Objective To study the effects of the Notch ligands Dlk1 and recombinant human nucleu factorκB (Jagged1) on the proliferation and transdifferentiation of the type Ⅱ alveolar epithelial cells when the Notch signaling pathway activated.Methods The primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) cultured with recombinant protein Dlk1 and recombinant human nucleu factor-κB (rhNF-κB) (activator of Jagged1),respectively,and then cultured with DMEM (containing 120 mL/L FBS) as controls.Proliferation and differentiation conditions of the AEC Ⅱ were observed at 48 h,72 h,96 h time point by the light microscope and electron microscopes separately.Cell number was counted with hemacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT) ; Immunofluorescence double standard method was used to detect the AEC Ⅱ specific surfactant protein C (SP-C) and AEC Ⅰ specific protein aquaporin5 (AQPS) ;the expression of SP-C,AQPS,Dlk1,Jagged1,Notch1 and Hes1 mRNA were detected by real time-PCR.Results The cell population and proliferation:compared with control group,AEC Ⅱ proliferation was promoted in the Dlk1 group [cell numbers (× 109/L) 9.05 ± 0.45 vs 7.95 ± 0.65,11.68 ± 0.43 vs 8.68 ± 0.52,11.55 ± 0.17 vs 8.73 ± 0.48,all P ＜ 0.05 ; MTT results (value A) 0.699 ± 0.050 vs 0.462 ± 0.080,0.912 ± 0.080 vs 0.535 ±0.040,0.726 ±0.050 vs 0.540 ±0.020,all P ＜0.05] and decelerated AEC Ⅱ transdifferentiation into AEC Ⅰ ; while AEC Ⅱ proliferation was inhibited in rhNF-κB group [cell numbers (× 109/L) 4.95 ± 0.33 vs 7.95 ± 0.65,4.73 ±0.71 vs 8.68 ± 0.52,4.04 ± 0.11 vs 8.73 ± 0.48,all P ＜ 0.05; MTT results (value A) 0.398 ± 0.030 vs 0.462 ± 0.080,0.402 ± 0.070 vs 0.535 ± 0.040,0.380 ± 0.110 vs 0.540 ± 0.020,all P ＜ 0.05] and accelerated AEC Ⅱ transdifferentiation into AEC Ⅰ.One-Way ANOVA showed that the difference among the 3 groups had statistical significance (cell numbers:F =486.73,P =0.02; cell proliferation:F =37.16,P =0.02).The mRNA expression:compared with control group,the expression of SP-C mRNA of Dlk1 group was significantly higher (P ＜ 0.05) while the expression of AQP5 mRNA was remarkably lower and delayed (P ＜ 0.05),the expression of Jagged1 mRNA was weak or little,Dlk1 and Notch1 mRNA were up-regulated (P ＜ 0.05),and the Hes1 mRNA was reduced (P ＜ 0.05) ; the expression of SP-C mRNA of rhNF-κB group was significantly reduced (P ＜ 0.05),while the AQP5 mRNA expressed ahead of time and increased (P ＜ 0.05),Jagged1,Hes1 and Notch1 mRNA were higher (P ＜ 0.05),and the Dlk1 mRNA was weak.One-Way ANOVA showed that the difference in the expressions of SP-C,AQP5,D1k1,Jagged1,Hes1 and Notch1 mRNA among the 3 groups had staistical significance (F =96.80,P =0.01 ; F =82.55,P =0.01 ; F =269.80,P=0.00;F =312.34,P =0.00;F =169.17,P =0.01;F =19.85,P =0.02).Conclusions There are varied effects on proliferation and differentiation of the AEC Ⅱ when the Notch signaling is activated by different ligands:Dlk1 promoted proliferation and inhibited differentiation,while Jagged1 inhibited proliferation and promoted transdifferentiation.

