The strategy of silico cloning based on the express sequence tags sequence (ESTs)and genome database is a new technique developed in recent years,and the core of the technique is to use bioinformatics technique to assemble and extend ESTs to get part of cDNA and even all sequence.It has the virtue of low investment,high speed,low and well-targeted technical requirements,etc.The relationship between human disease and genes has been widely confirmed,therefore,silico cloning technology will become the important means of disease research.This paper aims at elaborating the progress of silico cloning in medicine.

Objective To study influence of travoprost eye drops in treatment of after anti-glaucoma surgery and its effects on visual function, the tear film stability and inflammatory index. Methods 90 patients of anti glaucoma who received therapy from August 2014 to August 2015 in the first hospital of Ninghai County Zhejiang Province were selected as research objects. The control group was treated with timolol maleate, while the observation group was treated with travoprost eye drops, then the visual acuity, intraocular pressure, tear film stability (rupture time, schirmer), inflammation (flashlight, cell count), visual field defect percentage range, curative effect after treatment were compared. Results After treatment, the vision in observation group (0.95±0.26)D was better than the control group (0.76±0.21)D, intraocular pressure in observation group (11.29±3.23) mmHg was less than the control group (13.89±3.72)mmHg, the difference was statistically significant (P<0.05), rupture time in observation group (12.93±1.90)s was higher than the control group (10.36±1.80)s, schirmer in observation group (13.01±1.60)mm was higher than the control group (11.10±1.02)mm, the difference was statistically significant (P<0.05), TNF-α, IL-6 in observation group was less than the control group, the difference was statistically significant (P<0.05), field of visual field defect in observation group (38.96±10.21)% was less than the control group (47.37±11.35)%, the difference was statistically significant (P<0.05), the total effective rate of observation group 95.56%(43/45) was statistically higher than that in the control group 77.78%(35/45), the difference was statistically significant (P<0.05). Conclusion Travoprost eye drops can improve glaucoma postoperative visual function, tear film stability, reduce inflammation.

Objective To construct a naive human Fab fragment phage display library,from which the anti-gp96 antibodies may be panned by the gp96 purified from the tissue of urothelial carcinoma in the urinary bladder and provide a basis to new therapy for the malignant tumors.Methods Peripheral blood lymphocytes were isolated from 800 ml of blood,which was obtained from four healthy blood donors.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lympbocytes were amplified by PCR with variable regions 5' and 3' primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E.coli XL1-Blue by electroperation.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as Sac Ⅰ,Xba Ⅰ,Xho Ⅰ and Spe Ⅰ, and both the phage antibody Fabs and phage-raids abstracted from amplified E.coil were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment.The gp96 purified from the urothelial carcinoma tissue of the bladder by affinity chromatog-raphy on eoncanavalin-A sephnrose and DEAE-sephnrose ion exchange chromatography were utilized as antigens to process three rounds of panning to the original Fab antibody library.Results The quantity of total RNA and cDNA were qualified.By combination of light chain and heavy chain genes, an antibody library containing 6.6×106 clones was obtained, and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids.The gp96 protein was obtained from urothelial carcinoma tissue in the urinary bladder.After having been panned by gp96, the original antibody library gained enrichment by 68 times.Conclusion Utilizing the technology of phage surface display, specific antibody can be gained from the human naive Fab phage display library,which can be used for immunological therapy for tumors.

Objective To investigate the expression of β-catenin and Oct-4 in colonal carcinoma and explore the relationship with recurrence and metastasis after operation. MethodsImmunohistochemical analysis was used to evaluate the expression of β-catenin and Oct-4.The correlation of β-catenin and Oct-4 expression with tumor cell differentiation,T stage,N stage and metastasis was analyzed.The gene expression of Oct-4 was examined by RT-PCR in 20 frozen tumor tissues and normal tissues adjacent to tumor.Results Thirty-five patients had metastasis. The positive rates of β-catenin and Oct-4 expression were significantly higher in metastasis group than in the non-metastasis group (65.71％ vs 31.11％,51.43 ％vs 13.33 ％,x2 =9.843,P =0.002,x2 =13.605,P =0.001).Expression of β-catenin and Oct-4 was not associated with differentiation,T stage or N stage.The positive expression rate of Oct-4 in colonal carcinoma tissues was significantly higher than that in normal tissues.Metastatic rates in patients with positive expression of β-catenin and Oct-4 was higher than that in negative expression.The survival analysis showed that time of metastasis was significantly different in two groups of patients (P ＜0.05).Conclusion The expression of β-catenin and Oct-4 in tumor tissues is related to metastasis of colonal cancer after surgery and might be used to predict metastasis of colonal cancer after operation.

