Objective:To clone LIF gene for the construction of mammalian expression vector PCDNA3.1(+)-LIF.Methods:LIF gene was cloned from pregnant uterine decidua tissues by using RT-PCR method,the gene was ligated with T-vector,then LIF gene was excised from T-LIF plasmid and inserted into PCDNA3.1(+) to construct eukaryotic expression vector consisting of LIF gene.Results:LIF gene sequence and vector were verified correctly by enzyme digestion,sequence analysis.Conclusion:The successful clone of LIF gene and the construction of its mammalian expression vector has been obtained.The results have provided basis for the further studies on LIF.

Objective To study the risk factors for dose wearing off (WO)in PD patients .Methods One hundred and thirty-three PD patients were recruited in this study according to the UKPDBB criteria .Their dose WO was diagnosed according to the Wo questionnaire 9 (WOQ-9) .The pa-tients were divided into WOQ-9 (+ ) group (n=111) and WOQ-9 (-) group (n=22) .The pa-tients in WOQ-9 (+) group were further divided int WO (+ ) group (n=59) and WO (-) group (n=52) .The difference in their clinical and therapeutic parameters was compared .Results The dose WO was observed in 83 .5% of the 133 PD patients ,53 .2% of which accorded with the dose WO clinical definition .T he disease onset age ,disease course ,maximal levodopa daily dose and ac-cumulated levodopa dose differed greatly in WOQ-9 (+ ) group and WOQ-9 (-) group (P<005) . The disease course ,H-Y stage ,UPDRS score ,tetany score ,maximal levodopa daily dose ,levodopa dose per body weight and accumulated drug use time differed greatly in WOQ-9 (+ ) group and WOQ-9 (-) group (P< 0 .01) .The major risk of dose WO was the levodopa dose per body weight (OR=1.364 ,P<0 .05) .Conclusion Dose WO is related with the progress of disease and the use of levodopa .Levodopa dose per body weight is an independent risk factor for dose WO .

Objective To study the spatiotemporal distributions of epithelial proliferation and apoptosis,and the correlation of morphogenesis with them in developing stomach.Methods Serial sections of fetal mice aged 11-15.5 days were made.The morphogenesis of stomach was observed under light microscope.The density of mitotic cells(DMC) and apoptotic bodies(DAB) of epithelium in the developing cardia,pylorus,greater curvature,lesser curvature were measured with stereological method.Results The morphological development of stomach took place on the 11~(th) day of mouse embryo development.The fundus,greater curvature and lesser curvature were observed on the 12~(th) day.The peak value of DMC all appeared before the peak value of DAB.In the epithelium of cardia,no obvious peak value of DMC was observed and the peak value of DAB was on the 11.5~(th),12.5~(th),13.5~(th) day.In the epithelium of pylorus,the peak value of DMC was on the 12.5~(th) day,the peak value of DAB was on the 14~(th) day.In the epithelium of greater curvature,the peak value of DMC was on the 11.5~(th) and 12~(th) day,the DAB value was low.In the epithelium of lesser curvature,the peak value of DMC was on the 11.5~(th) day,the peak value of DAB was on the 12.5~(th) day.Conclusion There was very close spatiotemporal relationship between the cell proliferation,cell apoptosis and the morphological development of stomach.

