BACKGROUND: It has been confirmed that platelet aggregation defect presents in patients with hemorrhagic thrombopathy, and platelet membrane glycoproteins play a significant role in the process of platelet aggregation.OBJECTIVE: To observe the expression changes of platelet membrane glycoproteins between patients with hemorrhagic thrombopathy and healthy people.DESIGN: Case-control analysis.SETTING: Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology.PARTICIPANTS :① Seventy-nine patients with hemorrhagic thrombopathy who received treatment from January 2001 to March 2003 in the Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were enrolled in this study. The patients, including 31 male and48 femlae, averaged (35.76±14.14)years old. They all agreed to participate in this study and met with the diagnosis of hemorrhagic thrombopathy revised in 1996.Informed consents were obtained from all the patients and their relatives.The course of multiple bleeding symptoms was 1 to 14 years. And the bleeding sites were totally 167: extremity petechia was found in 64 cases, rhinorrhagia in 33 cases, hypermenorrhea (more than 150 mL in each cycle) in 29 cases, gingival bleeding in 28 cases, ocular fundus bleeding in 8 cases and conjunctival hemorrhage in 5 cases.②lnclusive criteria: All the cases presented clinical manifestation of multiple focal bleeding;The laboratory tests including platelet counts,bleeding time and coagulation profiles were examined with no marked abnormal changes;According to hemorrhage sites, the patients were examined by specialists of internal medicine,otorhinolaryngology, gynecology, stomatology and ophthalmology, and no specific diagnostic focuses of infection were found; They had normal blood pressure and heart rate,and those with thrombocytasthenia had been ruled out through ristocetin test. ③Another 34 healthy volunteer donors, 15 males and 19 females with an average age (30.12±7.14) years,were selected as normal control group.METHODS:① Detection of platelet aggregation ratio: Adenosine diphosphate (ADP, final concentration 1.90 μmol/L),arachidonic acid(AA, final concentration 0.37 μmol/L),and platelet activating factor(PAF, final concentration 150 nmol/L)were used as aggregation inductors, and Chronolog 430 type aggregometry was used to detect platelet maximum agglutination ratio.When maximum agglutination ratio was within 21％ and 40％, it was considered as aggregation defect, and when below 20％, it was considered as aggregation absence.② Test of platelet membrane glycoprotein: 3.6 mL cubital venous blood was drawn from the patients and mixed with 20 g/L disodium ethylenediamine tetraacetic acid(EDTA-NA2) for the purpose of anticoagulation at 1:9, and the same procedure was performed on the blood samples from the normal control group. The plasma was isolated and collected, after being centrifuged, the supernatant liquid was discarded, and paraform was added to fix the rest of the platelet solution. The platelet concentration was adjusted to 3×107 L-1. 100 μL regulated platelet solution was extracted, and fluorescein isothiocyanate (FITC)-marked CD42b (anti-GP Ⅰ b),CD41 (anti-GP Ⅱ b), CD61 (anti-GP Ⅲ a), CD42b/CD42a (anti-GP Ⅰ b/Ⅸ ), CD41/ CD61 (anti-GP Ⅱ b/Ⅲ a) and CD62p(anti-P-selectin)clonal antibodies 200 μL were added respectively and well mixed,and then cultured for 30 minutes at room temperature away from light. Finally phosphate buffer solution was added to the solution till the volume reached 1 mL,and FACS420 flow cytometer was used to analyze the samples, one sample for 10000 platelets, and the results were demonstrated by positive fluorescence percentage.MAIN OUTCOME MEASURES: ① Platelet aggregation ratio. ② Expression of platelet membrane glycoproteins.RESULTS:All the patients and the healthy subjects were involved in the analysis of results. ①All the cases were found to have platelet aggregation defect or absence which could be induced by single or more platelet aggregating agents. No abnormal platelet aggregations were found in the control group. ② The fluorescence intensities of GPIb/Ⅸ, GP Ⅱ b/Ⅲa complexes, GPIb, GPⅢa and P-selectin in patients with hemorrhagic thrombopathy were lower than those in the healthy subjects (t =2.50-5.57,all P ＜ 0.05). The fluorescence intensity of GP Ⅱb in patients with hemorrhagic thrombopathy was slightly higher than that in the healthy subjects, but no significant difference was found. (t =0.86, P ＞0.05).CONCLUSION: Abnormal expressions of platelet membrane glycoproteins may be partial pathogenesis of hemorrhagic thrombopathy.

