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- Fredriksson, Camilla, 1969-
(författare)
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Keratinocytes in tissue engineering of human skin: invitro and in vivo studies
- 2008
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Full thickness wounds, such as deep burns, need restoration of both the dermal and epidermal layers of the skin. In normal wound healing, re-epithelialization occurs by migration and proliferation of keratinocytes from the wound edges and by differentiation of stem cells from remaining hair follicles. Restoration of dermis occurs by influx of growth factors secreted by macrophages, platelets, and fibroblasts; by fibroblast proliferation and subsequent synthesis and remodeling of collagenous dermal matrix. In the case of full-thickness acute burn injuries and chronic wounds (e.g. pressure ulcers, venous ulcers and diabetic foot ulcers), these processes are defective. With the principles of tissue engineering in mind (to correct, improve and maintain tissues and their functions), researchers have developed promising materials and methods to make it possible to restore either the dermal (Integra® DRT, Alloderm®) or the epidermal layer (split thickness skin grafts (STSG), cultured epithelial autografts (CEA), autologous keratinocytes in single cell suspension). It is now well established that superior results are obtained if both dermal and epidermal components are combined, for example in a bilayered skin equivalent. Apligraf® is recommended for use on venous ulcers and is the only bilayered living skin equivalent currently approved by the FDA. Studies on different factors affecting the wound healing capacity as well as techniques in use provide valuable information for further development.In this licentiate thesis, we evaluated different transplantation techniques for delivering cultured human keratinocytes in single cell suspension, a measure becoming more frequently used in addition to STSG and CEA for restoring the epidermal layer of the skin. We found that the pressure device, commonly used to spray cell suspension onto the wound with pressures as high as 200 kPa, killed around 0% of the cells. In comparison, an ordinary syringe with the attachment of a spray nozzle showed almost 90% viable cells post transplantation and provided an equally good distribution of the cell suspension.We also studied different silver containing dressings regarding silver accumulation in human skin. In addition, we graded the re-epithelialization to evaluate whether the dressings caused any delay in the wound healing process. We found that the silver dressings tested, with few exceptions, caused dermal accumulation of silver, primarily aggregated around blood vessels. We could also show that most of the dressings had negative effect on the re-epithelialization.For the restoration of the dermal layer of the skin, Integra® DRT functions as a scaffold for guided tissue regeneration of the dermis. We had the possibility to study a case of necrotizing fasciitis were the treatment consisted of the use of Integra® DTR together with sub-atmospheric pressure (after initial surgical debridement) and later transplantation of split thickness skin grafts. This measure proved to be safe as well as giving satisfactory pliable and aesthetically acceptable result.
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| 2. |
- Wallin, Åsa, 1976-
(författare)
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Molecular Studies of Irradiation and SN-38 on Colorectal Cancer
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is one of most common cancer diseases worldwide. In Swedenapproximately 5,000 new cases of CRC are generated each year, which makes it the thirdmost common cancer disease among both men and women. The past decades ofimproved treatment strategies have considerably increased the five-year survival for CRCpatients. However more could be achieved in this area, in particular for metastatic CRC,which is the cause of most CRC-related deaths. Therefore it is important to study thebiological response to certain treatments induced in CRC to find valuable predictiveand/or prognostic factors to select patients for better suited treatments.The aim of this thesis was to gain insight into the molecular changes that occurfollowing irradiation or treatment with SN-38 in rectal cancer patients or colon cancercell lines by studying the RNA expression, protein expression, DNA cell cycledistribution and apoptotic response. The expression of phosphatase of regenerating liver(PRL) proteins was investigated in rectal cancers from 125 patients included in arandomized clinical trial of preoperative radiotherapy (RT). Increased expression of PRLswas seen at the invasive margin of primary and metastatic cancers compared with theinner area of the tumors. Moreover, strong PRL staining at the invasive margin correlatedto distant recurrence and worse survival of patients in the RT group but not in non-RTgroup (Paper I). Radiosensitivity was studied by treating KM12C, KM12SM andKM12L4a colon cancer cell lines with radiation. KM12C is of low metastatic naturecompared with the highly metastatic KM12SM and KM12L4a. Upregulation of ΔNp73and PRL-3 might contribute to the radioresistant phenotype in KM12C. In contrast,KM12L4a shows a high frequency of apoptosis and lack of upregulation of ΔNp73, PRL-3 and survivin, which might explain its radiosensitive phenotype (Paper II). KM12C,KM12SM and KM12L4a were treated with SN-38 which inhibits topoisomerase 1 (topo-1). The results show that SN-38 induces G2/S arrest and possess the capacity to triggerapoptosis in the three cell lines (Paper III). To further elucidate SN-38 effect on these celllines, the gene expression profile following SN-38 treatment was studied. Oligonucleotidearrays consisting of ~27,000 spots were hybridized with sample and reference cDNA.Both unsupervised and supervised hierarchical clustering analysis, and functional analysiswere performed. Supervised hierarchical clustering gives a strong signal of 1453discriminated genes, the vast majority being upregulated. Both upregulated anddownregulated genes point toward a favorable impact of SN-38 regarding the apoptoticpathways. For example RhoB and Bax are upregulated together with downregulation ofKras and survivin, which promotes apoptosis (Paper IV).In conclusion, PRLs may be valuable biomarkers for RT resistance, predicting apoor prognosis in rectal cancer patients. Targeting radio-resistance factors, such asΔNp73 and survivin may contribute to an increased sensitivity to RT. SN-38 affects cellproliferation and apoptosis.