Objective To investigate the effect of all-trans retinoic acid (at-RA) on fetal alveolar epithelial type Ⅱ cells (fAEC Ⅱ s) proliferation and the expression of pulmonary surfactant C (SPC) as well as aquaporin 5 (AQP5).Methods fAEC Ⅱ s were isolated and purified from fetal lung of pregnant SD rats (19 days).After being cultured for 1 day,and the fAEC Ⅱ s were interfered by at-RA for 1,2 and 3 days.Cell proliferation,viability as well as growth state,expressions of SPC mRNA as well as AQP5 mRNA and expressions of protein SPC as well as AQP5 were respectively detected by using 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT),inverted microscope,real-time fluorescence quantitative PCR (RT-PCR) and Western blot.Results (1) When fAEC Ⅱ s were treated with at-RA for 1 day,and the cell proliferation and viability did not change (P ＞ 0.05),while the proliferation and viability were significantly improved in 2 days (P ＜ 0.05),and the difference was the most obvious (P ＜ 0.05) at 3 days.(2)Compared with the control group,the cell growth state was better,and the cell adherence was tighter and the refraction was higher in at-RA group.(3) Compared with the control group,at-RA up-regulated the expressions of AQP5 mRNA and AQP5 protein(t =-19.58,-10.44,-16.01,-46.25,-12.79,-27.96,all P ＜ 0.05),and the percentages of control group were 281.07％,766.67％,1 163.33％ and 792.65％,1 310.52％,1 561.56％ in 1,2 and 3 days respectively.(4) Compared with control group,the expressions of SP-C mRNA and SPC protein were up regulated when cells were exposed to at-RA for 1 and 3 d,but while they were down-regulated at 2 days(protein:the percentages of control group were 615.480％,369.450％ and 11.269％,respectively ; mRNA:728.33 ％,400.83 ％,66.57％,respectively)(t=-26.34,-25.26,13.80,-25.25,-31.71,9.12,all P＜0.05).Conclusions at-RA can promote the proliferation and differentiation of fAEC Ⅱs,enhance the fAEC Ⅱ s viability,and improve the expression of SPC and AQP5.

Background and purpose:The pro-oncogene Bmi-1 is a member of the polycomb-group family, can regulation of the proliferation and self-renewal of normal and tumor stem cells. In recent years, Bmi-1 has been found that it is overexpressed in varieties of human malignant tumors. The study aimed to observe the effects of Bmi-1-siRNA on the growth capacity of lung cancer cell line A549 in vivo and in vivo, and explore its mechanism. Methods:The most effective one as a target sequence was chosen from four Bmi-1 siRNA sequences which were designed by our lab, and one random sequence was chosen as a negative control. In short, the chemically synthesized siRNA and control sequences were connected to a retrovirus expressing vector, pSUPERretro-Neo plasmid, and then transfected into A549 cells. The stably transfected cells were cultured and passed. The level of mRNA and protein of Bmi-1 in A549 cells were assessed by RT-PCR and Western blot respectively. The proliferations of A549 cells in vivo was analyzed with MTT, trypan blue exclusion and plate colony forming methods. Flow cytometry was used for cell cycle analysis. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of A549 cells. The expressions of cyclin D, p21/27, p-AKT and PTEN were analyzed by Western blot. Results:Compared to A549-ctr and A549-wt cells, Bmi-1 mRNA and protein levels all signiifcantly reduced in A549-Bmi-1-siRNA cells. Bmi-1-siRNA inhibited the growth, colony formation in vitro and tumorigenesis in vitro of A549 cells, and the interference cells cell cycle arrested in G1 phase. In A549-Bmi-1-siRNA cells, p-AKT and cyclinD1 expression were down-regulated while p21/p27 and PTEN were up-regulated. Conclusion:Silencing Bmi-1 gene inhibits the proliferation of A549 cells through G1 phase arrest, which involves the downregulation of cyclin D/p-AKT and upregulation of p21/p27/PTEN.

