RESUMO
Sepsisinduced myocardial dysfunction is one of the features of multiple organ dysfunction in sepsis, which is associated with extremely high mortality and is characterized by impaired myocardial compliance. To date, there are few effective treatment options available to cure sepsis. Tannic acid (TA) is reportedly protective during sepsis; however, the underlying mechanisms by which TA protects against septic heart injury remain elusive. The present study investigated the potential effects and underlying mechanisms of TA in alleviating lipopolysaccharide (LPS)induced H9C2 cardiomyocyte cell apoptosis. H9C2 cells were treated with LPS (15 µg/ml), TA (10 µM) and TA + LPS; control cells were treated with medium only. Apoptosis was measured using flow cytometry, reverse transcriptionquantitative PCR (RTqPCR) and western blot analysis. Additionally, the levels of cellular reactive oxygen species (ROS), malondialdehyde and nicotinamide adenine dinucleotide phosphate were evaluated. Western blotting and RTqPCR were also employed to detect the expression levels of endoplasmic reticulum (ER) stressassociated functional proteins. The present findings demonstrated that TA reduced the degree of LPSinduced H9C2 cell injury, including inhibition of ROS production and ER stress (ERS)associated apoptosis. ERSassociated functional proteins, including activating transcription factor 6, protein kinaselike ER kinase, inositolrequiring enzyme 1, spliced X boxbinding protein 1 and C/EBPhomologous protein were suppressed in response to TA treatment. Furthermore, the expression levels of ERSassociated apoptotic proteins, including cJun Nterminal kinase, Bax, cytochrome c, caspase3, caspase12 and caspase9 were reduced following treatment with TA. Additionally, the protective effects of TA on LPSinduced H9C2 cells were partially inhibited following treatment with the ROS inhibitor Nacetylcysteine, which demonstrated that ROS mediated ERSassociated apoptosis and TA was able to decrease ROSmediated ERSassociated apoptosis. Collectively, the present findings demonstrated that the protective effects of TA against LPSinduced H9C2 cell apoptosis may be associated with the amelioration of ROSmediated ERS. These findings may assist the development of potential novel therapeutic methods to inhibit the progression of myocardial cell injury.
Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Taninos/farmacologia , Acetilcisteína/farmacologia , Animais , Linhagem Celular , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , RatosRESUMO
BACKGROUND: The 8th American Joint Committee on Cancer tumor-node-metastasis (AJCC-TNM) staging system is based on a few retrospective single-center studies. We aimed to test the prognostic validity of the staging system and to determine whether a modified clinicopathological tumor staging system that includes lymphovascular embolization could increase the accuracy of prognostic prediction for patients with stage T2-3 penile cancer. METHODS: A training cohort of 411 patients who were treated at 2 centers in China and Brazil between 2000 and 2015 were staged according to the 8th AJCC-TNM staging system. The internal validation was analyzed by bootstrap-corrected C-indexes (resampled 1000 times). Data from 436 patients who were treated at 15 centers over four continents were used for external validation. RESULTS: A survivorship overlap was observed between T2 and T3 patients (P = 0.587) classified according to the 8th AJCC-TNM staging system. Lymphovascular embolization was a significant prognostic factor for metastasis and survival (all P < 0.001). Based on the multivariate analysis, only lymphovascular embolization showed a significant influence on cancer-specific survival (CSS) (hazard ratio = 1.587, 95% confidence interval = 1.253-2.011; P = 0.001). T2 and T3 patients with lymphovascular embolization showed significantly shorter CSS than did those without lymphovascular embolization (P < 0.001). Therefore, a modified clinicopathological staging system was proposed, with the T2 and T3 categories of the 8th AJCC-TNM staging system being subdivided into two new categories as follows: t2 tumors invade the corpus spongiosum and/or corpora cavernosa and/or urethra without lymphovascular invasion, and t3 tumors invade the corpus spongiosum and/or corpora cavernosa and/or urethra with lymphovascular invasion. The modified staging system involving lymphovascular embolization showed improved prognostic stratification with significant differences in CSS among all categories (all P < 0.005) and exhibited higher accuracy in predicting patient prognoses than did the 8th AJCC-TNM staging system (C-index, 0.739 vs. 0.696). These results were confirmed in the external validation cohort. CONCLUSIONS: T2-3 penile cancers are heterogeneous, and a modified clinicopathological staging system that incorporates lymphovascular embolization may better predict the prognosis of patients with penile cancer than does the 8th AJCC-TNM staging system. Trial registration This study was retrospectively registered on Chinese Clinical Trail Registry: ChiCTR16008041 (2016-03-02). http://www.chictr.org.cn.
