טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBen-Lulu Shani
SubjectEffect of Peptide Growth Factors on Intestinal Recovery and
Enterocyte Turnover after Chemotherapy Induced
Intestinal Injury in a Rat
DepartmentDepartment of Medicine
Supervisors Professor Igor Sukhotnik
Clinical Professor Jorge Mogilner
Full Thesis textFull thesis text - English Version


Abstract

The purpose of the present study was to evaluate the effect of TGF-α and TGF-β on enterocyte proliferation, apoptosis and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells.

   In Caco-2 cells we measured Cell proliferation, cell viability and apoptosis by FACS-cytometry and by AlamarBlue. For either TGF-α or TGF-β project, rats were randomized to four groups: 1- Control; 2- CONTR-TGF-α or TGF-β; 3- MTX; 4- MTX-TGF-α or TGF-β. Three days after MTX injection, rats were sacrificed. Body weight changes, intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and apoptosis were measured. RT-PCR and Western-Blotting used to determine levels of Bax and Bcl-2 mRNA and protein. Receptor expression along the villus-crypt axis was determined using microdissection and real-time PCR (for EGFr) or using immunohistochemistry and Western-Blotting (for TGF-βr). Western-blotting was used to determine the level of p-ERK and β-catenin.

   In vitro experiments with TGF-α of Caco-2 cells resulted in a significant stimulation of cell proliferation and inhibition of cell apoptosis. In vivo experiments showed a significant increase in bowel and mucosal weight, DNA and protein content, villus height, crypt depth, and cell proliferation in jejunum and ileum in MTX-TGF-α rats compared to MTX rats. Moreover, a decreased of apoptosis in MTX-TGF-α rats was coincided with decreased Bax and increased in Bcl-2 mRNA and protein levels. Inhibiting effect of TGF-α on enterocyte apoptosis was correlated with EGFr expression along the villus-crypt axis.

In vitro experiments of Caco-2 cells treated with TGF-β resulted in an increased of cell viability and decreased of cell apoptosis when treated with MTX. In vivo experiments showed that treatment with TGF-β resulted in a significant increase in bowel and mucosal weight, DNA and protein content, villus-height, crypt-depth, decreased intestinal-injury score, decreased level of apoptosis and increased cell proliferation in jejunum and ileum compared to the MTX group. TGF-β decreased Bax and increase Bcl-2 mRNA levels, decreased Bax protein levels in jejunum and ileum compared to MTX group. TGF-β also increased pERK and β-catenin protein levels in intestinal mucosa.

   Treatment with TGF-α prevents mucosal-injury, enhances enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal mucositis in rats. The effect of TGF-α on enterocyte turnover was correlated with EGF receptor expression along the villus-crypt axis.

Treatment with TGF-β prevents mucosal-injury, enhances p-ERK and β-catenin induced enterocyte proliferation, inhibits enterocyte apoptosis and improves intestinal recovery following MTX-induced intestinal-mucositis in rats.