The enantiomeric resolution of lactic acid was studied by reversed high performance liquid chromatography. β-cyclodextrin (β-CD), dimethyl (DM) and TM-β-CD were used as chiral mobile phase additives. The effects of different mobile phase, pH and concentration of chiral mobile phase additives on resolution of D,L-lactic acid were investigated. The results showed: the enantioselectivity of TM-β-CD was better than those of β-CD and DM-β-CD. D,L-lactic acid could be partly separated by DM-β-CD 　and could not be separated by β-CD, while it could be fully separated by TM-β-CD. As the concentration of TM-β-CD changed from 0.20　mmol/L to 1.00　mmol/L, The resolution varied slightly. The experiment of the effect of pH of mobile phase on Rs showed: as pH changed from 2.4 to 3.0, the Rs remain stable, and when pH＞3.1, the Rs decreased with the increase of pH. The RSD of retention time of L-lactic acid and D-lactic acid were 0.2% and 0.2%, respectively. The chromatographic systems with a dynamically-generated stationary phase with 2,3,6-tri-O-methyl-β-cyclodextrin proved to be an effective method for lactic acid enantiomer separation

Objective To study the genetic variation of two mitochondrial DNA molecules (CO1 and Cytb gene) of Oncomelania hupensis isolated from different areas. Methods Snails were collected from Jingxi of Guangxi,Yueyang of Hunan and Eryuan of Yunnan. Genomic DNA was extracted from the snails,Co1 and Cytb gene fragments were amplified by PCR,then purified and sequenced. Sequences of each isolates were edited by using Clustal W(1.82) software,and the nucleotide composition,transition and transversion were accounted by using MEGA(3.1) software. The genetic distances were computed with Kimura method and phylogenetic trees were constructed with UPGMA and MP method respectively. Results CO1 and Cytb gene fragments were about 700 bp and 600 bp(including 2 primers) respectively. A total of 106 mutation spots (15.9%) were tested in CO1 homological nucleotides,and 165 mutation spots (28.5%) were tested in Cytb homological nucleotides. The distance matrix between Guangxi isolate and Hunan isolate was 0.051 and 0.031 for CO1 gene and Cytb gene respectively;while that between Guangxi and Yunnan isolates was 0.158 and 0.405 respectively. Phylogenetic trees constructed by UPGMA and MP took on the similar topo-structure:isolates of Guangxi and Hunan clustered into one group,while the Yunnan isolate exhibited as another group. Conclusion Oncomelania hupensis in Guangxi,Hunan and Yunnan are of relatively rich polymorphism in CO1 and Cytb genes in general.

Objective To investigate the role of chemokine ligand 21 (CCL21) in the spinal cord in tibia bone cancer pain (BCP) in rats.Methods Forty adult female SD rats weighing 160-180 g were randomly divided into 5 groups (n=8 each):sham operation group (group Ⅰ ); sham operation + CCL21 neutralizing antibody group (groupⅡ); BCP group (group [); BCP + PBS group (group Ⅳ); BCP + control IgG group (groupⅤ)and BCP + CCL21 neutralizing antibody group (group Ⅵ).BCP was induced by inoculating Walker-256 mammary gland carcinoma cells into the rat tibia medullary cavity in groups Ⅲ-Ⅵ.PBS 15 μl,IgG 10 μg and CCL21 neutralizing antibody 10 μg were injected intrathecally (IT) at 14 days after intra-tibial injection of Walker-256 mammary gland cancer cells in groups Ⅳ- Ⅵ respectively.Mechanical withdrawal threshold to yon Frey filament stimulation (MWT) was measured at 1 day before (To,baseline) ; 7 and 14 d after Walker-256 cell injection (T1,T2)and at 0.5,1,2,4,8,12,24 and 48 h after intrathecal injection (T3-10 ).Results Intra-tibial injection of Walker-256 mammary gland cancer cells significantly decreased MWT as compared with the baseline values in administration of CCL21 neutralizing antibody at T5-8 as compared with MWT before intrathecal administration at T2 in group Ⅵ.MWT was significantly lower in groups Ⅲ- Ⅳ than in groups Ⅰ and Ⅱ.MWT was significantly higher at T5-8 in group Ⅵ than in groups Ⅲ - Ⅴ.Conclu]sion CCL21 in the spinal cord is involved in the maintenance of tibia BCP in rats.

