Objective To investigate the effect of bcl-2 gene tr ansfection on the apoptosis and bioactivity of cultured islet cells. Met hods Adenovirus-mediated gene transfection was employed to transfect b cl-2 into the islet cells, the expression of bcl-2 was determined by RT-PCR a nd immunocytochemical staining, and the apoptosis of islet cells was detected by TUNEL, the viability was determined by trypan blue staining and islets function was evaluated by insulin detection. Results Bcl-2 protein exp ressed in 70% of transfected rat islet cells. The apoptosis rate of transfected cells reduced significantly compared with untransfected cells (6% vs. 22%). The viability of transfected cells was higher than that of untransfected cells (91% vs. 68%). Insulin secrete capacity of transfected islet cells in high glucose me dium was superior to untransfected cells. Conclusions Islet cel ls may be successfully transfected with adenovirus-mediated gene transfection t echniques. The apoptosis may be declined by bcl-2 gene transfection, so the isl et cells' function improved.

BACKGROUND:Adipose-derived mesenchymal stem cells have the ability to self-renew and have pluripotent potential under specific conditions in vitro, which have broad application prospects in clinical practice. However, isolation and culture of adipose-derived mesenchymal stem cells stil appear to have many difficulties and shortcomings. OBJECTIVE:To isolate and culture rabbit adipose-derived mesenchymal stem cells in vitro in order to study their morphology, cellsurface markers and biological properties as wel as to investigate the osteogenic and adipogenic potentials of adipose-derived mesenchymal stem cells in vitro. METHODS:Primary adipose-derived mesenchymal stem cells were isolated from the subcutaneous adipose tissue of posterior cervical region from New Zealand white rabbits and digested by 0.1%col agenase I. The cells were passaged and amplified by the trypsin digestion. The passage 4 adipose-derived mesenchymal stem cells were induced to differentiate after exposure to adipogenic or osteogenic medium. The oil red O staining, alkaline phosphatase and alizarin red staining were used to detect the results. The cellviability was detected by the cellcounting kit 8 method to drawn the growth curve. cellsurface markers were examined using flow cytometry. RESULTS AND CONCLUSION:The adipose-derived mesenchymal stem cells isolated from the subcutaneous adipose tissue of rabbits exhibited a fusiform adherent growth in a vortex pattern, and had a strong capability of proliferation that could be passaged stably to the 10th generation. Flow cytometry results showed that the cells highly expressed CD29, CD90, CD44, but lowly expressed CD45 and CD34. After adipogenic induction, the adipose-derived mesenchymal stem cells were positive for oil red O staining;after osteogenic induction, the cells were both positive for alkaline phosphatase and alizarin red staining. These findings suggest that the adipose-derived mesenchymal stem cells were successful y isolated and cultured from the subcutaneous adipose tissue of rabbits, and these cells are pluripotent with the potential to differentiate into adipocytes and osteoblasts, which are expected to be ideal seed cells for bone tissue engineering.

Objective To investigate the impact of experimental stress on serum glucose , insulin, glucagon, cortisol, and ep-inephrine in diabetic rats and its intervention with insulin and fluoxetine .Methods Type 2 diabetic rats model was induced by feeding with high sugar and high fat diet , and intraperitoneal injection of streptozotocin (STZ).The rats were divided into normal group , dia-betic control group , diabetic treated with insulin , and diabetic treated with fluoxetine .All the rats were exposed to multiple stressors (forced swimming, cold stimulation, rotation, restrain, and crowding) for 8 weeks.The rat blood sera collected from each group were analyzed with multiple parameters including glucose , insulin, glucagon, cortisol, and epinephrine at the 4th and 8th week.Results After experimental stress, the levels of glucose [(16.7 ±3.5)mmol/L vs (5.1 ±1.1)mmol/L, t =13.9, P <0.01], glucagon [(158.5 ±50.2)ng/L vs (120.8 ±38.7)ng/L, t =2.5, P <0.05], epinephrine [(203.8 ±48.6)pg/ml vs (158.7 ±42.6)pg/ml, t =2.9, P <0.01], cortisol [(21.3 ±4.8)ng/ml vs (18.2 ±3.8)ng/ml, t =2.1, P <0.05], and HOMA-IR (10.9 ±2.6 vs 3.3 ±0.8 , t =12.3 , P <0.01 ) of diabetic rats were significantly increased .The glucose level [ ( 9.7 ±2.1 ) mmol/L vs ( 16.7 ± 3.5)mmol/L, t =7.0, P <0.01] was significantly improved after treated with insulin .The levels of epinephrine [(158.8 ±37.5 ) pg/ml vs (203.8 ±48.6)pg/ml, t =3.0, P <0.01], and cortisol [(15.7 ±4.2)ng/ml vs (21.3 ±4.8)ng/ml, t =3.5, P <0.01 ] were significantly improved after treated with fluoxetine .Conclusions Experimental stress increased the levels of glucose , en-docrine hormone , and insulin resistance of diabetic rats .Treatment with insulin improved the glucose level and treatment of fluoxetine improved the endocrine hormone level .

