Effects of porcine acellular dermal matrix combined with human epidermal stem cells on wound healing of full-thickness skin defect in nude mice / 中华烧伤杂志

Objective: To explore the effects of porcine acellular dermal matrix (ADM) combined with human epidermal stem cells (ESCs) on wound healing of full-thickness skin defect in nude mice. Methods: The morphology of porcine ADM was analyzed by photograph of digital camera, the cell residues in porcine ADM were observed by hematoxylin-eosin (HE) staining, the surface structure of porcine ADM was observed by scanning electron microscope, the secondary structure of porcine ADM was analyzed by infrared spectrometer, the porcine ADM particle size was analyzed by dynamic light scattering particle size analyzer, and the porcine ADM potential was analyzed by nano-particle size potentiometer. The morphology of porcine ADM was observed by inverted fluorescence microscope when it was placed in culture medium for 30 min, 1 d, and 5 d (n=2). The porcine ADM was divided into 5 min group, 10 min group, 20 min group, 30 min group, 60 min group, and 120 min group according to the random number table (the same grouping method below) in static state at normal temperature for the corresponding time to calculate the water absorption by weighing method (n=3). Swiss white mouse embryonic fibroblasts (Fbs) were divided into blank control group (culture medium only), and 50.0 g/L ADM extract group, 37.5 g/L ADM extract group, 25.0 g/L ADM extract group, 12.5 g/L ADM extract group, and 6.5 g/L ADM extract group which were added with the corresponding final concentrations of ADM extract respectively. At post culture hour (PCH) 24, 48, and 72, the cell survival rate was detected by cell counting kit 8 and the cytotoxicity was graded (n=5). The erythrocytes of a 6-week-old male Sprague-Dawley male rat were divided into normal saline group, ultra-pure water group, and 5 mg/mL ADM extract group, 10 mg/mL ADM extract group, and 15 mg/mL ADM extract group which were treated with the corresponding final concentrations of porcine ADM extract respectively. After reaction for 3 h, the absorbance value of hemoglobin was detected by microplate reader to represent the blood compatibility of porcine ADM (n=3). ESCs were isolated and cultured from the discarded prepuce of a 6-year-old healthy boy who was treated in the Department of Urology of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) in July 2020, and then identified by flow cytometry. The porcine ADM particles of composite ESC (hereinafter referred to as ESC/ADM) were constructed by mixed culture. After 3 days of culture, the composite effect of ESC/ADM was observed by HE staining and laser scanning confocal microscope. Thirty-six 7-8-week-old male non-thymic nude mice were divided into phosphate buffer solution (PBS) alone group, ADM alone group, ESC alone group, and ESC/ADM group, with 9 mice in each group, and the wound model of full-thickness skin defect was established. Immediately after injury, the wounds were treated with the corresponding reagents at one time. On post injury day (PID) 1, 7, 11, and 15, the wound healing was observed and the wound healing rate was counted (n=3). On PID 7, the epithelialization of wounds was observed by HE staining and the length of un-epithelialized wound was measured (with this and the following sample numbers of 4). On PID 11, the dermal area and collagen deposition of wounds were observed by Masson staining and the dermal area of wound section was calculated, the number of cells expressing CD49f, a specific marker of ESC, was calculated with immunofluorescence staining, the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in ESC after wound transplantation was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, and least significant difference t test. Results: The porcine ADM was white particles and composed of reticular structure, with no cells inside, disordered structure, and rough surface. The absorption peak of porcine ADM appeared at the wave numbers of 1 659, 1 549, and 1 239 cm-1, respectively. The main particle size distribution of porcine ADM in solution was 500 to 700 nm, with negative charge on the surface. The morphology of porcine ADM in static state at 30 min and on 1 and 5 d was relatively stable. The water absorption of porcine ADM remained relatively high level in static state from 30 min to 120 min. The cytotoxicity of mouse embryonic Fbs in 6.5 g/L ADM extract group, 12.5 g/L ADM extract group, and 25.0 g/L ADM extract group was grade 1 at PCH 24, and the cytotoxicity of the other groups was 0 grade at each time point. After reaction for 3 h, the absorbance value of hemoglobin of erythrocytes in ultra-pure water group was significantly higher than the values in normal saline group and 15 mg/mL ADM extract group (with t values of 8.14 and 7.96, respectively, P<0.01). After 3 days of culture, the cells of the fourth passage showed pebble-like morphology, with low expression of CD71 and high expression of CD49f, which were identified as ESCs. There was ESC attachment and growth on porcine ADM particles. On PID 1, the wound sizes of nude mice were almost the same in PBS alone group, ADM alone group, ESC alone group, and ESC/ADM group. On PID 7, 11, and 15, the wound contraction of nude mice in each group was observed, especially in ADM alone group, ESC alone group, and ESC/ADM group. On PID 7, the wound healing rates of nude mice in ESC alone group and ESC/ADM group were significantly higher than the rate in PBS alone group (with t values of 2.83 and 4.72 respectively, P<0.05 or P<0.01). On PID 11, the wound healing rate of nude mice in ESC/ADM group was significantly higher than that in PBS alone group (t=4.86, P<0.01). On PID 15, the wound healing rates of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly higher than the rate in PBS alone group (with t values of 2.71, 2.90, and 3.23 respectively, P<0.05). On PID 7, the length of un-epithelialized wound of nude mice in ADM alone group, ESC alone group, and ESC/ADM group was (816±85), (635±66), and (163±32) μm, respectively, which were significantly shorter than (1 199±43) μm in PBS alone group (with t values of 5.69, 10.19, and 27.54 respectively, P<0.01). On PID 11, the dermal areas of wound section of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly larger than the area in PBS alone group (with t values of 27.14, 5.29, and 15.90 respectively, P<0.01); the collagen production of nude mice in ADM alone group and ESC/ADM group was more obvious than that in PBS alone group, and the collagen production of nude mice in ESC alone group and PBS alone group was similar. On PID 11, in the wounds of nude mice in ESC alone group and ESC/ADM group, the cells with positive expression of CD49f were respectively 135±7 and 185±15, and the mRNA expressions of GAPDH were positive; while there were no expressions of CD49f nor mRNA of GAPDH in the wounds of nude mice in PBS alone group and ADM alone group. Conclusions: ESC/ADM particles can promote the wound healing of full-thickness skin defects in nude mice, which may be related to the improved survival rate of ESCs after transplantation and the promotion of dermal structure rearrangement and angiogenesis by ADM.

