Objective To develop a PCR-temperature gradient gel electrophoresis(TGGE) method applied to screening for the point mutations causing the non-deletion α-thalassemia.Methods The entire α2-globin gene fragment (1085 bp) was selectively amplified from human genomic DNA with different genotypes of α-thalassemia and a 543 bp fragment spanning the exon2 and exon3 of the α2-globin gene was amplified with a pair of nested primers. The condition of perpendicular and parallel TGGE for the two different fragments in length was optimized and the candidate mutant was confirmed by DNA sequencing. In pilot study, 15 samples with suspected non-deletion α-thalassemia were screened for point mutations in α-globin gene. Results Hb Constant Spring (Hb CS) and Hb Quong Sze (Hb QS), two most commonly types of the non-deletion α-thalassemia distributed in Chinese, and a rare type of the Hb Westmead mutation, α2 CD122 CAC→CAG(His→Gln) could be detected by PCR-TGGE. Among those 15 samples screened for α-thalassemia mutation were 10 samples with Hb CS mutation, 2 samples with Hb QS mutation, 2 samples with Hb Westmead mutation, and a previously unreported one, α2 CD31 AGG→AAG(Arg→Lys), confirmed by DNA sequencing.Conclusion The present PCR-TGGE method could be a useful tool for the molecular screening for the point mutations causing α-thalassemia and α-globin gene polymorphism.

AIM: To determine the effect of exogenous phosphocreatine (PCr) at different concentrations on transient outward potassium (Ito) current in rat ischemic ventricular mid-myocardial (M) cells and to explore the antiarrhythmia mechanism in the treatment of ischemic heart disease. METHODS: M cells were isolated enzymatically from left ventricular mid-myocardium of rats. Peak Ito current was recorded by patch-clamp technique in the whole-cell configuration when M cells were superfused with normal Tyrode solution,simple ischemic solution,and simulated ischemic solution containing PCr at concentrations of 5,10,20 and 30 mmol/L for 10 min. RESULTS: Peak Ito current density of M cells superfused with simple simulated ischemic solution was significantly reduced by (76.1±6.3)% (P<0.05) compared with M cells superfused with Tyrode solution. Ischemic solution containing 5,10,20 and 30 mmol/L PCr reduced peak Ito current density by (57.1±9.6)% (P<0.05),(40.3±10.3)% (P<0.05),(34.3±9.6)% (P<0.05) and (32.1±10.6)% (P<0.05),respectively. There was statistical difference among ischemic solution without PCr and containing PCr at concentrations of 5 and 10 mmol/L groups (P<0.05). No statistical difference among groups of 10,20 and 30 mmol/L PCr was observed (P>0.05). CONCLUSION: PCr reverses the inhibition of Ito current under ischemic condition in M cells,which may be the mechanism responsible for arrhythmia prevention in ischemic heart disease. PCr at concentrations of 0～10 mmol/L exerts significant dose-effect relationship.

Objective: To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability. Methods: Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was ?. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

Objective:To construct a gene of ScFv against human HAb18G molecule and secretively express it in E coli Methods:The V H and V L genes cloned from McAb HAb18 hybridoma cell were ligated with a flexible linker(Gly4Ser) 3 to construct ScFv gene Then corresponding restriction endonuclease digestion site was introduced into 5′and 3′ end of ScFv gene Finally, it was cloned into expression vector pCANTAB 5His and expressed in E coli HB2151 Expression proteins were purified by affinity chromatography and detected by SDS PAGE electrophoresis?Western blot and ELISA Results:Restriction endonuclease digestion and DNA sequencing proved that ScFv gene was correctly cloned into expression vector SDS PAGE electrophoresis and Western blot analysis showed that ScFv was successfully expressed in E coli HB2151 The relative molecular mass (Mr) of the expression products was 31 kD, according with its predicted Mr value And ELISA assured that expression products had antigen specific binding activity Conclusion:The successful expression of anti HAb18G ScFv gene in E coli laid a solid foundation for its further application in diagnosis and therapy of human hepatoma.

Objective To observe the effect of supernatant liquid from brain tissue on the differentiation of adipose-derived stem cells (ADSCs) into neural cells in normal brain tissues, the homogenate of infarcted cerebral hemisphere and the opposite side in the rats. Methods The ADSCs were obtained from rat retroperitoneal adipose tissue. The normal brain tissues, the homogenate of the infarcted cerebral hemisphere and thc opposite side got from middle cerebral artery occlusion in rats were used to induce ADSCs. Immunocytochemistry or immunofluorescence were used to identify the cell types at the 3rd day. Positive expression rate was counted by fluorescence microscope. Results (1)The neuron-specific enolase (NSE) positive cells, microtubule-associated protein 2 (MAP-2) positive cells and glial fibrillary acidic protein (GFAP) positive cells were much more in the homogenate of the infracted cerebral hemisphere than in others (P＜0.05). (2)The NSE positive cells, MAP-2 positive cells and GFAP positive cells were much more in the homogenate of the normal brain tissues and the opposite side than normal level ( P ＜ 0. 05 ) . Conclusions The homogenate of the infracted cerebral hemisphere and the opposite side can induce adipose-derived stem cells into neural-like cells and express neural cells markers in rats.