Objective To evaluate the role of spinal IKK2/NF-κB pathway in the maintenance of bone cancer pain (BCP) in rats.Methods Twenty-eight unmated adult female Sprague-Dawley rats,weighing 160-200 g,were randomly divided into 4 groups (n =7 each) using a random number table:sham operation group (group S),BCP group (group BP),BCP + normal saline group (group BN),and BCP + BMS345541 group (group BB).BCP was induced by injecting Walker 256 mammary gland cancer cell suspension 5 μl (4 × 105 cells/ml) into the bone marrow of the right tibia of rats anesthetized with chloral hydrate in BP,BN and BB groups,while the equal volume of normal saline was injected in group S.On 10-12 days after operation,selective IKK2 inhibitor BMS345541 (50 μg/10 μl) was intrathecally injected once a day in group BB,and the equal volume of normal saline (10μl) was given once a day in group BN.The mechanical paw withdrawal threshold (MWT) was measured before intra-tibia injection (T0),on 7 days after intra-tibia injection (T1),at 1 h before drug administration and 1,2,4,12 and 24 h after drug administration on day 10 after operation,and at 4 h after drug administration on day 12 after operation (T2-8).The rats were sacrificed after MWT was measured at Ts and L4-6 segments of the spinal cord were removed for determination of phosphorylated NF-κB (p-NF-κB) expression (using Western blot analysis).Results Compared with group S,MWT was significantly decreased at T1-2,and the expression of p-NF-κB was up-regulated in BP,BN,and BB groups.Compared with group BP,MWT was significantly increased at T4-6,and the expression of p-NF-κB in the spinal cord was down-regulated in group BB.Conclusion Spinal IKK2/NF-κB signaling pathway is involved in the maintenance of bone cancer pain in rats.

A total of 1512 adult inhabitants were randomly recruited in Zhaiji district of Guiyang city in September2009.The levels of triglyceride (TG),systolic blood pressure,diastolic blood pressure,and the prevalences of abdominal obesity and hypertension increased significantly in the subclinical hypothyroidism group conpared to the euthyroid group (P＜0.05).The prevalences of high TG,low high density lipoprotein-cholesterol,and metabolic syndrome (MS) in the subgroup Ⅳ were higher than the subgroup Ⅰ (P＜0.05).Correlation analysis revealed that TSH was positively related to TG (P＜0.05).Logistic regression demonstrated that TSH was a risk factor for MS.Either in the euthyroid or total subjects serum TSH levels in the MS group were significantly higher than those in non-MS group(P＜0.05).

Objective To evaluate the effect of intrathecal resveratrol on activation of Ca2+/ calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in spinal dorsal horn neurons of rats with bone cancer pain.Methods Thirty-two adult female Sprague-Dawley rats,weighing 180-220 g,were equally randomized into 4 groups using a random number table:sham operation group (group S),bone cancer pain group (B group),and bone cancer pain + solvent control group (BD group).Walker 256 mammary gland cancer cell suspension (4× 105 cells/ml) 5 μl was injected into the medullary cavity of the right tibia in B,BR and BD groups.Normal saline 5 μl was given in group S.On 12,13 and 14 days after injection of mammary gland cancer cells,resveratrol 200 μg/10 μl was injected intrathecally once a day in group BR,and 1％ dimethyl sulfoxide 10 μl was intrathecally injected once a day in group BD.Before injection of mammary gland cancer cells (T0) and on 3,5,7,10,12 and 14 days after injection of mammary gland cancer cells (T1-6),mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.The rats were then sacrificed and L4,5 segments of the spinal cord were removed for confirmation of the location of phosphorylated CaMK Ⅱ (p-CaMK Ⅱ) in spinal dorsal horn neurons (by immunofluorescence) and for detection of p-CaMK lⅡ expression (using Western blot).Results Compared with group S,MWT and TWL were significantly decreased at T2-6,and p-CaMK Ⅱ expression was upregulated at T6,and p-CaMK Ⅱ was mainly co-expressed with neurons in B,BR and BD groups.Compared with group B,MWT and TWL were significantly increased at T5,6,and p-CaMK Ⅱ expression was down-regulated at T6 in group BR.There was no significant difference in MWT,TWL,and p-CaMK Ⅱ expression at each time point between group B and group BD.Conclusion Resveratrol can alleviate hyperalgesia in rats with bone cancer pain and inhibited activation of CaMK Ⅱ in spinal dorsal horn neurons may be involved in the mechanism.

Objective To investigate effects of intrathecal methotrexate on mechanical allodynia in rats with tibial bone cancer pain.Methods Forty-eight female SD rats weighing 150-180 g were randomly divided into 6 groups ( n =8 each):group Ⅰ sham operation + artificial cerebrospinal fluid(SA group),group Ⅱ sham operation + methotrexate 200 μg(SM group),group Ⅲ bone cancer pain + artificial cerebrospinal fluid(CA group),group Ⅳ-Ⅵ bone cancer pain + different doses of methotrexate (CM1-3 groups).The model of tibial bone cancer pain was induced by injecting Walker-256 cell into the tibial marrow cavity.CA and CM1-3 groups were intrathecal injected artificial cerebrospinal fluid,methotrexate 50,100 and 200 μg.SA and SM200 groups were intrathecal injected artificial cerebrospinal fluid and methotrexate 200 μg.The mechanical withdrawl threshold (MWT) was measured at day 1 before Walker-256 injection (baseline),7 day after injection (T0 ) and 2,4,8,24 hour and 1,3,5,7 days after intrathecal injection ( T1-8 ).Results Compered with the baseline,MWT was decrease in CA and CM1-s groups.Competed with To,MWT was decreased at T5-8 in CA group,MWT was increased at T3-5 in CM1 group,at T2-6 in CM2 group and at T2-7 in CM3 groups.MWT was decrease in CA and CM1-3 groups as compered with SA group; MWT was increased at T4-7 in CM1 group and at T3-7 in CM2 and CM3 groups.Conclusion Intrathecal injection of methotrexate can reduce tibial bone cancer pain in rats.