Background and purpose:The human oncogene B-cell-speciifc moloney murine leukemia virus integration site 1 (Bmi-1) is an important member of the polycomb group family, and it regulates cell proliferation and senescence via INK4a/ARF locus. This study investigated the effects of Bmi-1-siRNA on the proliferation of lung adenocarcinoma cell line SPC-A1 cells with INK4a/ARF locus and clarify the mechanism of Bmi-1-mediated effect on proliferation of lung adenocarcinoma cells. Methods:In this study, we chose the most efifcient siRNA chain the pGeneshl-2-Bmi-1 sense-1 and inserted into a pSUPER-retro-neo retroviral vector. The packaged si-Bmi-1 pSUPERret-ro-neo retroviral vector was stably transfected into lung adenocarcinoma SPC-A1 cell line. The stably transfected cells were cultured and passed. After transfection, the levels of Bmi-1 mRNA and protein expression of SPC-A1 cells were analyzed by RT-PCR and Western blot respectively. Trypan blue, MTT and plate colony forming assay were performed to observe the proliferation capibility of SPC-A1 cells and evaluate the cloning forming ability in vitro. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of SPC-A1 cells. Cell cycle distribu-tion was analyzed by lfow cytometry (FCM) in SPC-A1 cells. The expression levels of proliferation proteins including p16INK4a, p53, Cyclin D1, PTEN, Akt and Ser473p-Akt were analyzed by Western blot. Results:The mRNA and protein expression levels of Bmi-1 were signiifcantly reduced in SPC-A1-Bmi-1-siRNA cells transfected with pSUPER-retro-neo retroviral vector. Knockdown of Bmi-1 could inhibit the growth, colony formation in vitro and tumorigenesis in vivo of SPC-A1 cells (P<0.01). The transfected SPC-A1 cells were arrested in G1 phase [(64.6±1.2)%, P<0.05]. Compared with two control groups, p16INK4a, p53 and Akt were not affected (P>0.05), while Cyclin D1 and Ser473p-Akt were downregulated (P<0.01) and PTEN was up-regulated (P<0.01) in the SPC-A1-Bmi-1-siRNA cells. SPC-A1-Bmi-1-siRNA cells were treated with various concentrations of PTEN inhibitor to determine expression levels of PTEN, Bmi-1 and Ser473p-Akt protein. Ablation of PTEN rescued Bmi-1 and Ser473p-Akt expression in SPC-A1-Bmi-1-siRNA cells. Conclusion:Knockdown of Bmi-1 gene can arrest the proliferation of SPC-A1 cells through G0/G1 phase arrest by inhibiting Cyclin D1 expression indirectly, which may be not associated with p16INK4a signaling pathway.

Objective: To investigate the clinical value of quantitative detection of DNA aneuploidy in the diagnosis and treatment of cervical lesions in middle-aged and senile women. Methods: A total of 1 404 middle-aged and elderly women who underwent screening for early cervical lesions were retrospectively studied.Patients were divided into the two groups: the 40-49 years old group(n=897)and the 50-78 years old group(n=507). Cervical lesions were screened by DNA ploidy analysis and the results were compared with those screened by liquid-based cytology, colposcopy and high-risk human papillomavirus(HPV). Results: The positive detection rate of HPV by DNA ploidy analysis was 54.4%(764/1 404). Of 1 404 patients, HPV16/18 infection accounted for 21.3%(299/1 404). The detection rate of heteroploid cells was 50.92%(715/1 404). There was a significant positive correlation between HPV infection type and cervical epithelial cell ploidy changes(r=870, P=0.001). The detection rate of HPV by liquid-based cytology was 45.08%, which was lower than that by DNA aneuploidy(χ²=9.594, P=0.002). The differences in the incidences of low-grade squamous intraepithelial lesion(LSIL)and high-grade squamous intraepithelial lesion(HSIL)and above categories of lesions were statistically significant(χ²=289.598, P=0.000)between patients with and without DNA aneuploidy.The statistically significant differences were found between the 40-49 years old group and 50-78 years old group(P<0.05)in the occurrence of abnormal DNA ploidy cells, HPV infection rate, the proportion of LSIL, HSIL and above categories of lesions. Conclusions Compared with the conventional cytology, DNA aneuploidy quantitative detection has higher sensitivity and better specificity, and has no significant difference from the high-risk HPV detection.It can be used as one of methods for screening cervical lesions in middle-aged and elderly women, especially those with high-risk HPV infection.