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| 3. |
- Pfeifer, Daniella
(författare)
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p73 in colorectal cancer
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Colorectal cancer (CRC) is the third most common cancer in the world, with about 5000 new cases in Sweden every year. CRC is caused by mutation (inherited or acquired) in genes, by gene variants and changed expression of proteins. The primary way to achieve a curative result for CRC is to remove the tumor by surgery. To reduce risk of recurrence chemo- or radiotherapy are given as a complement to surgery. p73 is a structural and functional homologue of tumor suppressor p53. However, p73 is rarely mutated in tumors, but rather overexpressed as compared to normal tissue. There are two main isoforms of p73, the transactivation capable TAp73 and the truncated ΔNp73, which are involved in an autoregulatory loop with TAp73 and p53.The aim of this study was to investigate the role of p73 and related proteins in the development and treatment of CRC. A G4C14-to-A4T14 polymorphism of p73 was studied in CRC patients and healthy controls (Paper I), and rectal cancer patients who were randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper II). The AT/AT genotype of the p73 polymorphism may increase risk of CRC development and CRC patients with the AT allele had a better prognosis. When dividing the cases into colon and rectal cancer it was seen that in colon cancer the AT allele tended to be more favorable for overall survival, while in rectal cancer the GC allele seemed to be more favorable. Rectal cancer patients, with a combination of GC/GC genotype, wild type p53 and weak survivin expression survived longer after preoperative radiotherapy. This was not observed in the patients only receiving surgery. The protein expression of p73 was further studied in the rectal cancer patients randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper III). p73 was expressed higher in tumor tissue than in normal mucosa. Patients with p73 negative tumors had a lower risk of local recurrence after radiotherapy, as opposed to patients that had p73 positive tumors or patients with p73 negative tumors that did not receive radiotherapy. Effects of γ-radiation was further studied in colon cancer cell lines KM12C, KM12SM and KM12L4a regarding cell cycle, survival fraction (clonogenicity), apoptosis and protein expression patterns of mutated p53, TAp73, ΔNp73, survivin and PRL-3 (Paper IV). KM12C displayed low survival fraction, low apoptosis, no cell cycle arrest and an upregulation of the antiapoptotic ΔNp73 after irradiation. KM12L4a showed a high survival fraction, but high apoptosis, arresting of the cell cycle and upregulation of the radio-resistance factor survivin. The effects of overexpression and knockdown of survivin on TAp73, ΔNp73 and p53 expression in colon cancer cell lines HCT-116p53+/+ and HCT-116p53-/- with and without γ-radiation were studied (Paper V). Overexpression of survivin decreased wild type p53, whilst downregulation of survivin lead to a simultaneous downregulation of TAp73 and ΔNp73, mRNA and protein, both with and without γ- radiation. Knockdown of survivin also demonstrated an increase in apoptosis.In conclusion, we showed that the G4C14-to-A4T14 polymorphism of p73 and p73 protein expression may be involved in CRC development, radiotherapy response and survival. We further showed that TAp73, ΔNp73 and p53 were regulated by survivin in colon cancer cells.
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