Progress in the study of choroidal thickness on diabetic retinopathy by OCT / 国际眼科杂志(Guoji Yanke Zazhi)

? The choroidal vasculature provides oxygen and nutrients to the outer retina, and is responsible for maintaining the highly metabolically active photoreceptor cells. The normal structure and function of its vascular system is very important for the retina. So it is more meaningful to observe the choroid morphology for tracking pathological changes in diabetic retinopathy. With the application of the high resolution optical coherence tomography ( OCT ) technology, spectral -domain optical coherence tomography ( SD - OCT ) enhanced depth imaging technology can be used to measure the choroidal thickness quantitatively, which provides a new idea for the diagnosis and treatment of diabetic retinopathy. Therefore, the research and progress of choroidal thickness on diabetic retinopathy by OCT are summarized as follows.

PITX2C loss-of-function mutations responsible for idiopathic atrial fibrillation

OBJECTIVE: This study aimed to identify novel PITX2c mutations responsible for idiopathic atrial fibrillation. METHODS: A cohort of 210 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy individuals used as controls were recruited. The whole coding exons and splice junctions of the PITX2c gene, which encodes a paired-like homeobox transcription factor required for normal cardiovascular morphogenesis, were sequenced in 210 patients and 200 control subjects. The causative potentials of the identified mutations were automatically predicted by MutationTaster and PolyPhen-2. The functional characteristics of the PITX2c mutations were explored using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous PITX2c mutations (p.Q105L and p.R122C) were identified in 2 of the 210 unrelated patients with idiopathic atrial fibrillation. These missense mutations were absent in the 400 control chromosomes and were both predicted to be pathogenic. Multiple alignments of PITX2c protein sequences across various species showed that the altered amino acids were highly evolutionarily conserved. A functional analysis demonstrated that the mutant PITX2c proteins were both associated with significantly reduced transcriptional activity compared with their wild-type counterparts. CONCLUSION: The findings of this study associate PITX2c loss-of-function mutations with atrial fibrillation, supporting the hypothesis that dysfunctional PITX2c confers enhanced susceptibility to atrial fibrillation and suggesting potential implications for early prophylaxis and allele-specific therapy for this common arrhythmia. .

