[Advances in the research of delivery system of growth factor and the gene for promoting wound healing].

Growth factors play critical roles in the process of wound healing. Application of growth factor locally is a good way of promoting wound healing, while it is easy to be hydrolyzed in wounds and its efficacy has dose- and time-dependent manner. Therefore, appropriate growth factor delivery system is needed to assist it to function in wounds. In addition to delivering growth factor directly to wounds, viral and non-viral vectors can be used for gene transfection of growth factor in wounds. The gene can be transformed to growth factor to promote wound healing by transcription and translation. This article reviews the advances in the research of delivery system of growth factor and the gene for promoting wound healing.

[Short-term clinical outcomes and risk factors associated with in-hospital mortality in patients undergoing off-pump coronary artery bypass grafting].

Objective: To explore short-term clinical outcomes and risk factors associated with in-hospital mortality in patients undergoing off-pump coronary artery bypass grafting (OPCABG) and establish a prediction model for in-hospital mortality. Methods: The clinical data of patients undergoing OPCABG in Beijing Anzhen Hospital between January 2014 and January 2016 was retrospectively studied. Univariate analysis and logistic regression were applied to determine the potential risk factors, and then a prediction model for mortality was confirmed. The calibration and discrimination of the prediction model was finally tested. Results: A total of 2 546 patients who underwent OPCABG were recruited. In-hospital mortality of OPCABG was 0.7% (17 cases). Seven variables: female, age, left main disease >50%, low left ventricular ejection fraction (LVEF), acute myocardial infarction before surgery, operative status (selective or emergent), moderate concomitant mitral valve regurgitation were independently correlated with OPCABG mortality (all P<0.05). The result of Hosmer-Lemeshow test was χ(2)=5.912, P=0.676. The area under receiver-operating characteristic curve (ROC) was 0.881. Conclusions: OPCABG is safe and effective for myocardial revascularization in a short term. The following risk factors are associated with an increased operative mortality of OPCABG: male, age, left main disease >50%, low LVEF, acute myocardial infarction before surgery, operative status (selective or emergent), moderate concomitant mitral valve regurgitation. The prediction model established by above-mentioned potential risk factors was proven to perform well by statistical tests.

[Advances in the research of three-dimensional skin printing].

As a new technology, three-dimensional printing possesses the characteristics of high precision and strong controllability, which has become a new technology and can be used in tissue engineering. Currently, using three-dimensional printing to build artificial skin has made certain achievement, and experiments in vitro have confirmed that the three-dimensional printing has the possibilities to build artificial skin whose structure and function are close to those of nature skin. However, the technology is not yet very mature and there are still some problems need to be solved, such as the recreation of the cutaneous appendages and the degradation and absorption of the extracellular matrix.

[Advances in the research of application of collagen in three-dimensional bioprinting].

As a new industrial technology with characteristics of high precision and accuracy, the application of three-dimensional bioprinting technology is increasingly wide in the field of medical research. Collagen is one of the most common ingredients in tissue, and it has good biological material properties. There are many reports of using collagen as main composition of " ink" of three-dimensional bioprinting technology. However, the applied collagen is mainly from heterogeneous sources, which may cause some problems in application. Recombinant human source collagen can be obtained from microorganism fermentation by transgenic technology, but more research should be done to confirm its property. This article reviews the advances in the research of collagen and its biological application in three-dimensional bioprinting.

[Efficacy of a hydrosurgery system applied in the debridement of extensive residual wounds of patients with severe burn].

