Aldefluor protocol to sort keratinocytes stem cells from skin

Abstract Purpose: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.

Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients

ABSTRACT PURPOSE: To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. METHODS: Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. RESULTS: Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. CONCLUSION: There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

Cancer stem cells: a new approach to tumor development / Células-tronco de câncer: um nova abordagem do desenvolvimento tumoral

Many theories have been proposed to explain the origins of cancer. Currently, evidences show that not every tumor cell is capable of initiating a tumor. Only a small part of the cancer cells, called cancer stem cells (CSCs), can generate a tumor identical to the original one, when removed from human tumors and transplanted into immunosuppressed mice. The name given to these cells comes from the resemblance to normal stem cells, except for the fact that their ability to divide is infinite. These cells are also affected by their microenvironment. Many of the signaling pathways, such as Wnt, Notch and Hedgehog, are altered in this tumoral subpopulation, which also contributes to abnormal proliferation. Researchers have found several markers for CSCs; however, much remains to be studied, or perhaps a universal marker does not even exist, since they vary among tumor types and even from patient to patient. It was also found that cancer stem cells are resistant to radiotherapy and chemotherapy. This may explain the re-emergence of the disease, since they are not completely eliminated and minimal amounts of CSCs can repopulate a tumor. Once the diagnosis in the early stages greatly increases the chances of curing cancer, identifying CSCs in tumors is a goal for the development of more effective treatments. The objective of this article is to discuss the origin of cancer according to the theory of stem cell cancer, as well as its markers and therapies used for treatment.

Diversas teorias buscam explicar a origem do câncer. Atualmente, há evidências de que nem todas as células tumorais têm poder de iniciar um tumor. Apenas uma pequena parte das células cancerígenas, chamadas de células-tronco de câncer (do inglês cancer stem cells - CSC), é capaz de iniciar um tumor idêntico ao original quando retirada de tumores humanos e enxertada em camundongos imunossuprimidos. Essas células foram assim denominadas por suas semelhanças com células-tronco normais, exceto pelo fato de que sua capacidade de dividir-se é infinita. Essas células também recebem influência de seu microambiente. Várias vias de sinalização, como WNT, NOTCH e Hedgehog, estão alteradas nessa subpopulação tumoral, contribuindo também para a desregulação de sua proliferação. Pesquisadores descobriram vários marcadores para as CSC, porém ainda há muito a ser pesquisado, ou talvez nem exista um marcador universal, já que eles variam entre cada tipo de tumor e até de paciente para paciente. Foi constatado também que as CSC são resistentes à radioterapia e à quimioterapia, podendo explicar o reaparecimento da doença, visto que, além de não eliminá-la completamente, quantidades mínimas das CSC podem repovoar um tumor. Como o diagnóstico em estágios iniciais aumenta muito as chances de cura do câncer, a identificação das CSC em meio a um tumor é alvo para o desenvolvimento de tratamentos mais eficazes. O objetivo deste artigo é discutir a origem do câncer segundo a teoria das CSC, bem como seus marcadores e as terapias utilizadas em seu tratamento.

Social inclusion and solidarity in strict post-graduation: experiencing the laboratory \"scientific wake for the future\" (primary and secondary education) / Inserção social e solidariedade na pós-graduação stricto sensu: vivenciando o laboratório "despertar científico para o futuro" (ensino médio e fundamental)

Objective: To stimulate and awakening the interest of students of high school or elementary public schools in research and science through scientific initiation stages in the Postgraduate Program in Translational Surgery. To stimulate and awakening the interest of students of high school or elementary public schools in research and science through scientific initiation stages in the Postgraduate Program in Translational Surgery. Method: The target audience for the development of scientific activities were students enrolled in mid-level course (second year initially) and have approval of their participation in this project by the school and by legal guardians. The inclusion criteria were: physical proximity to the higher education institution, signing the consent form by the legal responsible for the students, and for the board of the school unit and the researcher. Initially, students performed diagnostic evaluation about the prior knowledge of biology, science and scientific research. From there, the classes were prepared based on the result of this test, then started the activities of Junior Scientific Initiation in basic education. Results: The school chosen for this initial phase of the pilot project was the State School Rui Bloem which has 13 classrooms for the second year of medium education in a total of 390 students. Of these, 160 (41%) were interested but only 16 (10%) were eligible to start the pilot project in Translational Surgery Laboratory of Unifesp. These students showed average yield of 50% in diagnostic test and should start the next training in cell and molecular biology laboratory and also to attend scientific meetings. Conclusion: In the initial phase of the project, was observed the great student interest in scientific career, but at the same time, a great need for improvement. The choice of public school was for access to university and proximity. In addition, these students have more shortcomings and deficiencies. But this does not mean that the fascination for scientific career cannot turn them into great researchers thus contributing to the economic, social and intellectual growth of our country through scientific research.