Assuntos
Metástase Linfática/patologia , Neoplasias Penianas/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Penianas/patologia , Prognóstico , Análise de Sobrevida , Adulto JovemRESUMO
Long non-coding RNAs (lncRNAs) were playing critical roles in tumorigenesis. However, in prostate cancer, the roles and mechanisms of lncRNAs especially ANRIL were largely unknown. We investigated the effects of ANRIL on the proliferation and migration of prostate cancer cells using CCK-8 assay and Transwell migration assay. Real-time PCR and western blotting assays were used to analyze the levels of ANRIL, let-7a, TGF-ß1, p-Smad2 and p-Smad7. Our results showed that ANRIL was significantly overexpressed in prostate cancer tissues compared with corresponding normal tissues. Knockdown of ANRIL significantly inhibited the proliferation and migration of prostate cancer LNCap, PC3 and DU145 cells. Knockdown of ANRIL significantly decreased the levels of TGF-ß1 and p-Smad2, and increased the level of p-Smad7 in prostate cancer LNCap cells. We further found that knockdown of ANRIL significantly enhanced the expression of let-7a, and rescue experiment found that let-7a inhibitor recovered the suppressive effects of ANRIL silencing on the proliferation and migration of prostate cancer LNCap, PC3 and DU145 cells. And let-7a inhibitor recovered the suppressive effects of ANRIL silencing on the activity of TGF-ß1/Smad signaling pathway in prostate cancer LNCap cells. Taken together, our findings indicated that overexpression of lncRNA ANRIL promoted the proliferation and migration of prostate cancer cells via regulating let-7a/TGF-ß1/Smad signaling pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Longo não Codificante/genética , Transdução de Sinais , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Masculino , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Current guidelines recommend pelvic lymphadenectomy (PLND) for patients with pelvic lymph node metastasis and special state. However, these data and recommendations do not distinguish the role of PLND in different patient groups and confirm the final benefits. The aim of this study was to confirm the efficacy of pelvic lymphadenectomy (PLND) for the different groups of patients. METHODS: Data obtained from 7 centers were retrospectively analyzed. Of the patients, 190 pN2-3 penile carcinoma patients confirmed by bilateral inguinal lymph node excision were included in this study. Sixty-nine and 121 of these patients did and did not undergo bilateral PLND, respectively. The baseline differences from the patients were matched by propensity score analysis. RESULTS: In this study, the Kaplan-Meier estimated disease-specific survival (DSS) was not significantly different between the PLND and no-PLND groups (P = 0.796). According to the propensity score matching for T stage, N stage, grade, adjuvant therapies, and lymph node stage (number of inguinal lymph node metastasis and extranodal extension), 48 patients were selected for each group. Among the pN2 patients, the PLND group showed higher DSS rates than the no-surgery group (P = 0.030). However, even after matching, survival did not differ between the PLND and no-PLND patients among all patients (P = 0.609) and pN3 patients (P = 0.417) with comparable DSS. CONCLUSION: Bilateral PLND may improve survival in pN2 patients. Men with pN3 may not benefit from bilateral PLND.
