【Objective】In order to establish the foundation for transgenic mouse model,the human thalassemic gene(β654) was cloned and sequenced.【Methods】The human β654 gene was amplified by PCR,and cloned into the plasmid BGT51 in which the human β gene was cut out aforehand.The recombinant plasmid was certified by enzyme-digestion,reverse dot hybridization and sequencing.【Results】A recombinant plasmid was obtained,which contained the human β654 gene in the correct recombinant direction.Sequencing showed that the cloned insert was correct.【Conclusions】The recombinant plasmid constructed is useful for establishing a transgenic mouse.

Objective To investigate the clinical value of medical image-three dimensional visible system (MI-3DVS) in percutaneous transhepatic cholangioscopy lithotripsy (PTCSL) for the treatment of hepatolithiasis.Methods The clinical data of 66 patients with hepatolithiasis (55 were from the First People's Hospital of Shunde City and 11 were from the Zhujiang Hospital) were retrospectively analyzed.The images of computed tomography were three-dimensionally reconstructed with MI-3DVS.The location,number,size,shape of the stones were figured out,and the operation strategies of PTCSL were designed according to the types of the disease.The clinical value of MI-3DVS was evaluated according to the operation results and postoperative recovery of patients.All the patients were followed up via telephone or out-patient re-examination.Results Liver,biliary system,stones and blood vessels were three-dimensionally reconstructed with the MI-3 DVS,and the size,number,shape,location of the stones and location,degree,length of the biliary stricture and its anatomical relationship with adjacent blood vessels were clearly displayed.The coincidence rate of planned and actual operations was 95.5％ (63/66).The mean operation time,intraoperative blood loss,rate of stone clearance,complication rate and duration of hospital stay of 63 patients were (117 ± 9) minutes,(18 ± 1) ml,92.4％ (61/66),6.1％ (4/66) and (15 ± 4) days,respectively.All the patients were followed up till September 2012,the median survival time was 16 months (range,1-69 months),and the recurrence rate of hepatolithiasis was 9.1％ (6/66).One patient died of tumor metastasis and multi-organ dysfunction syndrome postoperatively.Conclusion MI-3DVS could effectively improve the safety and efficiency of PTCSL for patients with hepatolithiasis.

Objective To explore the association of anti-mullerian hormone(AMH)in seminal plasma and serum with sperm counts and energy for male.Methods For 215 cases of healthy male selected from our reproductive clinic,with women′s reason for infertility,seminal plasma and serum AMH were detected,as semen parameters(sperm density,living rate,vitality and malformation rate),6 items of serum sex hormone.In seminal plasma and serum AMH respectively as the dependent variable,using multiple line-ar regression model to explore its quantitative relation with semen parameters and sex hormone levels.Results 215 cases were en-rolled,aged 34.28±5.70 years,while the median of the seminal plasma AMH was 0.47,quartile 0.05-3.09 pmol/ejaculation.The median of the serum AMH was 53.07,quartile 32.32 -72.20 pmol/L.Through multiple linear regression analysis,after adjusted by age and BMI,the seminal plasma of AMH and total number of sperm,sperm concentration,dynamic motility,total sperm activi-ty,serum inhibin B were positively correlated(P <0.05);The correlation between sperm morphology and other serum sex hormone had no statistical significance(P >0.05);Serum AMH negatively correlated with serum FSH,with serum inhibin B positively(P <0.05);Seminal plasma in various parameters and other related serum sex hormone had no statistical significance(P >0.05).Conclu-sion The seminal plasma of AMH were positively correlated with sperm concentration,sperm counts,sperm vitality,with the asso-ciation for serum AMH not yet found.

