Objective: To study the characteristics of mouse embryonic stem (ES) cell line R1 expressing green fluorescent protein (GFP) efficiently and stably. Methods: Four kinds of biological characteristics: growth curve, express level of alkaline phosphatase, karyotype and pluripotentiality of the subclones of R1 ES cell expressing GFP were observed and analysed. Results: No obvious differences between R1 ES cells and the 4 ES cell clones expressing GFP［ESG（+）］ on the proliferation speed, differentiation state and the ratio of normal karyotype were observed. Teratocarcinoma concluded 3 germinal layers could form after inoculating ESG(+) cells to nude mice. Conclusion: The expression of GFP may not make any detectable effect on the proliferation speed, differentiation state and the pluripotentiality of R1 ES cells. This research work ensures efficient utilization of GFP as a reporter gene tracing ES cell in vivo.

Objective: To ascertain the gene flow tracks and patterns and the origin of HLA class Ⅱ in the Chinese Nation.Methods : HLA classⅡ in 11 Chinese groups and 5 reference populations was studied by clustering analysis of genetic structure and distribution comparison of gene frequencies.Results and Conclusion: These analyses revealed that these genes decreased southwards and originated probably in Caucasoid or Negroids (DQA 1*0501, DQB1*0501, etc.),and those decreased northwards and originated probably in Southeast Asia (DQA1* 0601, DRB1*1202, etc.).It was found that these genes decreased both southwards and northwards (DQB1*0303, DRB1*0901,*1501,etc.) and originated probably in certain groups in mainland of Chinese Nation,and that HLA genes in different Chinese groups migrated from each other and amalgamated deeply,but Dais, Buyis etc were isolated from outside in some degree. Our results also supported that the Chinese Nation comprises two major populations of north and south.We proposed that the migration and mixing are the dominant driving force of the HLA gene flow while the genetic drift caused by isolation and the nature selection have an important influence on HLA genetic structure.

p53 gene mutations and their expression in protein levels of colorectal adenomas, carcinomas and regional lymph nodes metastasis were detected by PCR-SSCP and AB-PAP immunohistochemical analysis, respectively. The results showed that p53 gene mutations were detected in 25% colorectal adenomas, 80% carcinomas and 100% regional lymph nodes metastasis. Meanwhile, p53 proteins were detected at rates of 75%, 60% and 57% in these three groups, respectively. It is suggested that p53 gene mutations occur frequently at late stage of progression to colorectal adenoma-carcinoma sequence and may relate to its metastasis, while the accumulation of p53 protein may be a specific genetic alteration initiated from adenomatous stage. Thus, the biological significances of the two genetic alterations are different. Investigations of these concomitantly would be helpful in understanding the malignant potential of adenoma and evaluation of its staging.

Objective To investigate the outcome of the urethral realignment simultaneous internal fixation in treatment of pelvic fracture associated with posterior urethral disruption. Methods The study included 33 patients with pelvic fracture associated with posterior urethral disruption treated simultaneously by urethral realignment and internal fixation from December 2003 to August 2009. According to AO classification, two patients were with type A2 fracture, six with type B1 ,seven with type B2,five with type B3 ,six with type C1 ,four with type C2 and three with type C3. All the patients were found with complete posterior urethral disruption. Primary urethral realignment combined with emergency open reduction and internal fixation of pelvic fracture were performed in 12 patients. Due to unstable condition, 21 patients underwent primary suprapubic cystostomy and external fixation, sequentially delayed urethral realignment and internal fixation.Results The mean follow-up time of all patients was 37 months ( range, 3-63 months). Of all the patients, 25 patients (76%) regained good without urethral dialation or needed only short term urethral dilatation, and eight patients (24%) suffered from urethral stricture and needed further complex surgery. The incidences of urinary incontinence and erectile dysfunction were 6%(2/33) and 18% (6/33). The erectile dysfunction of two patients was ascribed to sacral nerve injury.At the final follow-up, the mean score was 90.3 points (range, 66-100) according to the Majeed' s scoring system, which showed that the result was excellent in 24 patients, good in eight and fair in one.Conclusions Urethral realignment and simultaneous internal fixation can attain good clinical results for pelvic fracture associated with posterior urethral disruption. It takes advantages of minor surgical trauma,short treatment cycle, good outcome and low complication rate.