OBJECTIVE: To explore the efficacy of a hydrosurgery system applied in the debridement of extensive residual wounds of patients with severe burn prospectively. METHODS: Seventy-six severely burned patients with extensive residual wounds after treatment were admitted into Department of Burn Surgery of Changhai Hospital of the Second Military Medical University from May 2012 to November 2015. The patients were divided into hydrosurgery system debridement group (HD, n=34) and traditional debridement group (TD, n=42) according to the random number table, and their residual wounds were debrided by a hydrosurgery system and conventional surgical instruments respectively. The wounds of patients in two groups were repaired with stamp-like grafts made from split-thickness autoskin after debridement. Second time of debridement and skin grafting as above was performed if necessary. The time used in debriding 1% TBSA wound in the first surgery, positive rate of bacterial culture of wound exudate on post first surgery day (PFSD) 3, survival rate of skin grafts on PFSD 7, second debridement rate, and wound healing time of patients in two groups were compared. Data were processed with two independent-sample t test and chi-square test. RESULTS: The time used in debriding 1% TBSA wound of patients in the first surgery in group HD was (1.2±0.3) min, which was significantly shorter than that in group TD[(1.9±0.4) min, t=-9.098, P<0.001]. On PFSD 3, positive rate of bacterial culture of wound exudate of patients in group HD was 11.76% (4/34), which was significantly lower than that in group TD[38.10% (16/42), χ(2)=6.718, P=0.010]. On PFSD 7, the survival rate of skin grafts of patients in group HD was (90±8)%, which was significantly higher than that in group TD[(83±15)%, t=2.334, P=0.022]. The second debridement rate of patients in group HD was 5.88% (2/34), which was significantly lower than that in group TD[23.81% (10/42), χ(2)=4.542, P=0.033]. The wound healing time of patients in group HD was (10.6±1.5) d, which was significantly shorter than that in group TD[(13.3±2.5) d, t=-5.442, P<0.001]. CONCLUSIONS: The hydrosurgery system applied in the debridement of extensive residual wounds of patients with severe burn is thorough and highly efficient, which is beneficial to improving the survival rate of skin grafts and accelerating wound healing.

Mechanism of lower genotoxicity of toremifene compared with tamoxifen.

An increased incidence of endometrial cancer has been reported in breast cancer patients taking tamoxifen (TAM) and in healthy women participating in the TAM chemoprevention trials. Because TAM-DNA adducts are mutagenic and detected in the endometrium of women treated with TAM, TAM adducts are suspected to initiate the development of endometrial cancer. Treatment with TAM has been known to promote hepatocarcinoma in rats, but toremifene (TOR), a chlorinated TAM analogue, did not. TAM adducts are primarily formed via sulfonation of the alpha-hydroxylated TAM metabolites. To explore the mechanism of the lower genotoxicity of TOR, the formation of DNA adducts induced by TOR metabolites was measured using (32)P-postlabeling/ high-performance liquid chromatography analysis and compared with that of TAM metabolites. When alpha-hydroxytoremifene was incubated with DNA, 3'-phosphoadenosine 5'-phosphosulfate, and either rat or human hydroxysteroid sulfotransferase, the formation of DNA adducts was two orders of magnitude lower than that of alpha-hydroxytamoxifen. alpha-hydroxytoremifene was a poor substrate for rat and human hydroxysteroid sulfotransferases. In addition, the reactivity of alpha-acetoxytoremifene, a model activated form of TOR, with DNA was much lower than that of alpha-acetoxytamoxifen. Thus, TOR is likely to have lower genotoxicity than TAM. TOR may be a safer alternative by avoiding the development of endometrial cancer.

Bacterial expression, purification, and characterization of rat hydroxysteroid sulfotransferase STa.

Hydroxysteroid (alcohol) sulfotransferase catalyzes numerous reactions that are important to our understanding of the metabolism of both endogenous steroids and exogenous alcohols. Here we report a method for prokaryotic expression and rapid purification of the recombinant hydroxysteroid sulfotransferase STa, a major isoform of hydroxysteroid sulfotransferase in the rat. The cDNA encoding STa was cloned into a pET-3c vector and expressed in Escherichia coli BL21 cells. After disruption of the cells by sonication, the enzyme was purified in one step by affinity chromatography on adenosine 3',5'-diphosphate-agarose. The purified recombinant STa had a relative molecular mass on SDS-PAGE that was identical with the native hepatic STa in rat liver. The expressed enzyme displayed similar substrate inhibition characteristics with dehydroepiandrosterone as have been noted previously with the native enzyme purified from rat liver. Furthermore, the catalytic efficiency in sulfation of 7-hydroxymethyl-12-methylbenz[a]anthracene, as well as the stereoselectivity of sulfation of the enantiomers of 1-phenyl-1-heptanol and 1-naphthyl-1-ethanol, catalyzed by the recombinant STa were consistent with characteristics of the STa isolated from rat liver.

Measurement of aryl and alcohol sulfotransferase activity.