Objective To investigate the CT findings of cardiac rhabdomyoma in childhood. Methods Five children with cardiac rhabdomyoma confirmed by operation was retrospectively analysed. Enhanced electron beam CT (EBCT) was performed in 4 children and enhanced 64-slice helical CT (MSCT) scan was performed in 1 infant. Three dimensional reconstruction was applied for the full display of the lesions based on the transverse section images. Results ( 1 ) Multiple masses or nodules originated from atrioventricular septum were observed in 4 children including 1 nodule from the anterior walls of the left and right ventricles. A mass was derived from the mitral valve in 1 infant (2) The CT value of the lesions after enhancement ranged from 80 to 180 HU and was similar to neighbouring left ventricular myocardium. (3) The ventricular outlet obstruction was found in 3 children including 1 infant with pericardial effusion and pulmonary inflamation of the right lower lobe. Conclusion The rhabdomyoma should be considered first when masses or nodules originated from atrioventricular septum and with the enhancement similar to neigbouring left ventricular myocardium in children.

BACKGROUND: The occurrence and severity of acute high-altitude disease(AHAD) are determined by the speed entering the highland, the altitude of highland and seasons. The association between obesity and AHAD has not been fully investigated.OBJECTIVE: To investigate the association between obesity and acute high-altitude in people exposed rapidly to the highland.DESIGN: Comparative observation.SETTING: Department of Cardiology, General Hospital of Chinese PLA and Department of Cardiology, Tibetan General Hospital of Chinese PLA.PARTICIPANTS: The experiment was conducted at the Department of Cardiology, General Hospital of Tibetan Military Area Command of Chinese PLA in August 2006. Totally 82 Henan subjects aged 28-45 years were selected from the male workers constructing the Qinghai-Tibet Railway and their managers with acute high-altitude exposure. They had never been to the highland before, and all agreed to the detection.METHODS: ①Every subject completed the AHAD self-report questionnaire at sea level and 12 hours and 24 hours after ascending high-altitude. The items in the questionnaire included symptoms of headache, gastrointestinal symptoms,fatigue or weakness, dizziness, and insomnia. Each symptom was graded from 0-3 with 0 as no symptoms, 1 as mild symptoms, 2 as moderate symptoms, and 3 as severe symptoms and a total score of 15. A score of 4 or more could by identified as AHAD. ②The height and body mass were measured to calculate the body mass (BMI). Those with BMI≥28 kg/m2 served as the obesity group [n =39, mean age (35±8) years], and those with BMI of 18.5-23.9 kg/m2 as the normal body mass group [n =43, mean age (35±8) years]. ③Arterial blood was taken to evaluate arterial oxygen saturation (SO2), arterial oxygen pressure (PaO2) and arterial carbon dioxide pressure (PaCO2) at baseline and 24 hours after ascending high-altitude. ④The measurement data was compared by t test.MAIN OUTCOME MEASURES: BMI, vital capacity of lungs, SO2, PaO2 and PaCO2 levels of obese and normal people.RESULTS: Totally 39 obese people and 43 normal people were involved in the result analysis. ①AHAD score: No symptom was reported at sea level in all participants (scored 0), but the AHAD scores in the obesity group were significantly higher than those in normal group 12-hour and 24-hour after ascending high-altitude. ②Blood gas analysis:At sea level, there were no statistical differences in the levels of SO2, PaO2, and PaCO2 between two groups (P ＞ 0.05).But 24 hours after ascending high-altitude, SO2 and PaO2 of the obesity group were much lower than in the normal group (P＜ 0.01), and PaCO2 was significantly higher than in the normal group (P＜ 0.01).CONCLUSION: Obese men are more vulnerable to high-altitude hypoxia than people with normal body mass. Obesity is an important risk factor for the development of acute high-altitude disease.

Objective To investigate the clinical application of electron-beam CT(EBCT)in the diagnosis of congenital cardiovascular diverticula. Methotis Retrospective analysis of 9 patients with congenital cardiovascular diverticula confirmed by operation and pathology was done.Of them,enhanced continuous volume scan was performed on 8 patients and enhanced single slice scan was performed on one patient with an Imatron C-150 scanner.Results The group of 9 pailents included one patient with diverticulum of the left ventricle.3 patients with diverticulum of the atria and 5 patients with diverticulum of the aorta.EBCT scan and three dimensional reconstruction could demonstrate not only the origin,size,shape,Location and adjacent structure of diverticula,but also other important complicated abnormalities such as ventrieuloarterial connection disorder,cardiac sepud defect,aortic coarctation and even dissection.Conclusion EBCT is an ideal noninvasive technique in the diagnosis of congenital cardiovascular diverticula

Objective To investigate the application of CT in the diagnosis of cardiac lipoma.Methods Retrospective analysis of 6 patients with cardiac lipoma confirmed by operation and pathology was done. Four patients had singles slice electron beam CT plain and contrast and movie scan. Two patients had 64-slice CT plain and enhanced scan. Results (1) One patient was isolated intracavitary lipoma in the right artium, 1 patient was isolated intrapericardial lipoma and 4 patients were intramural lipomas. Of the 4 intramural lipoma, 2 were infiltrative lipomas located in the left ventricle wall or the right ventricle and septum, 2 patients were isolated in the atrio-ventricular septums. (2) CT and three-dimensional reconstruction could depict the location, shape, size, margin and characteristic fat density of lipoma,indicating the diagnosis and classifications. The displacement of coronary artery, pulmonary inflammation and effusions of pericardium and pleural cavity could also be revealed. Conclusion Cardiac lipoma can be accurately diagnosed and classified by CT.