MicroRNA-138 Aggravates Inflammatory Responses of Macrophages by Targeting SIRT1 and Regulating the NF-κB and AKT Pathways.

BACKGROUND/AIMS: With increased understanding of sepsis, mortality is decreasing. However, there is still a lack of effective therapeutic strategy. The inflammatory response of macrophages is critical during sepsis. METHODS: Macrophages were stimulated with LPS. Western blotting and qRT-PCR were used to detect inflammatory responses. Then, the inhibitor of microRNA-138 was transfected and Western blotting, qRT-PCR, H&E staining and ELISA were used to verify the role of microRNA-138 in inflammation. Then target gene prediction databases were used to predict the potential target of microRNA-138. Both animal and cell models under LPS challenges were established to verify the regulation of SIRT1 and microRNA-138 during inflammation. RESULTS: The present study showed that microRNA-138 was increased in macrophages stimulated with LPS. Additionally, the NF-κB and AKT pathways were both activated. The pre-treatment of microRNA-138 inhibitor decreased inflammatory factors, downregulated the NF-κB pathway, activated the AKT pathway and protected against organ damage in mice challenged with LPS. SIRT1 was demonstrated as a potential target of microRNA-138In macrophages stimulated with LPS, the inhibition effect of microRNA-138 inhibitor on inflammation was lost by SIRT1 siRNA pre-treatment. In the animal model, the protective effect of microRNA-138 antagomir disappeared in SIRT1 knockout mice. CONCLUSION: We demonstrated that miR-138 participated in the inflammatory process by inhibiting SIRT1 and activating the NF-κB pathway.

Acute downregulation of miR-155 at wound sites leads to a reduced fibrosis through attenuating inflammatory response.

Fibrosis, tightly associated with wound healing, is a significant symptomatic clinical problem. Inflammatory response was reported to be one of the reasons. MiR-155 is relatively related with the development and requirement of inflammatory cells, so we thought reduce the expression of miR-155 in wound sites could improve the quality of healing through reduce inflammatory response. To test this hypothesis, locally antagonizing miR-155 by directly injecting antagomir to wound edge was used to reduce the expression of miR-155. We found wounds treated with miR-155 antagomir had an obvious defect in immune cells requirements, pro-inflammatory factors IL-1ß and TNF-α reduced while anti-inflammatory factor IL-10 increased. With treatment of miR-155 antagomir, the expression of α-smooth muscle actin (α-SMA), Col1 and Col3 at wound sites all reduced both from mRNA levels and protein expressions. Wounds injected with antagomir resulted in the structure improvement of collagen, the collagen fibers were more regularly arranged. Meanwhile the rate of healing did not change significantly. These results provide direct evidences that miR-155 play an important role in the pathogenesis of fibrosis and show that miR-155 antagomir has the potential therapy in prevention and reduction of skin fibrosis.

[Impact of resting heart rate on new-onset diabetes in population without hypertension].

