- Email: [email protected]
359 Platelets promote hepatocyte regeneration
04D. Molecular and cellular biology
(d) Liuer regeneration and experimental oncology
S137
Effect of platelet Col|tact o1| hepatocyte prolifel'atiolt
regeneration of the liver. Claudins, the main proteins of tight junction, are involved in changes of cell adhesion during liver regeneration. Out of the 24 types of recently known claudins, type 7 has been associated with bile duct cells (BDCs). For this reason, the aim of our study was to analyse the expression of claudin-7 in HSCs and to compare the found expression with mature HCs and BDCs. Methods: Rats were treated with 2-acetylaminofluorene (AAF) followed by 70% partial hepatectomy (PH). Claudin-7 expression was detected in paraffin sections by immunohistochemistry and further investigated on snap frozen specimens by Western immunoblot analysis. Double immunofluorescence-staining of claudin-7 and of oval cell markers (CK7, OV-6) was performed on frozen sections and detected by confocal microscopy. Results: Weak immunoreactivity of claudin-7 was found in normal BDCs whereas HCs were negative. Large number of oval cells with strong claudin-7 immunostaining was observed in regenerating liver. Double immunofluorescence revealed that the cells expressing claudin-7 were indeed stem cells, since they also expressed CK7 and OV-6. Western blot confirmed the increased expression of claudin-7 in the AAF/PH treated rat liver. Conclusions: Our data demonstrate a strong expression of claudin-7 in HSCs during hepatic regeneration. Based on our results claudin-7 protein could well become a new marker for the identification of hepatic stem cells. The project was supported by grants: NKFP-1/0023/2002, NKFP1A/002/2004, ETT- 228/2001, OTKA-T037838.
2..~ 2
:t:
1.~
1 0.~
0
platelet(-)
platelet(+)
platelet separated
Figure 1
]~ffeet of platelet on hepatocyte prolifer,~tion 2.5
0.5
•
PLATELETS PROMOTE
HEPATOCYTE
REGENERATION
c o n t r ol
FL_~u~ 2
R. Matsuo, S. Murata, O. Ikeda, N. Ohkohchi. Institute of Clinical
xl xlO ]R.atio of platelets,'hepatocytes
xIO0
Medicine, Gastroenterological Surgery, Uniuersity of Tsukuba, Tsukuba city, Ibaraki, Japan ]~ffect o f fr;~rtiolt;~tion of pl;~telet olt hep;~toryte prolJfer;~tion
Background and Objectives: Mechanism of liver regeneration after hepatectomy is still not known precisely. Many studies have been carried out focusing on hepatotropic factors to prevent hepatic failure after extended hepatectomy. However, no substance can be applied in the clinical field yet. We found that platelets promote liver regeneration after hepatectomy. The objective of this study is to investigate which component of platelets promotes hepatocyte regeneration in vitro. Methods: Hepatocytes of male BALB/c mice were obtained by the collagenase perfusion method. Study A: Effect ofplatelet contact on hepatocyte proliferation; three groups were prepared as follows: (i) a platelet(+) group, in which hepatocytes were cultured with platelets; (ii) a platelet( ) group, in which hepatocytes were cultured without platelets; and (iii) a platelet separated group, in which hepatocytes were cultured in a chamber separated from hepatocytes by a permeable membrane with 0.2 m pore size. Examination item: DNA synthesis of hepatocytes was measured by 3H-thymidine and bromodeoxyuridine (BrdU). Study B: Which component of platelet is effective for hepatocyte proliferation, membrane or contents? By freezing and thawing, we disrupted platelets and isolated the membrane fraction and supernatant by centrifugation. To investigate their proliferative effects, we added each fraction to cultured hepatocytes and DNA synthesis of hepatocytes was measured by 3H-thymidine. Results: Study A: In the platelet(+) group, significant increment of 3H-thymidine uptake was recognized in comparison to the platelet( ) group (p <0.001). This phenomenon was not observed in the platelet separated group (Figure 1). Effect of platelet increased in proportion to platelet concentration in medium (Figure 2). Study B: Significant increment of 3H-thymidine uptake was observed in cultures with whole disrupted platelets and supernatant groups (p < 0.0001), but not with membrane fraction group (Figure 3).