Low back pain caused by intervertebral disc degeneration is a common clinical chronic disease. The regenerative ability of intervertebral disc tissue is extremely poor. Meanwhile, current treating methods can not fundamentally solve such problems. With the increasing awareness of the mechanism of disc degeneration and the rapid development of the fields of cellular and molecular biology, gene and materials engineering, using stem cells and tissue engineering technology to slow down or reverse the progress of disc degeneration may become possible. The author reviewed the application of stem cells for treating degenerative discs from present researching status and concepts for the future in the combination of researches reported both at home and abroad.
Humans ; Intervertebral Disc ; pathology ; Intervertebral Disc Degeneration ; therapy ; Low Back Pain ; therapy ; Regeneration ; Stem Cell Transplantation ; Tissue Engineering

To compare the efficiency of methods of DNA extraction from lungs of Pomacea canaliculataused in PCR assay, 80 P.canaliculata collected in field were divided into 8 groups and the lungs of each snail were separated from the soft body. Six methods of DNA extraction from lungs of P. canaliculata were used to extract DNA from lungs, i.e. With Qiagen, Tiangen,and Omega commercial DNA extraction kits, guanidine thiocyanate method, Chelex 100 resin method and Chelex-silica particle method. The 16S rDNA of C.canaliculata was amplified by PCR and the concentration of PCR-products relative to marker was determined in order to evaluate the efficiency of each method. It was demonstrated that each method was valid to extract DNA from lungs used in PCR assay, but the concentrations of PCR-products were different. The concentrations of PCR-products obtained by Qiangen kit, Omega kit, Chelex 100 resin method and Chelex-silica particle method were significantly higher than those of other 4 methods of DNA extraction, in which Qiangen and Omega kits were suitable for small sample size. In term of efficiency and cost, Chelex 100 method and Chelex-silica particle method were feasible for large sample scale, while the guanidine thiocyanate method was preferred due to its fast extraction and low cost, but on account of its toxicity, it is used in urgent status or in large scale of sample extraction.

Part of undergraduates were recruited from the universities round Xueyuan Road, Haidian district in Beijing. Self-administered questionnaire were conducted, on the basis of the status quo of students in understanding the environmental and health knowledge, attitude, behavior (KAP), to analyse the need for environmental health educational courses setting among undergraduates and to afford related suggestions on contents and methods for the course.

Objective:Influence of clinicopathological characteristics and different therapy patterns on the overall survival of patients with gastric cancer with bone metastasis was investigated. Methods:A total of 146 gastric cancer patients with bone metastasis were enrolled from December 1996 to December 2014. Data of clinicopathological characteristics, treatment methods, and overall survival were collected. Univariate and multivariate analyses were performed using log-rank tests and Cox's proportional hazard model, respec-tively. Results:A total of 51 (34.9%) patients had synchronous metastasis, while 95 (65.1%) had metachronous metastasis. Moreover, 35 (24.0%) patients only had bone metastasis, while 111 (76.0%) patients were complicated with other organ metastases, such as liver (30.0%), peritoneal (24.0%), lung (15.1%), and bone marrow (7.5%). After diagnosis of bone metastasis, bisphosphonates, bone radio-therapy and bone surgery were applied in 99 (67.8%), 34 (23.3%), and 5 (3.4%) patients, respectively. Additionally, 96 (65.6%) patients received palliative chemotherapy. The median overall survival was 5.8 months (95%CI:4.284-7.316). Multivariate analysis revealed that KPS<80 (P=0.030), bone marrow metastasis (P<0.001), elevated serum CA199 (P<0.001), and without palliative chemotherapy (P<0.001) were independent poor prognostic factors. Conclusion:The outcome of gastric cancer with bone metastasis is very poor, espe-cially in patients with bone marrow metastasis, worse KPS, and elevated CA199. Palliative chemotherapy may be beneficial for the sur-vival of these patients.

