Separation of the epidermal sheet by dispase

Kitano, Yukio; Okada, Natsuko

doi:10.1111/j.1365-2133.1983.tb01056.x

Cited by 176 publications

(102 citation statements)

References 12 publications

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“…Primary keratinocytes were prepared from newborn transgenic mice and from their control littermates by ā otation procedure in dispase (0.1 g dispase/ml PBS) at 48C overnight (Kitano and Okada, 1983). The keratinocytes were grown in MDCB 153 Medium (Seromed, Berlin, Germany) containing the following supplements: 0.05 mM Calcium, 0.3 ng/ml EGF (Sigma, Deisenhofen, Germany), 0.5 mg/ml Insulin (Seromed), 0.05 mg/ml hydrocortisone (Seromed), 7.5 mg/ml bovine pituitary extract (Sigma) and 3% dialyzed FCS (Drozdo and Pledger, 1993).…”

Section: Primary Cell Culture and [ 3 H]-thymidine Incorporationmentioning

confidence: 99%

Expression of a dominant negative type II TGF-β receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development

et al. 1998

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The role of Transforming growth factor beta (TGF-b) in carcinogenesis is complex. There are reports on both tumor inhibition and tumor promotion by TGF-b. To elucidate the complex role of TGF-b in epithelial carcinogenesis, we generated transgenic mice overexpressing a dominant negative type II TGF-b receptor in the basal cell compartment and in follicular cells of the skin. Despite the reduced responsiveness of transgenic keratinocytes to TGF-b, both proliferation and di erentiation were normal in non-irritated epidermis of these transgenic mice. Thus, interruption of signaling of all three isoforms of TGF-b in basal and follicular cells does not disturb tissue homeostasis. However, during tumor promotion transgenic mice showed an elevated level of proliferation in the epidermis. This hyperproliferation correlated with a very early onset of carcinoma development and a malignant conversion frequency of 30% from benign papillomas to carcinomas. By comparison, the conversion frequency in wild-type mice of this strain has previously been reported as 5.5%. Even without induction of hyperproliferation by tumor promoters, transgenic mice developed far more carcinomas as controls when treated with a carcinogen. This result indicates that there is a synergistic e ect between loss of TGF-b responsiveness and mutations caused by initiation with a carcinogen leading to an endogeneous tumor promotion in initiated cells only.

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Section: Primary Cell Culture and [ 3 H]-thymidine Incorporationmentioning

confidence: 99%

Expression of a dominant negative type II TGF-β receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development

et al. 1998

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“…Normal human keratinocytes (NHK) were isolated from foreskins of young donors as described. 36 NHK cells were grown in keratinocyte serum-free medium (Life Technologies, Paisley, UK) with a calcium concentration of 0.09 mM and were supplemented with 50 mg/ml of bovine pituitary extract and 5 ng/ml of human epidermal growth factor (Life Technologies, Paisley, UK). Third to fifth passage cells were used.…”

Section: Keratinocyte Culture and Differentiationmentioning

confidence: 99%

Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing

et al. 2000

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The epidermis is a stratified squamous epithelium in which keratinocytes progressively undergo terminal differentiation towards the skin surface leading to programmed cell death. In this respect we studied the role of caspases. Here, we show that caspase-14 synthesis in the skin is restricted to differentiating keratinocytes and that caspase-14 processing is associated with terminal epidermal differentiation. The proapoptotic executioner caspases-3, -6, and -7 are not activated during epidermal differentiation. Caspase-14 does not participate in apoptotic pathways elicited by treatment of differentiated keratinocytes with various death-inducing stimuli, in contrast to caspase-3. In addition, we show that non-cornifying oral keratinocyte epithelium does not express caspase-14 and that the parakeratotic regions of psoriatic skin lesions contain very low levels of caspase-14 as compared to normal stratum corneum. These observations strongly suggest that caspase-14 is involved in the keratinocyte terminal differentiation program leading to normal skin cornification, while the executioner caspases are not

