Objective To investigate the role of Wnt signal pathway related proteins in the tumorigenesis of sponta-neous breast cancer in TA2 mice. Methods Mammary glands were collected from tissues of normal 5 cases of TA2 mice. Spontaneous breast cancers and relevant mammary glands precancerous lesions were harvested from 5 cases of TA2 mice. Mammary tissue samples from normal TA2 mice, precancerous lesions and cancer tissues of spontaneous breast cancer-bearing TA2 mice were subsequently used to detect the expression of Wnt1,Wnt5a andβ-catenin via immunohistochemical staining. Results ① Results of immunohistochemical staining showed that the positive ex-pression of Wnt1 and Wnt5 a located in cytoplasm in spontaneous breast cancers and mammary gland precancerous lesions from TA2 mice. The normal mammary glands were negative for Wnt1 and Wnt5a staining. The cytoplasm of precancerous lesions showed higher expression for Wnt1 and Wnt5a than them in the breast cancer. β-catenin in breast cancer and precancerous lesions were abnormal cytoplasmic expression. The expression level in breast cancer was higher than in the precancerous lesions. In normal mammary gland, the positive expression ofβ-catenin mainly located in the cytomembrane. ② The positive expression rates of Wnt1, Wnt5a and β-catenin in normal breast group,precancerous group and breast cancer group were ( 4. 46 ± 3. 57 )%, ( 96. 50 ± 6. 30 )%, ( 90. 00 ± 1. 17 )%, ( 2. 27 ± 4. 55 )%, ( 96. 24 ± 6. 00 )%, ( 95. 90 ± 4. 32 )%, ( 4. 78 ± 3. 57 )%, ( 60. 59 ± 5. 63 )%, (84. 87 ± 2. 56)%,respectively. The positive expression rates of the three in precancerous group and breast cancer group were significantly higher than those in normal breast group(P<0. 01). The positive expression rates of Wnt1, Wnt5a in precancerous group were higher than those in the tumor group,and the differences of Wnt1 had statistical significance (P<0. 05) while Wnt5a did not (P>0. 05). Compared with breast cancer group,the precancerous group had lower positive expression rate of β-catenin and the differences also had statistical significance ( P <0. 05 ) . Conclusion Wnt1 , Wnt5 a and β-catenin promote the progression of spontaneous breast cancer in TA2 mice.

Objective To explore the clonality of the T cells and the role of cellular immunological pathogenesis in chronic asymptomatic HBV carriers (AsC) by TCR CDR3 size spectratyping and determining sequence. Methods The TCR CDR3 region genes of 24 BV families were amplified by utilizing inverse polymerase chain reaction (RT-PCR) technology, and the CDR3 size lengths of T cell receptor (TCR) ?-chain were analyzed with Genescan technology for 4 healthy individuals and 9 AsCs. The clonality of T cells presumed by spectratyping was further confirmed by CDR3 sequencing. Results The CDR3 repertoire of 4 healthy individuals showed Gaussian distribution. The clonal expansions of T cell were observed in 8 out of 9 AsCs. The expanded T cells have different CDR3 sequences. Conclusion There is significantly clonal expansion in the compartment of the peripheral blood T lymphocyte in AsCs. The expanded T cells do not have homogenicity.

Objective To construct the recombinant adenovirus of mouse Osf2/Cbfal gene and to observe its ability to infect NIH3T3 fibroblasts. Methods The Osf2/Cbfal gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into NIH3T3 cells using Lipofectine DOTAP, The target gene was detected by poly-merase chain reaction (PCR). The titer and its infection rate were determined using the green fluorescent protein (GFP) expression in the shuttle plasmid. Results Restriction endonuclease and PCR analyses confirmed that the Osf2/Cbfal gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.6?1012 pfu/ml. The adenovirus had a strong effect on NIH3T3 cells. Conclusion The recombinant adenovirus containing Osf2/Cbfal gene was successfully constructed by the method of homogenous recombination in bacteria.