Assuntos
Carcinoma de Células Escamosas/cirurgia , Linfonodos/cirurgia , Neoplasias Penianas/cirurgia , Adulto , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/secundário , Terapia Combinada , Intervalo Livre de Doença , Humanos , Estimativa de Kaplan-Meier , Excisão de Linfonodo , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Pelve , Neoplasias Penianas/mortalidade , Neoplasias Penianas/patologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Mesenchymal stem/stromal cells (MSCs) possess some characteristics of immune cells, including a pro-inflammatory phenotype, an immunosuppressive phenotype, antibacterial properties and the expression of Toll-like receptor proteins. Here we show that, similar to immune cells, MSCs retain information from danger signals or environmental stimuli for a period of time. When treated with the pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), MSCs display increased expression of IL-6, IL-8 and MCP-1. Following re-plating and several rounds of cell division in the absence of stimulating factors, the expression of IL-6, IL-8 and MCP-1 remained higher than in untreated cells for over 7 days. A spike in cytokine secretion occurred when cells were exposed to a second round of stimulation. We primed MSCs with LPS and LPS-primed MSCs had better therapeutic efficacy at promoting skin flap survival in a diabetic rat model than did unprimed MSCs. Finally, we found that several microRNAs, including miR146a, miR150 and miR155, along with the modification of DNA by 5-hydroxymethylcytosine (5hmC), mediate the MSC response to LPS and TNF-α stimulation. Collectively, our data suggest that MSCs have a short-term memory of environmental signals, which may impact their therapeutic potential.
Assuntos
Memória Imunológica , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Tecido Adiposo/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Quimiocina CCL2/metabolismo , Metilação de DNA/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Ensaio de Imunoadsorção Enzimática , Memória Imunológica/efeitos dos fármacos , Imunofenotipagem , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Retalhos Cirúrgicos/fisiologia , Sobrevivência de Tecidos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: To evaluate the correlation of pulmonary embolism (PE) and original diseases by retrospectively analysis of the patients for 20 years in single medical center. METHODS: Five hundred and five patients with PE were admitted and treated in General Hospital of Chinese PLA from January 1989 to January 2009, and their clinical data were retrospectively reviewed to analyze the risk factors of PE and the correlations of PE with the original diseases. RESULTS: Of the 505 patients with PE in the past 20 years, the incidence of PE was increased year by year, especially it increased spectacularly after the year of 2004 [61.2% (309) vs. 38.8% (196)]. It was found to be most prevalent in patients of 4160 years old. Its incidence in males was 1.52 folds higher than that of the females [60.4% (305) vs. 39.6% (200)]. Dyspnea, chest pain and hemoptysis were the initial symptoms in the PE patients. Among the 505 patients, 40.0% of them complained dyspnea with chest pain and hemoptysis. Among them, dyspnea occurred in 100.0% of patients, hemoptysis in 52.1%, and chest pain in 40.0%. In 31.1% of the patients if was complicated with deep venous thrombosis (DVT), 19.8% of them suffering from varicosity, 9.5% of them had the history of surgery less than 30 days before, 22.0% of them suffering from neoplasm, 3.6% of them were accompanied with cerebrovascular disease within 4 days, 17.4% of them were accompanied with infection, 10.1% of them were accompanied with primary pulmonary hypertension, and 16.8% of them were accompanied with heart diseases. Multivariate analysis showed that the history of surgery, DVT and neoplasm had significant correlation with the occurrence of PE [odds ratio (95% confidence interval), OR (95%CI) was 4.540 (2.186-9.443), 0.325 (0.155-0.682), 2.610 (1.020-6.708), P<0.05 or P<0.01], while oral contraception, primary pulmonary hypertension and cerebrovascular disease showed a less significant correlation with the occurrence of PE [OR (95%CI) was 0.297 (0.078-1.126), 3.210 (0.855-12.110), 2.939 (0.862-10.020), all P>0.05]. The age and infection did not show significant correlation with the occurrence of PE [OR (95%CI) was 1.041 (0.674-1.607) and 0.820 (0.410-1.665), both P>0.05]. CONCLUSION: The PE is difficult in diagnosis, but with increasing cognizance, the diagnostic rate of PE has been increased. Patients with history of surgical operation, DVT or neoplasm, who complain dyspnea without known cause, chest pain or hemoptysis, should be subjected to further examinations, as to confirm the diagnosis of PE, then the survival rate of the patients with PE may be elevated.