Objective To study the relationship between cleanliness of children′s hands and diminution of Ascaris lumbricoides infection. Methods Before the study all persons positive for ascaris eggs in the preliminary survey were treated with albendazole. Hand washing habit before meal and after defecation was kept in children of experimental group, but not in the control group. Kato thick smear stool examination was done once every two months for one year to compare the new infection rates in children without ascaris infection in the two groups, and the reinfection rates in the cured negative cases were also compared between them in half a month after chemotherapy. Results All the new infection rates as well as reinfection rates of each reexamination in the experimental group were significantly lower than that of the control group ( P

Objective To explore the role of visual oculomotor-vestibular eye balance function in the diagnosis of central and peripheral vertigo. Methods From January 2015 to June 2016,one hundred and sixty-two patients with central vertigo who were treated at Kailuan General Hospital were enrolled in the study, including 124 males and 38 females, aged ( 64. 09 ± 10. 98 ) years old; there were 166 cases of peripheral vertigo,75 males and 91 females,aged (52. 13±12. 20) years old. Spontaneous nystagmus test,gaze test,position test, saccade test, smooth visual tracking test, visual single-speed test, visual sinus test, swivel chair rotation- emergency stop test using infrared video nystagmus and static balance posture instrument,open-closed eye hard plate erect test, open-closed eye sponge soft bottom erect test balance function electrophysiological test were conducted. Results The detection rate of pathological spontaneous nystagmus and pathological gaze nystagmus was higher in the central vertigo group than that in the peripheral vertigo group (χ2=5. 674,16. 458,P<0. 05) . The occurrence rate of positional nystagmus was higher in peripheral vertigo group than that in central vertigo group (χ2=48. 896,P<0. 001). The abnormal rate of scanning test,stable visual tracking test,visual movement single speed and sinusoidal test,and static balance posture test were higher in the central vertigo group than those in the peripheral vertigo group (χ2 =137. 169, 166. 972, 150. 877, 150. 877, 27. 273, P<0. 001 ) , while the abnormal rate of rotating chair sudden stop test was higher in the central vertigo group than that in the peripheral vertigo group (χ2=51. 000,P<0. 001) . The abnormal results were mainly scanned underflush and slow scan in central vertigo group (χ2=103. 846,4. 296,P<0. 05),stable visual tracking curve (χ2=147. 389,4. 296,P<0. 05) in type III-IV,and the gain of nystagmus decreased unilaterally and bilaterally (χ2=47. 531,44. 477, 52. 529,53. 255,P<0. 001) . Anomalies of proprioception in reverse and vertical nystagmus and static balance posture were induced by rotating chair sudden stop test (χ2=11. 847, 23. 778, P<0. 001 ) , while peripheral vertigo group showed unilateral decrease of nystagmus gain induced by rotating chair sudden stop test. (χ2=79. 771, P < 0. 001 ) . Conclusion The patients with peripheral vertigo have obvious body position spontaneous vestibular response and vestibular oculomotor system dysfunction, while the patients with central vertigo mainly have visual and oculomotor system dysfunction,and may be accompanied by vestibular oculomotor system and vestibular spinal reflex dysfunction.

Objective: To evaluate the safety and effectiveness of repeated transcranial magnetic stimulation (rTMS) for treating non-fluent aphasia after stroke. Methods: Forty-five stroke survivors with non-fluent aphasia were randomly divided into a 0.5 Hz group, a 1 Hz group and a sham group, each of 15. In addition to routine linguistic training, the three groups were given rTMS over the inferior frontal gyrus of the right hemisphere at the corresponding frequency or sham stimulation. Before as well as 5 and 10 days after the treatment, all of the subjects were evaluated using the Chinese version of the Western Aphasia Battery (WAB). The occurrence of adverse events was also observed. Results: Before treatment, no significant differences were observed in the groups′ average aphasia ratio, spontaneous speech, listening comprehension, retelling and naming using the WAB. After 5 and 10 days significant increases were observed in the average WAB scores of all three groups, but the listening comprehension of the 0.5 Hz group was significantly better than that of the sham group 10 days later, as was the spontaneous speech of the 1 Hz group. Conclusion rTMS at either 1 Hz or 0.5 Hz can improve the linguistic functioning of stroke survivors with non-fluent aphasia. Both 0.5 Hz and 1 Hz rTMS are safe, but the latter is more effective.