Objective To explore the correlation between the sperm indicators and early spontaneous abortion after in vitro ferti ‐lization(IVF) .Methods 251 couples receiving IVF ,were retrospectively analyzed and divided into the research group (98 couples) and control group (153 couples) according to whether spontaneous abortion occurring .The male′s indicators of sperm DNA frag‐mentation index (DFI) ,sperm nucleoprotein immaturity rate ,percentage of normally morphologic sperms ,etc .in the two groups were analyzed .Results DFI ,percentage of normally morphologic sperms ,sperm concentration ,motility and forward motile sperm had no statistical differences between the research group and the control group (P> 0 .05) ;while the sperm nucleoprotein immaturi‐ty rate of the research group was significantly higher than that of control group ,the difference was statistically significant (P <0 .05) ;the sperm nucleoprotein immaturity rate had weak correlation with spontaneous abortion in early pregnancy (P < 0 .05) . Conclusion The sperm nucleoprotein dysmaturity is correlated with the spontaneous abortion in early pregnancy .The sperm nucle‐oprotein immaturity rate could be used to evaluate the risk of spontaneous abortion in early pregnancy after IVF .

Objective To obtain information on the application value of percutaneous epididymal sperm aspiration(PESA) and percutaneous testicle sperm aspiration(PTSA) in the differentiating diagnosis of obstructive and nonobstructive azoospermia.Methods Sperm recovery procedures were done in infertile men with obstructive azoospermia(OA)(n=37) and nonobstructive azoospermia(NOA)(n=28) by PESA or PTSA.Cytological smears were analysed.Results Sperm was found in the 32 epididymides and 5 testicles of OA group and in the 7 epididymides and 11 testicles of NOA group.Sperm counts were significantly different in two groups.Conclusion PESA and PTSA are efficient methods in differentiating OA and NOA.

AIM: To clone human ?-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human ? IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human ? 654 gene isolated from the ? thalassemia patients homozygous for the ? 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human ? LCR and ? 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3.1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human ? 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human ? thalassemic mutation ?IVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human ? 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human ? thalassemic mutation ?IVS II654(C→T).

Objective To evaluate the treatment of advanced liver cancer by intraoperative argon super cryosurgery system (ASCS) Methods A total of 24 cases suffering from advanced liver cancer were treated by ASCS during operation Nine cases were treated by ASCS only, nine by ASCS in combination with liver artery or portal vein chemotherapeutic pump, four were treated by ASCS with tumor resection and chemotherapeutic pump placement, other two cases received tumor resection together with lymph nodes′ ASCS and pump placement Results The procedure was successful in all 24 cases, complications such as bleeding of puncture aperture, thorax effusion, bile leakage and hepatic coma, were successfully managed Twenty two cases have survived 6 12 months, 17 cases for 12 17 months 9 cases for 18 24 months, 5 cases for more than 24 months Conclusion Intraoperative ASCS provides a new alternative to the treatment of advanced liver cancer with acceptable complication rates

Objective To review the treatment results of intracytoplasmic injection(ICSI) of epididymal or testicular sperm obtained from 38 obstructive azoospermic patients.Methods Sperm was retrieved by percutaneous epididymal sperm aspiration(PESA) or testicular sperm extraction(TESE).Intracytoplasmic injection was performed.The rates of fertilization and clinical pregnancy were evaluated.Control group was set up in which intracytoplasmic injection was performed using sperm of ejaculation.Results Forty-one treatment cycles were performed in the 38 obstructive azoospermc patients.The rates of fertilization and clinical pregnancy were 73.3% and 53.6%.Thirty-three treatment cycles were done in the 31 ejaculatory ones.The rates of fertilization and clinical pregnancy were 75.1% and 48.4%.No significant difference was seen between the two groups.In the obstructive azoospermia group,22 clinical pregnancies were achieved including 13 live deliveries and 3 ongoing pregnancies and 6 miscarriages.In the ejaculatory group,16 clinical pregnancies were achieved including 10 live deliveries and 5 ongoing pregnancies and 1 miscarriages.Conclusions ICSI with PESA or TESE is an effective method for treatment of obstructive azoospermic patients.