Assuntos
Embolia Pulmonar/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/terapia , Estudos Retrospectivos , Adulto JovemRESUMO
OBJECTIVE: To investigate the possibility of adipose derived stem cells (ADSCs) for wound healing by detecting cellular phenotype conversion of ADSCs into endothelial cells (ECs). METHODS: ADSCs were isolated and cultured from adipose tissue derived from SD rats (n = 8), and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) in vitro. The marker antigen of P3 ADSCs was detected by analysis CD49d and CD106 antigens expression using flow cytometry, and the multipotential differentiation of P3 ADSCs were identified by specific medium inducing to differentiate into osteoblasts and adipocytes. And then, the ADSCs were cultured and induced for 3 days by condition culture medium (containing 30% superior of homogenating rat blood vessels in 10%FBS DMEM) as experimental group, and were cultured by 10% FBS DMEM as control group, and the expression of CD34 and von Willebrand factor (vWF) in ADSCs were analyzed by flow cytometry. RESULTS: Flow cytometry analysis showed that the expression of CD49d and CD106 in ADSCs were positive (98.32 ± 0.37)% and negative (1.67 ± 0.61)%, respectively. The multipotential differentiation experiment demonstrated that the cultured P3 ADSCs can be induced to differentiate into osteoblasts and adipocytes in vitro. The positive rate of CD34 and vWF were (77.14 ± 0.76)% and (75.46 ± 0.37)% in condition medium group, higher than (1.38 ± 0.31)% and (1.70 ± 0.23)% in 10% FBS DMEM control group, respectively (P < 0.01). CONCLUSION: The ADSCs can be induced to differentiated into ECs, suggesting that ADSCs have potential to take part in wound repair and angiogenesis.
Assuntos
Tecido Adiposo/citologia , Células Endoteliais/citologia , Endotélio Vascular/citologia , Células-Tronco/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Transdiferenciação Celular , Células Cultivadas , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: coronary artery ligation to induce myocardial infarction (MI) in mice is typically performed by an invasive and time-consuming approach that requires ventilation and chest opening (classic method), often resulting in extensive tissue damage and high mortality. We developed a novel and rapid surgical method to induce MI that does not require ventilation. OBJECTIVE: the purpose of this study was to develop and comprehensively describe this method and directly compare it to the classic method. METHODS AND RESULTS: male C57/B6 mice were grouped into 4 groups: new method MI (MI-N) or sham (S-N) and classic method MI (MI-C) or sham (S-C). In the new method, heart was manually exposed without intubation through a small incision and MI was induced. In the classic method, MI was induced through a ventilated thoracotomy. Similar groups were used in an ischemia/reperfusion injury model. This novel MI procedure is rapid, with an average procedure time of 1.22 ± 0.05 minutes, whereas the classic method requires 23.2 ± 0.6 minutes per procedure. Surgical mortality was 3% in MI-N and 15.9% in MI-C. The rate of arrhythmia was significantly lower in MI-N. The postsurgical levels of tumor necrosis factor-α and myeloperoxidase were lower in new method, indicating less inflammation. Overall, 28-day post-MI survival rate was 68% with MI-N and 48% with MI-C. Importantly, there was no difference in infarct size or post-MI cardiac function between the methods. CONCLUSIONS: this new rapid method of MI in mice represents a more efficient and less damaging model of myocardial ischemic injury compared with the classic method.