ObjectiveTo evaluate the effects on serum homocysteine(Hcy) level,vascular function and carotid intima-media thickness(IMT) in metformin-treated diabetic patients with or without supplementation with folate and Vitamin B12.MethodsA total of 100 newly diagnosed diabetic type 2patients were divided into two groups by random digits table with 50 cases each,90 patients completed study.Forty-seven participants (control group) received a 6-month course of metformin treatment,43 patients (treatment group) received mefformin,folate and Vitamin B12 treatment.The levels of serum Hcy,endothelin-1 (ET-1),carotid IMT,large or small arterial elasticity index (C1,C2),flow-mediated dilatation (FMD) of the brachial artery were evaluated before and after treatment.ResultsThe level of serum Hcy in control group significantly increased compared with before treatment[ (13.4 ± 2.7)μ mol/L vs.(11.1 ± 1.9)μ mol/L],hut the level of serum Hcy in treatment group significantly decreased compared with before treatment [ (9.2 ± 1.8 ) μ mol/L vs. ( 11.3 ± 2.0) μ mol/L ],there was significant difference(P ＜ 0.05 ).A beneficial effect was observed in the serum ET-1,FMD,carotid IMT and C2 in treatment group[ (20.0 ± 6.2)ng/L,( 15.8 ± 7.6)％,(0.8 ± 0.2) mm,(4.1 ± 2.1 ) ml/mm Hg ( 1 mm Hg =0.133 kPa) × 100 vs. (31.3 ±10.1 ) ng/L,(9.7 ± 4.5)％,( 1.1 ± 0.4) mm,(2.3 ± 1.0) ml/mm Hg × 100 ] (P ＜ 0.05).The levels of ET-1,FMD,carotid IMT and C2 after treatment in control group [ (24.8 ± 6.8) ng/L,( 12.9 ± 6.3 )％,(0.9 ± 0.3)mm,(3.0 ± 1.4) ml/mm Hg × 100] had significant difference compared with before treatment [ (30.6 ± 8.7)ng/L,(9.8 ± 4.6)％,( 1.0 ± 0.3) mm,(2.2 ± 0.9) ml/mm Hg × 100](P＜ 0.05).However,the results were improved significantly in treatment group than those in control group (P ＜0.05).In treatment group,significant correlation were detected between changes of Hcy and ET- 1 (r =0.43,P ＜ 0.05 ),carotid IMT(r =0.56,P ＜ 0.05),FMD (r =-0.54,P ＜ 0.05 ),C2 (r =-0.37,P ＜ 0.05 ).ConclusionsAdministration of folate and Vitamin B12 can reduce the levels of serum Hcy and ET-1 in metformin-treated type 2 diabetic patients,which exert beneficial effect on carotid IMT,FMD and C2.