OBJECTIVE: To investigate the impact of resting heart rate (RHR) on new-onset diabetes (NOD) in population without hypertension. METHODS: This prospective cohort study was performed in 2006 and 2007 and screened 101 510 participants. All subjects were employees of the Kailuan Group, a state-run coal mining company. The observation cohort included 48 926 subjects with normal fasting blood glucose (FBG) <7.0 mmol/L, no history of diabetes, complete FBG and RHR examination data, systolic blood pressure <140 mm Hg (1 mm Hg = 0.133 kPa) , diastolic blood pressure <90 mm Hg, no history of hypertension, and no use of hypoglycemic agents or antihypertensive drugs.We excluded participants without a health examination in 2008-2009 or 2010-2011 and those with incomplete examination data. A total of 29 910 participants were included in the final analysis. The observation population was divided into four groups according to RHR data collected during 2006-2007 health examinations: quartile 1 (RHR<63 beats/min) ; quartile 2 (63 beats/min ≤ RHR<70 beats/min) ; quartile 3 (70 beats/min ≤ RHR<75 beats/min) ; quartile 4 ( RHR ≥ 75 beats/min). Kaplan-Meier analysis was used to calculate the incidence of NOD. The relationship between RHR and NOD was estimated using Cox proportional hazard analysis. RESULTS: The incidences of NOD/1000 person-years for the above quartiles of RHR were 11.22, 13.58, 13.96, and 17.55, respectively in the total observational population; the corresponding incidences were 12.17, 15.20, 16.08, 20.44, and 8.29, 9.38, 8.86, and 9.60 in men and women, respectively. Compared with quartile 1, Cox proportional hazard regression analysis showed that the other three RHR groups had an increased risk of NOD after adjusting for age, gender, systolic blood pressure, diastolic blood pressure, and other risk factors. The hazard ratio values for these groups were 1.20 (95%CI:1.04-1.40, P < 0.05), 1.25 (95%CI:1.07-1.45, P < 0.01) and 1.58 (95%CI:1.36-1.82, P < 0.01), respectively. Furthermore, after adjusted the FBG, risk of NOD was significantly higher in quartile 2 (HR = 1.21, 95%CI:1.04-1.40, P < 0.01) and quartile 4 (HR = 1.22, 95%CI:1.06-1.41, P < 0.01 compared that in quartile 1. After adjusting for the factors listed above, the influence of RHR on NOD was not significant in women (P > 0.05) , but there was still an increased risk of NOD in men compared with quartile 1 with hazard ratio values of 1.21 (95%CI:1.02-1.43, P < 0.05) , and 1.27 (95%CI:1.09-1.49, P < 0.01) for quartile 2 and quartile 4, respectively. CONCLUSION: Higher RHR is linked with higher risk of NOD in population without hypertension.

[Effects of Angelica dahurica extract on biological behavior of dermal fibroblasts].

OBJECTIVE: To observe the effects of Angelica dahurica extracts on the biological characteristics of human dermal fibroblasts in vitro and to preliminary explore its possible therapeutic mechanism for wound healing. METHODS: The optimal concentration of Angelica dahurica extracts was identified by analysing of proliferation activity of human normal fibroblasts (Fb) that treated with different concentration of Angelica dahurica extracts through thiazole blue (MTT) colorimetric assay. Cell cycle, collagen I and collagen III mRNA levels of the optimal Angelica dahurica extracts treated Fb were detected by flow cytometry (FCM) and real-time PCR techniques. RESULTS: At concentrations of 5 × 10(-4) to 5 × 10(-2) g/L, the Angelica dahurica extracts significantly enhanced the proliferation of Fb. The most significant concentration was 5 × 10(-3) g/L (t = 5.79, P < 0.01), at which an increased percentage of G1 to S and S to G2 phase cells (t = 11.2, 5.69, 2.44, P < 0.05) as well as an increased level of collagen I (1.61 ± 0.26 vs. 1.00 ± 0.16) and collagen III mRNA (3.36 ± 0.40 vs. 1.00 ± 0.14) were obtained compared to the control group (t = 6.69, 7.64, P < 0.01). CONCLUSIONS: Angelica dahurica extracts can notably promote the proliferation of Fb and accelerating the cell cycle of Fb as well as up-regulating the expression of collagen I and collagen III, which may enhance the process of wound healing.

[The prevalence and distribution of ideal cardiovascular health behavior in employees of Kailuan Group cohort].

OBJECTIVE: To observe the prevalence and distribution of ideal cardiovascular health behavior. METHODS: Health examination data between 2008 to 2009 from the employees of Kailuan Group were analyzed. RESULTS: A total of 101 333 subjects took part in the health examination, subjects with previous myocardial infarction (n = 871), stroke (n = 2255), myocardial infarction and stroke (n = 162) and subjects with incomplete examination data (n = 9311) were excluded and 88 534 subjects were included for final analysis [mean age (50.6 ± 12.3) years, male 69 916]. (1) Body mass index (BMI), systolic and diastolic pressure, cholesterol (TC) and triglyceride were significantly higher in males than in females (all P < 0.05), women's income and the education lever were significantly higher than men (P < 0.05). (2) The distribution of ideal cardiovascular health behavior (smoking, BMI, physical exercise, salt intake) was 55.8%, 41.4%, 18.9% and 14.0% respectively among the population; the ideal cardiovascular factors (fasting blood glucose, TC, blood pressure) was 80.9%, 61.8% and 18.5%, respectively. (3) The subjects with distribution of seven, six, five, four ideal cardiovascular health behavior and factors was 0.1%, 1.9%, 9.1%, 20.3%, respectively. (4) Multiple logistic regression analysis showed that female, age < 55 and high education level were associated with the ideal cardiovascular health status with a RR value (95%CI) of 4.52 (4.32 - 4.72), 1.46 (1.39 - 1.53) and 2.23 (2.10 - 2.37), respectively. CONCLUSION: The prevalence of ideal cardiovascular health is extremely low in the study population, most persons were not in the ideal cardiovascular health behavior and factors and female, age < 55 and high education level are linked with ideal cardiovascular health status.

