BACKGROUND:It is accepted that the best method for spermatogonial stem cells separation is using artificial cryptorchism model combined with surface makers.OBJECTIVE:To explore the feasibility of separation spermatogonial stem cells with ?6-integrin and c-kit as specific surface markers.DESIGN,TIME AND SETTING:The randomized control experiment was performed at the Renmin Hospital of Wuhan University from May to December 2006.MATERIALS:Forty adult,white Kunming mice with 6 weeks old were randomly divided into cryptorchidism and control groups,with 20 animals in each group.METHODS:Artificial cryptorchidism model was prepared by made an incision at the median of abdomen,and testis was pulled into abdominal cavity,which was fixed at the each side of lateral abdominal wall.There was no treatment in the control group.The single cell suspension of seminiferous epithelium was obtained by traditional two step enzyme digestion at 2-3 months after operation.FITC-conjugated anti-?6-intergrin antibody and PE-conjugated anti-c-kit antibodies were added.Then the cells with low side scatter light-scattering properties were sorted and positively stained for ?6-intergrin and negative c-kit expression.Meanwhile,the viability of the isolated cells was assessed by trypan blue staining.MAIN OUTCOME MEASURES:The morphological changes of cryptorchidism,and the sorting results of spermatogonial stem cells.RESULTS:Cell distribution in seminiferous tubule was disorder with reduced numbers.The layer and lumens were disappeared,and cell division phase could be seen in the center of tubules.Compared to the control group,the testicular cells in the cryptorchidism group were increased in the side scatterlow,Forward scatterhi areas,with figure left-upward displacement.The distribution of ?6-integrin+ and c-kit cells were deviated each other,it named that most ?6-integrin+ cell were not spermatogonial stem cells,so do the c-kit-cells.Only 2.8% of testicular cells exhibited side scatterlow,?6-integrin+,and c-kit-,which were spermatogonial stem cells in the cryptorchidism group.And trypan blue staining showed that over 95% of them were viable.CONCLUSION:Using the two surface markers to sort spermatogonial stem cells can advance the purity of the spermatogonial stem cells in cell suspension,but the specificity is insufficient.

The effect of paper publication time on knowledge discovery in biomedical literature was studied by analyzing the role of time factor in indirect link mining and tested using the PubMed-covered data set,which showed that recently published papers played a better role than early published papers in knowledge discovery,indicating that the time factor plays a certain role in knowledge discovery in biomedical literature and can thus improve the efficiency of knowledge discovery in biomedical literature.

Objective To evaluate the safety and efficacy of sorafenib for patients with advanced stage renal cell carcinoma.Methods The clinical data of 85 patients with advanced renal cell carcinoma were reviewed.These patients were treated by sorafenib 400 mg Bid,dose escalation of sorafenib(400 mg Bid 1-4 weeks;600 mg Bid 5-8 weeks;800 mg Bid since then) or sorafenib 400 mg Bid+NF-α,respectively,until intolerance or disease progression occurred.The primary end points were objective response,disease control rate and adverse effects rate.Results The data of 80 patients can be evaluated.The median follow-up duration was 72 weeks (4-108 weeks).One patient (1.2%) reached complete remission(CR),17 cases(21.2%) reached partial remission(PR),50 cases (62.5%) maintained stable disease (SD),and 12 cases (15%) progressed.The objective response (CR+PR) was 22.5%,disease control rate (CR+PR-SD)was 85.0%.By May 2009,only 18 patients died,progression free survival and overall survival were not available.The common side effects included hand-foot skin reaction (55.0%),mucosa hemorrhage (52.5%),diarrhea(40.0%),lassitude (35.0%),anorexia(22.5%),mucosa ulcer(20.0%),hypertension(15.0%) and baldness(15.0%)etc.Most of these side effects could be released by symptomatic treatment.Conclusion Sorafenib has good short term effect for patients with advanced renal cell carcinoma and is well tolerated.

Objective To study the status and risk factors for falling down in elderly people in rural areas, and to provide evidences for preventions. Methods A total of 1 003 farmers aged 60 years old and over were surveyed to study the falls occurred during the previous 12 months before March 2001 in the elderly in a town of Laizhou City by cluster sampling method. Results The average incidence of falling down was 22.6%, 15.9% for men and 28.4% for women, respectively. The risk factors of falls included gender, age, single-living, falling history in the previous year, at least combined two kinds of chronic diseases, manifesting at least two symptoms, gait abnormalities, as well as complaining poor physical status and poorer general conditions than the previous year. Less social actions was negatively associated with the falls. Conclusions The falling in the elderly walkers results from an outcome of combination of multifactors. Since the complex pathogenesis, it should be prevented by a combination of several methods.