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“…[12][13][14][15] In in vitro permeation studies, a significant increase in the permeated amount of bovine insulin was obtained from pH 3.0 solution after pretreatment with 0.25% trypsin, but no enhancement was observed with pH 6.0 solution. It is known that insulin forms aggregates to varying degrees from dimers to hexamers depending on the pH, concentration, and ionic strength of the solution.…”

mentioning

confidence: 99%

“…For more than 50 years, proteolytic enzymes such as trypsin have been extensively used in laboratory settings for in vitro epidermal separation and keratinocyte isolation. [12][13][14][15] The unique ability of proteases to cause selective epidermal separation has been in part explained by the proteolytic degradation of desmosomal proteins in the SC, which leads to cell dissociation. 16,17) Recently, several endogenous proteases occurring in the epidermis have been found to play important roles in regulating epidermal cell desquamation.…”

mentioning

confidence: 99%

Transdermal Delivery of Insulin Using Trypsin as a Biochemical Enhancer

Quan

Zang

et al. 2008

Biological & Pharmaceutical Bulletin

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There are 30 million patients with diabetes mellitus worldwide, and 1.2 million new patients are diagnosed each year, including a growing number of children and young adults. 1)However, insulin has been administered only by parenteral routes for the therapeutic control of type 1 diabetes; many patients typically require injections of insulin a minimum of two to three times per day. To improve patient convenience and compliance, alternative routes have been investigated including nasal, oral, transdermal, buccal, ocular, rectal, and vaginal pathways. 2) Transdermal delivery of insulin can prevent its metabolization and the first-pass effect and may thus be an optimal route of administration. However, delivery of large molecules by the transdermal route is also limited by the unique bioarchitecture of skin, which has primarily evolved as a protective barrier against the entry of microorganisms and water.3)The stratum corneum (SC) barrier can be partially overcome by disruption of the lipid structure using fatty acids and surfactants as chemical penetration enhancers. [4][5][6] However, these enhancers are only useful for small molecules, 6,7) and it is difficult to achieve meaningful permeation of large peptides. Most recent research has focused on the use of electrical energy to increase drug flux across the skin, including techniques such as iontophoresis, sonophoresis, electroporation, etc. [8][9][10] Unfortunately, the large, complex, and relatively uncharged insulin molecule still cannot across the intercellular lipid layer of the SC. The transport rate of hexameric insulin by iontophoresis across mouse skin has been low and variable.11) Monomeric insulin analogues carrying two additional negative charges have demonstrated transport rates 50-100-fold higher than those of hexameric insulin. However, the quantity of delivery is still not sufficiently high to provide an effective basal insulin level (1 IU/h). 11)To overcome the barrier presented by the SC, proteolytic enzymes maybe used as skin penetration enhancers because their action is highly specific toward proteins and their molecular size is too large to permeate the viable epidermis. Such enzymes perhaps alter skin permeability by provoking biochemical and metabolic events within skin. For more than 50 years, proteolytic enzymes such as trypsin have been extensively used in laboratory settings for in vitro epidermal separation and keratinocyte isolation.12-15) The unique ability of proteases to cause selective epidermal separation has been in part explained by the proteolytic degradation of desmosomal proteins in the SC, which leads to cell dissociation. 16,17) Recently, several endogenous proteases occurring in the epidermis have been found to play important roles in regulating epidermal cell desquamation.18-21) Based on these findings, several therapeutic applications have been attempted for wound debridement and epidermal ablation. [22][23][24] However, no studies have examined the use of such proteolytic enzymes as a penetration enhancers for t...

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Separation of the epidermal sheet by dispase

Cited by 176 publications

References 12 publications

Expression of a dominant negative type II TGF-β receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development

Expression of a dominant negative type II TGF-β receptor in mouse skin results in an increase in carcinoma incidence and an acceleration of carcinoma development

Epidermal differentiation does not involve the pro-apoptotic executioner caspases, but is associated with caspase-14 induction and processing

Transdermal Delivery of Insulin Using Trypsin as a Biochemical Enhancer

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