AIM:To clone the SPA gene promoter and construct its luciferase report vector of SPA gene and to study its transcriptional targeting activity.METHODS:① The SPA gene sequence was acquired from GenBank,of which the upstream was analyzed according to bioinformatics.The results showed that the upstream region of SPA gene sequence about 163bp has the function of promoter.② The SPA gene promoter fragment was generated by polymerase chain reaction and then subcloned into the multiple clone site(MCS)of luciferase report gene vector pGL3-basic to generate the recombined plasmid pGL3-SPA.This fragment was also subcloned into pGL3-control to generate recombined plasmid pGL3-SPA-enhancer by replacing its primary SV40 promoter.③ pGL3-SPA,pGL3-SPA-enhancer,pGL3-control,pGL3-basic were cotransfected with pRL-TK into A549 cells and H441 cells.The luciferase activities were measured by dual luciferase reportor(DLR)system.RESULTS:Sequencing and restricted digestive results showed that SPA gene promoter was successfully cloned and identified,and also correctly subcloned into plasmid pGL3-basic and pGL3-control to construct its luciferase report plasmid pGL3-SPA and pGL3-SPA-enhancer,respectively.The transcriptional activity was high in H441 cells.CONCLUSION:The luciferase report system of SPA gene promoter is successfully constructed with high transcriptional activity.

Objective To construct the eukaryotic expression vector harboring the fragment of Alia gene, and to investigate the effects of it on the signal of quorum sensing and virulence factors producted by Pseudomonas aeruginosa(Pa). Methods The plasmid pET-AiiA was cutted by Nhe Ⅰ and Xho Ⅰ , then the AiiA fragment was cloned into eukaryotic expression vector pEGFP-N2. After the plasmid was transfected into A549 cells, the protein was extracted and AiiA protein was found in it by Western blot. After the extrac- tion was admixed into the LB broth, from culture supernatant extracts of Pa, the N-acylhomoserine lactone (AHL) was detected by bioassay, and the expression of pyocyanin and elastase were assayed by RT-PCR and optical density. Results The fragment of AiiA gene was cutted and then cloned into pEGFP-N2. AiiA protein was found in the transfected cells. After admixed with the extract harboring AiiA protein, in Pa medium, the AHL was hydrolyzed, and the expression of pyocyanin and elastase were reduced. Conclusion The virulence factors synthesized by Pa were reduced by the AiiA protein expressed in eukaryotic cell.

Objective To investigate the feasibility of NR2B small interference RNA(NR2B siRNA)carried by water-soluble lipopolymer(WSLP)for treatment of neuropathic pain in rats.Methods One hundred healthy male SD rats weighing 180-200 g were randomly divided into 5 groups(n=20 each):normal control group (group C),sham operation group(group S),neuropathic pain group(group NP),group WSLP-NR2B siRNA (group W)and group WSLP-negative NR2B siRNA(group WN).Neuropathic pain was induced by partial ligation of sciatic nenre.WSLP-NR2B siRNA complex was formed by binding WSLP and NR2B siRNA.Normal saline.WSLP-NR2B siRNA complex and WSLP-negative NR2B siRNA 20μl were injected intrathecally after operation in NP,W and WN groups respectively.Mechanical withdrawal threshold(MWT)and thermal withdrawal duration (TWD)were measured before(baseline)and at 3,7,14 and 21 days after operation.Ten animals in each group were sacrificed on the 3rd day after operation and the lumbar segment(L4-6)of the dorsal root ganglia was removed for determination of the expression of NR2B mRNA and protein using RT-PCR and Western blot analysis.Results Sciatic nerve ligation significantly decreased MWT and prolonged TWD and increased NR2B mRNA and protein expression in group NP as compared with group C.WSLP-NR2B siRNA complex significantly reduced sciatic nerve ligation-induced hyperalgesia and decreased NR2B mRNA and protein expression in group W as compared with group NP.Conclusion WSLP not only mediates NR2B siRNA successfully and inhibits the expression of NR2B,but also reduces neuropathic pain in rats.