Objective To study whether Clonorchis sinensis eggs were involved in the pathogen esis of gallbladder stones.Method From March to August 2011,the gallbladder stones from 183 patients were studied using microscopic examination and FTIR spectroscopy.Twenty egg-positive and 20egg-negative gallbladder stones found under microscopic examination were chosen randomly and analyzed through real-time fluorescent PCR and scanning electron microscopy.Results Eggs were detected in 118 of 183 gallbladder stones.The eggs were preliminarily diagnosed as Clonorchis sinensis eggs using the morphologic characteristics under light microscope.The results of real-time fluorescent PCR showed eggs in the gallbladder stones were Clonorchis sinensis eggs.The 118 egg-positive gallbladder stones were composed of 97 pigment stones,21 mixed stones and no cholesterol stones.The detection rate of Clonorchis sinensis eggs in pigment stones were the highest among the three types of gallbladder stones (P＜0.001).The 20 egg-positive stones were observed by scanning electronic microscopy:dozens or even hundreds of Clonorchis sinensis eggs were visible under 400 times magnification; and a dozen or even dozens of Clonorchis sinensis eggs and the surrounding particles were adherent to or parceled with each other under 1000 times magnification.While under 3000 times magnification,uneven texture was seen on the surface of eggs with pieces being shed from some eggs.Some eggs were hollow or without cover.Most of them were adherent to or wrapped with amorphous particles or mucoid matter.Conclusions A large amount of Clonorchis sinensis eggs were detected in gallbladder stones.Clonorchis sinensis eggs were associated with the formation of gallbladder stones,especially with pig ment stones.

Objective A systematic study to classify mixed gallbladder stones to determine their relationship with Clonorchis sinensis infection.Methods 349 consecutive patients with mixed gallbladder stones were enrolled into this study and their gallstones were studied.The material composition of gallbladder stones was analyzed using Fourier Transform Infrared spectroscopy,and the distribution and microstructure of the material components were studied using Scanning Electron Microscopy.The composition and distribution of the elements were analyzed by an X-ray energy spectrometer.Gallbladder stones were classified accordingly,and the stones were then grinded for microscopic examination.The stones were divided into two groups based on the results of detection of clonorchis sinensis eggs.Patients were also divided into two groups according to the patients' region (high-incidence and low-incidence regions of Clonorchis sinensis infection).The diversity of the subtypes of mixed gallbladder stones was compared.Results 14 subtypes were found,including Bilirubinate-calcium carbonate mixed stones (n =134),Cholesterol-bilirubinate mixed stones (n =87),Bilirubinate-phosphate mixed stones (n =39),Cholesterol-calcium carbonate mixed stones (n =30),Cholesterol-bilirubinate-calcium carbonate mixed stones (n =15),etc.Bilirubinate-calcium carbonate mixed stones were the main subtype of egg-positive mixed gallbladder stones and the patients were from high-incidence regions.Cholesterol-bilirubinate mixed stones were the main subtype of egg-negative mixed gallbladder stones and patients were from low-incidence regions.The differences of the subtypes constituent between the high-incidence and low-incidence regions groups were significant (P ＜ 0.05),and between the egg-positive and egg-negative groups were also significant (P ＜ 0.05).Conclusions The most common subtypes of mixed gallbladder stones were bilirubinate-calcium carbonate mixed stones,while the second was cholesterol-bilirubinate mixed stones.Clonorchis sinensis infection was mainly associated with Bilirubinatecalcium carbonate mixed stones.

Objective To analyze subclinical changes of left ventricular deformation by using two-dimensional (2-D) speckle tracking imaging (STI) in primary mitral regurgitation (MR) and to identify sensitive parameters of left ventricular deformation that reflect reduction of left ventricu-lar function after mitral valve repair.Methods We evaluated patients with primary MR who underwent mitral valve repair between May 2013 and June 2015.According to the reduction in ejection fraction (EF),the MR patients were divided into MR1 (EF reduction of ≤10％) and MR2 groups (EF reduction of ＞10％).The parameters of left ventricular deformation were measured by using 2-D STI.Results Compared with the MR1 group,the MR2 group had increased global longitudinal strain (GLS;P ＜ 0.05).A positive correlation was found between postoperative EF reduction and left ventricular intemal dimension at end-diastole,left ventricular internal dimension at end-systole,end-diastohc volume,left ventricular mass index,regurgitant volume,and GLS (P ＜ 0.05).GLS showed a diagnostic value in predicting EF reductions of ＞ 10％.Conclusion STI is useful for evaluating the postoperative reduction of left ventricular function in primary MR patients and thus is helpful for optimizing surgical timing.

