Objective:On the basis of synthetic antigens on minoxidil ,we prepared its polyclonal antibody and established a ELISA method to detect minoxidil .Methods:We synthesized minoxidil artificial antigen with glutaraldehyde and identified by UV scan -ning.We had established a detection of minoxidil competition ELISA .Results: The UV scan showed that the minoxidil successfully coupled to a carrier protein.The conjugate of minoxidil-BSA was used to immunize BALB/c mice.And the produced antiserum showed high titer of 1:12 800 in the indirect ELISA.Its ELISA curve equation:y=-0.082 3 x+0.938 ( R2 =0.9811 ) , minoxidil mass concentration and the absorbance in 0.001-10 μg/ml showed a good linearity .Conclusion: This method has been successfully preparing minoxidil artificial antigen and polyclonal antibodies and established ELISA detection method for minoxidil ,which provides a basis for the practical application of minoxidil immunoassay method .

Objective To investigate the species,density and distribution of the overwintering adult mosquitoes in Taibaihu District,Jining City. Methods The overwintering adult mosquitoes were monitored by visual observation in Taibaihu District, Jining City from December 2015 to March 2016. The mosquitoes were collected and dissected to observe the body fat accumula?tion and ovarian development situation. Results Totally 1 677 mosquitoes were captured and all of them were Culex pipiens pal?lens. The highest positive rate of overwintering mosquitoes was in sweet potato cellars and greenhouses,with the positive rates of mosquitoes of 98.25%(56/57)and 81.36%(48/59)respectively. The accumulation of the fat body in the female mosquitoes plummeted from late February,and disappeared in middle March. Conclusions The adults of Culex pipiens pallens can over?winter in Taibaihu District,Jining City,and locate in sweet potato cellars and greenhouses mainly. The control work of adult mosquitoes should be strengthened in the overwintering period.

OBJECTIVE The antagonism of obidoxi me on sarin induced miosis and visual impair-ment was evaluated and its antagonistic mechanism was investigated.METHODS ① 30 min after sarin (2 μg /0.1 mL per eye)was given as an eyedrop,the ability of the 2.5%,5.0%,7.5% obidoxi me and 1 .0% atropine to reverse effects of sarin on pupil dia meter and light reflex were evaluated at different ti mes.② Another 36 rabbits received sarin and at 30 min afer sarin exposure,the drugs above were ad-ministrated and their effects on pupillary light reflex,as well as the AChE activity of cornea,iris and reti-na were recorded 4h after the treatment.RESULTS ① Miosis and impaired pupillary light reflex oc-curred soon after sarin exposure but the abnormal pupil width and pupillary light reflex had disappeared by 48 h after sarin exposure;Subcequent to 1 .0% atropine treatment,the pupil dilatedinstead while the impaired light reflex did not i mprove significantly;unlike atropine,soon after ad ministration of 2.5%, 5.0%,7.5% obidoxi me,the pupil dia meter and light reflex were significantly increased(P <0.01 )and then had beco me normal totally by 24 h post-dose,much faster than those of the control and atropine treatment group.However,there was no significant difference in the recovery ti me between the different dose groups of obidoxi me.② 4h after treatment,the AChE activity in cornea and irisof sarin-treated group were (42 ±4)%,(26 ±2)%,respectively;the AChE activity in cornea of 2.5%,5.0%,7.5%obidoxi me were (74 ±1 1 )%,(81 ±10)% and (74 ±7)%,respectively,and the AChE activity in iris were(39 ±10)%,(43 ±8)% and (43 ±8)%,respectively ,co mpared with sarin-treated group,AChE activities of cornea and iris as well as light reflex of the obidoxi me-treated group were significantly increased(P<0.01 ).But there was no difference in light reflex and AChE activity between the sarin-treated and atropine-treated groups.CONCLUSION Obidoxi me showed better antagonism of sarin-induced ocular effects than that of the commonly used drug,atropine;the antagonistic mechanism is likely closely related to its rapid reactivation of the inhibited AChE in the cornea and iris.

OBJECTIVETo examine the flora samples from the tongue dorsum of the atrophic glossitis group and to discuss the relationship so as to provide a thinking pattern for therapy and a clue for deeper research.

METHODSTo collect personal information on 60 cases of atrophic glossitis and 40 cases of volunteers as control. The main items include general status, oral examination, salivary flow rate, pH value and bacteria test. All data were analyzed statistically.