Objective To study the biodistribution of anti-HER-2/neu monoclonal antibody Herceptin labeled by 131I(131I-Herceptin) in healthy KM mice and nude mice bearing human ovarian cancer xenografts and radioimmunoimaging (RII) of the nude xenografts-bearing mice.Methods 131I-Herceptin was prepared using Iodogen method.The labeling efficiency, radiochemical purity, stability and immunocompetence were measured.The percentage activity of injection dose per gram of tissue (%ID/g) and the radioactivity ratio of tumor to non-tumor tissue (T/NT) were calculated for each time point.The optimal time for imaging was investigated by comparing the 131I-Herceptin SPECT for the nude mouse models bearing ovarian cancer xenografts at different time points.Results The labeling efficiency and radiochemical purity of 131I-Herceptin were 89.8% and 98.4%, respectively.The labeling was stable and had good immunocompetence.131 I-Herceptin was cleared rapidly mainly through liver, spleen and kidneys, consistent with first order two-compartment model.The uptake of 131I-Herceptin in the tumors bearing human SKOV-3 xenografts was much higher than that in nontumor tissue.The% ID/g was 18.08 in the tumor at 24 h post injection.The T/NT ratio increased with time and was 27.27 at 72 h post injection.The tumors in nude mice bearing SKOV-3 xenografts could be visualized on 131I-Herceptin SPECT imaging 2 h post injection; definitely identiffed 48 h post injection and the radioactivity ratio of tumor to contralateral tissue was 11.44 at 120 h post injection.However, the tumor in nude mice bearing HO-8910 xenografts did not show abnormal uptake of 131 I-Herceptin at each time point.Conclusions 131 I-Herceptin is a good radiopharmaceutical targeting SK-OV-3 xeuografts and it may be useful in imaging carcinoma of ovary and target therapy of its metastases with high HER-2/neu expression.

The misuse of toxic drugsisseriousone of the threats of public health. In this study, toxic Hyoscyami Semen and its adulterants were identified by DNA barcoding. The genomic DNA was extracted from 61 samples including Hyoscyami Semen and its adulterants by reagent kit method. Their ITS2 sequences were amplified, and purified PCR products were sequenced. Sequence assembly and consensus sequence generation were performed using CodonCode Aligner v 4.25. The genetic distances, variable sites and the neighbor-joning (NJ) phylogenetic tree were computed by MEGA 6.0 in accordance with the Kimura 2-parameter(K2P) model. The results showed that the intra-specific genetic distances of Hyoscyamusniger were 0.005 which were smaller than inter -specific ones (0.360) of H. niger and their adulterants. The NJ tree showed that H. niger was clustered into one monophyletic branch, and clearly separated with other species. Therefore, ITS2 sequence was able to identify Hyoscyami Semen and its adulterants to ensure the safty of medicines.

In order to confirm the alteration and significance of cigarette smoke exposure on SP-A in rats, 20 Wistar rats were assigned randomly to two groups: an N group (n=10), and an S group (n=10). The ultra-structural change was observed by electron microscopy. The number of cells positive for SPA was by immunohistochemically measured. The mRNA expression in the lung tissues was determined by reverse transcription polymerase chain reaction (RT-PCR). The number of cells positive for SPA of the S group (0.52 +/- 0.05) was lower than that of the N group (0.72+/-0.06) (P<0.05). The levels of mRNA of SPA in the lung tissues of the S group (0.3522+/-0.0512) was significantly lower than that of the N group (0.4432+/-0.05628) (P<0.05). It is concluded that cigarette smoke alone decreased the level of SP-A and that might have an important effect on surfactant metabolism and the host defense functions of surfactant in the peripheral airways, which might play a crucial role in the development of chronic obstructive lung disease.
Gene Expression Regulation ; Immunohistochemistry/methods ; Lung/metabolism ; Microscopy, Electron ; Pulmonary Surfactant-Associated Protein A/*biosynthesis ; RNA, Messenger/metabolism ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Smoking/*adverse effects ; Tobacco Smoke Pollution/*adverse effects

Objective:To investigate variety pattern of expression level of TCRV?subfamilies in mononuclear cell in spleen tissue of rats with dampness pathogenic factors and normal rats by using FQ-PCR technique. Methods:32 SD rats were divided into four groups: normal group, external dampness group, internal dampness group, external and internal dampness group. Observing period was 20 days. 3 Rats were randomly selected from each group in order to exam the TCRV? subfamilies expression level. Results:The expression of TCRV?1, TCRV?7, TCRV?9 and TCRV?13 in external dampness group were higher than those in normal group (P

OBJECTIVETo study the effect of Tongluo Xingnao effervescent tablets on learning and memory capacity and expression of Na(+)-K(+)-ATPase in hippocampus of rats with chronic cerebral ischemia-induced learning and memory dysfunction model.