Objective To explore the clinicopathological characteristics and prognosis of invasive micropapillary carcinoma of the breast (IMPC),and the distinction between IMPC and invasive ductal carcinoma of the breast (IDC).Methods From February 2004 to November 2013,195 IMPC patients and 420 IDC patients were analyzed retrospectively.Results There were significant differences in mammilla invasion,lymph vessel invasion,orange peel sign,soft tissue encroachment,neoadjuvant chemotherapy,radical mastcctomy,lymph node metastasis,clinical stages,tumor size,lymph node staging,estrogen receptor (ER),progestin receptor (PR),human epidermal growth factor receptor 2 (HER2),molecular subtyping,ratio of radiation,ratio of endocrine therapy,disease-free survival (DFS),overall survival (OS)between the two groups,all P ＜0.05.Patients with IMPC had lower 5-year DFS and OS rates (68.2％ and 73.8％,respectively) than IDC patients (85.7％ and 88.6％,respectively),all P ＜ 0.05.In IMPC patients with positive ER/PR,HER2-negative,smaller tumor volume,less lymph node metastasis,negative nipple invasion,negative lymphatic vessel tumor thrombus,negative orange peel change had higher 5-year DFS and OS rates than those with negative ER/PR,HER2 overexpression,larger tumor volume,more lymph node metastasis,positive nipple invasion,positive lymphatic vessel tumor thrombus,positive orange peel change,all P ＜ 0.05.Besides,the patients with pathologic stage Ⅰ had higher OS than those with stage Ⅲ (P ＜ 0.05).Cox regression analysis found that orange peel change,lymph vessel invasion and HER2 were the independent risk factors for the survival time of patients with IMPC.Conclusions IMPC patients have lower DFS and OS compared with IDC.

Objective To investigate the clinical and pathologic features of squamous cell carcinoma of the breast.Methods The clinical and pathologic data of 23 squamous cell carcinoma of the breast patients admitted between 1984 and 2013 to Tianjin Medical University Cancer Hospital was analyzed retrospectively.Results Primary squamous cell carcinoma of the breast was a very rare tumor accounting for 0.06％ of all breast cancers.All of the 23 patients were females aged 28 years to 87 years(median age 49 years).Average tumor size was 4.5 cm.9 patients suffered from lymph node metastasis at admission (39.1％).The positive rates of estrogen receptor (ER),progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) were 13.6％ (3/23),4.5％ (1/23) and 0 (0/20) respectively.With a follow-up time varying from 5 months to 36 months recurrence or metastasis were found in 8 patients,and another 1 patient was found having distant metastasis at admission.Lung metastasis (7/9) was most common.6 patients died.Conclusions Squamous cell carcinoma of the breast is highly invasive,with low rate of positive receptors and early distant metastasis or recurrence after operation,and poor patients' survival.

Objective To search for the neutralizing epitopos on hepatitis E virus (HEV) capsid besides the known neutralizing epitope (aa459-606). Methods By analysis of several strains of monoclonal antibodies against HEV capsid and their recognized epitopes, the neutralizing activity of epitope (aa394-458) at N-terminus was compared with that of an immunodominant neutralizing epitope (an459-606). Re-suits The research showed a novel potential neutralizing epitope in aa423-437 of HEV ORF2 though detec-ting and comparing the characteristics of several antibodies and corresponding determinations. The epitope is a linear non-immunodominant epitope which is different from the other neutralizing epitope in aa459-606.And the amino acids sequence of this novel epitope is conservative. Conclusion ORF2 aa423-437 is a no-vel potential neutralizing leaner epitope of HEV. It is believed that the present work adds fundamental knowl-edge to our understanding of HEV capsid domain and contributes to the prevention and control of this dis-ease.

Objective To express the recombinant caspid of genotype 4 hepatitis E virus(HEV) ORF2. Methods HEV recombinant capsid protein D66 was expressed in E. coli, using the ORF2 fragment (aa368-606, obtained from swine bile) of genotype 4 HEV. Results The recombinant capsid proteins D66 self-assemble to be particle with a radius of 13 nm through dimeric form in neutral solution. Coated particles reacted well with sera obtained from patients during acute or recovered phase of HEV infection. Immunofluorescence and immnoblot assay suggested that D66 bound and penetrated HepG2 cell lines, and the process of attachment was blocked by sera collected from patients during acute or recovered phase of HEV infection.Conclusion Recombinant D66 particles simulate the structure at the surface of genotype 4 HEV well and specifically adhere and penetrate the host cells, which lays the foundation for the investigation of the molecular mechanism of genotype 4 HEV infection.