RESULTS1) Among the 60 cases, 75.00% were female patients. Glosso-pain, dry mouth and taste loss were the most common symptoms. 2) In regard to the pathogenic factors, the systematic diseases were often visible, i.e. gastritis, coronary disease and anemia. 3) Oral hygiene of the patients was worse than that of the control group, the saliva flow rate and pH value were lower than that of the control. 4)The statistic analysis showed that the quantity of some bacteria of tongue dorsum and their detectable rate were different between the glossitis group and the control one, between the patients with atrophic glossitis who also suffered from different systematic diseases and the control group, and between the complete type and the partial type. These bacteria included Streptococcus sanguis, Stomatococcus, Staphylococcus aureus, Saccharomyces, etc.

CONCLUSIONAtrophic glossitis is the consequence co-affected by host, circumstances of oral cavity and bacteria. The tangible relationship between atrophic glossitis and micro-ecological changes on glossal dorsum has not been confirmed yet, however, flora change on dorsum may have relations with occurrence, and development of the disease.

ObjectiveTo determine thallium in whole blood by atomic absorption detection method, and to investigate the eliminating effect of hemoperfusion (HP) for thallium in blood.Methods The blood of Beagle dogs which had not exposed to thallium before were obtained for preparation of thallium nitrate (TlNO3)-containing solution in three concentrations according to the conversion formula based on animal weight and volume of blood. HP was performed in the simulated in vivo environment. The content of TlNO3 in blood of the next group was determined on the amount of TlNO3 for the last HP of the former dose group. Thallium quantity in different samples was measured with atomic absorption spectrometer blood samples before and after HP. Finally, the thallium concentration in blood was analyzed statistically.Results Thallium concentrations showed a good linear relationship in the range of 0-200μg/L (r = 0.998 4). The intra-day precision (RSD) was lower than 4.913%, the intra-day recovery rate was 96.2%-111.9%; the inter-day precision (RSD) was lower than 7.502%, the inter-day recovery rate was 89.6%-105.2%. The concentration of thallium in blood was significantly reduced after HP per time in high, middle, and low dose groups [(453.43±27.80) mg/L to (56.09±14.44) mg/L in high dose group,F = 8.820,P = 0.003;(64.51±13.60) mg/L to (3.19±0.23) mg/L in middle dose group,F = 36.312,P = 0.000; (5.40±0.98) mg/L to (0.38±0.25) mg/L in low dose group,F = 46.240,P = 0.000]. The adsorption rate of four times of HP in high, middle and low dose group were (87.63±2.48)%, (95.06±1.54)% and (92.76±4.87)%, respectively, without significant difference (F = 4.231,P = 0.070 ).Conclusions The method for measuring thallium was established, and it shows a very stable, simple, sensitive for determination of thallium. HP can effectively remove thallium from blood. Thallium concentration can be reduced by 90% after four times of HP. HP is also effective even when thallium concentration is not high.

BACKGROUNDTo explore the possibility of targeting blockage of Wnt signal transduction pathway of nm23-H1 gene transfection in human high-metastatic large cell lung cancer cell line L9981, and to provide evidence to elucidate the signal conductive mechanism of nm23-H1 mediated tumor metastasis suppression.

METHODSThe expression of GSK-3β and β-catenin of Wnt signal pathway was detected in cytoplasm and nucleus in L9981 cell line with nm23-H1 deletion, L9981-pLXSN cell line transfected with vector and L9981-nm23-H1 cell line transfected with nm23-H1 gene by Western blot.

RESULTS(1)GSK-3β expression in L9981-nm23-H1 cytoplasm (6 341±541) was significantly higher than those in L9981 (3 736±298) and L9981-pLXSN (3 613±383) cell lines ( P < 0.001); (2)GSK-3β expression in L9981-nm23-H1 nucleus (4 356±490) was significantly higher than those in L9981 (657±57) and L9981-pLXSN (705±75) cell lines ( P < 0.001); (3)β-catenin expression in L9981-nm23-H1 cytoplasm (3 649±118) was significantly higher than those in L9981 (1 401±31) and L9981-pLXSN (1 350±55) cell lines ( P < 0.001); (4)No statistical difference of the β-catenin expression in nucleus was observed among L9981-nm23-H1 (2 945±68), L9981 (2 604±23) and L9981-pLXSN (2 652±53)( P > 0.05); (5)No significant difference of GSK-3β or β-catenin expression in cytoplasm and nucleus was observed between L9981 and L9981-pLXSN ( P > 0.05).

CONCLUSIONS(1)nm23-H1 gene can remarkably upregulate the expression of GSK-3β in cytoplasm and nucleus, and β-catenin expression in cytoplasm in L9981-nm23-H1 cell, but can not induce the nucleus accumulation of β-catenin. (2)Regulation of GSK-3β and β-catenin expression, and targeting blockage of Wnt signaling pathway may be one of molecular mechanisms that nm23-H1 contributes to play a vital role in the "Lung Cancer Metastasis Suppressive Cascade".