Assuntos
Vasos Coronários/cirurgia , Modelos Animais de Doenças , Infarto do Miocárdio/etiologia , Projetos de Pesquisa/normas , Animais , Ligadura/efeitos adversos , Ligadura/métodos , Masculino , Métodos , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
OBJECTIVE: To explore a new method of isolation and culture of eccrine sweat gland ductal cells from human split-thickness skin graft in vitro. METHODS: Human split-thickness skin graft which was presented by volunteer (n = 10) was digested with type II collagenase, and then sweat gland duct were isolated from the split-thickness skin graft, primary cultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2, 95% O2. The cultured eccrine sweat gland ductal cells were identified by analysis CEA, CK8, CK18, CK19 antigens expression with flow cytometry, RT-PCR and Western Blot, and by detecting the electrophysiology with whole cell patch clamp technology. RESULTS: The isolated eccrine sweat gland ductal cells could grow by adhering to the wall, proliferate in vitro after 48 h of adhering to the wall, and confluens after 2 - 4 weeks of adhering to the wall. The FACs analysis showed the expression of CEA was (90.26 +/- 1.12)%, (89.70 +/- 1.43)%, and CK8 was (94.41 +/- 1.84)%, (93.65 +/- 1.63)% in primary cultured sweat gland ductal cells and primary cultured eccrine sweat gland cells, respectively, and there is no significant difference between the two groups (P > 0.05). Immunocytochemistry staining showed CEA, CK8, CK18, CK19 was positive in sweat gland duct cells, RT-PCR revealed that CEA, CK8, CK18 and CK19 gene expression in sweat gland ductal cells, and Western Blot analysis showed the expression of CEA brand, CK8 brand, CK18 brand, and CK19 brand in sweat gland ductal cells, patch clamp indicated that this cells has distinct amiloride sensitive Na(+) channels. CONCLUSIONS: The cultured human eccrine sweat gland duct cells in vitro display the markers and biological characteristics of sweat gland epithelial lineage, and this method of digest the split-thickness skin graft to get the sweat gland duct cells is better than classical dissect sweat gland under dissect microscope.
Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Glândulas Sudoríparas/citologia , Células Cultivadas , HumanosRESUMO
OBJECTIVE: To investigate the migrating effect of adipose derived stem cells (ADSCs) on the wound model of human epidermal keratinocyte (HEKa). METHODS: Rat ADSCs (rADSCs) were isolated and cultured (n = 10), rADSCs were direct co-cultured with HEKa cells in experiment group (experimental group, n = 10). In the control groups, rADSCs were indirect co-cultured with HEKa cells in transwell chamber (indirect group, n = 8), or HEKa was cultured alone (single group, n = 8). Then confluent HEKa cells were scraped to establish a wound model under invert microscope. After scraped 24, 48, and 72 h, cell numbers of which migrated across the edge of the wound was measured, the rate of wound healing was calculated by using SigmaScan Pro 5 software, and the proliferating effect of rADSCs on HEKa were examined by incorporation of [(3)H] thymidine. RESULTS: The cells migrated across the edge of wound after 24 hours in experimental group, indirect group, and single group were (9.2 + or - 0.2), (5.0 + or - 0.3), (4.2 + or - 0.3), and were (58.5 + or - 0.4), (26.5 + or - 0.3), (20.7 + or - 0.5) 48 hours after, and were (125.8 + or - 0.4), (43.0 + or - 0.5), (35.6 + or - 0.5) cells/HP 72 hours after, respectively; the numbers were all significantly higher in experimental group than those in control groups (P < 0.05). The rates of wound healing after scraped 72 hours were 61.0% + or - 3.0%, 35.0% + or - 2.5% and 32.0 + or - 2.1%, the outcome in experimental group was significantly better than in the control groups (P < 0.05). And the thymidine feeding displayed the proliferation of HEKa in the three groups were (1440 + or - 210), (1050 + or - 280) and (1130 + or - 390) cpm/10(5) cell, and there was significant difference between the experimental and the control groups (P < 0.05). CONCLUSIONS: The rADSCs can promote the migration of HEKa by direct contact with it.