Objective To approach the brain protective effect and mechanism of Sini decoction on rats with cardiopulmonary resuscitation (CPR) syndrome.Methods Fifty Sprague-Dawley (SD) rats were divided into sham operation group (n = 10), CPR model group (n = 20) and Sini decoction treatment group (n = 20) by random number table. The rat models were established by trachea clipping to induce cardiac arrest, and after heart beat stopped for 5 minutes, CPR was carried out. In the Sini decoction group, Sini decoction 5 g/kg was given through a stomach tube, once per 24 hours, while in the sham and CPR model groups, the same amount of normal saline was given by the same method at the same time. Venous blood was collected before CPR and 6, 12, 24, 48 and 72 hours after CPR, and the levels of serum interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent test (ELISA); after CPR for 72 hours, the rat brain tissue was obtained from all the groups, the content of caspase-3 in brain tissue was detected by immunohistochemistry method, and its protein expression caspase-3 was detected by Western Blot; the apoptosis situation of brain tissue was detected by terminal deoxynucleotidyl transferase-mediated duTP nick end labeling (TUNEL).Results With the prolongation of time, the levels of IL-6 and TNF-α in CPR model and Sini decoction groups showed a tendency firstly increased and then decreased, IL-6 reached its peak value after resuscitation for 24 hours, while TNF-α reached its peak value after resuscitation for 48 hours, and both IL-6 and TNF-α were decreased after resuscitation for 72 hours ; beginning from 6 hours after resuscitation, the levels of serum IL-6 (ng/L: 61.79±1.31, 62.49±1.42 vs. 21.48±0.79) and TNF-α (ng/L: 48.32±1.98, 25.32±1.96 vs. 18.34±2.45) in CPR model and Sini decoction treatment groups were all significantly higher than those in sham group, since 12 hours after resuscitation, the level of IL-6 was significantly lower in Sini decoction than that in CPR model group (ng/L: 70.41±2.21 vs. 88.32±1.59), and since 6 hours after resuscitation, TNF-α was obviously lower in Sini decoction group than that in CPR model group (ng/L: 25.32±1.96 vs. 48.32±1.98, allP < 0.05), both IL-6, TNF-α persisting to 72 hours after resuscitation, and their levels did not return to normal at the end of experiment in the two groups. After the end of resuscitation, the content and protein expression of caspase-3 and rate of cell apoptosis in the brain tissue in CPR model group were significantly higher than those in the sham group [caspase-3 content (A value,×103): 2.59±0.26 vs. 1.57±0.06, caspase-3 protein (gray value): 0.80±0.08 vs. 0.43±0.04, apoptosis rate: (2.01±0.08)% vs. (0.26±0.02)%, allP < 0.05], above indexes in the Sini decoction treatment group were significantly lower than those in the CPR model group [caspase-3 content (A value,×103): 1.89±0.08 vs. 2.59±0.26, caspase-3 protein (gray value): 0.57±0.02 vs. 0.80±0.08, apoptosis rate: (1.03±0.05)% vs. (2.01±0.08)%, allP < 0.05).Conclusion The Sini decoction has a protective effect on rats with post-resuscitation syndrome, and its mechanism is possibly realized by the inhibition of inflammatory factors and reduction of cell apoptosis.

Objective: To study the effects of endostatin and doxycycline on microcirculatory patterns in the melanoma transplant model of the chorioallantoic membrane of the chicken embryo and the experimental evidences for melanoma therapy thereof. Methods: Endostatin was dripped on the chorioallantoic membrane after melanoma was transplanted 3 days (A group) and 5 days (B group). Doxycycline was dripped on the chorioallantoic membrane after melanoma was transplanted 3 days (C group) and 5 days (D group). The PBS solution was dripped on the chorioallantoic membrane in control group (E group). The hematoxylin-eosin（HE）staining and immunohistochemical staining were performed to observe microcirculation patterns in sections. Results: Compared with the E group, the area of endothelium-dependent vessels were significantly decreased in A group and B group (P ＜ 0.01). But the areas of vasculogenic mimicry were bigger in the two groups(P ＜ 0.05). Compared with E group, the areas of endothelium-dependent vessels and vasculogenic mimicry were significantly decreased in D group(P ＜ 0.05). Conclusion: Endostatin can inhibit angiogenesis. But it has no effect on vasculogenic mimicry in the transplant melanoma. And Doxycycline can inhibit vasculogenic mimicry and angiogenesis in the transplant melanoma. The results provided experimental evidences for melanoma therapy.

Objective: To investigate the clinicopathological features, diagnosis, and differential diagnosis of gallbladder sarcomatoid carcinoma. Methods: The clinical manifestations and the microscopic and immunohistochemical characteristics of six patients with gallbladder sarcomatoid carcinoma were analyzed with a follow-up period. Related literature was reviewed. Results:Immunohistochemical markers of gallbladder sarcomatoid carcinoma with spindle cell morphology were epithelial and mesenchymal positivity. Conclusion: Gallbladder sarcomatoid carcinoma could be firmly diagnosed by microscopic morphology and immunohistochemistry. Radical resection is currently used to treat such patients. However, more cases with longer follow-up period are needed to discover better treatments and improve the survival of these patients.