Objective To discuss the clinical value of the suture of sacral ligament and pubovaginalis fascia plus sacrospinous ligament fixation in the treatment of moderate and severe pelvic organ prolapse ( POP) . Methods Thirty-two cases with moderate or severe POP,who were treated by the suture of sacral ligament and pubovaginalis fascia besides sacrospinous ligament fixation,were retrospectively analyzed. Results The locations of POP in the 32 patients were mainly in the anterior and middle pelvis cavity. The degree of uterine prolapse was not less than POP-Q Ⅲ phase. The anterior and/or posterior vaginal walls were also prolapsed. The patients were treated by the transvaginal panhysterectomy,vaginal wall neoplasty,sacrospinous ligament fixation and the suture of sacral ligament and pubovaginalis fascia. Those with stress incontinence were also treated with urethral posterior ligament plication. The operation time was 60~120 min,and the hemorrhage vol-ume was 100~300 mL. There were no severe complications or recurrence in all patients. Conclusion The suture of sacral ligament and pubovaginalis fascia could greatly reduce the hazard rate of the recurrence of anterior pelvic organ defects in POP patients treated by sacrospi-nous ligament fixation. The suture operation could strengthen the anterior pelvis cavity,and was proven to be simple,secure and effective. Therefore,the operation is valuable to be used in clinical application.

Objective To investigate the influence of clinical results of early low frequency training for acute Achilles tendon rupture after anchors repair.Methods A total of 35 patients with acute Achilles tendon rupture were treated with early low frequency training after anchors repair,including 28 males and 7 females.Aged 18 to 37 years, mean 26 years old.Duration 2h to 5d from injury to therapy.All patients palpated the discontinuity of Achilles tendon, limitative activity of ankle plantar flexion,Thompson test was positive,and MRI examination was taken.Results 35 patients were followed up for 12 to 18 months,with an average follow -up of 14.5 months.According to the Amer-Lindholm score,excellent in 30 cases,good in 5 cases,the good rate was 100%.All the wounds were healed, without infection,dehiscence,sural nerve injury and re -rupture occurred.Conclusion Early low frequency training after anchors repair for acute Achilles tendon rupture is safe and reliable,patients can receive a better functional outcome.

Objective To explore the effect of different ages on behavioral dimensions of anxiety animal models in Kunming mice.Methods Young/Old,male,Kunming mice were recorded twice,for five minutes,with a one-week inter-trial interval in open field test (OFT),elevated plus maze (EPM) and light/dark box (LDB).The following parameters were evaluated:percentage of time spent in the OFT central area (Ctime％),percentage of number of squares visited in the OFT central area (Ccross％),the total number of horizontal squares in OFT (Cross),the total number of rears in OFT (Rear),percentage of time spent in the EPM open arms (Otime％),percentage of number of entries into the EPM open arms (Oentries％),the total number of entries into the EPM open and closed arms (Entries),percentage of time exploring in the LDB light area (Ltime％),percentage of number of squares crossing in the LDB light area (Lcross％),percentage of rears in the LDB light area (Lrear％),the total number of transition in LDB (Transition),the total number of horizontal squares in LDB (Cross) and the total number of rears in LDB (Rear).Results OFT-Ctime％,OFT-Ccross％,EPM-Otime％,EPM-Oentries％,LDB-Ltime％,LDB-Lcross％ and LDB-Lrear％ had significant loadings on anxiety factor; OFTCross,EPM-Entries,LDB-Transition and LDB-Cross loaded on locomotion-exploration factor.Old mice had higher anxiety emotion (Ctime％,t =3.12,P＜ 0.01; Ccross％,t =2.37,P＜ 0.01; Otime％,t =1.31,P＜ 0.05; Oentries％,t=1.57,P＜0.05; Ltime％,t=4.15,P＜0.01 ; Lcross％,t=3.28,P＜0.01 ; Lrear％,t=2.73,P＜0.01) and lower locomotion and exploratory activity (Cross,t =2.05,P ＜ 0.01 ; Entries,t =2.09,P ＜ 0.05 ;Transition,t=2.51,P＜0.01; Cross,t=3.02,P＜0.01),compared with Young.Conclusion Ages have no effect on structural dimensions of anxiety animal models.With the growth of ages,old mice have higher anxiety emotion but lower locomotion and exploratory activity.