BACKGROUNDTo explore the effects of nm23-H1 gene transfection and forskolin on PKA activity in human high-metastasis large cell lung cancer cell line L9981.

METHODSThree cell lines, primary human large cell lung cancer cell line (L9981), vector transfection cell line (L9981-pLXSN) and nm23-H1 gene transfection cell line (L9981-nm23-H1-pLXSN), were treated with PKA activator forskolin. The PKA activity at different time points after treatment with forskolin was detected in the three lung cancer cell lines by radioimmunological method with SignaTECT cAMP-dependent PKA assay system.

RESULTS(1) Before forskolin treatment, the activity of PKA of L9981-nm23-H1-pLXSN was remarkably higher than those of L9981 and L9981-pLXSN (P < 0.01), but no significant difference in the PKA activity was observed between L9981 and L9981-pLXSN (P > 0.05). (2)The PKA activity was remarkably increased in all the three lung cancer cell lines after treatment with different concentration of forskolin (P < 0.01), and up to the highest level at the concentration of 100 μmol/L. It showed a dose-dependent relationship between the PKA activity and forskolin concentration; (3) The PKA activity in all the three cell lines was elevated to the highest level at 30 minutes after treatment with forskolin of 100 μmol/L, and it showed a time-dependent relationship between the PKA activity and action time of forskolin.

CONCLUSIONS(1)Transfection of nm23-H1 gene can up-regulate the PKA activity of human high-metastasis large cell lung cancer cell line L9981, and its function as a tumor metastasis suppressor gene may be related to its effects on regulation of PKA signal transduction pathways; (2)Forskolin can remarkably up-regulate the PKA activity of L9981 cell line, and the elevation of PKA activity has a time-dependent and dose-dependent relation to forskolin.

BACKGROUNDTo explore the influences of nm23-H1 gene transfection and protein kinase C (PKC) inhibitor Calphostin C on PKC signal transduction pathway in human high-metastasis large cell lung cancer cell line L9981, and to evaluate the effects of nm23-H1 gene on translocation and activation in subcellular region.

METHODSThe translocation of PKC in subcellular region was observed in L9981 before and after nm23-H1 gene transfection and Calphostin C treatment by Laser scanning confocal microscope (LSCM) method.

RESULTSPKC-α and PKC-βII were found to locate in different subcellular site in L9981 before and after nm23-H1 gene transfection. PKC-α and PKC-βII mainly located in nucleus and perinucleus in L9981 and L9981-pLXSN cell lines, which were in active status. PKC-α and PKC-βII mainly located in soluble cytosolic fraction in L9981-nm23-H1 cell line and were inactive status. PKC-α and PKC-βII mainly located in cytosolic fraction and were in inactive status in all the three cell lines after treatment with Calphostin C.

CONCLUSIONSThe results suggest that nm23-H1 gene might make PKC to translocate from nucleus and perinucleus to soluble cytosolic fraction in L9981 cell line. PKC inhibitor, Calphostin C, can also make PKC to translocate from nucleus and perinucleus to soluble cytosolic fraction in L9981, L9981-pLXSN cell lines. Both transfection of nm23-H1 gene and treatment with Calphostin C can suppress the PKC signal transduction in L9981 cell line.

BACKGROUNDTo investigate the influence of tumor metastasis suppressor gene nm23-H1 on the activity of glycogen synthase kinase 3β (GSK-3β) in human high-metastasis large cell lung cancer cell line L9981.

METHODSThe levels of GSK-3β expression in cytoplasm and nucleus were determined with anti- GSK-3β antibody in human high-metastasis large cell lung cancer cell line L9981 (cell line with nm23-H1 gene deletion), L9981-nm23-H1 (cell line with nm23-H1 transfected) and L9981-pLXSN (cell line with vector transfected) by Western blot method. The activity of GSK-3β among those three cell lines was detected by immunoprecipitation and analysed by a radioactive isotope scintillation counter before and after treating with 20 mmol/L LiCl.