Assuntos
Tecido Adiposo/citologia , Células Epidérmicas , Queratinócitos/citologia , Células-Tronco/citologia , Animais , Contagem de Células , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Humanos , Ratos , Ratos Sprague-Dawley , CicatrizaçãoRESUMO
Recent studies have shown that insulin growth factor-1 (IGF-1) and either erythropoietin (EPO) or the long-acting EPO analog Darbepoetin alfa (DA) protect the heart against ischemia/reperfusion (I/R) and myocardial infarction (MI). The present study examined the cardioprotective effect of simultaneous treatments with IGF-1 and DA in these models of cardiac injury. Rats were subjected to I/R or MI and were treated with IGF-1, DA, and a combination of IGF-1 and DA, or vehicle treatment. IGF-1 and DA treatments imparted similar protective effect by reducing infarct size. Moreover, these treatments led to improvement of cardiac function after I/R or MI compared to vehicle. In the reperfused heart, apoptosis was reduced with either or both IGF-1 and DA treatments as measured by reduced TUNEL staining and caspase-3 activity. In addition, after MI, treatment with IGF-1 or DA significantly induced angiogenesis. This angiogenic effect was enhanced significantly when IGF-1 and DA were given simultaneously compared to vehicle or either agents alone. These data indicate simultaneous pharmacological treatments with IGF-1 and DA protect the heart against I/R and MI injuries. This protection results in reduced infarct size and improved cardiac function. Moreover, this treatment reduces apoptosis and enhances angiogenesis in the ischemic heart.
Assuntos
Eritropoetina/análogos & derivados , Fator de Crescimento Insulin-Like I/uso terapêutico , Isquemia Miocárdica/patologia , Neovascularização Patológica , Traumatismo por Reperfusão/patologia , Animais , Apoptose , Caspase 3/metabolismo , Darbepoetina alfa , Eritropoetina/uso terapêutico , Marcação In Situ das Extremidades Cortadas , Masculino , Modelos Biológicos , Infarto do Miocárdio/patologia , Miocárdio , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To isolate and culture mesenchymal stem cells ( MSC) from different sources and to investigate the chemotactic effects of burn rat serum on MSC derived from different sources. METHODS: Seventy-two Wistar rats were randomly divided into burn( n = 36, with 30% TBSA full-thickness burns on the back) and sham burn (n = 36, without burns) groups. Bone marrow and peripheral blood of the rats in both groups were collected to isolate and culture MSC. Ratio of MSC, growth speed and cell morphology were observed with inverted microscope. Effects of different serum ( fetal bovine serum, normal rat serum and burn rat serum) on chemotaxis of MSC derived from different sources and their migration ability were subsequently examined with a transwell system. Results MSC were obtained from bone marrow of the rats in both groups. MSC were successfully obtained from bone marrow of all burn rats(100% , P <0.05) , but only from peripheral blood of 7 burn rat(58% ) , and no MSCs were obtained from peripheral blood of 12 rats in sham group( P <0.05). There was small amount of adherent cells 24 hrs after culture, and fusiform shaped adherent cells were sporadically observed in scattered distribution 2-3 days later with inverted microscope. There was no obvious difference in the cell morphology between the 2 groups. In the sham group, the number of MSC migrating to the lower surface of transwell after burn serum treatment [ (94 Il ) cells/ high power field] was significantly greater than that after the treatment with normal rat serum and fetal bovine serum [ (37 +/- 6) , (38 +/- 11) cells/high power field , P <0.01 ] , while no difference in migration ability was found after normal serum treatment compared with that after fetal bovine serum treatment ( P >0. 05). The migration rate of MSCs which were derived from bone marrow in sham group was obviously lower than those derived from bone marrow and peripheral blood from burn rats ( P <0. 05 or 0. 01). Though some difference of the migration ability existed between MSC derived from bone marrow and peripheral blood, there was no statistically significant difference ( P >0. 05). CONCLUSION: MSC can be isolated and cultured from bone marrow and peripheral blood of burn rat, but not from peripheral blood of normal rat. Burn rat serum has a stronger chemotactic effect on MSC. Moreover, the migration ability of MSC derived from burn rat is stronger than that of MSC derived from normal rat.
