To establish a better method of primary culture for alveolar epithelial type II cells (AEC II) and to study its bionomics, alveolar epithelial type II cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin G. The purified AEC II were identified by alkaline phosphatase staining, electron microscopy, immunocytochemical staining of pulmonary surfactant protein A (SPA). The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC II could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 x 10(7), and a purity of about 75%-84%. Cells could be quickly identified with AKP staining. AEC II were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC II, and AKP staining is simple in cell identification. AEC II can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.
Cell Culture Techniques/*methods ; Cell Separation/*methods ; Cells, Cultured ; Ecology ; Epithelial Cells/*cytology ; Immunoglobulin G/pharmacology ; Pulmonary Alveoli/*cytology ; Pulmonary Surfactant-Associated Protein A/biosynthesis ; Rats, Wistar

Voriconazole is a broad spectrum triazole antifungal agent, widely used in the prevention and treatment of invasive fungal diseases.Long-term use of voriconazole can induce periostitis and cutaneous squamous cell carcinoma, accounting for serious adverse effects on patients, which has been reported in increasing clinical cases.This review is to characterize the epidemiological and clinical manifestations of periostitis and squamous cell carcinoma induced by voriconazole, and to analyze the mechanisms of triggering periostitis and squamous cell carcinoma, so as to promote the rational use of voriconazole in clinics.

OBJECTIVE:To observe clinical efficacy and safety of salmeterol fluticasone combined with tiotropium bromide in the treatment of COPD via different inhalation devices.METHODS:Eighty COPD patients were selected from our hospital during Jan.2014 to Jan.2015,and then divided into trial group and control group according to random number table,with 40 cases in each group.Both groups were given Salmeterol fluticasone inhalant 500 μg,bid+Tiotropium bromide inhalant 18 μg,qd.Control group was given medicine via inhalation device coming with medicine,while trial group was given medicine via gas compression type ultrasonic spray inhalator.Both groups were treated for 1 year.Blood concentration of medicine 0.5 h after medication,mMRC score and COPD acsessment test (CAT) score 3,6,9 months after treatment,the times of acute exacerbation during treatment,FEV1％ before and af ter treatment were all observed in 2 groups.The occurrence of ADR was recorded.RESULTS:Four cases withdrew from trial group and 1 case from control group.After medication,there was no statistical significance in blood concentration of fluticasone,salmeterol and tiotropium bromide between 2 groups (P＞0.05).0.5 h after medication,mMRC score of trial group was slightly lower than that of control group,without statistical significance (P＞0.05);CAT score of it was significantly lower than that of control group,with statistical significance (P＜0.05).The times of acute exacerbation in trial group during treatment was significantly less than control group,with statistical significance (P＜0.05).The decrease of FEV1％ in trial group was slightly lower than control group,without statistical significance (P＞0.05).The incidence of ADR in trial group was significantly lower than control group,with statistical significance (P＜0.05).CONCLUSIONS:For COPD patients,salmeterol fluticasone combined with tiotropium bromide via gas compression type ultrasonic spray inhalator is better than inhalation device coming with medicine in clinical efficacy and safety.

This paper explored the multi-angle, comprehensive and systematic clinical prac-tice teaching system for international students. Six teaching methods were integrated: bedside teach-ing, standardized patients teaching, electronic patients clinic teaching, Chinese students one-to-one help foreign students, a variety of English clinic small presentation and many after-school social activi-ties. The multi-angle, comprehensive and systematic clinical practice teaching system was evidently a better choice than traditional teaching system for international students.

Objective To explore the characteristics of blood oxygen level dependent (BOLD) magnetic resonance imaging (MRI) in healthy native kidneys. To investigate the relationship between BOLD-MRI and common physiological in-dexes. Methods GE 3.0T MRI scanner and Torsopa phased-array coil were employed to acquire renal coronal T1WI and BOLD image. Ninety patients who were ruled out chronic kidney diseases underwent BOLD-MRI with T2*-spoiled gradient recalled echo (T2*SPGR) sequence. BOLD images were analyzed on R2*map software. Cortical and medullary R2*values were analyzed in bilateral kidneys and in different gender. Different regional R2*values in cortex and medulla were also ana-lyzed. Physiological indices including age, body height and weight, body mass index (BMI), body surface area (BSA), estimat-ed glomerular filtration rate (eGFR) were recorded. Correlation between R2*value and physiological indices were analyzed. Results Bilateral renal cortical R2*values [left (16.56±1.40) Hz and right (16.66±1.28)Hz, respectively] were less than val-ues in medulla [left (28.82±3.71)Hz and right (28.36±3.72)Hz, respectively]. Female and male cortical R2*values [female (16.55±1.30Hz) and male (16.66±1.38)Hz, respectively] were also less than corresponding values in medulla [female (28.46± 3.64) Hz and male (28.70±3.78) Hz, respectively]. In bilateral renal medullary region, R2*values in low pole (27.29±3.05) Hz was less than values in middle (29.32±3.47) Hz and upper pole (29.16±4.21)Hz (F=15.184, P<0.001). Age was positive-ly correlated with R2* values in medulla (r =0.284, P =0.002). However, eGFR was negatively correlated with medullary R2*value (r=-0.232, P=0.007). Conclusion R2*values reflected the levels of renal partial pressure of oxygen and as-sessed the degree of renal ischemia. BOLD MRI could offer a simple, convenient and noninvasive method for to evaluate renal oxygen metabolism in cortex and medulla.