Objective To explore the risk factors of dyskinesias in Parkinson' s disease (PD) patients.Methods Patients with PD who had taken levodopa at least 1 month were recruited according to the United Kingdom Brain Bank (UKBB) Criteria..All patients were divided into dyskinesias group and non-dyskinesias group according to the clinical definition of dyskinesia.The parameters including gender,age,age onset,duration,body weight,UPDRS scale score,and treatment parameter,such as the maximum daily dose of levodopa,the cumulative time of medication of levodopa,levodopa dose per weight of patients with dyskinesias were recorded.Multivariate logistic regression analysis was used to analyze risk factors of dyskinesias.The patients were divided into dyskinesias and no dyskinesias groups based on presence of motor complications.Results The incidence rate of dyskinesias was 7.8％ (11/142) in all 142 patients.Of which,9 cases were with peak-dose dyskinesia,and 2 cases with both wearing-off and dyskinesia.There were statistically significant difference between dyskinesias group and non-dyskinesias group in terms of sex,weight,the maximum daily dose of levodopa and levodopa dose per weight(P ＜ 0.05).The same results appeared between wearing-off group and wearing-off with dyskinesia group (P ＜ 0.05).Multivariate Logistic regression analysis showed that total dose of levodopa(OR =1.846,95％ CI:1.234-2.762,P =0.003) and levodopa dose per weight were independentriskfactors(OR=0.991,95％CI:0.984-0.999,P=0.033).Conclusion The risk factors of dyskinesias in Parkinson's disease is closely linked to total dose of levodopa and levodopa dose per weight.

Background Posterior capsular opacification (PCO) is a primary complication after extracapsular cataract extraction.The mechanism of PCO is associated with proliferation,migration and epithelialmesenchymal transition (EMT) of human lens epithelial cells (LECs).To explore the target treatment of PCO is very important.Objective This study was to investigate the biological effects of second mitochondria-derived activator of caspases (Smac) on the proliferation and apoptosis of LECs.Methods Human LECs line (HLE-B3) and Smac-overexpressed LECs line were cultured,and the cells were transfected using small interfering RNA (siRNA)-Smac3 plasmid with green fluorescent protein (GFP) for 24 hours.Different concentration of transforming growth factor-β2 (TGF-β2) (5,10,20 and 50 μg/ml) or 200 μmol/L H2O2 were added respectively into the culture medium to establish PCO model and oxidative stress model.Cell counting kit-8 (CCK-8) assay was used to compare the cell proliferative activity among PBS group,TGF-β2 group and Smac-hyperexpression +TGF-β2 group.Flow cytometry was used to evaluate the apoptotic rate of the PBS group,H2 O2 group and siRNA-Smac+H2 O2 group.The expressions of Smac,caspase-3 and proliferating cell nuclear antigen (PCNA) mRNA and their proteins in the cells were detected by real-time quantitative PCR (RT-PCR) and Western blot.Results The GFP+ cells were≥ 80％ 12 hours after siRNA-Smac3 transfection,with the optimal plasmid of siRNA-Smac3.GFP+ cell rate was (72.32 ± 2.31)％ in the siRNA-Smac3 transfection group,which was significantly higher than that in the blank plasmid group ([4.91 ±0.24] ％) (t=116.342,P＜0.001).The relevant expression levels of Smac was 35.21 ±4.11 in the Smachyperexpression group,and that in the blank plasmid group was 15.24±2.48,with a significant difference between them (t =215.47,P＜0.05).The cell viability of 20 ng/ml TGF-β2 affected PBS group,TGF-β2 group and Smachyperepression+TGF-β2 group was (98.4 ± 1.7) ％,(98.9 ± 0.1) ％ and (64.2 ± 3.1) ％,and the cell viability of Smac-hyperepression+TGF-β2 group was significantly lower in the Smac-hyperepression+TGF-β2 group than that in the TGF-β2 group (P＜0.05).The apoptotic rate in the PBS group,H2 O2 group and siRNA-Smac+H2 O2 group were (2.9 ± 1.2) ％,(45.1 ±4.5) ％ and (27.5 ± 1.8) ％,and the apoptotic rate was evidently lower in the siRNA-Smac +H2O2 group than that in the H2O2 group (P＜0.05).RT-PCR results showed that the expression levels of caspase-3 mRNA in PBS group,H2 O2 group and siRNA-Smac + H2 O2 group were 0.321 ± 0.103,0.715 ± 0.112 and 0.479 ±0.209,respectively.Compared with the H2 O2 group,the relative expression level of caspase-3 mRNA in siRNA-Smac+ H2O2 group was significantly decreased,the difference was statistically significant (P＜ 0.05).The PCNA mRNA expression levels in PBS group,TGF-β2 group and Smac-hyperepression+TGF-β2 group were 0.299±0.013,0.645± 0.102 and 0.490±0.209,respectively.Western blot results showed that the relative expression of caspase-3 protein in siRNA-Smac+H2O2 group and H2O2 group was 0.712±0.012 and 0.973±0.051,with significant difference between the two groups (t =132.52,P＜0.05).The relative expression of PCNA protein in Smac-hyperepression+TGF-β2 group was 0.782±0.212,which was lower than 1.126±0.251 in the TGF-β2 group (P＜0.05).Conclusions Smac may prevent and treat PCO by inhibiting the proliferation and promoting apoptosis of human LECs.