Objetivo: Estimular e despertar o interesse dos alunos do ensino médio ou fundamental de escolas públicas na pesquisa e na ciência por meio de estágios de Iniciação Científica Junior no Programa de Pós-Graduação em Cirurgia Translacional da Unifesp. Método: O público alvo para o desenvolvimento das atividades científicas foram alunos regularmente matriculados em curso de nível médio (segundos anos inicialmente) e que tivessem aprovação de sua participação pela direção da escola e responsáveis legais. Quanto aos critérios de inclusão: proximidade física com a instituição de ensino superior, assinatura do termo de consentimento pelos responsáveis dos alunos, pela diretoria da unidade escolar e pelo pesquisador. Inicialmente, os alunos realizaram avaliação diagnóstica acerca dos conhecimentos prévios de biologia, ciências e pesquisa científica. A partir daí as aulas eram elaboradas com base no resultado deste teste, para então iniciar as atividades de Iniciação Científica Junior. Resultados: A escola escolhida para esta fase inicial do projeto piloto foi a Escola Estadual Rui Bloem que possui 13 salas de aula para o segundo ano do ensino médio com total de 390 alunos. Destes, 160 (41%) apresentaram-se interessados; porém, somente 16 (10%) foram elegíveis para iniciar o projeto piloto no Laboratório de Cirurgia Translacional da Unifesp. Estes alunos apresentaram rendimento médio de 50% na prova diagnóstica e deverão iniciar os próximos treinamentos no Laboratório de Biologia Celular e Molecular e também a frequentar as reuniões científicas. Conclusão: Nesta fase inicial houve interesse dos alunos do ensino médio e o projeto piloto apresentado estimulou e despertou interesse dos alunos de escola pública na pesquisa e na ciência. A escolha de uma escola pública foi para o acesso à universidade e proximidade. Além disso, esses alunos têm mais carências e deficiências. Mas isso não significa que o fascínio pela carreira científica não pode transformá-los em grandes pesquisadores, contribuindo assim para o crescimento econômico, social e intelectual do nosso país através da pesquisa científica.

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury

PURPOSE: To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). RESULTS: After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome. .

Human beta defensin-4 and keratinocyte growth factor gene expression in cultured keratinocyte and fibroblasts of burned patients

PURPOSE: To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. METHODS: Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. RESULTS: In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. CONCLUSIONS: KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients. .

Innate and adaptive immunity gene expression of human keratinocytes cultured of severe burn injury

PURPOSE: Evaluate the expression profile of genes related to Innate and Adaptive Immune System (IAIS) of human Primary Epidermal keratinocytes (hPEKP) of patients with severe burns. METHODS: After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific IAIS PCR Arrays plates (SA Biosciences). RESULTS: After the analysis of gene expression we found that 63% of these genes were differentially expressed, of which 77% were repressed and 23% were hyper-regulated. Among these, the following genes (fold increase or decrease): IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (- 73), CD86 (-41) and HLA-A (-35). CONCLUSIONS: This study contributes to the understanding of the molecular mechanisms underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome. .

Keratinocyte growth factor, interleukins (1 beta, 6, 8, 10, 12), and tumor necrosis factor alpha in culture medium of dermal fibroblast of burned patients

PURPOSE: To evaluate the level of cytokines and keratinocyte growth factor (KGF) or Fibroblast Growth Factor 7 (FGF-7) in the culture medium of cultured human dermal fibroblasts from patients with large burn in comparison to small burn. METHODS: Fibroblasts of 10 patients (four large burns, four small burns and two controls) were initiated by the enzymatic method using collagenase. Cytokines and KGF in the supernatant of the culture medium was measured by, respectively, flow cytometry using Cytometric Bead Array Human Inflammation kit (CBA, BD Biosciences, USA) and the enzyme immunoassay method using the Quantikine (r) Human KGF. The experiments were performed in triplicate. RESULTS: The expression of IL-12 protein in patients with large burns showed a tendency to increase. IL- 6, IL- 10, and IL- 1beta were observed no difference. For IL - 8, TNF - alpha and KGF was observed a significant difference between the expression in large and small burned patient. CONCLUSION: That IL-8, TNF-alpha and KGF showed higher expression in cultured fibroblasts of large burned patients. .

Keratinocyte growth factor, tumor necrosis factor-alpha and interleukin-1 beta gene expression in cultured fibroblasts and keratinocytes from burned patients

PURPOSE: To evaluate the gene expression of KGF, TNF-alpha and IL-1 beta in skin fibroblasts and keratinocytes cultured from burned patients. METHODS: Three patients with large burns and three patients with small burns, as well as two controls, were included. The cell culture was initiated by the enzymatic method. After extraction and purification of mRNA, qPCR was used to assess the gene expression of KGF, TNF-alpha and IL-1 beta. RESULTS: The expression of KGF was increased on average 220-fold in large burns and 33.33-fold in small burns in fibroblasts, and 11.2-fold in large burns and 3.45-fold in small burns in keratinocytes compared to healthy patients (p<0.05). Expression of TNF-alpha was not observed. IL-1 beta is down-regulated in fibroblasts of burned patients, and much more repressed in small burns (687-fold, p<0.05). In keratinocytes, the repression of IL-1 beta expression occurs in patients with small burns (28-fold), while patients with large burns express this gene intensively (15-fold). CONCLUSIONS: The study showed a quantitative pattern in the expression of KGF gene, which is more expressed according to the size of the burn. TNF-alpha was not expressed. A qualitative pattern in the expression of IL-1 beta gene was demonstrated.