Objective To investigate the clinical efficacy of ozone injection combined with radiofrequency ablation (RFA) for the treatment of far lateral lumbar disc herniation (FLLDH). Methods A total of 60 patients with FLLDH, who were admitted to authors’ hospital during the period from March 2013 to March 2014, were randomly and equally divided into the study group (n=30) and the control group (n=30). Simple ozone injection treatment was employed for the patients of the control group, while ozone injection combined with radiofrequency ablation was adopted for the patients of the study group. The same other adjunctive medication was used in both groups. All the patients were followed up at one week as well as at one, 3 and 6 months after the treatment. The clinical effect and the complications of both g roups were recorded. Results Successful operation was obtained in all 60 patients, no perioperative complications occurred, and the technical success rate was 100%. The excellent clinical results evaluated at one week, and one, 3 and 6 months after the treatment in the control group and in the study group were 73.3%, 76.7%, 70%, 60% and 70%, 76.7%, 83.3%, 90% respectively. The 3-month and 6-month clinical results of the study group were significantly better than those of the control group. Conclusion For the treatment of FLLDH, ozone injection combined with radiofrequency ablation is safe and effective. This technique should be recommended in clinical practice.

Purpose To detect the expression of autophagic genes Beclin 1 and MAP1LC3 in cutaneous malignant melanoma and to ex-plore the relationship between autophagia and malignant melanoma. Methods 85 cases of speicmens including normal skin tissue, in-tradermal nevi, radial growth phase melanomas, vertical growth phase melanomas, and metastatic melanoma were collected, and the protein expression of Beclin 1 and MAP1LC3 were evaluated by immunohistochemistry of SP methods. Results The Beclin 1 and MAP1LC3 expression were pretended to be 100% in normal skin tissue, and they were declined to 85% and 95% in intradermal nevi, 58% and 50% in radial growth phase melanomas, 49. 5% and 44. 4% in vertical growth phase melanomas, both of 17% in melanoma metastases (P<0. 05). Conclusion Beclin 1 and MAP1LC3 autophagic gene expression were significantly decreased with tumor pro-gression, as well as was correlated with conventional histopathologic prognostic factors.

Objective To study and summary the treatment of talus fracture. Methods Fifteen patients with talus fracture were treated by limited cut off reduction by leverage and hollow lag screw under C arm X-Ray machine January 2008 to November 2014, of whom there were 11 males and 4 females, aged 15-61 years old. Seven patients sufferred from talus fracture because of traffic accidents, 4 patients because of falls, 2 patients because of crush injury, and 2 patients because of sprains. Hawkins typing:Ⅰtype in 1 patient, Ⅱ type in 6 patients, Ⅲ type in 6 patients of Ⅲ type, and Ⅳ type in 2 patients. Results Followed up for 6 months to 4 years (mean 1.5 years), fracture healed better. Hawkins grading standard:9 patients showed excellent (1 patient ofⅠtype, 5 patients ofⅡtype, 3 patients ofⅢtype), and two patients showed good (Ⅲtype). The excellent and good rate was 11/15. Necrosis of talus occurred in one patient, and degenerative joint disease occured in one patient. Conclusions Minimally invasive internal fixation with hollow lag screw under C arm X-Ray machine in the treatment of talus fracture has a small injury to the periosteum and the blood supply of small soft tissue. It can fix reliably and can pressurize the fracture fragments, and is conducive to fracture healing.

Objective: To investigate the effect of Twist, β-catenin and E-cadherin on the recurrence and metastasis of hepatocellular carcinoma (HCC) and its correlation with clinical prognosis.Methods; Immunohistochemical staining (SP) was conducted to detect the expression of Twist, β-catenin and E-cadherin in 97 hepatocellular carcinoma patients (49 with recurrence and metastasis and 48 without recurrence and metastasis).Results: The recurrence and metastasis of HCC were correlated with clinical stage (P=0.000), but were not correlated with sex or age (P=0.424, P=0.738).The abnormal expression of Twist and β-catenin in the recurrence and metastasis group was higher than that in the group without recur-rence or metastasis (,0=-0.000, P=0.000).The positive ratio of E-cadherin in the recurrence and metastasis group was lower than that in the group without recurrence or metastasis (P=0.027).The mean survival was 28.880±3.285 months in patients with positive expression of Twist and 31.477±3.359 months in patients with positive expression of β-catenin.The median survival time of them was 26 and 28 months, respctively.The mean survival was 44.603±3.521 months in patients without Twist expression and 42.009±3.720 months in patients without β-catenin expression.The median survival time of them was 49 and 45 months, respectively.The survival of patients with positive expression of Twist and β-catenin was shorter than that in patients without expression of Twist and β-catenin (P=0.002, P=0.029).The mean survival time and median survival time of patients with Twist and β-catenin expression were 44.514±3.447 months and 49 months, respectively, significantly higher than those in patients without Twist and β-catenin expression (P=0.002), which were 29.110±3.581 months and 25 months, respectively.Conclusion: The overexpression of Twist and β-catenin as well as decreased expression of E-cad-herin may play critical roles in the recurrence and metastasis of HCC and indicate poor prognosis.