Objective To evaluate the clinical safety and efficacy of focused ultrasound body contouring apparatus in abdominal fat reduction and body contouring.Methods Forty healthy subjeets were equally randomized into treatment group and control group,and received two treatments with the JCS-01 device (3H Medical Technology Co,Ltd,Beijing,China) in the abdominal region at one-week interval.During each treatment,the subject was placed on the treatment bed and the region of interest was spread with an acoustic coupling medium.Then the ultrasonic transducer was fixed,and the operator controlled the computerized system to move the bed in a designed regon.The control group would not receive any energy therapy during treatment time.Subjects were followed for 28 days after the last treatment (day 35).Abdominal circumference,regional photos and safety parameters were recorded at the time instantly before and after treatment and days 14 and 35.Subject satisfaction survey was conducted at day 35.Results One subject in control group was loss of follow-up.No local skin reactions,such as erythema edema or papules,and no changes with clinical significance in laboratory examinations occurred in all other 39 subjects.The proportion of subjects with over 0.5 cm abdominal circumference reduction in the treatment group was significantly higher than the control group at all time points (P＜0.05).Peak abdominal circumference reduction was on day 35.The abdominal circumferences of 19/20 subjects in the treatment group were reduced by 0.83-6.33 cm,and the mean abdominal circumference was reduced by 3.09 cm on day 35.The total effective rate was 95 ％,and the subject satisfactory rate was 75 ％.Conclusions The focused ultrasound treatments for body contouring is safe and tolerable and also significantly reduces abdominal circumference.

AIM:Studies on totipotential stem cells using primordial germ cells (PGCs) have the same application prospect as embryonic stem cells, especially for the animals difficult to isolate embryonic stem cells from blastula. In vitro culture condition of embryo germ cells is the key to control this. This study designs a method to effectively amplify PGCs of mice in vitro and establishes an effective culture method of PGCs. METHODS: Experiments were carried out in the Department of Histology and Embryology of Chongqing Medical University and Key Laboratory of Biochemistry and Molecular Pharmacology from October 2006 to August 2007. ①Clean Kunming pregnant mice (embryos 0.5 dpc) were provided by Animal Experimental Center of Chongqing Medical University. Experimental procedures met the animal ethical standards. ②Allantois and surrounding tissues of 8.5 day post coitum (dpc) embryos, the hindhut and surrounding tissues of 9.5 dpc and 10.5 dpc embryos, the gonadal ridges and surrounding tissues of 11.5 dpc and 12.5 dpc embryos were collected and digested with 0.25%pancreatin-0.02%EDTA, then the cells were cultured in the plastic petridishes which are pretreated with 0.1% gelatine. The formation ratio of primary and the first passaged Embryonic germ (EG) clones were compared among those different time points. The SSEA-1 positive ratio of isolated cells was compared between the two days by immunohistochemical method. The assays of clone numbers counting and MTT were used to compare the different proliferation ability of PGCs from the 11.5 dpc and 12.5 dpc embryos. The effects of expanding PGCs from 11.5 dpc and 12.5 dpc embryos by those two ways above were compared. The EG clones were analyzed by the expression of alkaline phosphatase and the immunologic marker SSEA-1,the undifferentiated marker Nanog and the differentiation ability in vitro were also tested. RESULTS:①The formation ratio of primary and the first passaged EG clone from 11.5 and 12.5 dpc embryos was higher than that from 8.5 to 10.5 dpc embryos and efficiency of expansion was increased (P 0.05). On the third day, the number of the primary EG clones from 11.5 dpc embryos was higher than that from 12.5 dpc embryos (P 0.05), but the two groups had different characteristics. ④The EG clones have representative morphology with high level of alkaline phosphatase activity and SSEA-1, Nanog expression, which can differentiate into the cystic embryoid body. CONCLUSION: 11.5 dpc and 12.5 dpc mouse embryos, especially 11.5 dpc embryos are the optional time points to expand PGCs efficiently. Co-culturing with the tissue and isolating the gonadal ridges both of these two ways are practicable.