Clinical anatomy study of autonomic nerve with respective to the anterior approach lumbar surgery.

INTRODUCTION: Male genital dysfunction was recognized as a complication following anterior approach lumbar surgery. Disruption of efferent sympathetic pathways such as the abdominal aortic plexus (AAP) and superior hypogastric plexus (SHP) which lied pre-abdominal aorta and iliac artery had been thought as the main reason. Though there were some clinical reports of retrograde ejaculation, the applied anatomic study of the autonomic nerve anterior to the lumbar was little. The purpose was to find out a lumbar surgery approach which was ejaculation preservation through the detailed study of the anatomy and histology observation of the autonomic nerve anterior to the lumbar vertebrae. METHODS: The lumbar region of ten male cadavers was dissected and analyzed. We investigated the relationship between the peritoneum and abdominal aorta, iliac artery and sacral promontory fascia, as well as the trend and distribution of the autonomic nerve and SHP anterior to the L5-S1. We also observed the distribution of autonomic nerve at retroperitoneum through hematoxylin and eosin (HE)-stained tissues pre-aorta, para-aorta, and pre-vertebrae sacrales. RESULTS: Superior hypogastric plexus, which deviated to left, located in a triangle formed by the common iliac arteries and its bilateral branches, its truck sited anterior to the lumbarsacral space in seven cases (70%), and anterior to sacrum in three cases (30%); at the aortic bifurcation, SHP strided over left iliac artery from left-hand side, then located in front of sacrum in four cases (40%), and sifted to the left at the lumbar sacral promontory in six cases (60%); from both anatomic and histological view, the autonomic nerve plexus lying in an fascia layer of retroperitoneum. CONCLUSION: At the anterior approach lumbar surgery of trans-peritoneum, we should choose the right-hand side incision; the SHP should be pushed aside carefully from right to left along intervertebral disc. The accurate surgical plane was at the deeper layer of autonomical nerve fascia; we also could lift the complete autonomical nerve layer which lies behind the aorta and lumbar sacral promontory, so that the autonomic nerve could be preserved.

Rapid prototyping drill guide template for lumbar pedicle screw placement.

OBJECTIVE: To develop a novel method of spinal pedical stereotaxy by reverse engineering and rapid prototyping techniques, and to validate its accuracy by experimental and clinical studies. METHODS: A 3D reconstruction model for the desired lumbar vertebra was generated by using the Mimics 10.11 software, and the optimal screw size and orientation were determined using the reverse engineering software. Afterwards, a drill template was created by reverse engineering principle, whose surface was the antitemplate of the vertebral surface. The drill template and its corresponding vertebra were manufactured using the rapid prototyping technique. RESULTS: The accuracy of the drill template was confirmed by drilling screw trajectory into the vertebral biomodel preoperatively. This method also showed its ability to customize the placement and size of each screw based on the unique morphology of the lumbar vertebra.The drill template fits the postural surface of the vertebra very well in the cadaver experiment. Postoperative CT scans for controlling the pedicle bore showed that the personalized template had a high precision in cadaver experiment and clinical application. No misplacement occurred by using the personalized template. During surgery, no additional computer assistance was needed. CONCLUSIONS: The authors have developed a novel drill template for lumbar pedicle screw placement with good applicability and high accuracy. The potential use of drill templates to place lumbar pedicle screws is promising. Our methodology appears to provide an accurate technique and trajectory for pedicle screw placement in the lumbar spine.