METHODThe 2-VO method was used to establish sd rat model learning and memory dysfunction induced by chronic cerebral ischemia. The 50 rats in the successfully established model were randomly divided into the model control group, the Dihydroergotoxine Mesylate tablets group (0.7 mg x kg(-1), Tongluo Xingnao effervescent tablets high dose (7.56 g x kg(-1)), middle dose (3.78 g x kg(-1)) and low dose (1.59 g x kg(-1)) groups and the sham operation group (n = 10) as the control group. The groups were orally given 10 ml x kg(-1) x d(-1) drugs for consecutively 90 days. On the 86th day, Morris water maze was adopted for them. On the 90th day, a leaning and memory capacity test was held. The brain tissues were fixed with 10% formaldehyde and observed for pathomorphism after routine slide preparation and staining. The expression of hippocampal Na(+)-K(+)-ATPase was detected with immunohistochemistry and image quantitative analysis.

RESULTCompared with the model group, all of Tongluo Xingnao effervescent tablets groups showed significant decrease in the escape latency at the 5th day in the Morris water maze, and notable increase in the frequency of the first quadrant dwell, the frequency passing the escape platform and the frequency entering effective area (p < 0.05). According to the pathomorphological detection, the control group showed a significantly higher pathological score than the sham operation group (p < 0.01), the middle dose group showed a significantly lower pathological score than the model group (p < 0.05). According to the immunohistochemistical detection, the model control group showed a remarkably lower mean OD value of Na(+)-K(+)-ATPase than the sham operation group (p < 0.05), high and middle dose groups showed a significantly higher mean od value than the model control group (p < 0.01).

CONCLUSIONTongluo Xingnao effervescent tablets can improve the learning and memory capacity, reduce pathological changes of hippocampal tissues of rats with chronic cerebral ischemia-induced learning and memory dysfunction model, and promote the expression of Na(+)-K(+)-ATPase in hippocampus.

Animals ; Brain Ischemia ; drug therapy ; enzymology ; genetics ; psychology ; Chronic Disease ; drug therapy ; psychology ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hippocampus ; drug effects ; enzymology ; Humans ; Learning ; drug effects ; Male ; Memory ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase ; genetics ; metabolism ; Tablets ; administration & dosage

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.

Objective To evaluate five years results of laser in situ keratomileusis(LASIK)for correction of myopia.Design Ret- rospective case series.Participant 64 patients(126 eyes)with myopia received operation of LASIK.Method 126 myopic eyes were treated with LASIK.Based on the preoperative spherical equivalent refraction(SER),the patients were divided into Group A,B and C(-3.00D～6.00D,-6.25D～-10.00D and -10.38D～-19.88D).Before and after operation,the visual acuity,refraction,anterior seg- ment,funduscopy,intraocular pressures of patients were measured,and the patients were followed-up five years.Main Outcome Measure Visual acuity,intraocular pressure,refraction and complication.Result At five years,in Group A,B,C,uncorrected visual acuity was≥1.0 in 87.2%,69.1%,31.3% patients,and 100%,98.2%,75% had vision of≥0.5.After five years of operation,the re- fraction was -0.70D?0.52D,-1.06?0.13D,-2.46?2.14D in three groups respectively,there was no significant difference between Group A and B(P=0.23);But there was significant statistical difference between Gruop A and C(P=0.008),Group B and C(P=0.021)respec- tively.At the fifth-year,68.3%,45.0% and 23.5% of refraction in Group A,B,C were within?1.00D.Intraocular pressure at the fifth year was 11.91?2.35mmHg,11.31?2.20 mmHg and 9.24?2.20 mmHg respectively.In Gruop B and C,one patient complained glare re- spectively.In Gruop B,one patient's intraecular pressure was elevated after using glueocorticoid.In Group C,one eye was complicated with rbegmatogenous retinal detachment at the second year after LASIK.Conclusion In the follow-up of five years,LASIK is effective and safe.The results in middle and high myopia are better than that in extreme high myopia.(Ophthalmol CHN,2006,15:312-314)