delay the collapse of femoral head.

Animals ; Diphosphonates ; therapeutic use ; Disease Models, Animal ; Femur Head ; drug effects ; pathology ; Femur Head Necrosis ; drug therapy ; pathology ; Imidazoles ; therapeutic use ; Male ; Osteoblasts ; drug effects ; pathology ; Osteoclasts ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley

Ischemia-reperfusion injury (IRI) has been recognized as a serious problem for therapy of cardiovascular diseases. Calcium regulation appears to be an important issue in the study of IRI. This article reviews calcium regulation in myocardial and vascular IRI, including the calcium overload and calcium sensitivity in IRI. This review is focused on the key players in Ca(2+) handling in IRI, including membrane damage resulting in increase in Ca(2+) influx, reverse-mode of Na(+)-Ca(2+) exchangers leading to increased Ca(2+) entry, the decreased activity of sarcoplasmic reticulum (SR) Ca(2+)-ATPase causing SR Ca(2+) uptake dysfunction, and increased activity of Rho kinase. These key players in Ca(2+) homeostasis will provide promising strategies and potential targets for therapy of cardiovascular IRI.
Animals ; Calcium ; metabolism ; Heart ; physiopathology ; Homeostasis ; Humans ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism