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A novel protein encoded by the open reading frame 4 (ORF4) was recently discovered in porcine circovirus type 2 (PCV2). However, little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2. In the present study, the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST, yielding recombinant plasmid pCMV-N-Flag-GST-ORF4. The recombinant plasmid was transfected into 293T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE, sequentially. The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry (MS). Finally, five candidate ORF4-interacting proteins, including Serine/threonine-protein phosphatase 6 catalytic subunit, alpha cardiac muscle 1, actin, SEC14-like protein 5 and myosin 9 were identified. These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.
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Animais , Humanos , Infecções por Circoviridae , Circovirus , Células HEK293 , Espectrometria de Massas , Fases de Leitura Aberta , Suínos , Proteínas ViraisRESUMO
BACKGROUND: Adipose-derived stem cells (ADSCs), a kind of adult stem cells, possess plasticity and can be induced into myocardial cells under certain conditions. Autologous ADSCs transplanted into the infarct area can differentiate into myocardial cells and vascular endothelial cells to construct new vessels and thereby improve cardiac pump function. OBJECTIVE: To study the factors that influence ADSCs differentiation and transplantation and the current clinical and laboratory research progress of ADSCs transplantation for treatment of cardiomyopathy.METHODS: A computer-based retrieval was performed in Medline (between January 1990 and April 2010), PubMed database, the China Biological Medicine Database (CBM) (between January 1990 and April 2010), and China National Knowledge Infrastructure (CNKI) with the keywords adipose-derived stem cells, myocardial cells, cell differentiation, cell transplantation, cardiomyopathy treatment.RESULTS AND CONCLUSION: A total of 30 articles, consisting of 6 reviews and 24 randomized controlled trials, were obtained. At present, there have been uniform methods of ADSCs isolation and culture, and ADSCs can be effectively proliferated in vitro, but there have been no direct methods to identify these stem cells. ADSCs differentiation can be induced both in vitro and in vivo, besides, with a characteristic of early differentiation. ADSCs transplantation is a more conductive therapy for myocardial disease compared with bone marrow stem cells (BMSCs) transplantation. Different ADSCs transplantation methods should be carried out in different types of cardiomyopathy. Stem cell labeling technique can help to dynamically monitor implanted in vivo. Transplantion of autologous ADSCs is a new way to treating cardiomyopathy. However, for successes in clinical practice, the method to inhibit tumor cells-promoting characteristics is needed to ensure long-term safety of the patients receiving ADSCs transplantation.
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BACKGROUND: Based on our previous researches in mechanism studies and clinical applications of human hair keratin (HHK), a new concept "in vivol in situ tissue engineering" has been proposed. Under the guidance of this theory, a scaffold of HHK-collagan sponge (inner layer) combined with poly (2-hydroxyethyl methacrylate) (PHEMA) (outer layer as a drug delivery carrier) would be developed to investigate its feasibility to be as a dermal dressing. OBJECTIVE: To develop a scaffold composed of HHK-collagan sponge (inner layer) combined with PHEMA film containing polydatin(PD)(outer layer as a drug delivery carrier) and to evaluate the therapeutic efficacy of the HHK-collagen sponge-PHEMA/PD complex on burn wound healing. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the Department of Histology and Embryology, Southern Medical University between March and December 2005. MATERIALS: Burn was induced in 15 male Sprague-Dawiey (SD) rats, Rat models of burn were evenly randomized to 3 groups: experimental, positive control, and negative control. METHODS: ①HHK-collagen sponge was prepared through combination of a HHK meshwork (1mm × 1 mm in size for each grid) made up of three components (determined according to biochemical procedures of various degrees, i.e., light, medial, and severe) at a ratio of 4:3:3 with primary collagen sponge extracted from bovine tendons in a mould. Sponge film (used as inner layer dressing) was made by vacuum freeze-drying. ② PHEMA was prepared by polymerization. Than PD was added to prepare PHEMNPD film (used as outer layer dressing).