Objective To find out whether NOTCH receptors can serve as direct downstream targets of transforming growth factor β(TGF-β)/Smad4 signaling in endothelial cells.Methods Real-time PCR and Western blotting were performed to verify whether the expression of notch1 and notch4 was regulated by TGF-β pathway.Luciferase reporter assay was employed to investigate how the promoter of notch1 and notch4 was regulated by TGF-β.Then, ChIP assay was used confirm whether the promoter of notch1 and notch4 physically interacted with SMAD protein.Results TGF-β1 and bone morphogenetic protein 4 (BMP4) treatment increased the expression of both notch1 and notch4 at the transcriptional level.In addition, SMAD4 was physically associated with the SMAD binding sites on the notch4 promoter, which was largely enhanced under the treatment of TGF-β1 and BMP4.Importantly, TGF-β1 and BMP4 failed to transactivate notch4 in the absence of endogenous SMAD4 or the SMAD binding regions on the notch4 promoter.Conclusion The expression of NOTCH receptor can be directly up-regulated by SMAD4-mediated TGF-β/BMP signaling in cerebrovascular endothelial cells.

Objective To investigate the isolation, purification and culture methods of human embryonic germ cells (EGCs) on feeder layer cells of human embryonic fibroblasts. Methods Primordial germ cells(PGCs) from the genital ridges of 6-11 weeks(post fertilization) human embryos were cultrued on feeder layer cells of human embryonic fibroblasts(HEFs) which were isolated from the 3-4 month embryos and routinely cultured for over 25 passages. The medium composed of growth factors and differentiation inhibitory factors. The cultures were analyzed with the expression of alkaline phosphatase, immunologic markers (SSEA-1,SSEA-4) and the transcription factor Oct-4 that have been used to routinely to characterize EGCS. Results A large-scale EG cells were obtained and meintained on feeder layers for over 8 passages. The cell surface marker showed that the EGCs possess high levels of AP activity. EGCs colonies stained strongly for SSEA-4,SSEA-1 and they expressed the transcription factor Oct-4.Conclusion EGCs have potential to maintain long term proliferation and undifferentiation state on human embryonic fibroblasts in vitro.;

Objective To study the temporal and spatial expression of Nanog in developing primordial germ cells(PGCs) of mouse.Methods Immunofluorescence technique was taken to qualitatively analyze the expression of Nanog in PGCs of 7.5days(post-coitus days)-15.5days mouse embryo.A further quantitative analysis of Nanog expression change in PGCs of 11.5days-15.5days mouse embryo was done by using SSEA-1 and Nanog double labelling immunofluorescence staining with confocal microscopy.Results The PGCs in mouse allantois,hindgut,dorsal mesentery and genital ridge were Nanog positive.Both of the highest positive ratio and the highest fluorescence intensity appeared in PGCs of 11.5days mouse embryo.For the female mice,Nanog expression drops dramatically after 12.5days,and for the male mice,a noticeable decline of Nanog expression was occurred after 13.5 postcoitus days.Conclusion\ Nanog expresses stably in the proliferating PGCs.The down-regulation of its expression may be related with the differentiation of PGCs.