Objective 4-Hydroxyisoleucine (4-HIL) is the active component isolated from the seeds of Trigonella foenum-graecum. It exerts broad pharmacological effects such as anti-hyperglycemia,blood lipid lowing,improvement of insulin resistance,etc. As the Chinese traditional medicine,it can be used clinically in treating many diseases such as type 2 diabetes mellitus,dyslipidemia,metabolic syndrome,fatty liver and other diseases. Here the current research status and application prospect on 4-Hydroxyisoleucine were summarized.

AIM: To develop a method to detemine pinostrobin in Weitengning Talets (Lindera reflexae Hemsl.) by HPLC. METHODS:A C_(18) column was used,methyl alcohol-water(80∶20) was used as a mobile phase and the wavelength of UV detector was set at 290 nm. RESULTS:The linearity of this method was good with the average recovery of 97.9%,RSD was 0.74%(n=5). CONCLUSION:The methed is simple,reliable,sensitivity,and with good reproducibility.It can be used in quality control of Weitengning Tablets.

BACKGROUND: It has been proved that platelet activation is involved in the development of diabetic angiopathy. Glycoprotein (GP) Ib/IX/V complex is one of the main platelet membrane glycoproteins, and the receptor of both von Willebrand Factor and thrombin. It plays a key role in the process of platelet activation.OBJECTIVE: To observe the expression changes of GP Ib/IX/V complex and its component GP Ibα in patients with type 2 diabetes mellitus. DESIGN: Case-control study. SETTING: Department of Integrated Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.PARTICIPANTS: A total of 51 type 2 diabetic outpatients who visited Union Hospital were enrolled from December 2005 to January 2007. The diagnosis was based on the independent criteria from WHO in 1999. Of all the 51 patients, there were 23 females and 28 males, with a peripheral platelet count of over 50×109 L-1. All the subjects had no history of administrating drugs two weeks before the examination, which would potentially influence platelet count. According to disease controlling condition, the patients were assigned to well controlled patient (WCP) group (n = 25) and poorly controlled patient (PCP) group (n =26); and according to whether angiopathy was accompanied, diabetic patients were divided into vascular disease (VD) group (n =27) and non-vascular disease (NVD) group (n =24). Meanwhile 23 healthy subjects were enrolled as normal control group. Informed consents were obtained from subjects and their relatives. The experiments were approved by the ethical committee of Union Hospital.METHODS: Fasting venous blood was harvested from all the subjects' elbow on the early morning of visit day.①Biochemical analysis: Fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c) and fasting plasma insulin (FINS) was measured by the Clinical Chemistry Department in Union Hospital.② Measurement of platelet membrane GP Ib/IX/V complex and its component GP Ibα: First, 3 mL cubital fasting blood was drawn from each subject and was anti-coagulated with 38 g/L natrium citricum. After that, all samples were fixed with 10 g/L paraform for 45 minutes. Then 50 μL well-fixed blood was added into the polystyrene tube, meanwhile 20 μL monoclonal antibody, such as CD42a-b-c-d and PE-labeled CD42b, was respectively mixed gently with the blood sample and incubated at room temperature in dark for 30 minutes. Next, 20 μL FITC-labeled rat IgG was mixed with the sample containing CD42a-b-c-d and incubated equally. In the end all blood samples were analyzed by FACS420 flow cytometry and the results were expressed as mean fluorescence intensity (MFI). ③ Platelet maximun aggregation rate (MAR) was detected according to reference.MAIN OUTCOME MEASURES: ①Biochemical indicators;②The expressions of GP Ib/IX/V complex and GP Ibα;③Platelet MAR.RESULTS: Fifty-one patients with type 2 diabetes mellitus and twenty-three healthy subjects were all involved in the result analysis.①There were significant differences in FINS in WCP group and PCP group compared with normal controls (P ＜ 0.01). FPG and HbA1c were significantly higher in PCP group compared with normal control group and WCP group (P ＜ 0.01).②Expressions of GP Ib/IX/V complex and GP Ibα were significantly lower in WCP group and PCP group compared with normal control group (P ＜ 0.01), significantly lower in PCP group than in WCP group (P ＜ 0.05), and also significantly lower in VD group and NVD group compared with normal control group (P ＜ 0.01). Moreover the expression of GP Ibα in VD group and NVD group was significantly lower than that of normal control group (P ＜ 0.05), and it also significantly decreased in VD group compared with NVD group (P ＜ 0.05). MFI of GP Ib/IX/V complex had an obvious negative correlation with FBG, HbA1c and FINS (r =-0.634, -0.573, -0.649, P ＜ 0.05), and GP Ibα MFI was obviously negatively correlated with FBG and HbA1c (r =-0.602, -0.543, P ＜ 0.05).③Platelet MAR of diabetic patients were remarkably higher than in healthy subjects (t =-3.852, P ＜ 0.01). Platelet MAR in PCP and VD groups were respectively higher than those in WCP and NVD groups (P ＜ 0.05). CONCLUSION: Platelet activation exists in the early stage of type 2 diabetes mellitus without diabetic angiopathy, and is more obvious after diabetic angiopathy. There is a positive correlation between platelet activation and blood glucose. As a receptor of thrombin and von Willebrand Factor, GP Ib/IX/V complex may be involved in the development of diabetic angiopathy.