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

AIM: To design,prepare and screen out functional small interfering RNA(siRNA) for specifically silencing proliferating cell nuclear antigen(PCNA) gene expression and effectively inhibiting cell proliferation on human hepatoma cell line SMMC-7721,human gastric carcinoma cell line SGC-7901 and human colorectal carcinoma cell line Caco2.METHODS: PCNA siRNA was designed based on previous studies about design guidelines and synthesized in vitro by T7 Mega short script reaction kit according to the manufacturer's instructions.After purification,the integrities of siRNA were identified through 19% denaturing polyacrylamide gel electrophoresis,and the concentrations of the generated siRNA were measured by testing the absorbance at 260 nm using a spectrophotometer.Four synthesized double-strand siRNA were transfected into three types of carcinoma cell lines by LipofectamineTM2000 reagent,respectively.WST-8 assay was employed to examine the proliferative inhibitions of these three cell lines.The subsequent alterations on PCNA mRNA and protein levels were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) and immunocytochemistry,respectively.RESULTS: 3 sequences,No.2,No.3 and No.4 PCNA siRNA showed effective inhibitions on tumor cells among the 4 candidate siRNA,and a single dose of 50 nmol/L of No.2,No.4 PCNA siRNA showed the most effective inhibitory rates as more than 62% at 48 h after transfection.50 nmol/L of No.2 and No.4 PCNA siRNA transfection caused 72% decrease of PCNA mRNA level and almost completely loss of the protein in human Caco2 cells.CONCLUSION: No.2 and No.4 PCNA siRNA have been screened out in this study,which show the capability to effectively down-regulated PCNA expression and significantly inhibit the proliferation of carcinoma cells.The optimal concentration is 50 nmol/L and satisfactory effects are achieved 48 h after transfection in vitro.

AIM: To investigate p16 gene alterations in gastric carcinoma. METHODS: 36 fresh tumor specimens taken from gastric cancer patients were analyzed for p16 gene deletion and mutation by polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Homozygous deletion of exon 1 and exon 3 was observed in 2 cases and 3 cases of diffuse carcinoma, respectively. The frequency of homozygous deletion was 13.89%. No p16 gene point mutation was detected. CONCLUSION: The deletion of p16 genes may be related to gastric carcinogenesis.

Objective To profile the omp1 genotypes of Chlamydia trachomatis (Ct) in patients with nongonococcal urethritis (cervicitis) in Guangzhou region. Methods Swab samples were obtained from the urethra of males and cervix of females in clinical settings of venereology and gynecology as well as at outreach sites for the prevention and control of sexually transmitted diseases (STDs). DNA was extracted from the swabs and nested PCR was performed to amplify the variable domain (VD) 1 - 3 of omp1 gene of Ct followed by gene sequencing. The genotypes of Ct were determined based on the amino acid mutation in VD 1 - 2 of omp1 gene. Results Totally, 1208 swabs were collected. Of them, 132 were Ct positive, and 130 positive samples underwent genotyping. Ten ompl genotypes were determined in total, including serotype E (38, 29.23%), D (25, 19.23%), J(24, 18.46%), F(21, 16.15%), G(7, 5.38%), H(5, 3.85%), K(5, 3.85%), B(2, 1.54%), Ja (2, 1.54%), I (1, 0.77%). E, D, J and F were the dominant type of Ct in this region, and amounted to 83% of all the Ct isolates. Mutations were observed within VD 1 and 2 of omp1 gene in serotype D, B and K.Serotypes were undetermined for Ct in 2 patients with mixed infection. Conclusions In Guangzhou region, E,D, F and J are the predominant genotypes of Ct, and amount to 83% of all the Ct isolates. Ct serotype B is also observed in the urethra of males and cervix of females in this region.

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.