ObjectiveTo investigate the results of open reduction with internal fixation for high anterior column fracture of acetabulum with intact true pelvic brim.MethodsFrom January 2006 to January 2010,12 patients suffered high anterior column fracture of acetabulum without involvement of true pelvic brim were identified.There were 9 males and 3 females,with the average of 35.6 years(range,29-46).The injury was caused by crush in 7 cases,smash of heavy object in 3 cases,and fall-down from height in 2 cases.These fractures were classified into three types:isolated high anterior column fracture of acetabulum without involvement of true pelvic brim,high anterior column fracture of acetabulum without involvement of true pelvic brim with posterior wall fracture of acetabulum and commminuted high anterior column fracture of acetabulum without involvement of true pelvic brim according to present and unpresent of posterior wall fracture.Five cases suffered isolated fracture and 2 cases associated smaller and nondisplacement fracture fragment of posterior wall were reduced and fixed by buttress plate of iliac crest and lag screw of anterior border of ilium through an iliofemoral approach; 4 cases with displacement posterior wall fracture of acetabulum were reduced and reconstructed by buttress plate and lag screws of through combinations of anterior and posterior approaches.One case suffered comminuted fracture were reduced and reconstructed by plates and screws of through extended iliofemoral approach.ResultsThe mean follow-up time of all patients was 26.7 months (range,14-37months).The quality of reduction was grade as anatomical in 8 patients,imperfect in 3,poor in 1 by Matta's score system.The fracture union was uneventful.There was nonunion and loss of internal fixation.At the final follow-up,the mean score was 16.8 (range,11-18),7 cases were graded as excellent,4 good,1 fair,according to modified Merle d'Aubigne and Postel score system.Heterotopic ossification and traumatic osteoarthritis were recorded in 1 patient.ConclusionGood clinical results can be achieved by anatomical reduction and rigid fixation through optimal approach.

Objective: To study the factors effecting the expression of the reporter green fluorescent protein (GFP) gene in the mouse embryo stem cell line R1. Methods: Three different kinds of GFP euko-expression vectors were constructed, and the expression efficiency was contrasted both at mRNA and protein levels after they were integrated into the chromosomes of host cells. Results: At protein level, the GFP expression level of the colonies transfected by the expression vector-pEF-GFP with the promoter of the peptide elongation factor (EF) were significantly higher than that of the colonies transfected by pCMV-GFP with CMV promoter and by pdCMV-GFP with double copies of CMV-GFP expression unit. There was no significant difference between the colonies transfected by pCMV-GFP and pdCMV-GFP. The detection results on mRNA level of GFP had the same tendency as that at protein level. Conclusion: (1) GFP gene expression efficiency controlled by EF promoter is distinctly higher than that by CMV promoter in NIH3T3 and R1 ES cell line.(2) A slight increase of the copy number of the foreign gene expression units in the host chromosome can not make obvious increase of its expression efficiency. (3) The vector express GFP in R1 ES cell line efficiently and stablely is obtained.

Objective: To establish C57BL/6 transgenic mice lineages containing pMTR1 plasmids as an efficient animal model for studying gene mutation in vivo . Methods: The linearized pMTR1 DNAs, constructed by molecular cloning, were injected into 572 fertilized eggs of C57BL/6 mice by microinjection. The manipulated embryos were transferred into the oviducts of 45 pseudopregnant mice respectively, from which 35 offsprings were obtained. The genomic DNAs of these offsprings were analyzed with PCR and genomic Southern blotting. Four mice whose genomes integrated with copies of intact pMTR1 vectors were chosen as founders to establish transgenic mice lineages. The recovery of pMTR1 plasmid were conducted on the genomic DNAs of F1 or F2 transgenic offsprings. Results: It was showed that intact and functional pMTR1 plasmid could be efficiently recovered from the genomic DNAs of these mice. Conclusion: (1) The transgenic mouse lineages containing copies of a stably integrated pMTR1 plasmid is established. (2) The transgenic mice are suitable models for studying gene mutation in vivo .

Objective: To study the construction and application of a vector with Cre recombinase recognition site lox66 for mouse HPRT gene targeting in embryonic stem(ES) cells. Methods: Using the HPRT genomic DNA fragment and synthesized oligonucleotides, pSP HPRT lox66 Neo was designed and constructed as a replacement vector by common molecular cloning techniques. After the linearized pSP HPRT lox66 Neo DNA was electroporated into ES cells, and transfected cells were cultured in G418/6 TG drug selection medium. The recombination efficiency of this vector was tested. Results: The main components of pSP HPRT lox66 Neo were a positive selection gene Neo, lox66, long and short homologous fragments of mouse HPRT gene and plasmid backbone. Twenty ES cell clones with HPRT gene inactivated were obtained. Conclusion: An effective replacement vector with Cre recombinase recognition site lox66 is constructed and applied to HPRT gene targeting in ES cells.

To study the significance of the expression of splice variants of CD44 (CD44v) in tu-m0r-adjacent tissue of the patients with primary liver cancer(PLC). Methods: To research into the signifi-cance of the expression of CD44v mRNA in tumor-adjacent tissue of 30 patients with PLC by RT-PCR andfollow-up. Results: In the patients that the expression of CD44v mRNA of tumor-adjacent tissue washigher than those of tumor tissue(group I ), clinical pathological indexes were higher than in the patientsthat the expression of CD44v mRNA was higher than the tumor tissue(group I ). The recurrent rate ofgroup I was higher than that of group n (P

Mammalian central nervous system is a very heterogenous and complicated system. The mechanism of neural-specific gene expression is of considerable general interest to scientists. The present paper as a review will focus on the progress in this research field.