RESULTS(1) The expression indensity of GSK-3β of cytoplasm and nucleus was (6 341±541) and (4 356±490) IOD in L9981-nm23-H1, (3 613±383) and (705±75) IOD in L9981-pLXSN, and (3 736±298) and (675±57) IOD in L9981, respectively. A high significance in GSK-3β expressive indensity of both cytoplasm and nucleus existed among L9981-nm23-H1, L9981-pLXSN and L9981 (P < 0.01); Multiple comparison: A highly significant difference was observed when L9981-nm23-H1 was compared with L9981-pLXSN or L9981 (P < 0.01), but no significant difference was observed between L9981-pLXSN and L9981 (P > 0.05). (2) The GSK-3β activity of cytoplasm and nucleus was (28 955±2 509) and (9 247±924) CPM in L9981-nm23-H1, (11 241±1 495) and (1 492±176) CPM in L9981-pLXSN, and (12 505±1 469) and (1 763±125) CPM in L9981, respectively. A highly significant difference in GSK-3β activity of both cytoplasm and nucleus existed among L9981-nm23-H1, L9981-pLXSN and L9981 (P < 0.01); Multiple comparison: the GSK-3β activity in L9981-nm23-H1 was significantly higher than that in L9981-pLXSN and L9981 (P < 0.01), but no significant difference was observed between the L9981-pLXSN and L9981 (P > 0.05). (3) After treatment with 20 mmol/L LiCl, the expressive indensity of GSK-3β of cytoplasm and nucleus was (4 718±549) and (3 823±350) IOD in L9981-nm23-H1, (2 030±155) and (217±15) IOD in L9981-pLXSN, and (2 164±151) and (224±19) IOD in L9981, respectively. No significant difference in GSK-3β expressive indensity existed between before and after treatment with LiCl in L9981-nm23-H1 (P > 0.05). However, the GSK-3β expressive indensity in cytoplasm and nucleus before treatment was remarkably higher than those after treatment in both L9981-pLXSN and L9981 (P < 0.05). (4) After treatment with 20 mmol/L LiCl, the GSK-3β activity in cytoplasm and nucleus was (11 099±1 112) and (3 748±215) CPM in L9981-nm23-H1, (4 447±430) and (1067±159) CPM in L9981, and (4 435±427) and (909±156) CPM in L9981-pLXSN, respectively. The GSK-3β activity both in cytoplasm and nucleus after treatment with LiCl was remarkably lower than that before treatment in L9981-nm23-H1, L9981-pLXSN and L9981 (P < 0.01 or P < 0.05).

CONCLUSIONS(1) Transfection of nm23-H1 gene can significantly up-regulate the expression level and activity of GSK-3β in human high-metastasis large cell lung cancer cell line L9981; (2) LiCl can remarkably suppress the upregulation effects of nm23-H1 gene on GSK-3β activity in L9981 cell line; (3) The effects of nm23-H1 gene on suppressing the signal transduction of Wnt pathway might be carried out through upregulating GSK-3β expression and activity in human high-metastasis large cell lung cancer cell line L9981.

BACKGROUNDTo explore the effects of transfection of nm23-H1 gene on expression of β-catenin and phospho-β-catenin in human high-metastatic large cell lung cancer line L9981, and to provide evidence to elucidate the molecular mechanism of nm23-H1 mediated tumor metastatic suppression.

METHODSTo determine whether nm23-H1 contributes to cytoplasm and nuclear β-catenin and phospho-β-catenin expression, the expression level of β-catenin and phospho-β-catenin in cytoplasm and nucleus was detected in human high-metastatic large cell lung carcinoma cell lines including primary cell line L9981 with nm23-H1 gene deletion, L9981-nm23-H1 transfected with nm23-H1 gene, and L9981-pLXSN transfected with vector by Western blot.

RESULTS(1)β-catenin expression in L9981-nm23-H1 cytoplasm (IOD) (3 649±118) was significantly higher than that in L9981 (1 401±31) and L9981-pLXSN (1 350±55) cell lines (P < 0.001);(2)There was no statistical diffe-rence of the β-catenin expression in nucleus among L9981-nm23-H1 (2 945±68), L9981 (2 604±23) and L9981-pLXSN (2 652±53) cell lines (P > 0.05);(3)Phospho-β-cetenin expression of cytoplasm in L9981-nm23-H1 cell line (3 123±102) was significantly lower than that in L9981 (4 362±131) and L9981-pLXSN ( 4 500 ±117) cell lines (P < 0.001);(4)Phospho-β-catenin expression of nucleus in L9981-nm23-H1 (5 136±112) was significantly higher than that in L9981 (2 666±116) and L9981-pLXSN (2 661±66) cell lines (P < 0.001);(5)There was no statistical difference of β-catenin or phospho-β-catenin expression in cytoplasm and nucleus between L9981 and L9981-pLXSN cell lines (P > 0.05).

CONCLUSIONS(1)nm23-H1 gene transfection can remarkably upregulates the expression of cytoplasm β-catenin in human high-metastatic large cell lung cancer cell line L9981, but do not induce the nucleus accumulation of β-catenin; (2)Transfection of nm23-H1 gene can significantly upregulate the expression of phospho-β-catenin in nucleus and remarkably downregulate the expression of phospho-β-catenin in cytoplasm of L9981; (3)Regulation of the expression of the key modecule, β-catenin, in Wnt signal pathway might be the important melecular mechanisms which nm23-H1 gene controls "Lung Cancer Metastatic Suppresive Cascade" and reverves cancer metastasis in L9981.