Two methods are presented for determining the catalytic activity of aryl and alcohol sulfotransferases. The first assay is a simple and rapid procedure that is based on the extraction of a paired ion formed between the product organic sulfate and methylene blue. The second method employs HPLC analysis of the substrate-dependent formation of PAP, an assay that is particularly useful when the sulfuric acid ester product is chemically unstable.

Expression of CYP2A genes in rodent and human nasal mucosa.

CYP2A10 and CYP2A11, which are abundant in olfactory microsomes from rabbits, are active in the metabolic activation of a number of nasal toxicants, such as hexamethylphosphoramide and N-nitrosodiethylamine. Previous immunohistochemical studies indicated that CYP2A-related cytochromes P450 may also be present in rodent and human olfactory tissue. In the present study, the expression of cytochromes P450 highly homologous to rabbit CYP2A10/11 in rat, mouse, and human nasal mucosa was studied. In Sprague-Dawley rats, CYP2A3 mRNA was detected in olfactory mucosa at levels much higher than those found in total RNA from lung. Similar observations were made for the level of microsomal CYP2A3 protein with the use of antibodies to rabbit CYP2A10/11. However, mRNAs for two other rat cytochrome P450 genes in the CYP2A subfamily, CYP2A1 and CYP2A2, were not detected in nasal tissue by RNA-polymerase chain reaction analysis. In C57BL/6 mice, both CYP2A4 and CYP2A5 mRNAs were detected in the olfactory mucosa by RNA-polymerase chain reaction, but the CYP2A5 transcript was present at a level much higher than that of CYP2A4. The expression of another mouse gene in CYP2A subfamily, CYP2A12, was not detected in nasal tissue. CYP2A5 protein was also detected in mouse olfactory microsomes at higher levels than in liver, lung, or kidney microsomes. However, no significant sex differences in the levels of CYP2A4/5 mRNA or microsomal coumarin 7-hydroxylase activity were found with the nasal tissue. In addition, consistent with previous immunohistochemical studies, the expression of CYP2A6 in human nasal mucosa was detected by RNA-polymerase chain reaction as well as RNA blot analysis. The identification of CYP2A6 in human nasal tissues may have important implications for risk assessment of potential nasal toxicants, and the abundant expression of the CYP2A genes in rat and mouse olfactory tissue suggests a molecular basis for the known tissue-specific toxicity of numerous inhaled compounds in rodents.

Metabolic activation of 2,6-dichlorobenzonitrile, an olfactory-specific toxicant, by rat, rabbit, and human cytochromes P450.

The herbicide 2,6-dichlorobenzonitrile (DCBN) is known to cause tissue-specific toxicity at very low doses in the olfactory mucosa of rodents. The toxicity of DCBN is reportedly cytochrome P450 (P450) dependent, but the isoforms involved have not been identified, and the effects of this agent on humans are not known. In the present study, DCBN metabolism was examined with microsomes and with purified P450s in a reconstituted system. Rat and rabbit olfactory microsomes act on DCBN to form DCBN-protein adducts as well as two metabolite peaks, designated M1 and M2, identified through high performance liquid chromatography with radiometric detection. The activity of rat olfactory microsomes in DCBN metabolism is much higher than that of liver or lung microsomes. Of seven purified rabbit P450s known to be expressed in the olfactory mucosa, including 1A2, 2A10/11, 2B4, 2E1, 2G1, and 3A6, the 2A10/11 preparation is the most active, producing M2 as well as DCBN-protein adducts; P450 2E1 is the only other active isoform. The addition of purified epoxide hydrolase (EC 4.2.1.63) to the reconstituted enzyme system leads to the formation of M1 and decreased formation of M2. It seems that M1 and M2 are derived from an epoxide intermediate that also forms covalent protein adducts. Gas chromatography- and liquid chromatography-mass spectrometry analyses of nasal microsomal DCBN metabolites and DCBN-glutathione conjugates indicated that the major reactive intermediate may be 2,3-oxo-DCBN and that M1 may be 2,3-dihydroxy-6-chlorobenzonitrile, whereas M2 may correspond to a monohydroxy-DCBN. Interestingly, heterologously expressed human P450s 2A6 and 2E1, but not 1A2, are active in the metabolism of DCBN, forming protein adducts as well as M2. Thus, the preferential expression of P450s of the 2A subfamily in olfactory tissue suggests a molecular basis for the tissue-specific toxicity of the herbicide and may have important implications for risk assessment in humans.