Μοριακή ανάλυση της ηπατικής αναγέννησης (κυτταρικός πολλαπλασιασμός/απόπτωση) υπό συνθήκες οκτρεοτίδης μετά ηπατεκτομή

Abstract

ABSTRACT: Introduction: To investigate the effects of somatostatin after hepatectomy and liver regeneration in rats. Materials and methods: Seventy five male Wistar rats weighing 300-350g underwent hepatectomy, by resection of anterior and left lateral lobes of the liver. They were randomly divided into receiving octreotide group and control group. Each group had seven time points: 24h, 48h, 72h, 96h, 168h, one month, two months. Results: Ki67 and PCNA positive nuclei numbers were significanly higher in the control group, compared to the octreotide administration group. Conclusions: Exogenous somatostatin (octreotide administration) suppressed the proliferation of hepatocytes from day 1 to two months, whereas increased the number of Kupffer cells after hepatectomy, therefore inhibits normal liver regeneration and increases connective tissue. INTRODUCTION: Somatostatin inhibits gastric acid secretion and motility, intestinal absorption, pancreatic bicarbonate and enzyme secretion and selectively decreases splanchnic and portal blood flow. Octreotide (the active peptide analog of somatostatin) has been administered to ameliorate the symptoms associated with endocrine-overproducing tumors, pancreatic fistulas, enterocutaneous fistulas. Octreotide administration in pancreatitis is not very well documented. There are studies about the interference of octreotide in the treatment of hepatocellular carcinoma. It has been reported that octreotide is effective in inhibiting growth of hepatocellular carcinoma both in vivo and in vitro significantly. The antineoplastic effect mechanisms of action may be involved in inhibiting DNA synthesize and inducing apoptosis of tumor cells. The results proved that octreotide inhibits the proliferation of cholangiocarcinoma cells through GO/G1 cell cycle arrest rather than through the process of apoptosis. Somatostatin analogue octreotide is able to inhibit angiogenesis induced by HCC in vivo. It seems that octreotide can promote the effects of hepatic arterial occlusion therapy for transplanted cancer in rat's liver. Decreasing the blood perfusion of tumor after hepatic arterial blockage maybe one of its major mechanisms. We also see reports referring to the beneficial work of octreotide in liver micrometastases. We are investigating possible complications that come with the wide use of octreotide and somatostatin. Pro and anti studies of the real effects of somatostatin are being investicated. There are reports to support extensive cholangiolar and fibrous tissue proliferation, hypertrophy and swelling of Kupffer cells in the liver parenchyma of rats receiving octreotide. Whereas there are reports that octreotide can inhibit hepatic stellate cells transforming into myofibroblasts, down-regulate TGFbetal, collagen type 1 and PCIII transcriptions, so that it has therapeutic effects on experimental hepatic fibrosis especially in obstructive jaundice. MATERIALS AND METHODS: Eighty male Wistar rats weighing 300-350g underwent hepatectomy, by resection of anterior and left lateral lobes of the liver. They were randomly divided into receiving octreotide group and control group. Each group had seven time points: 24h, 48h, 72h, 96h, 168h, one month, two months. They were randomly divided into receiving octreotide group (50pg/kg subcutaneous injection twice a day) and control group (receiving 1ml of N/S subcutaneasly twice a day). Tissue sections were fixed in 4% parafomaldehyde, embedded in paraffin and stained with antisera against Ki67 and PCNA. A total of ten tissue sections were analyzed for each animal. Ki67 and PCNA labelling indices were estimated in reactive nuclei in 1000 cells. Western-blotting was also used for frozen tissue. RESULTS: Positive hepatocytes nuclei were significantly higher in 24h and 48h control group in both Ki67 and PCNA stains. Ki67 and PCNA positive nuclei numbers were significantly higher in control group 168h after hepatectomy, as well. Kupffer positive cells with either Ki67 or PCNA were higher in octreotide groups, except on 72h octreotide group. Western-blot assay supported the results of the two other techniques. CONCLUSIONS: Exogenous somatostatin (octreotide administration) suppressed the proliferation of hepatocytes from day 1 to two months, whereas increased the number of Kupffer cells after hepatectomy, therefore inhibits normal liver regeneration and is against fibroinosis as new aspects proved that Kupffer cells help fibroinosis to resolve.

Σκοπός: Η μελέτη της κίνησης των κυτταρικών πληθυσμών του ήπατος, μετά από υπατεκτομή υπό την επίδραση του φαρμάκου της οκτρεοτίδης. Υλικό και Μέθοδος: Για την εκτέλεση του πειράματος αποκτήθηκαν 80 αρσενικοί επίμυες Wistar, ηλικίας 8-12 εβδομάδων και βάρους 300-350g. Υπεβλήθηκαν σε ηπατεκτομή 213 κατά Higgins και Anderson. Χωρίστηκαν σε ομάδες χορήγησης οκτρεοτίδης και ομάδα μαρτύρων. Κάθε ομάδα είχε επτά ομάδες θυσίας, στις 24h, 48h, 72h, 96h, 168h, ένας μήνας, δύο μήνες. Η ομάδα χορήγησης της οκτρεοτίδης λάμβανε 50μg/ng υποδόριων ανά 12h και η ομάδα των μαρτύρων 1ml N/S υποδορίως ανά 12h. Επίσης, μετά τη βιοψία των ιστών οι μισές μονιμοποιήθηκαν σε παραφολμλδεϋδη, σκηνώθηκαν σε μπλόκ παραφίνης και χρησιμοποιήθηκε στις τομές των ιστών για χρώση με αντιγόνο Μ-67 και PCNA. Στους υπόλοιπους ιστούς πραγματοποιήθηκε Western-Blot με τη χρήση του αντιγόνου PCNA. Αποτελέσματα: Με τη μέθοδο Ki-67 όσο και με τη μέθοδο PCNA, παρατηρήθηκε ελάττωση του πολλαπλασιασμού των ηπατοκυττάρων. Όσο αφορά τα κύτταρα του Kupffer καταμετρήθηκαν αυξημένα στις ομάδες χορήγησης του φαρμάκου της οκτρεοτίδης. Η τεχνική Western-Blot υποστηρίζει τα αποτελέσματα αυτά. Συμπέρασμα: Η χορήγηση οκτρεοτίδης καταστέλλει τον πολλαπλασιασμό των κυττάρων, ενώ αυξάνει τον αριθμό των κυττάρων του Kupffer, κατά συνέπεια την ινωδόλυση.