Application of navigation template to fixation of sacral fracture using three-dimensional reconstruction and reverse engineering technique.

OBJECTIVE: To provide a new method in the fixation of sacral fracture by means of three-dimensional reconstruction and reverse engineering technique. METHODS: Pelvis image data were obtained from three-dimensional CT scan in patients with sacral fracture. The data were transferred into a computer workstation. The three-dimensional models of pelvis were reconstructed using Amira 3.1 software and saved in STL format. Then the three-dimensional fracture models were imported into Imageware 9.0 software. Different situations of reduction (total reduction, half reduction and non-reduction) were simulated using Imageware 9.0 software. The best direction and location of extract iliosacral lag screws were defined using reverse engineering according to these three situations and navigation templates were designed according to the anatomic features of the postero-iliac part and the channel. The exact navigational template was made by rapid prototyping. Drill guides were sterilized and used intraoperatively to assist in surgical navigation and the placement of iliosacral lag screws. RESULTS: Accurate screw placement was confirmed with postoperative X-ray and CT scanning. The navigation template was found to be highly accurate. CONCLUSION: The navigation template may be a useful method in minimal-invasive fixation of sacroiliac joint fracture.

[Primary clinical result of digital template as navigation to supper cervical pedicle instrumentation].

OBJECTIVE: To observe the primary clinical result of digital template as navigation to the upper cervical pedicle instrumentation. METHODS: CT scan of upper cervical vertebrae was performed. 3-D model of upper cervical vertebrae was reconstructed by software Amira 3.1 and was preserved in STL format. Then 3-D model was run in software UG Imageware 12.0, the best pedicle channel was extracted according to the reverse engineering principle. A virtual navigational template was established according to he lamina anatomic trait, and the best pedicle channel. The virtual vertebrae and navigational template were manufactured using rapid prototyping. The navigational template was sterilized and used intra operative to assist with the placement of pedicle screw. The Accuracy of screw placement was confirmed with postoperative X-ray and CT scanning. RESULTS: The digital navigational template had been established and used in the 3 cases, the good trajectory of cervical pedicle had been showed by the CT scan of post operation. There were not complications of related pedicle screw insertion. CONCLUSIONS: A novel method of upper cervical pedicle location using Reverse Engineering and rapid prototyping has been developed; the navigational template is found to be highly accuracy and has great expectation.

Reproductive toxicity of perchlorate in rats.

Perchlorate, as an oxidizer, has many applications such as explosives and pyrotechnics, especially in rocket propellants and missile motors. Because it was found in water including wells and drinking water in the US, its effect on human health was being noted. However, the reproductive toxic effect on perchlorate is still unclear. In present study, the effects of repeated exposure to perchlorate on reproductive toxicity were evaluated in Wistar rats. The rats were treated orally with perchlorate at doses of 0.05, 1.00 or 10.00 mg/kg body weight (b.w.) daily for 8 weeks. The levels of T3 and T4 hormones in the rat serum were detected by radioimmunoassay kit. The indexes of reproduction, percentage of organ in body weight (%) and frequency of abnormal sperm cells were also analyzed in this study. DNA damage in testicular cells was evaluated by Comet assay. The levels of MDA, GSH and SOD were examined in testicle tissues of rats by ELISA. The expression of c-fos and fas protein was examined in testicle tissues by immunohistochemistry. The results showed that perchlorate did not affect the body weight of rats. Perchlorate also significantly decreased indexes of live birth and weaning in the groups of 1.00 and 10.00 mg/kg, and viability index only in the 10.00 mg/kg group (P < 0.05). Perchlorate also significantly decreased the serum level of T3 in male rats of 1.00 and 10.00 mg/kg groups, increased the rate of sperm abnormality (10.00 mg/kg), potentially caused DNA damage in testicular cells and altered the status of oxidative stress in male rats. In addition, because of the increase in the expression of fas and c-fos protein in testicle tissues, perchlorate could induce apoptosis in spermatogenesis. Thus, these findings indicate that perchlorate could cause DNA damage in testicular tissues and reduce testicular spermatogenic ability, resulting in reproductive toxicity.

