| Abstract | Cilj istraživanja: Koštani morfogenetski proteini (eng. bone morphogenetic proteins, BMPs) jesu članovi TGF– β nadobitelji i djeluju na mjestima epitelno-mezenhimalne tranzicije, a neki članovi u tom smislu imaju sposobnost potaknuti regeneraciju organa. S obzirom da BMP-9 izaziva proliferaciju stanica ljudskog hepatoma te pokazuje hipoglikemijski učinak sličan inzulinu, moguće je da BMP-9, baš poput inzulina pomaže regeneraciji oštećene jetre. Pretpostavka je da se ekspresija BMP-9 mijenja sukladno oštećenju jetrenog parenhima.
Ispitanici i metode: Ovim istraživanjem analizira se uzorak oštećenog tkiva jetre čovjeka. Uzorci humane jetre dobiveni su iz arhive Zavoda za sudsku medicinu i kriminalistiku Medicinskog fakulteta u Rijeci (N=40). Patohistološkim pregledom je ustanovljena početna ciroza i nodularna ciroza. Uzorke smo analizirali imunohistokemijskom metodom, metodom Western blot i zimografijom gelatinaza. Kontrolnu skupinu čine ispitanici kod kojih na obdukciji i histološkim pregledom nisu nađene patološke promjene u jetri (N=20). Istraživanjem su obuhvaćeni ispitanici stari od 25 do 50 godina, oba spola čiji su uzorci tkiva jetre prikupljeni u razdoblju od 2005.-2012. godine.
Rezultati: Kad govorimo o ekspresiji i lokalizaciji BMP-9 i TGF-β1, našli smo ih u hepatocitima i u epitelu žučnih kanalića zdrave jetre te u: hepatocitima, epitelu žučnih kanalića i Kupferovim stanicama različitim stupnjem ciroze zahvaćene jetre čovjeka. Kvantitativnom analizom intenziteta obojenja BMP-9 ekspresije utvrđena je statistički značajna razlika između nodularne ciroze i početne ciroze (P<0,05) te između nodularne ciroze i zdrave jetre (P<0,05). Kvantitativnom analizom intenziteta TGF-β1 ekspresije, utvrđena je statistički značajna razlika između nodularne ciroze i zdrave jetre (P<0.05) te između početne ciroze i zdrave jetre (P<0,05). Western blot analizom utvrđena je najveća razina ekspresije BMP-9 i TGF-β1 u nodularnoj cirozi. Statistički značajno jači intenzitet ekspresije MMP-2 utvrđen je u nodularnoj cirozi, u odnosu na zdravu jetru (P<0,05). MMP-9 ima statistički značajno veću ekspresiju u početnoj cirozi, u odnosu na zdravu jetru (P<0,05).
Zaključak: Imunohistokemijskom analizom utvrdili smo ekspresiju i lokalizaciju BMP-9 u zdravoj jetri, početnoj i nodularnoj cirozi. U hepatocitima se ekspresija BMP-9 mijenja sukladno stadijima razvoja ciroze jetre. Najjači intenzitet imunohistokemijskog obojenja BMP-9 ekspresije utvrđen je u nodularnoj cirozi, dočim početna ciroza i zdrava jetra pokazuju podjednak intenzitet ekspresije. Najjači intenzitet imunohistokemijskog obojenja TGF – β1 ekspresije utvrđen je također u nodularnoj cirozi, što je podjednako intenzitetu obojenja u početnoj cirozi. Zdrava jetra pokazuje najslabiji intenzitet TGF-β1 ekspresije. Western blot analiza potvrđuje nalaz imunohistokemije, što upućuje na finu ravnotežu između BMP-9 i TGF-β1 u jetri čovjeka. Naš nalaz ekspresije MMP–2 i MMP–9 govori o tome da stupanj oštećenosti uzorka u različitim stadijima razvoja ciroze humane jetre korelira s ekspresijom enzima gelatinaza. |
| Abstract (english) | Objective: Bone morphogenetic proteins (BMPs) are members of the TGF – β superfamily and play a role in places of epithelial - mesenchymal transition, and some BMPs exhibit the ability to regenerate organs. Since BMP-9 induces proliferation of human hepatoma cells and shows hypoglycemic effect like the one of insulin, it is possible that BMP - 9 helps regeneration of the damaged liver. The assumption was that the expression of BMP - 9 is corresponding to the liver damage.
Patients and Methods: We analyzed, in this study, samples of human damaged liver tissue. Human liver samples were obtained from the Department of Forensic Medicine and Criminalistics, School of Medicine in Rijeka (N = 40). Pathohistologically, all samples were grouped whether they represented initial or nodular cirrhosis. Samples were further processed by immunohistochemistry, Western blot and gelatinase zymography. The control group consisted of subjects in whom the histological examination failed to find pathological changes in the liver (N = 20). Study included subjects 25-50 years old, both sexes whose liver tissue samples were collected during the period from 2005th to 2012th.
Results: In regard of the expression and localization of BMP - 9 and TGF – β1, we found them in the hepatocytes and bile duct epithelium of the healthy liver and in: hepatocytes, bile duct epithelium and Kupfer cells of varying degrees of the liver cirrhosis in humans. Quantitative analysis of the intensity of staining of BMP-9 expression, showed statistically significant difference between the nodular cirrhosis and initial cirrhosis (P <0,05) and between nodular cirrhosis and healthy liver (P<0,05). Quantitative analysis of the intensity of TGF-β1 expression, revealed a statistically significant difference between nodular cirrhosis and healthy liver (P <0,05) and between the initial cirrhosis and healthy liver (P<0,05). Western blot analysis revealed the highest level of expression of BMP-9 and TGF-β1 in nodular cirrhosis. Significantly higher intensity of MMP - 2 expression was found in the nodular cirrhosis compared to healthy liver (P<0,05). MMP-9 has significantly higher expression in the initial cirrhosis, compared to healthy liver (P<0,05).
Conclusion: Using immunohistochemistry we determined the expression and localization of BMP - 9 in the healthy liver, initial and nodular cirrhosis. In hepatocytes the expression of BMP - 9 is corroborating the stages of development of liver cirrhosis. The strongest intensity of immunohistochemical staining of BMP-9 expression was found in nodular cirrhosis, whereas the initial and healthy liver cirrhosis show an equal intensity of expression. The strongest intensity of immunohistochemical staining of TGF-β1 expression was found also in nodular cirrhosis, which is equal to the intensity of staining in initial cirrhosis. A healthy liver showed the weakest intensity of TGF-β1 expression. Western blot analysis confirmed immunohistochemistry findings, suggesting a fine balance between BMP-9 and TGF-β1 in the human liver. Our findings of expression of the MMP- 2 and MMP-9 indicate that the degree of damage at different stages of development of cirrhosis of the human liver, correlated with the expression of the enzyme gelatinase. |
