Objective:To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor (CTGF) by RNA interference and verified its function. Methods:A specific sequence, which was verified to be able to silence CTGF gene with high efficiency, was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF. The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF. After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation. The recombinant adenovirus was packaged. The titer of the Ad-siCTGF was increased after three times of cross-infection. 4T1 cells were infected with the adenovirus. The silencing efficiency was tested by real-time fluorescence quantitative (RFQ)-PCR and Western blotting.Results:Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed. The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10~(10) pfu/mL after amplification and purification. The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%, respectively, compared with the control groups.Conclusion:The recombinant adenovirus which can silence the expression of CTGF was successfully constructed. It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.

Abstract: The loop-mediated isothermal amplification (LAMP) technique is a technique for the specific and efficient amplification of target fragments at a constant temperature using two pairs of specially designed primers and a strand displacement activity DNA polymerase. LAMP technique is a simple, rapid, specific, sensitive and cost-effective nucleic acid amplification method, and therefore has a promising future in the field rapid detection of Mycobacterium tuberculosis and grassroots applications. In this review, the basic principles and characteristics of the LAMP technique, the main molecular markers for the diagnosis of tuberculosis, and the use of different molecular markers and various types of novel techniques in the diagnosis of pulmonary tuberculosis, extrapulmonary tuberculosis, and drug-resistant tuberculosis were described. The LAMP technique has been widely used in the diagnosis of tuberculosis with high sensitivity and specificity, but the technique still has some shortcomings. This paper reviews the progress of its application in tuberculosis in recent years and provides an outlook on its development, with a view to providing a rational research direction for rapid diagnosis of tuberculosis in a resource-limited environment.

Huanglian is a commonly used Chinese medicinal herb in the ancient and the present. It has a history of 2000 years in clinical application, having the efficacy of "Clear away heat and remove dampness, purge the sthenic fire and eliminate toxic materials", therefore can be used for various diseases or syndromes in types of dampness-heat and fire-toxin by internal or external use. Compound prescriptions mainly based on Huanglian or prescribed by Huanglian, such as Puji Xiaodu Yin, Huanglian Jiedu Tang, Zhusha Anshen Wan, Qingying Tang, Angong Niuhuang Wan, Niuhuang Qingxin Wan, Jiaotai Wan, Huanglian Ejiao Tang, Zuojin Wan, Danggui Longhui Wan, Huanglian Yanggan Wan, Wu Xiexin Tang, Lianpu Yin, Gegen Huangqin Huanglian Tang, Baitouweng Tang, Xianglian Wan etc. All of these are well-known formulas for clearing away toxin of heat-fire of heart and liver, as well as dampness-heat of stomach and intestines. Nowadays, Huanglian is generally considered as a kind of antibiotic and antivirus herb and is widely used for many infective diseases. In fact, it is also used to cure cardiovascular and cerebrovascular diseases, diabetes and cancer based on pharmacological studies. Having been using Huanglian in treating the above diseases and having conducted clinical and experimental research on cancer and liver diseases, the author observed that Huanglian and its compound prescriptions have obvious effects on liver diseases such as acute or chronic hepatitis, liver fibrosis, liver cirrhosis and liver cancer due to types of dampness-heat and fire-toxin. Part of the effects has been proved by experimental research and it is worth carrying out more research in this area for development and clinical application.
Adult ; China ; Digestive System Diseases ; drug therapy ; Drug Prescriptions ; history ; Drugs, Chinese Herbal ; therapeutic use ; Female ; History, 20th Century ; History, 21st Century ; History, Ancient ; Humans ; Liver Diseases ; drug therapy ; Male ; Medicine, Chinese Traditional ; history ; Middle Aged

BACKGROUNDAlthough video-assisted radical operation for lung cancer has been widely accepted for treatment of nonsmall cell lung cancer (NSCLC), the debate over video-assisted thoracic surgery (VATS) segmentectomy still remains. This study analyzed the clinical outcomes using VATS segmentectomy for stage I NSCLC patients to explore the safety and efficacy of VATS segmentectomy for Ia NSCLC.

METHODSRetrospective review was conducted of patients who underwent VATS segmentectomy for clinical stage I NSCLC at Shanghai Chest Hospital between November 2009 and May 2012. VATS segmentectomy was performed on 36 patients. Analyses of the patient group were performed on patient demographics and clinical characteristics, intraoperative parameters, complications, and postoperative survival.