Clinical anatomic study of the lower lumbar anterolateral vein: with respect to retroperitoneal endoscopic surgery.

OBJECTIVE: To study the anatomy of veins of the lower lumbar spine and provide the anatomic basement for laparoscopic lumbar surgery. METHODS: A total of 15 formaldehyde-preserved cadavers were studied with special attention to the variety and surrounding structure of ascending lumbar vein (ALV) and iliolumbar veins (ILV), and their relationship with lumbar plexus. RESULTS: ALV and ILV can be found on every sides, which have four variants including separate entry and common entry. The ascending vein and iliolumbar vein separately enter common iliac vein in 18 cases, and as a common stem enter the common iliac vein in 12 cases. Retracting common iliac vein medially both the ascending lumbar and the iliolumbar veins are always at risk of avulsion on exposure of the disc space. The injury of obturator nerve and lumbosacral trunk of lumbar plexus should be avoided. CONCLUSION: Awareness of these anatomic variation can prevent the hemorrhage and be helpful for the surgeon in performing a careful ligation of these veins before medial retraction of the common iliac vein. Our findings emphasize the need for proper dissection of ALV and ILV before ligature during exposure of the lower lumbar spine.

[The clinic anatomy of lumbar plexus in the lumbar anterolaterally approach minimally invasive surgery].

OBJECTIVE: To provide anatomic data for reducing lumbar plexus nerve injury. METHODS: The applied anatomy of lumbar plexus was studied by 15 formaldehyde-preserved cadavers, two groups of sectional images of lumbar segment and three series of virtual chinese human dataset. RESULTS: Arrangement of the lumbar nerve was regular. From anterior view, lumbar plexus nerve arranged from lateral to medial from L2 to L5; from lateral view, lumbar nerve arrange from ventral to dorsal from L2 to L5. The angle degree between the lumbar nerve and lumbar increased from L1 to L5. The lumbar plexus nerve was revealed to be in close contact with transverse process. By sectional anatomy, all parts of the lumbar plexus nerve were located in the dorsal third of the psoas major. The safety zone of the psoas major to prevent nerve injuries was ventrally 2/3. CONCLUSIONS: Psoas major can be considered as surgery landmark when expose the lateral anterior of lumbar by incising the psoas muscle. Incising the psoas muscle ventral 2/3 can prevent lumbar plexus injury. Transverse process can be considered as landmark for the position of lumbar plexus in operation.

Repair and reconstruction of severe leg injuries: retrospective review of eighty-five patients.

OBJECTIVE: To explore a good way of the reconstruction of severe tibial shaft fractures by using different flaps and external fixators. METHODS: Eighty-five patients of Type IIIC tibial shaft fractures with average age of 42.5 years were treated in our hospital from 1990 to 2005. Injuries were caused by motorcycle accidents in 66 patients, by machine accidents in 16 patients, and by stone bruise in 3 patients. The management procedures consisted of administration of antibiotics, serial debridment, bone grafting if needed, application of different flaps, such as free thoracoumbilical flaps, fasciocutaneous flaps, saphenous neurocutaneous vascular flaps, sural neurocutaneous vascular flaps and gastrocnemius muscular flaps, and different external fixations, for instance, half-ring fixators, unilateral axial dynamic fixators, AO fixators, Weifang fixators, and Hybrid fixators. The average follow up was 6.3 years. RESULTS: All flaps survived. Eighty-three cases had bone healed. The average bone healing time of different external fixations was 5.5 months in 47 cases with half-ring fixators, 9.2 months in 4 cases treated with unilateral axial dynamic fixators, 8.5 months in 6 cases with AO fixators, 10.7 months in 16 cases with Weifang fixators, and 7.8 months in 10 cases with assembly fixators. Except half-ring fixation, other fixations all needed necessary bone graft. Two cases treated with unilateral axial dynamic fixators had nonunion of bone and developed osteomyelitis. The wounds healed after the removal of the fixators and immobilization by plaster. The last follow up examination showed ankle and knee motion was normal and no pain was noted. CONCLUSIONS: The combination of half-ring external fixators with various flaps provides good results for Type IIIC tibial shaft fractures.

