Expression Of Haptoglobin In Normaland Lesional Skin

1. To investigate the expression of haptoglobin ( Hp) mRNA in the epidermis , dermis, whole skin( excluding subcutis) in mouse under normal and inflammatory conditions. Localization study was also performed by in situ hybridization in order to observe the expressing sites of Hp mRNA in skin and its correlation with inflammation.2. To elucidate the expression of Hp in normal human skin from mRNA level in order to confirm the exact expressing sites of Hp mRNA in the skin and the differences of Hp mRNA expression in different parts of human skin. Immuno-histochemical study was also performed to investigate the expression of Hp in normal human skin from protein level.3. To investigate the expression of Hp in some skin diseases including pso-riatic lesion and normal - looking perilesional skin, lesions with lichen planus, erythroderma, seborrheic keratosis, verruca vulgaris, basal cell carcinoma, squamous carcinoma, lesions and sun - exposed normal - looking skin with systemic lupus erythematosus (SLE) , lesions with pemphigus and bullous pemphig-oid from mRNA level in order to reassure the expression sites of Hp mRNA in skin and the different expressing intensities in these skin diseases. Hp expression in skin lesions was also carried out by immunohistochemistry from protein level.Materials and Methods1. Samples1.1 AnimalsTwenty C57BL/6 mice were evenly distributed into two groups as saline or LPS - treated group. They are all female and two to four months old. The epidermis, dermis and whole skin(exluding subcutis) were derived respectively.1.2 Normal human skinBiopsies of normal skin were obtained from nineteen men and eleven women, two from scalp, six from face, thirty from trunk and nine from extremities. No tumor or inflammation involved in the biopsies and all biopsies were ex-luded of subcutis.1. 3 Lesions from skin diseasesPsoriatic lesions from twenty - two patients of psoriasis vulgaris, fourteen in progressing phase and eight in stationary phase. Sixteen normal - looking perile-sional skin from these psoriasis patients. Biopsies from sixteen with lichen pla-nus, eight with erythroderma(two induced by mycosis fungoid, three induced by psoriasis and three induced by eczema) , fifteen with seborrheic keratosis, ten with verruca vulgaris, ten with basal cell carcinoma, ten with squamous carcinoma , twelve lesion and ten sun - exposed normal - looking skin from systemic lupus erythematosus, ten with pemphigus including seven pemphigus vulgaris and three pemphigus erythematosus, ten with bullous pemphigoid were also obtained. All were performed diagnosis confirmation by clinical, pathologic and immunopathologic features, etc.2. Methods2.1 Semi - quantity reverse transcriptase - polymerase chain reaction ( RT -PCR)2. 1. 1 Extraction of RNA from mouse skin and liverTotal tissues RNA were extracted from C57BL6 mice skin and liver tissues by homogenizing the tissues in guanidinium thiocyanate, followed by phenol -chloroform extraction.2.1.2 Identification the concentration, purity and integrity of total RNAThe optical densities (OD) of RNA were measured by violet - spectropho-tometer at 260nm and 280nm. Then the concentration and purity of total RNA were determined. 1.5% agarose formaldehyde electrophoresis was used to evaluate the integrity of total RNA.2.1.3RT-PCREvery sample was amplified by Hp and B - actin primers under certain con-ditions. The products were 475bp and 540bp respectively.2.2 In situ hybridization for Hp mRNA2.2.1 Serial 12|jun thick frozen cryostat sections from mouse, normal human skin and dermatoses lesions were prepared.2. 2.2 Haptoglobin in situ hybridization kit was used for detection. The sequence of Hp oligonucleotide probes as follows: 5'- AGTGC TCCAC ATAGC CATGT GCAAT CTCGG -3;5'- CCTGC CAGGG AAAGC TGCCT TTGGC ATCCA -3',5'-CATAC CAGGT GTCCT CCTCC AGGTC GTGAA -3'.2.2.3 In the meanwhile, positive control(mouse liver) and negative controls ( omitting biotin - conjugated mouse anti - digoxin or prob...