Comparison of adenosine stress and dobutamine stress by real-time myocardial contrast echocardiography in detecting myocardium ischemia in dogs / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Real-time perfusion imaging (RTPI) using ultrasound contrast agents has shown good "accuracy" in detecting myocardial infarction, however its accuracy in the assessment of peri-infarct ischemia and stress echocardiography are not known. The aim of this study was to determine the accuracy of RTPI in assessment of peri-infarct ischemia during dobutamine and adenosine stress.</p><p><b>METHODS</b>We employed the RTPI modality (Agilent and ATL Philips) in a canine model (18 dogs) of distal coronary occlusion and proximal coronary stenosis. Using coronary flow probe recordings, the physiologic significance of proximal coronary stenosis was established by confirming abolition of the coronary reserve. The contrast agent Optison was given as a slow bolus injection at baseline, during prolonged distal coronary occlusion, during adenosine bolus stress and during dobutamine stress. Triphenyltetrazolium chloride (TTC) staining was used to verify a distal infarction. RTPI recordings at baseline, the distal coronary occlusion and stress protocols were randomly mixed and reviewed blindly.</p><p><b>RESULTS</b>In all but one dog, RTPI detected a distal infarct as small as 9% of the left ventricle. The sensitivity, specificity and overall diagnostic accuracy of RTPI in the detection of distal infarcts were: 94%, 89% and 92%, respectively. The sensitivity, specificity, and overall diagnostic accuracy of RTPI in the assessment of peri-infarction ischemia were 83%, 92% and 88% for adenosine stress and 95%, 86% and 91% for dobutamine stress, respectively.</p><p><b>CONCLUSIONS</b>Even small distal infarcts can be detected by RTPI; peri-infarct ischemia can be accurately recognized by RTPI during stress; adenosine and dobutamine stress appear equally reliable in the RTPI evaluation of peri-infarct ischemia.</p>

Comparison of the veracity of real-time perfusion, harmonic angio, and ultraharmonic mocardial contrast imaging modes in evaluation of acute myocardial infarction area / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Innovative advancements in ultrasound instrumentation present a number of imaging modalities for myocardial contrast echocardiography (MCE) in ischemic syndromes. How well they compare to each other in diagnostic accuracy in the detection of acute myocardial infarction is unclear. The purpose of this study was to assess the relative accuracy of 3 different imaging modes of MCE, low mechanical index (MI) real-time perfusion imaging (RTPI), triggered harmonic angio mode (HA), and ultraharmonic imaging mode (UH) in the detection of acute experimental myocardial infarction within the time frame suitable for potential reperfusion.</p><p><b>METHODS</b>MCE was performed in 10 open-chest dogs using RTPI, triggered HA and triggered UH modes at baseline and one hour after occlusion of left anterior descending coronary artery. Presence or absence of perfusion defects, and the perfusion defect size when present, were analyzed and compared with the infarct size delineated by triphenyltetrazolium chloride (TTC) staining.</p><p><b>RESULTS</b>The infarct area was (15.8 +/- 2.4)% by TTC staining; Perfusion defect area by MCE was similar to anatomic infarct area in all the three MCE approaches: (16.1 +/- 2.7)% by RTPI mode, (15.5 +/- 2.9)% by HA mode, and (15.5 +/- 3.0)% by UH mode. The sensitivity, specificity and overall diagnostic accuracy in the detection of myocardial infarction were 100%, 88%, and 94% for RTPI mode, 88%, 100%, and 94% for HA mode, and 100%, 75%, and 88% for UH mode.</p><p><b>CONCLUSION</b>All modes of MCE, RTPI, triggered HA mode and triggered UH mode have excellent diagnostic accuracy in the immediate hour of acute coronary occlusion within the optimal time frame suitable for reperfusion therapy.</p>