Objective:To investigate the effect of piggyBac transpon,as a carrier of four defined transcription factors Oct4,Sox2,Klf4 and c-Myc,in the reprogramming of mouse embryonic fibroblasts (MEFs)to induced pluripotent stem cells (iPSCs).Methods:The MEFs were isolated from Oct4-GFP fetal mice and transfected by piggyBac transposon with four factors (Oct4,Sox2,Klf4 and c-Myc).The morphological changes of clones were traced with microscope during the process of induction.The chromosomes were analyzed to evaluate the karyotypic variation of iPSCs.The mRNA expressions of Oct4, Nanog and FGF4 associated with embryonic stem cells (ESCs)in the iPSCs of mice were tested by RT-PCR;the protein expressions of SSEA-1,Nanog and Alkaline phosphatase in the iPSCs of mice were determined by flow cytometry,immunofluorescence and AP staining.The iPSCs were transplanted into the NOD-SCID mouse groin,4 weeks later,the teratomas were removed for HE staining and the differentiation of tissue was observed.Results:The iPSCs were successfully obtained from MEFs by piggyBac carrying Oct4,Sox2,Klf4,and c-Myc.The round or oval iPSCs clones were similar to ESCs with clear boundry and large dense nuleus.The iPSCs showed the normal karyotypic and expressed the marker genes (Oct4,Nanog and FGF4)and proteins (SSEA-1,Nanog and AP)of ESCs.Teratomas containing three germ layers were formed in NOD-SCID mice after tanspalantation of iPSCs.Conclusion:The iPSCs are reprogrammed from MEFs by piggyBac transposon with four transcription factors-Oct4,Sox2,Klf4 and c-Myc,and the iPSCs with normal karyotype possess the characteristics of ESCs.

Objective To investigate the clinical values of laryngeal electromyography (LEMG) for evaluating the amyotrophic lateral sclerosis(ALS) patients with swallowing disorder .Methods A total of 25 ALS patients with or without complaining of swallowing disorder were recruited for the study .Their right cricothyroid muscles were taken unilaterally with concentric needle electrodes by anterior cervical approach .Each patient was tested with Kub-ota drinking test too .Results Of all the 25 patients ,11 were found abnormal in the right cricothyroid muscle which presented with typical degeneration patterns ,15 cases were found abnormal by Kubota drinking test .The coinci-dence rate of the two methods was 60% ,the poisitive coincidence rate was 32% .The McNemar test showed there was some consistency between the drinking water test and LEMG (P=0 .344) ,and the Kappa coefficient was 0 .219 . Conclusion The cricothyroid muscle electromyography may also be a potential method to evaluate ALS patients since it seemed to offer an evidence of dysphagia related with abnormal sensory function of laryngeal mucosa .But there may be some limitations .