Stand-by training is an important step for a medical contingent to realize medical support in peacekeeping operations.In line with Draft MOU and General Guidelines for Peacekeeping OPNS,we need to shape a feasible standby training blueprint,and make the training plan for the medical contingent as a whole with consideration of tasks on our contingent and the actual situation of our army.

Objective To evaluate the effects of intrathecal methotrexate on the activation of microglia in spinal cord in a rat model of tibial cancer pain (TCP).Methods Thirty female Sprague-Dawley rats,aged 5-7 weeks,weighing 150-180 g,were randomly divided into 3 groups (n =10 each):sham operation + artificial cerebrospinal fluid (group SA),TCP + artificial cerebrospinal fluid (group CA),and TCP + methotrexate (group CM).TCP was induced by injecting Walker-256 cancer cells into the medullary cavity of tibia.Artificial cerebrospinal fluid or methotrexate 100μg (15μl) was injected intrathecally over 10 min on 7th day after TCP.Mechanical pain threshold (MPT) was measured before TCP,at 1,3,5 and 7 days after TCP and 2,4,8 and 24 h after administration (T0-8).The rats were sacrificed after measurement of the pain threshold at T8 and the spinal cord was isolated for detection of the activation of microglia (by immunofluorescence) and content of tumor necrosis factor-α (TNF-α) and IL-1β (by ELISA).Results Compared with group SA,MPT was significantly decreased,and the number of activated microglia cells in the spinal cord was increased,and the contents of TNF-α and IL-1β were increased in groups CA and CM (P ＜ 0.05).Compared with group CA,MPT was significantly increased,and the number of activated microglia cells in the spinal cord was decreased,and the contents of TNF-α and IL-1β were decreased in group CM (P ＜ 0.05).Conclusion The mechanism by which intrathecal methotrexate reduces TCP in rats is related to inhibition of the activation of microglia and reduction of the secretion of proinflammatory cytokines TNF-α and IL-1β in the spinal cord.

Objective To investigate the antitumor effects and the possible mechanisms of dendrit-ic cells co-cultured with cytokine-induced killer cells(DC-CIK)in combination with sorafenib on two lung adenocarcinoma cell lines,A549 cells(harboring KRAS gene mutation)and PC-9 cells(harboring EGFR gene mutation). Methods DC and CIK cells were routinely generated in vitro by stimulating PBMCs isola-ted from lung cancer patients with different cytokines and then co-cultured after a week of culturing. Flow cy-tometry analysis(FCM)was used to analyze the phenotype of DC-CIK cells after 7 days of co-culturing. The 50% inhibitory concentration(IC50 )of sorafenib against tumor cells was detected by MTT assay. The tumor cells were treated with DC-CIK cells alone or in combination with sorafenib. The proliferation of tumor cells was tested by CCK-8 kit and dynamically monitored by real-time cellular analysis(RTCA). Annexin-V/ PI staining was used to examine the apoptosis rates in each group. Real-time fluorescent quantitative PCR and FCM were respectively performed to detect the expression of natural killer group 2 member D ligands (NKG2DLs)at mRNA and protein levels after the treatment with sorafenib for 24 h. Results There was no significant difference between the IC50 of sorafenib against A549 and PC-9 cells after a 24-hour exposure(P﹥0. 05). Compared with the DC-CIK biotherapy,treating the tumor cells with DC-CIK cells in combination with sorafenib significantly inhibited the cell proliferation and increased the total apoptosis rates of tumor cells(P﹤0. 05). Moreover,the inhibition rates to tumor cell proliferation were enhanced along with the in-crease of effect-to-target ratio(E/ T). Compared with the single-factor treatment groups,the normalized cell index(NCI)in the combined treatment group was significantly decreased. Blocking NKG2D could abate the inhibitory effect of DC-CIK cells on tumor cell proliferation(P﹤0. 05). The expression of NKG2DLs(inclu-ding ULBP1,UBLP2 and ULBP3)on tumor cells at mRNA and protein levels were increased to different ex-tent after treating with 5 μmol/ L of sorafenib for 24 h. Conclusion There was no significant different be-tween the inhibitory effects of sorafenib on the proliferation of lung adenocarcinoma cancer cells harboring KRAS or EGFR gene mutation. The antitumor effects of DC-CIK cells combined with sorafenib on lung ade-nocarcinoma cells might be induced by regulating the NKG2D-NKG2DLs pathway and enhancing apoptosis. Moreover,the antitumor effects of the combined treatment were better than those of single-factor treatments.