Objective To evaluate left ventricular myocardial function altemation by three-dimensional speckle tracking imaging (3D-STI) after surgical aortic valve replacement (AVR) in severe aortic stenosis (AS) patients with preserved left ventricular ejection fraction (LVEF),and discuss its clinical value.Methods Forty patients with severe aortic stenosis who were hospitalized or outpatient in Nanjing First Hospital Affiliated to Nanjing Medical University during the period of October 2014 to October 2016 (AS group),and forty healthy volunteers (normal control group) were enrolled in this study.Normal control group underwent conventional echocardiography and 3D-STI measurement,while the AS group underwent conventional echocardiography and 3D-STI measurement at preoperative,1 week postoperative and 3 months postoperative,then we obtained left ventricular end-diastolic diameter (LVEDD),left ventricular end-systolic diameter (LVESD),interventricular septum thickness diameter (IVSd),left ventricular posterior wall thickness diameter (LVPWd),left ventricular ejection fraction (LVEF),global longitudinal strain (GLS),global circumferential strain (GCS),global radial strain (GRS),global area strain (GAS) strain and 3D-strain.The independent sample t test was used to compare the difference of conventional ultrasound parameters and 3D-STI parameters between AS preoperative patients and healthy controls,AS patients at 3 months postoperative and healthy controls.The single factor analysis of variance was used to compare the difference of conventional ultrasound parameters and 3D-STI parameters in severe patients at preoperative,1 week postoperative and 3 months postoperative.LSD-t test was used to compare in different AS groups.Results Compared with normal control group,IVSD,LVPWD and GCS of severe AS patients increased significantly (t=13.824,11.298 and-6.584,all P ＜ 0.001),GLS,GRS and 3D-Strain decreased significantly (t=10.221,-6.237 and-5.674,all P ＜ 0.001),LVEDD,LVESDand GAS had no significant difference.Compared with preoperative AS patients,LVEF,GLS,GAS and GCS decreased significantly (t=-2.205,-2.093,-2.034 and-3.152,all P ＜ 0.05 or 0.01) at 1 week postoperative,LVEDD,LVESD,IVSD,LVPWD,GRS and 3D-strain had no significant difference at 1 week postoperative;GLS,GRS and 3D-strain increased significantly (t=5.446,-4.923 and-4.388,all P ＜ 0.05 or 0.01) at 3 months postoperative,GCS,IVSD and LVPWD decreased significantly (t=-3.988,4.794 and 4.211,all P ＜ 0.05 or 0.01) at 3 months postoperative,LVEDD,LVESD,LVEF and GAS had no significant difference at 3 months postoperative.Compared with AS patients at 1 week postoperative,LVEF,GLS,GRS,GAS and 3D-strain increased significantly (t=-2.631,7.383,-4.719,2.923 and-4.154,all P ＜ 0.05 or 0.01) at 3 months postoperative,GCS,IVSD and LVPWD decreased significantly (t=-2.109,4.747 and 4.323,all P ＜ 0.05 or0.01) at 3 months postoperative.But in AS patients at 3 months postoperative,IVSD,LVPWD,GLS and GCS were still higher than those of normal control group (t=9.809,7.066,4.752 and-2.553,all P ＜ 0.001 or ＜ 0.05),LVEDD,LVESD,LVEF,GRS,GAS and 3D-strain had no significant difference.Conclusion The alternation of left ventricular myocardial function have a certain characteristic before and after aortic valve replacement in severe aortic stenosis patients with preserved LVEF,and 3D-STI can evaluate it more accurately.

Lymphocyte subsets in peripheral blood and function of CD4+CD25+ regulatory T cells(Tregs) were assayed in patients during different periods of Graves' disease ( GD ).Breakdown of Tregs' function lead to the wild proliferation of CD4+T lymphocyte,and may play an important role in the pathogenesis of GD.It is difficult to fully restore the Tregs' function to normal after successful medication in Graves' disease,this phenomenon may lead to easy relapse of GD.