With the improvement of people's living standard and changes of their lifestyle, the incidence of coronary artery disease (CAD) has increased quite significantly, which has attracted great concern. Examination methods for CAD are more and more advanced, while the cost of inspection is rising. As a traditional test, the exercise treadmill test (ETT) is convenient, safe, and cheap. ETT can be conducted in almost every hospital as it doesn't need advanced equipments. The values of some new evaluation indexes for diagnosis and prognosis of CAD are reviewed in this article.

Objective To explore the diagnosis and differential diagnosis of tricholemmal carcinoma and the significance of expression of cytokeratin(CK)and oncogene protein(c-erbB-2)in it.Method Twenty-two cases of tricholemmal carcinoma were studied with histologic and immunohistochemical tech-niques,and compared with those in squamous cell carcinoma.The neoplastic cells were stained with cytok-eratin-H,cytokeratin-L and c-erbB-2immunohistochemically(S-P method).Results The morphological characteristics of the tricholemmal carcinoma were the lobular structure and sudden keratinization in the cen-ter of the lobule,which were the key points to be differentiated with squamous cell carcinoma.The positive rate of cytokeratin-H of tricholemmal carcinoma was81.8%(18/22),and the positive cells distributed in the middle zone between the keratinized lobular center and tumor cells in the peripheral area,which differed from squamous cell carcinoma.Cytokeratin-L expression was not found in tricholemmal carcinoma.The posi-tive rate of c-erbB-2was50%(11/22)in tricholemmal carcinoma,with positive cells distributed to the mar-gin of the carcinoma cell nests,and increased with the poor differentiation.Conclusions The particular dis-tribution of cytokeratin-H suggests the tricholemmal origin of tricholemmal carcinoma.C-erbB-2may take part in the activation and differentiation of this tumor.

Objective To investigate whether there has any difference of gastric and small bowel transit time and completion rates between two capsule endoscopes with different size and weight. Methods Clinical data of patients who had undergone OMOM or MiroCam (smaller and lighter than OMOM) capsule endoscopy were retrospectively studied. Comparison of gastric and small bowel transit time and completion rates were made between the two kinds of capsule endoscopy. Results 1, 448 patients (628 in OMOM group and 820 in MiroCam group) were finally includ-ed. In patients with Crohn's disease or suspected Crohn's disease, gastric transit time of OMOM was significantly longer than that of MiroCam [(53.4 ± 52.6) minutes vs (41.1 ± 47.9) minutes, = 0.022]. In patients with gastroin-testinal bleeding, gastric transit time in OMOM was significantly shorter than that in MiroCam [(42.1 ± 44.8) minutes vs (62.0 ± 78.6) minutes, = 0.016). No significant difference in small bowel transit time or completion rate was found. Conclusions We conclude that the differences of gastric transit time, small bowel transit time and completion rates between the two kinds of capsule endoscopy with different size and weight are not significantly. Whereas, in patients with Crohn's disease or suspected Crohn's disease, gastric transit time of smaller and lighter capsule en-doscopy is shorter in patients with gastrointestinal bleeding, but longer of gastric transit time in smaller and lighter capsule endoscopy.

Objective To evaluate the efficacy and safety of high-frequency electric dissection for colorectal cysts.Methods The personal information,clinical data,operation methods and postoperative complications of patients who were diagnosed as having colorectal cysts and underwent high-frequency electric dissection in Nanfang hospital and Zhujiang Hospital of Southern Medical University between January 1st,2005 and July 1st,2015 were analysed.All patients enrolled in the study were followed up to obtain their resuits of colonoscopy.Results A total of 63 patients were enrolled into our study,9 lesions located in the ileocecus,17 in the ascending colon,19 in the transverse colon,10 in the descending colon,7 in the sigmoid colon and 1 in the rectum.The maximum diameter of the cysts was 20.2+7.5 mm (5-40 mm).All patients underwent high-frequency electric dissection to remove the cysts completely or part of the cyst wall for drainage.Hemorrhage occurred in only one patient and bleeding stopped after being clipped by Titanium clip.Forty-five patients were followed up and there were no delayed complications or recurrence during a postoperative follow-up of 24.1 + 14.3 months (6-87 months).Conclusion High-frequency electric dissection is a safe and effective procedure for the treatment of colorectal cysts.

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein- mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-tansfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors,encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.

Objective To establish a simple and effective method for isolation and culture of mouse adipose-derived stem cells (mASCs)in vitro,in order to provide the sufficient sources of seed cells for the research of mesenchymal stem cells.Methods The mouse inguinal fat tissues were isolated in vitro and performed a digestion with 0.1% collagenase type NB4,then adipose-derived stem cells(ASCs)were seeded and adhered to the culture dishes in low glucose DMEM containing 10% fetal calf serum.The cellu-lar morphology,in vitro proliferation capacity,multidifferentiation potential and immunophenotype were assessed.Results The mASCs showed good cell morphology,extremely strong proliferation capacity and potential of adipogenesis,osteogenesis and chon-drogenesis via in vitro three-dimensional induction.The cellular surface antigen phenotype was consistent with that reported by lit-erature,and the expression of CD34 and CD105 was positive,Sca-1 was highly expressed,CD45 and SSEA-1 were not expressed. Conclusion Using the experimental methods in this research can culture the high purity of mASCs with the excellent stem cell properties and extremely strong proliferative ability.

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.