OBJECTIVETo investigate the association of mutations and expression of MUS81 gene with the tumorigenesis and progression of laryngeal squamous cell carcinoma (LSCC).

METHODSPCR-SSCP and DNA sequencing were carried out to examine mutations at exons 9 and 10 of MUS81 gene in 42 LSCC samples, with paired adjacent normal laryngeal tissues (PANLs) as control. Semi-quantitative RT-PCR and Western blot were used to detect the expression of MUS81 gene in the specimens.

RESULTSNo mutation was detected in the control group. Among the 42 LSCC specimens, nineteen (45.2%) were found to harbor mutations, including 11(26.2%) occurring within exon 9, and 8 (19%) within exon 10. Seventeen (40.48%) samples showed lower mRNA level of the MUS81 gene (P<0.01), and same proportion of samples had lower protein level (P<0.01), suggesting that MUS81 gene was similarly down-regulated at both mRNA and protein levels in the LSCC samples. Furthermore, mutations of MUS81 gene did not significantly correlate with TNM stages, age and lymphoid node metastasis (P>0.05). Nor did the expression of MUS81 gene with the TNM stages, age and lymphoid node metastasis in LSCC (P>0.05).

CONCLUSIONMutations and abnormal expression of MUS81 gene in the LSCC tissues were observed, which suggested that abnormalities of MUS81 gene may play an important role in the tumorigenesis of LSCC.

Age Factors ; Base Sequence ; Carcinoma, Squamous Cell ; genetics ; pathology ; DNA-Binding Proteins ; genetics ; Endonucleases ; genetics ; Exons ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Laryngeal Neoplasms ; genetics ; pathology ; Lymphatic Metastasis ; genetics ; Molecular Sequence Data ; Mutation ; Neoplasm Staging ; RNA, Messenger ; genetics ; metabolism

Objective:To explore the role of virtual reduction and design of 3D printed guide template in assisting reduction and internal fixation of comminuted patellar fractures.Methods:A retrospective study was conducted of 12 patients who had been treated for closed patellar fractures from March 2016 to April 2019 at Department of Orthopaedics, Jiangxi Provincial People's Hospital. They were 8 males and 4 females with an average age of 35.4 years (from 22 to 51). All their fractures were type 34-C3 (more than 3 fragments) according to the AO/OTA classification. Their preoperative CT data were imported into software Mimics to print a full patella, upper and lower halves of the patella, and a guide template for reduction. During operation, bone fragments were first temporarily fixated with a fine Kirschner wire after combined together with the assistance of reduction templates for upper and lower hemi-patellas. Next, the whole patella was precisely reset and definitely fixated using patellar ring ligation and tension band wire with the assistance of the reduction guide template for full patellar. Recorded were operation time, fluoroscopic frequency, and visual analogue scale (VAS), knee range of motion (ROM) and knee function B?stman scores at the last follow-up.Results:In this group of patients, operation time averaged 90 minutes (from 75 to 120 minutes), fluoroscopic frequency 4.5 times (from 3 to 8 times), follow-up duration 19 weeks (from 16 to 22 weeks), and fracture healing time 14 weeks (from 11 to 17 weeks). At their last follow-up, X-ray showed good patellar morphology, fine internal fixation positions and an average VAS pain scores of 0.3 (from 0 to 1), their knee joints could be fully straightened, their knee ROM averaged 130°(from 100° to 138°), and their knee B?stman scores 28.8 points (from 21 to 30 points), giving 9 excellent and 3 good cases.Conclusion:3D printed reduction guide templates can significantly improve the efficiency and quality of surgical reduction in comminuted patella fractures, enhancing fixation strength and facilitating early and full range exercise of the knee joint to achieve excellent recovery.