Assuntos
Queimaduras/sangue , Quimiotaxia , Células-Tronco Mesenquimais/citologia , Soro , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Movimento Celular , Separação Celular , Células Cultivadas , Modelos Animais de Doenças , Masculino , Ratos , Ratos WistarRESUMO
OBJECTIVE: To investigate the transdifferentiation of the ADSCs to epidermal cells. METHODS: ADSCs were isolated and cultured from rat adipose tissue by digestion of enzyme. ADSCs was identified by immunocytochemistry and flow cytometry. ADSCs were divided into four groups in order to induce: the condition medium (containing 30% superior of homogenizing rat skin in 10% FBS/DMEM) group, 7 days; 10% FBS/DMEM with EGF (20 ng/ml) group, 7 days; the condition medium for 4 days and then 10% FBS/DEME instead of the condition medium for 3 days group; 10% FBS/DMEM for 7 days group (control group). Cytokeratin 19 and cytokeratin 10 expressions in ADSCs were detected by flow cytometry. RESULTS: (1) The results of immunocytochemistry showed that ADSCs were positive for CD49d and negative for CD106, CD34, CD19, CD10. The results of flow cytometry showed ADSCs were positive for CD49d and CD44. (2) The CK19 expression of ADSCs was 45.32% in the condition medium group, 26.58% in the condition medium with EGF group, 23.37% in te condition medium for 4 days and then 10% FBS/DMEM instead of the condition medium for 3 days gropu and 18.53% in control group, P <0.01. The CK10 expression of ADSCs was 43.56% in the condition medium group, 25.54% in the condition medium with EGF group, 18.20% in the condition medium for 4 days and then 10% FBS/DMEM instead of the condition medium for 3 days group and 2.46% in control group, P < 0.01. CONCLUSIONS: The superior of homogenizing rat skin can induce CK19 and CK10 expressing in ADSCs, and thereby demonstrating ADSCs can differentiate to epidermal cell phenotype in vitro.
Assuntos
Adipócitos/citologia , Transdiferenciação Celular , Células-Tronco/citologia , Animais , Células Cultivadas , Queratina-19/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To get the full length of human METH1 cDNA and express it steadily in mammalian cell stably. METHODS: METH1 was amplified by RT-PCR, and cloned into pCDNA3.0 after confirmed by sequence analysis. HepG2 cells were transfected by Lipofectamine reagent and then selected in medium with G418. The expression level of METH1 was detected by RT-PCR and Western blot. RESULTS: METH1 with expected length was effectively amplified, and completely matched the published sequence of encoding mature peptide [GI:5725505] as shown by sequence analysis. Eukaryotic vector expressing METH1 was obtained by gene cloning, cells expressing METH1 was got by selection with G418 at 3 weeks after transfection. RT-PCR and Western blot showed high level expression of METH1. CONCLUSION: Full length of human METH1 gene is cloned successfully and expressed in HepG2 steadily, The results set up a basis for the study of effects of METH1 on hypertrophic scar angiogenesis.
Assuntos
Inibidores da Angiogênese/genética , Desintegrinas/genética , Metaloendopeptidases/genética , Proteínas ADAM , Proteína ADAMTS1 , Inibidores da Angiogênese/metabolismo , Western Blotting , Linhagem Celular Tumoral , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Desintegrinas/metabolismo , Humanos , Metaloendopeptidases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção/métodosRESUMO
OBJECTIVE: To investigate the effect of local application of bFGF and sucralfate during continuous tissue expansion (CTE). METHODS: CTE combined with local administration of bFGF and sucralfate was used in twelve patients with scar and nasal tip defects. Twenty three expanders were placed in the subcutaneous pockets through intralesion short incisions. Continuous expansion began at 1-3 days after expander implantation. The histomorphological changes and epidermal cell proliferation were observed. The clinical results were investigated. RESULTS: The average inflation time was 8.9 days. The average interval of the two operations was 13.5 days. The average hospitalization was 28.4 days. The average immediate stretch-back rate of the expanded skin was 25.7%. The clinical results were satisfactory without any complications. Histological examinations showed that the epidermal, granular and spinous layer became thicker. The basal cells increased significantly. The dermis thinned slightly and the collagen fibers became thicker. The elastic fiber regenerated significantly. Fibroblast and capillary density increased obviously. The immunohistochemistry analysis showed that the proliferation of epidemic basal cells was significant postoperatively. CONCLUSION: Local application of exogenous bFGF and sucralfate during CTE was feasible in patients. It could accelerate tissue expansion and improve the quality of expanded skin flap.