③ Degree Ⅱ burn wound models were established in SD rats by scalding, Superficial necrotic tissue was removed from burn wounds at postnatal 3 days and leave the denatured dermis remained. The wounds were either covered with human HHK-collagen- PHEMNPD complex in the experimental group, or with glutaraldehyde-treated porcine skin in the positive control group, and sterile absorbent gauze was used in the negative control group. MAIN OUTCOME MEASURES: ① Complete epithelization was taken as the standards, and at postoperative 7, 14, and 21 days, wound healing was respectively calculated. ② At postoperative 1, 2, 4, 6, and 8 weeks, the whole wound surface and its peripheral tissue were dissected for observing granulation tissue growing under an optical microscope and detecting the collagen fiber and elastic fiber in the newly formed tissue by immunohistochemical staining. RESULTS: ① Gross observation results revealed that in the experimental group, the volume of the diffusate under the ideal moisture was less compared with the positive control group; the healing time was slightly shorter in both the experimental group and the positive control group than in the negative control group (P= 0.000); At postoperative 7, 14, and 21 days, the healing rate was higher in the experimental and positive control groups than in the negative control group (P=0.000), in addition, the experimental group exhibited higher healing rate than the positive control group at postoperative 14 days ( P ＜ 0.05). ②Optical microscope results showed that at postoperative 2 weeks, a small quantity of collagen fibers were found in the wound granulation tissue in all 3 groups, in particular in the experimental group. Immunohistochemical staining results regarding collagen protein and elastin revealed that at postoperative 2 weeks, both the fine strip-like type Ⅰ collagen fibers and a few silk-like elastic fibers were stained yellowish-brown in the dermal matrix in the experimental group, which were weakly positive in the positive control group, while there was no elastin detectable in the negative control group; at postoperative 8 weeks, burn wounds in all the 3 groups werefully recovered. Remodeling of collagen fibers was more obvious in the experimental and positive control groups than in thenegative control group, while the tendency to scar formation with derangement of epithelial cells and collagen fibers in dermis was more prominent in the negative control group than in the remaining two groups.CONCLUSION: HHK-collagen sponge-PHEMA/PD complex may be a new burn dressing via in vivo construction of tissueengineered epidermis, in which PHEMA may be a feasible drug-delivery carrier.
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BACKGROUND: The grafted human hair keratin(HHK) artificial tendon,being degradable and absorbable, can induce formation of neotendon. In the present study, the main biomechanical indexes of HHK artificial tendon and its components were measured so as to determine a proper ratio for the artificial tendon composition according to different needs of the body sites and make it possess tensile stress required for functional activities.OBJECTIVE: To determine the main biomechanical indexes of HHK artificial tendon and its components.DESIGN: An observational study based on HHK artificial tendon.SETTING: Department of Biochemistry and Molecular Biology, Medical Biomechanical Key Laboratory of Chinese PLA, and Department of Histology and Embryology, a military medical university; Foshan Kangxing Medical Technology Co., Ltd.MATERIALS: The experiment was completed at the Medical Biomechanical Key Laboratory of the First Military Medical University of Chinese PLA in October 2001. The material of HHK artificial tendons was uncontaminated black hair collected from healthy young women in remote mountainous areas free from endemic diseases. There were three components of HHK artificial tendons with different in vivo absorption velocities Z, B and F. The in vivo absorption was the slowest for Z, faster for B, and the fastest for F. Different kinds of HHK artificial tendon were the mixture of Z, B and F at a proper ratio. Each group had 3 kinds, namely, Z3.0-20, B3.0-20, and F3.0-20. HHK artificial tendon consisted of 6 kinds: ZBF1. 0-20,ZBF1.5-20, ZBF3.0-20, ZBF6. 0-20, ZBF9. 0-20, and ZBF12.0-20.METHODS: Both ends of an HHK artificial tendon or each of its components were fixed on MTS 858 Moni Bionix checker to determine such biomechanical indexes as fracturing pull and tensile stress.MAIN OUTCOME MEASURES: Fracturing pull and tensile stress of HHK artificial tendon of different kinds and its components.RESULTS: Fracturing pull in Z3.0-20, B3.0-20 and F3.0-20 groups was (211.8 ± 12. 1 ), ( 178.8 ± 8.2), and ( 151.3 ± 6. 7) N, respectively; tensile stress was(71.3 ±3.9), (59.9 ±2. 7), and(50. 3 ±2.3) Mpa, respectively. Fracturing pull and tensile stress were decreased gradually in the same cross-section area of the 3 kinds of materials; Fracturing pull in ZBF1.0-20,ZBF1.5-20, ZBF3.0-20, ZBF6.0-20, ZBF9.0-20, and ZBF12.0-20 groups was(75.0 ± 3.0), (107.8±4.2), (194.1±5.3), (375.9±12.7),(508.2 ±21.3), and(670.8 ±25.4) N, respectively, while tensile stress was(75.0 ±3.0), (72.0 ±2.8), (65.1 ± 1.8), (62.9 ±2.2), (56. 3 ±2.4),and(55.8 ± 1.5) Mpa, respectively.CONCLUSION: Fracturing pull of HHK artificial tendon is increased with the increase of cross-section area, whereas tensile stress is slightly decreased with the increase of cross section area. With the increased extent of human hair treatment, its absorption in vivo is accelerated, and fracturing pull and tensile stress of HHK artificial tendon are decreased.