RESULTSThirty-five of thirty-six patients underwent VATS segmentectomy with only one conversion to open thoracic surgery. There was one peri-operative mortality from the segmentectomy group and all other patients are alive with a median follow up of 327 days. The mean volume of chest tube drainage after operation for segmentectomy was 1021.4 ml. Among other parameters, the mean blood loss was 162.5 ml (50.0 - 1600.0 ml), the mean operation time 124.8 minutes (75.0 - 271.0 minutes), chest tube duration 4.1 days (2 - 8 days), and the mean length of hospital stay 6.2 days (4 - 11 days). There was one (2.8%) locoregional recurrence after segmentectomy. Two patients successfully underwent bilateral segmentectomies and are still disease free.

CONCLUSIONFor patients with stage I NSCLC, VATS segmentectomy offers a safe and equally effective option and can be applied to complicated operations such as bilateral segmentectomy.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; mortality ; pathology ; surgery ; Female ; Humans ; Lung Neoplasms ; mortality ; pathology ; surgery ; Male ; Middle Aged ; Neoplasm Staging ; Pneumonectomy ; methods ; Retrospective Studies ; Thoracic Surgery, Video-Assisted ; methods

BackgroundThe immunotherapy for retinoblastoma(RB) is gradually concerned recent year.To seek relative immune-associated antigen is a basis of immunotherapy.NY-ESO-1 and NY-SAR-35 are two kinds of genes of cancer testis antigen( CTA ).To understand their expressions in RB tissue can offer index for relative study.ObjectiveThis study was to investigate the expressions of two CTA NY-ESO-1 and NY-SAR-35 in RB and explore the possibility of them as potentially promising targets for antigen-specific immunotherapy of RB.Methods The samples were obtained from 15 RB eyes,12 non-tumor retinopathy eyes and 22 normal eyes with other benign eye diseases.Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expressions of NY-ESO-1 mRNA and NY-SAR-35 mRNA in the samples.Genes of positive PCR results were sequenced randomly.The relevance of the expression of the two cancer-testis antigen genes with the clinical characteristics such as tumor stage,tumor size and clinical stage were analyzed.This study was approved by Ethic Committee of Guangxi Medical University.Written informed consent was obtained from each patient before operation. Results NY-ESO-1 mRNA was positively expressed in 6 RB samples and NY-SAR-35 mRNA was expressed in 9 RB samples.In the non-tumor retinopathy samples and normal eye tissues,NY-ESO-1 mRNA and NY-SAR-35 mRNA were absent.No significant relevances were found between the expressions of the NY-ESO-1 mRNA and NY-SAR-35 mRNA with clinical characteristics such as age ( P =0.426,0.822 ),gender ( P =0.180,0.464 ),pathological classification ( P =0.744,0.582 ),tumor size ( P =0.760,0.790),and clinical stage ( P =0.868,0.707 ).Conclusions NY-ESO-1 and NY-SAR-35 have high expressing frequencies in RB tissue and their expressions in RB have specificity.These results offer a clue for the identification of targets antigen of RB.

OBJECTIVETo analyze the embryo development potential after intracytoplasmic injection of sperm from azoospermia patients with different spermatogenic functions.

METHODSWe performed ICSI with sperm retrieved from azoospermia patients with different spermatogenic functions using percutaneous epididymal sperm aspiration (PESA) and testicular sperm aspiration (TESA). Then we recorded and analyzed the rates of normal fertilization, cleavages, excellent embryos and pregnancies.

RESULTSNo statistically significant differences were found between the PESA and TESA groups in the rates of normal fertilization ([74.9 +/- 19.6] vs [66.3 +/- 22.7]%, P > 0.05), cleavages ([96.7 +/- 8.6] vs [92.8 +/- 19.8]%, P > 0.05), excellent embryos ([43.5 +/- 26.2] vs [35.0 +/- 29.4]%, P > 0.05) and pregnancies (44.0 vs 52.0%, P > 0.05). The normal fertilization rates in the patients with normal spermatogenesis, mild spermatogenic dysfunction (SD), moderate SD and severe SD were (77.8 +/- 18.4), (68.4 +/- 18.5), (73.5 +/- 19.8) and (51.4 +/- 27.9)%, respectively, with significant difference between the normal spermatogenesis and mild SD groups (P < 0.05) as well as between the severe SD and the other groups (P < 0.05); the cleavage rates were (96.7 +/- 9.2), (96.5 +/- 15.0), (93.9 +/- 12.1) and (93.7 +/- 11.1)%, respectively, with no significant difference among the four groups; the excellent embryo rates were (47.1 +/- 25.8), (40.3 +/- 27.6), (36.2 +/- 23.1) and (15.0 +/- 24.6)%, respectively, with significant difference between the severe SD and the other groups; the pregnancy rates were 54.8, 50.0, 13.6 and 10.0%, respectively, with significant differences among the four groups (P < 0.001).

