الفهرس 
Snake venomsOnly 14 pages are availabe for public view
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Abstract
This study was took place in the research unit of natural toxins, biochemistry department, faculty of medicine, Ain shams university.
The aim of the work is to study the effect of purified disintegrin fraction and / or Mesenchymal Stem Cells on liver regeneration in white mice liver injury model induced by CCl¬4.
The current study includes; purification of Cerastes cersates crude venom according to Zaki et al., (2011) through 3 fractionation steps:
1- Gel filtration chromatography on Sephadex G-100 column using ammonium acetate buffer 0.02 M, pH 4.8. Five fractions were obtained and designated A1, A2, B, C and D. The platelet aggregation inhibitory activity has been detected in fraction A2.
2- Ion exchange chromatography on DEAE Sepharose eluted using a linear gradient of ammonium acetate buffer (0.01 M, pH 6.5 and 0.5 M, pH 4.9).Three peaks were obtained and designated A2a, A2b, and A2c .The platelet aggregation inhibitory activity has been detected in A2c fraction.
3- A2c fraction was auto-proteolysed by incubating it ;45μl (0.75 mg/ml) with 5 μl Tris-HCl (50mM)-CaCl2 (10mM) , pH 8.5 for different time intervals ( 24 hours , 48 hours, 72 hours) at 37°C . Products of Auto-proteolysis were detected and their molecular weights were determined by SDS-disc gel electrophoresis (phosphate buffer 0.2 M, pH 7.2). Fraction A2c incubated at 37 C° for 24 hours,48 hours and 72 hours was auto-proteolysed showing 3 bands of molecular weights ; 19 KDa , 15 KDa and 6 KDa respectively.
4- Re-fractionation of products of auto-proteolysed A2c fraction by Gel filtration chromatography on Sephadex G-50 column using ammonium acetate buffer 0.02 M, pH 4.8 , 3 fractions were obtained and designated P¬1 , P¬2 and P¬3.
5- P¬1 fraction which had a molecular weight of 19 KDa showed platelet aggregation inhibitory activity and it had no proteolytic activity.
6- Prepared and labeled BM- derived stem cells with PKH26 were obtained as injectable preparations from suppliers [Unit of biochemistry &molecular biology (UBMB)-Cairo University].
CCl4, P¬1 fraction and/or labeled BM- derived stem cells were injected in 5groups of white albino mice of average weight 25 grams as follows:
Group1. Injected intra-peritoneally with normal saline as control once weekly for 2 weeks in a dose of 0.8 ml/kg body weight.
Group2. Injected intra-peritoneally with CCl4 dose 0.8 ml/kg body weight as modified from Abdel-Aziz et al., (2005). Once weekly for 2 successive weeks
Group 3. Injected intra-peritoneally with CCl4 0.8 ml/kg once weekly for 2 successive weeks followed one week later by P1 fraction 0.3 mg/kg according to Zaki, et al., ( 2011) once weekly for 2 successive weeks .
Group 4. Injected intra-peritoneally with CCl4 0.8 ml/kg once weekly for 2 successive weeks followed one week later by intra-venous injection of labeled BM- derived stem cells at a concentration of 1 ×106 cells as modified from Abdel Aziz et al., (2007) once.
Group 5. Injected intra-peritoneally with CCl4 0.8 ml/kg once weekly for 2 successive weeks followed one week later by injection with labeled BM- derived stem cells 1 ×106 cells intra-venously and P1 fraction 0.3 mg/kg intra-peritoneally.
Serum samples were taken from the previous groups to measure Albumin, AST and ALT concentrations in serum.
It was found that ALT and AST serum levels (U/L) were significantly increased (86. 2 ± 7.9 U/L; 288.2 ± 53.5, respectively) in animals injected intra-peritoneally with CCl4 alone as compared to that in the control group (40.8 ± 8.3U/L; 69.0 ± 7.0 respectively) (p < 0.05). Moreover, ALT and AST serum levels (U/L) were significantly decreased in animals injected intra-peritoneally with P1 fraction ( 64.0 ± 7.8;155.6 ± 52.0 ,respectively) or injected intra-venously with labeled BM- derived stem cells (67.0 ± 10.0; 156.0 ± 52.4, respectively) after intra-peritoneal injection with CCl4 and in animals injected with both P1fraction intra-peritoneally and labeled BM- derived stem cells intra-venously after intra-peritoneal injection with CCl4 (66.0 ± 7.6; 110.4 ± 31.4, respectively) as compared to animals injected intra-peritoneally with CCl4 alone (86. 2 ± 7.9 U/L; 288.2 ± 53.5, respectively) (p < 0.05) .
Regarding Albumin serum levels (g/dl), they were significantly decreased (2.9 ± 0 .3) in animals injected intra-peritoneally with CCl4 alone and animals injected intra-venously with labeled BM-derived stem cells after intra-peritoneal injection with CCl4 (2.9 ± 0 .2) as compared to that in the control group (3.8 ± 0.4) (p < 0.05).
Liver tissue samples from previously mentioned groups examined for expression of TNF-α, HO-1, VEGF, β-catenin and Caspase-3 genes.
TNF-α and HO-1 genes were expressed in animal groups injected intra-peritoneally with CCl4 and animal groups injected with P1 fraction intra-peritoneally and /or labeled BM- derived stem cells intra-venously after intra-peritoneal injection with CCl4.
VEGF gene was expressed in animal groups injected intra-peritoneally with CCl4 and in animal groups injected intra-peritoneally with P1 fraction alone or with labeled BM- derived stem cells intra-venously after intra-peritoneal injection of CCl4.
β-Catenin gene was expressed in animal groups injected intra-peritoneally with CCl4 and in animal groups injected with labeled BM- derived stem cells intra-venously alone or with P1 fraction intra-peritoneally after intra-peritoneal injection of CCl4.
Moreover, Caspase-3 gene was expressed in animals group injected with labeled BM- derived stem cells intra-venously alone or with P1 fraction intra-peritoneally after intra-peritoneal injection with CCl4.
BM- derived stem cells labeled with PKH26 fluorescent dye showed stronger red auto-fluorescence after transplantation into mice suggesting that these cells were more seeded into the liver tissue in animals injected with CCl4 followed by both P1 fraction and BM- derived stem cells than animals injected with CCl4 followed by BM- derived stem cells alone.
Also, histo-pathological examination of from the same previous groups showed that:
Liver tissue samples from animals injected with saline Group 1 (control) showed cords of normal cells radiating from normal none dilated non congested central vein, normal blood sinusoids.
While liver tissue samples from animals injected intra-peritoneally with CCl4 (Group 2) showed distorted hepatic lobular architecture, dilated central vein surrounded by inflammatory cells, most hepatocytes are swollen, vacuolated, and ballooned with degenerated nuclei leading to obliteration of some blood sinusoids.
For liver tissue samples from animals injected with CCl4 fraction followed by P1 fraction (group 3), animals injected with CCl4 fraction followed by BM- derived stem cells (group 4) and animals injected with CCl4 fraction followed by both P1 fraction and BM- derived stem cells (group 5) showed non dilated central vein, non-obliterated blood sinusoids, few inflammatory cells, surrounding normal hepatocytes with some cells showing signs of regeneration as bi-nucleated cells with dense chromatin.