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BACKGROUND: Numerous experiments and clinical practice show that human hair keratin artificial tendon induces the organism to form autogenous tendon. The process of autogenous tendon formation mainly involves the synthesis, secretion and package of collagen subtype I.OBJECTIVE: To explore the role of collagen subtype I mRNA expression in autologous tendon formation after human hair keratin artificial tendon implantation.DESIGN: A completely randomized controlled experiment based on the experimental animals.SETTING: Department of Histology and Embryology and Department of Biochemistry and Molecular Biology of Southern Medical University.MATERIALS: The experiment was conducted in the Experimental Animal Center of the First Military Medical University of Chinese PLA from May 2003 to September 2004. Totally 33 New Zealand rabbits of either gender,weighing 2.0 to 2. 5 kg, were provided by the center. The animals were randomly divided into experiment 3, 6, 9, 12, 16 and 20 weeks groups, negative control 9 and 20 weeks groups and normal control group. Among them,experiment 3 and 6 weeks group and normal control group had 3 rabbits in each and the other groups had 4 rabbits. Human hair keratin artificial tendons were normal human hair treated by a series of biochemical methods and were supplied by the Department of Biochemistry & Molecular Biology of the university. The human hair keratin artificial tendons were divided into three groups with different degradation rates, namely, fast(F), medium(B) and slow(Z). The tendons were made up of the fast, medium and slow degradation groups mixed at the ratio of 4: 3: 3.off by 1.0- 2.0 cm, human hair keratin artificial tendon was grafted by end-to-end anastomosis with both ends of the broken tendon before sewing control group, no artificial tendon was implanted although the animals ungroup was normal rabbits' tendon. Sampling was carried out at 3, 6, 9, 12, 16and 20 weeks after human hair keratin artificial tendon implantation in experiment groups, and at 9 and 20 weeks after operation in negative control group, respectively. The expression of collagen subtype I mRNA was detected at weeks 3, 6, 9, 12, 16 and 20 after grafting using reverse transcription polymerase chain reaction technique.MAIN OUTCOME MEASURES: The ratio of collagen subtype I mRNA to Glyceraldehyde-3-phosphate dehydrogenase(GADPH) mRNA in normal tendon and autogenous tendon induced by human hair keratin artificial tendon at all time points was calculated, and significance test between all these paired groups were performed.collagen subtype I mRNA/GADPH mRNA expression was 0.96 ±0.02 in expression of collagen subtype I mRNA/GADPH mRNA in autogenous tendon induced by human hair keratin artificial tendon in experiment group appeared at week 3, increased rapidly at week 3 to 6, peaked at week 6, and remained stable at week 9 to 20. The expression at week 6 was significantly higher in experiment group than in normal control group( F = 6. 254, P ＜ 0.05); the expression at other weeks was also significantly higher in experiment group than in normal control group( F= 1. 258 - 1. 987, P ＞ 0.05).CONCLUSION: The activation, proliferation and secretion of collagen protein as well as the synthesis of collagen subtype I by tenocytes may be responsible for autologous tendon formation after human hair keratin artificial tendon implantation.
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Objective To prepare huperzine A-carrier protein conjugate and determine its immunogenicity for later production of monoclonal antibody against HupA and establishing an enzyme-linked immunosorbent assay ( ELISA) -based detection method for HupA. Methods HupA was conjugated with bovine serum albumin ( BSA) using glutaraldehyde ( GA) method to obtain the HupA-GA-BSA conjugate,and the hapten number in the conjugate was determined by matrix-assisted laser absorption ionization time-of-flight mass spectrometry ( MALDI-TOF-MS) . BALB /c mice were immunized with HupA-GA-BSA to prepare the antiserum against HupA. The serum titer and specificity of the HupA antibodies were detected by indirect ELISA and competitive ELISA,respectively. Results The conjugation ratio of HupA and the carrier protein BSA was 8∶ 1. The antiserum against HupA with a titer of 1∶ 62 500 was obtained after immunizing the mice with the conjugate,and the antiserum reacted specifically to HupA. Conclusion The synthesized HupA-GA-BSA conjugate possesses good immunogenicity in mice,suggesting the feasibility of preparing the monoclonal antibody against HupA using this conjugate.
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The effect of plasma middle molecule substances (MMS) on the Ca2+-ATPase activity in human erythrocyte membranes was studied. The results showed that the Ca2+-ATPase activity was significantly inhibited when the membrane preparation was incubated with MMS at 37℃. The longer the period of time for incubation, the lower the Ca2+-ATPase activity. The activity of Ca2+-ATPase could be significantly inhibited by plasma MMS of both normal person and burn patient. The inhibitory rate (%) of Ca2+-ATPase activity was elevated markedly when MMS concentration was increased (P