CONCLUSIONICSI by PESA or TESA is an effective approach to azoospermia. There are no significant differences between PESA and TESA in the rates of normal fertilization, cleavages, excellent embryos and pregnancies. The severity of spermatogenic dysfunction affects fertilization and initial development of embryos, which were shown in the rates of normal fertilization, excellent embryos and pregnancies but not that of cleavages.

Adult ; Azoospermia ; physiopathology ; therapy ; Embryonic Development ; Epididymis ; Female ; Humans ; Male ; Pregnancy ; Pregnancy Rate ; Sperm Injections, Intracytoplasmic ; Sperm Retrieval ; Spermatogenesis ; Young Adult

Eleven flavonol glycosides were isolated from the ethanol extract of Lysimachia clethroides by a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, and reversed-phase HPLC. Their structures were identified as astragalin (1), isoquercitrin (2), isorhamnetin-3-O-β-D-glucopyranoside (3), quercetin-3-O-β-D-6"-acetylglucopyranoside (4), quercetin-7-O-β-D-glucopyranoside (5), prunin (6), 2-hydroxynaringin-5-O-β-D-glucopyranoside (7), kaempferol-3-O-rutinonoside (8), kaempferol-3-O-robinobioside (9), rutin (10) and kaempferol-3,7-di-O-β-D-glucopyranoside (11). Among them, compounds 4, 7 and 11 were obtained from the Lysimachia genus for the first time, while compounds 3, 5 and 9 were firstly reported from this plant. In the preliminary assays, compounds 2, 6 and 8 possessed significant inhibition against aldose reduc- tase, with IC50 values of 2.69, 1.00, 1.80 μmol · L(-1), respectively; none of compounds 1-11 exhibited obvious cytotoxic activity (IC50 > 10 μmol · L(-1)).
Drugs, Chinese Herbal ; chemistry ; Flavonols ; chemistry ; Glycosides ; chemistry ; Molecular Structure ; Primulaceae ; chemistry ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVETo explore the expression of connective tissue growth factor (CTGF) in pancreatic cancer and its influence on the proliferation and migration of cancer cells.

METHODSThe expression of CTGF in pancreatic cell line PANC-1 cells was analyzed by real-time PCR and in pancreatic carcinoma (50 cases) tissues by immunohistochemistry. The ability of proliferation and migration in vitro of PANC-1 cells was tested by MTT assay, scratch test and Boyden chamber test after the CTGF gene was overexpressed by Ad5-CTGF or silenced with Ad5-siCTGF transfection.

RESULTSCTGF was overexpressed in both pancreatic cancer cells and tissues. Overxpression of CTGF leads to increased proliferation and migration of PANC-1 cells. The CTGF-transfected PANC-1 cells showed apparent stronger proliferation ability and scratch-repair ability than that of empty vector controls. The results of Boyden chamber test showed that there were 34 cells/field (200× magnificantion) of the CTGF-transfected overexpressing cells, much more than the 11 cells/field of the empty vector control cells; and 6 cells/microscopic field of the Ad5-siCTGF-transfected silenced cells, much less than the 15 cells/field of the control cells.

CONCLUSIONSCTGF is overexpressed in both pancreatic cancer cells in vitro and in vivo, indicating that it may play an important role in the cell proliferation and migration in pancreatic cancer.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Connective Tissue Growth Factor ; genetics ; metabolism ; Humans ; Pancreatic Neoplasms ; metabolism ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Transfection

A high resolution Micro-CT system for small animal imaging is introduced in this paper. Micro-focus X-ray tube with focal diameter of 100 microm and flat panel detector with imaging area of 13cm x 13cm are adopted in this system. The data acquired in rotation scanning is reconstructed with cone beam algorithm. The resolving power of the detector is measured to be 31 lp/cm at 10% of the MTF. The resolution of the Micro-CT system could achieve 185 m when the magnification factor is 1.94. Thighbone of a rabbit is used as sample imaging with the system. The trabecular bone could be imaged clearly. And the ability of small animal imaging of the system has been demonstrated.
Animals ; Equipment Design ; Rabbits ; X-Ray Microtomography ; instrumentation

Female ; Humans ; Immunoglobulins, Intravenous ; therapeutic use ; Middle Aged ; Pyoderma Gangrenosum ; drug therapy ; pathology ; Skin ; pathology