Identification of sirtuin 1 as a promising therapeutic target for hypertrophic scars.

BACKGROUND AND PURPOSE: Sirtuin1 (SIRT1), the founding member of mammalian class III histone deacetylases, is reported to be a drug target involved in fibrotic diseases. However, whether it is an effective drug target in hypertrophic scar treatment is still not known. EXPERIMENTAL APPROACH: In the present study, we observed that SIRT1 localized to both the epidermis and the dermis of skin tissues by immunohistochemistry. After knock-down of SIRT1 by shRNA or up-regulating SIRT1 by resveratrol, the expression of α-SMA, Col1 and Col3 in fibroblasts were detected by western blots. A mouse excision wound healing model was used to observe the changes in collagen fibre associated with the different expression levels of SIRT1. KEY RESULTS: SIRT1 expression was inhibited in hypertrophic scar tissue. The down-regulation of SIRT1 resulted in an increased expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts. In contrast, the up-regulation of SIRT1 not only inhibited the expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts but also blocked the activation of TGFß1-induced normal skin-derived fibroblasts. In the mouse model of wound healing, the deletion of SIRT1 resulted in denser collagen fibres and a more disordered structure, whereas resveratrol treatment led to a more organized and thinner collagen fibre, which was similar to that observed during normal wound healing. CONCLUSIONS AND IMPLICATIONS: The results revealed that SIRT1 negatively regulates TGFß1-induced fibroblast activation and inhibits excessive scar formation and is, therefore, a promising drug target for hypertrophic scar formation.

Melatonin prevents acute kidney injury in severely burned rats via the activation of SIRT1.

Acute kidney injury (AKI) is a common complication after severe burns. Melatonin has been reported to protect against multiple organ injuries by increasing the expression of SIRT1, a silent information regulator that regulates stress responses, inflammation, cellular senescence and apoptosis. This study aimed to investigate the protective effects of melatonin on renal tissues of burned rats and the role of SIRT1 involving the effects. Rat severely burned model was established, with or without the administration of melatonin and SIRT1 inhibitor. The renal function and histological manifestations were determined to evaluate the severity of kidney injury. The levels of acetylated-p53 (Ac-p53), acetylated-p65 (Ac-p65), NF-κB, acetylated-forkhead box O1 (Ac-FoxO1), Bcl-2 and Bax were analyzed to study the underlying mechanisms. Our results suggested that severe burns could induce acute kidney injury, which could be partially reversed by melatonin. Melatonin attenuated oxidative stress, inflammation and apoptosis accompanied by the increased expression of SIRT1. The protective effects of melatonin were abrogated by the inhibition of SIRT1. In conclusion, we demonstrate that melatonin improves severe burn-induced AKI via the activation of SIRT1 signaling.

[A review of research on Schmallenberg virus].

Schmallenberg virus (SBV), a novel orthobunyavirus, was first isolated in 2011. SBV preferentially infects the central nervous system of cattle and sheep and causes fever, diarrhea, a drop in milk yields, congenital malformations and stillbirths. Until June 2014, more than 200 scientific publications regarding SBV have been published. Although more than 20 articles on SVB were published in China, most of these articles provided only a brief introduction of the disease without fully discussing the associated disease characteristics. As a new disease, it has been made a focus of the National Research Center for Exotic Animal Diseases at the China Animal Health and Epidemiology Center. In this review, in order to provide a reference for research into SBV in China, we have reviewed the state of current research progress on the etiology, diagnosis and epidemiology of SBV, and vaccine development.

MicroRNA-21 regulates hTERT via PTEN in hypertrophic scar fibroblasts.

BACKGROUND: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues. CONCLUSIONS/SIGNIFICANCE: These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring.

Construction of the tissue engineering seed cell (HaCaT-EGF) and analysis of its biological characteristics.