Objectives To explore the route of ESBL producing bacteria in neonatal faeces, and to investigate the gene and drug resistance of ESBL producing bacteria in intestinal tract of neonates. Methods Fecal samples of healthy newborns and their mothers were collected, and bacterial cultures were carried out using selective ESBL medium. The positive strains were identified by Time-of-flight mass spectrometry. ESBL genotyping and resistance gene detection were performed by whole genome sequencing technique. Results In 146 neonatal fecal specimens, the positive rate of ESBL producing bacteria was 8.90％,and the positive rate in the first time stool was 3.23％. Seventy-two hours after birth, the positive rate of fecal ESBL producing bacteria was 13.10％. Among the 13 ESBL producing strains, there were 9 strains of CTX type, 3 strains of TEM type and 1 strain of SHV type. Nine strains of CTX include five types such as CTX-M-24, CTX-M-18, CTX-M-27, CTX-M-42 and CTX-M-15. The positive rate of ESBL producing bacteria was 21.6％ in 167 mothers' fecal specimens. The ESBL genotype included 24 strains of CTX type, 6 strains of TEM type, 4 strains of SHV type and 2 strains of QnrS type. Twenty-four strains of CTX include CTX-M-24, CTX-M-14, CTX-M-18, CTX-M-27, CTX-M-42 and CTX-M-15. There were 2 or 3 ESBL genotypes in 12 maternal and neonatal specimens. It was detected to have 6 types of resistance gene such as aadA5, strA, strB, sul1, sul2 and dfrA17 in 49 strains of ESBL producing bacteria in maternal and neonatal strains. Resistance genes were exactly the same in the neonates as in mothers who were detected to have ESBL producing bacteria. A variety of resistance genes were detected in feces in 7 neonates and 23 mothers. Conclusions The neonates in hospital may be detected to have ESBL produing bacteria in the intestinal tract at the same time as their mothers or separately. However, there are many ways for neonates to have ESBL producing bacteria in intestinal tract. There are many genotypes and resistance genes of ESBL producing bacteria.

Objective To explore the intervention mechanism of Yiqi Huoxue Fang on deep venous thrombosis ( DVT) of rabbit model,by observing its effects on serum t-PA and PAI-1,D-D,NO,CRP,PT,blood rheology and vascular pathology. Methods Fifty New Zealand rabbits were randomly selected.Ten rabbits were served as blank control group.DVT animal models were established in the remaining 40 rabbits, which were randomly divided into model control group, Yiqi Fang group, Huoxue Fang group and Yiqi Huoxue Fang group (n=10 each group).The changes of serum t-PA,PAI-1,D-D,NO,CRP,PT and pathological changes of blood vessel were observed after 7-days treatment. Results As compared with the blank control group, the serum t-PA,PAI-1,D-D,CRP and PT were significantly increased in the model control group,Yiqi Fang group,Huoxue Fang group and Yiqi Huoxue Fang group (all P<0.01); NO was decreased (P<0.01 or P<0.05 in model control group,Yiqi Fang group and Huoxue Fang group;P>0.05 in Yiqi Huoxue Fang group vs.the blank control group).As compared with model control group,the serum t-PA,NO,D-D increased,PAI-1 and CRP value reduced in Yiqi Huoxue Fang group and Huoxue fang group (P<0.01 or P<0.05).As compared with Yiqi Huoxue Fang group,serum t-PA in both Yiqi Fang group and Huoxue Fang group was significantly lower (P<0.01 and P<0.05) ,other indexes were significantly higher than that in Yiqi Huoxue Fang group (serum PAI-1,P<0.01 in Yiqi Fang group;NO and D-D,P<0.01 or P<0.05 in Yiqi Fang group;CRP value,P<0.01).Vascular pathology showed that blood vessels,especially vascular endothelia,of Yiqi Huoxue Fang group were most slightly injured,with slightest infiltration of inflammatory cells. Conclusion Yiqi Huoxue Fang can prevent DVT through regulating t-PA and PAI-1,keeping the fibrinolytic system function at a relatively high level,protecting vascular intima and relieving the inflammation in venous thrombosis.

Objective To investigate the molecular mechanism of resistance to carbapenems in Pseudomonas aeruginasa from 5 teaching hospitals in Beijing. Methods A total of 213 non-duplicate imipenem-resistant Pseudomonas aernginosa (IRPA) isolates were collected from 5 hospitals in Beijing from June 2004 to December 2005. The minimal inhibitory concentrations (MICs) of meropenem, imipenem and others antibiotics were determined by agar dilution method. Disk diffusion test was used for screening metalloenzymes. The bla IMP,bla VIM and OprD2 genes were amplified by PCR and only sequenced. Results Out of 213 isolates, OprD2 loss was detected in 84 isolates and IMP-1 enzyme was detected in 6 isolates simultaneously. Thirteen IRPAs only produced IMP-1 and 2 isolates only produced VIM-2. Conclusion OprD2 loss and metallo-β-lactamuse production are the parts of the mechanisms of resistance to carbapenems in Pseudomonas aeruginosa in Beijing.