OBJECTIVE: To construct the tissue engineering seed cell (HaCaT cell line) with stable expression of the human epidermal growth factor (EGF), and analyze the changes of its biological characteristics. METHODS: PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell, and G418 was utilized to select the HaCaT-EGF cell line. Using an inverted microscope, PCR, ELISA method to detect the changes of the cell morphology, the expression of the EGF gene and protein, and the mRNA expression levels of apoptosis related molecule Caspase-3, the cell cycle related protein cyclin D1. RESULTS: The mRNA expression levels of the obtained HaCaT-EGF cell were more than 100 times higher than the level of ordinary HaCaT cell. The colony of the HaCaT-EGF cells was more focused and tight compared to the empty vector transfected HaCaT cells and normal HaCaT cells. The expression levels of apoptotic factor Caspase-3 and cyclin D1 in HaCaT-EGF cell were significantly higher than those in the empty vector HaCaT- pcDNA3.1 cell, and the differences were statistically significant (P<0.01), but there was no significant difference compared to the normal HaCaT cells (P>0.05). CONCLUSIONS: HaCaT-EGF cell can continuously secrete EGF, and the biological characteristic is stable. It can be used for tissue engineering experiment and is an ideal seed cell for constructing tissue engineered skin.

[Effects of adipose-derived stem cells on renal injury in burn mice with sepsis].

OBJECTIVE: To investigate the effect of adipose-derived stem cells (ADSC) on renal injury in mice with burn injury and sepsis and its underlying mechanism. METHODS: (1) Adipose tissue was collected from both inguinal regions of 5 C57BL/6J mice to isolate, culture and purify ADSC through enzyme digestion, density gradient centrifugation, and adherence method. Cells of the third passage were used in the experiment. The morphologic change in cells was observed and the growth curve of cells was determined. The expression of cell surface antigen phenotype was analyzed by flow cytometry, and the cells were identified by adipogenic and osteogenic differentiation. (2) Another 37 C57BL/6J mice were divided into normal control group (n = 5), saline group (n = 16), and group ADSC (n = 16) according to the random number table. The mice in saline group and group ADSC were injected with Pseudomonas aeruginosa after being subjected to 15% TBSA full-thickness burn on the back to reproduce septic burn model. Then the mice were injected with saline and ADSC through tail vein respectively. At post burn hour (PBH) 12, 24, 48, and 72, the pathological change in kidney tissue was observed, the levels of blood urea nitrogen and serum creatinine were determined, and the levels of TNF-α, IL-12, IL-10, and cyclooxygenase-2 (COX2) mRNA were determined with real-time fluorescence quantitative PCR in both groups. Above-mentioned indexes were also examined in the normal control group (without burn). Data were processed with multifactor analysis of variance and LSD- t test. RESULTS: (1) Cells in the third passage were orderly arranged with the shape similar to fibroblasts. The percentages of CD90(+), CD105(+), CD34(-), and CD45(-) cells were all above 90%. The cells could differentiate into osteoblasts and adipocytes. The cells were identified to be ADSC. (2) From PBH 12 to PBH 72, the neutrophil infiltration gradually increased, and the structure of kidney tubules and glomeruli were deranged in saline group. The pathological change in kidney tissue in group ADSC was less serious than that of normal control group at each time point. From PBH 12 to PBH 72, the levels of blood urea nitrogen and serum creatinine in saline group were significantly higher than those of normal control group and group ADSC (P values all below 0.01). Compared with those of the normal control group, the levels of TNF-α and IL-12 mRNA were higher in group ADSC and saline group at PBH 24 (P values all below 0.05). At PBH 24, the level of TNF-α mRNA in group ADSC (1.58 ± 0.19) was lower than that of saline group (3.36 ± 0.30, P < 0.05). At PBH 24, the levels of IL-10 and COX2 mRNA in group ADSC (2.89 ± 0.47, 4.90 ± 0.59) were higher than those in normal control group (1.00 ± 0.15, 1.00 ± 0.27) and saline group (1.32 ± 0.38, 1.57 ± 0.38, P values all below 0.05). CONCLUSIONS: ADSC can decrease the levels of blood urea nitrogen and serum creatinine, promote the production of anti-inflammatory cytokines IL-10 and COX2, and reduce the release of the pro-inflammatory cytokines TNF-α and IL-12 to offer protective effects against renal injury in burn mice with sepsis.

Protection against TGF-ß1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring.

Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-ß1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.