Objective To investigate the status of depression with anxiety symptoms, and analyze the influencing factors of anxiety symptoms from demographic data and social psychological factors. Methods Hamilton depression rat?ing scale (HAMD), Hamilton anxiety rating scale (HAMA), Eysenck personality questionnaire (EPQ), life event scale (LES), trait coping style questionnaire (TCSQ) and social support scale (SSS) were used to evaluate 729 patients with de?pression. According to HAMA scores, patients were divided into non anxiety symptoms group (HAMA<7) and anxiety symptoms group (HAMA>14). Social psychological factors were compared between two groups, and the influencing fac?tors of anxiety symptoms were analyzed. Results The incidence of anxiety symptoms in depression was 58.85% (429/729), and 119 cases (16.32%) were certainly without anxiety symptoms. Compared with the group without anxiety symp?toms, the anxiety symptoms group had higher scores on neuroticism, psychoticism, negative life events and negative cop?ing style (P<0.001), but lower scores on introversion and extroversion (P=0.010). Degree of depression (OR=9.255, 95%CI:4.726~18.127), neuroticism (OR=1.595, 95%CI:1.197~2.125), negative life events (OR=1.009, 95%CI:1.001~1.017) and negative coping style (OR=1.046, 95%CI:1.013~1.080) were the risk factors of anxiety symptoms (P<0.05). Conclu?sion The incidence of anxiety symptoms in patients with depression is high. Patients with higher degree of depression and typical neurotic personality experiencing more negative life events and those with tendency to adopt negative coping style are more susceptible to anxiety symptoms.

Objective To investigate the change characteristics of the brain neurotransmitters of human with stress events by the Encephaloflucgram technology (ET) at the noninvasive condition. Methods Extract shocking informations with neurotransmitter requlation systems in EEG ( S spectral line) by ET and analyze thechange characteristics of the brain neurotransmitters of human with stress events, the clincial symptons of the patients were evaluated by the post-traumatic stress disorder-check scale (PCL-C). Results ①Compared with expected number,the activity of neurotransmitters such as γ-aminobutyric acid (GABA) were significantly decreased (4.64 ±2.88,8.45 ±0.42, P＜0. 01 ) and the activity of neurotransmitters such as dopamine( DA ) ( 17.01 ±7.41,7.59±0.55, P＜0. 01),acetylcholine(Ach) (17.01 ±7.41,14.95 ±0.65, P＜0.05) ,norepinephrine (NE ) ( 13.07 ± 4.33,11.82 ± 0.84, P ＜ 0. 05 ) were increased. ②There was a significant difference on GABA ( t =6.902, P ＜ 0. 01 ) between suspect of post-traumatic stress disorder and non-post-traumatic stress disorder. ③In PCL-C scale score, intrusion factor had negative correlation to the activity of GABA ( r = - 0.777, P ＜ 0.01 ), and positive correlation to the activity of DA ( r = 0.360, P ＜ 0.01 ), hyper-arousal factor was positive correlated with the activity of NE ( r=0.221, P＜0.05) ,escaping/numbness factor was negative correlated with the activity ofGlu( r= -0.274, P＜0.05). Conclusion In traumatic stress events GABA,Ach,DA,NE neurotransmitters aresignificantly changed ,and meybe participat stress responses.

Objective To establish normally immortalized porcine hepatocyte lines by ectopic expression of simian virus 40 large T (SV40LT) antigen and the human telomerase reverse transcriptase(hTERT). Methods Primary porcine Hepatoeyte cells were transfeeted with recombinant retrovirus containing SV40LT or hTERT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Immortalized porcine hepatocyte was confirmed by examination. Results The morphological phenotype of the transfected cells was similar to the primary porcine hepatocyte. One clone, HepLP, has been maintained in cultue for half year, and expanded by more than 60 passages. SV40 LT and hTERT could be detected in transfected porcine hepatocyte. Pig albumin mRNA was also detected by RT-PCR. No tumor formation occurred when HepLP cells were injected into Balb/c nude mice. Conclusions The immortalized, nontumorigenic, porcine hepatoeytes maintained the properties of porcine primary hepatocytes such as the albumin secretion. This generation of immortalized porcine hepatocyte may be helpful for bioartifical liver support system, hepatocytes transplantation, drug/toxicological studies, and liver biologic studies.