3 ~.,25
:1:
~'
:1:
~- 1.5
~
0.~
0 contlol Figme 3
disnlpted
supematant
lnembl ane
Conclusions: Our results suggest that direct contact of platelets induces hepatocyte regeneration and platelets contain a substance which strongly promotes hepatocyte regeneration.
•
PLATELETS PROMOTE LIVER REGENERATION EXTENSIVE HEPATECTOMY
AFTER
S. Murata, R. Matsuo, O. Ikeda, N. Ohkohchi. Institute of Clinical Medicine, Gm'troenterological Surgery, Uniuersity of Tsukuba, Tsukuba, Ibaraki, Japan
Background and Aims: Platelets contain many growth factors in alpha granules, such as, platelet derived growth factor, hepatocyte growth factor, and epidermal growth factor. In previous in vitro study we reported
(d) Liuer regeneration and experimental oncology
S137
Effect of platelet Col|tact o1| hepatocyte prolifel'atiolt
regeneration of the liver. Claudins, the main proteins of tight junction, are involved in changes of cell adhesion during liver regeneration. Out of the 24 types of recently known claudins, type 7 has been associated with bile duct cells (BDCs). For this reason, the aim of our study was to analyse the expression of claudin-7 in HSCs and to compare the found expression with mature HCs and BDCs. Methods: Rats were treated with 2-acetylaminofluorene (AAF) followed by 70% partial hepatectomy (PH). Claudin-7 expression was detected in paraffin sections by immunohistochemistry and further investigated on snap frozen specimens by Western immunoblot analysis. Double immunofluorescence-staining of claudin-7 and of oval cell markers (CK7, OV-6) was performed on frozen sections and detected by confocal microscopy. Results: Weak immunoreactivity of claudin-7 was found in normal BDCs whereas HCs were negative. Large number of oval cells with strong claudin-7 immunostaining was observed in regenerating liver. Double immunofluorescence revealed that the cells expressing claudin-7 were indeed stem cells, since they also expressed CK7 and OV-6. Western blot confirmed the increased expression of claudin-7 in the AAF/PH treated rat liver. Conclusions: Our data demonstrate a strong expression of claudin-7 in HSCs during hepatic regeneration. Based on our results claudin-7 protein could well become a new marker for the identification of hepatic stem cells. The project was supported by grants: NKFP-1/0023/2002, NKFP1A/002/2004, ETT- 228/2001, OTKA-T037838.
2..~ 2
:t:
1.~
1 0.~
0
platelet(-)
platelet(+)
platelet separated
Figure 1
]~ffeet of platelet on hepatocyte prolifer,~tion 2.5
0.5
•
PLATELETS PROMOTE
HEPATOCYTE
REGENERATION
c o n t r ol
FL_~u~ 2
R. Matsuo, S. Murata, O. Ikeda, N. Ohkohchi. Institute of Clinical
xl xlO ]R.atio of platelets,'hepatocytes
xIO0
Medicine, Gastroenterological Surgery, Uniuersity of Tsukuba, Tsukuba city, Ibaraki, Japan ]~ffect o f fr;~rtiolt;~tion of pl;~telet olt hep;~toryte prolJfer;~tion
Background and Objectives: Mechanism of liver regeneration after hepatectomy is still not known precisely. Many studies have been carried out focusing on hepatotropic factors to prevent hepatic failure after extended hepatectomy. However, no substance can be applied in the clinical field yet. We found that platelets promote liver regeneration after hepatectomy. The objective of this study is to investigate which component of platelets promotes hepatocyte regeneration in vitro. Methods: Hepatocytes of male BALB/c mice were obtained by the collagenase perfusion method. Study A: Effect ofplatelet contact on hepatocyte proliferation; three groups were prepared as follows: (i) a platelet(+) group, in which hepatocytes were cultured with platelets; (ii) a platelet( ) group, in which hepatocytes were cultured without platelets; and (iii) a platelet separated group, in which hepatocytes were cultured in a chamber separated from hepatocytes by a permeable membrane with 0.2 m pore size. Examination item: DNA synthesis of hepatocytes was measured by 3H-thymidine and bromodeoxyuridine (BrdU). Study B: Which component of platelet is effective for hepatocyte proliferation, membrane or contents? By freezing and thawing, we disrupted platelets and isolated the membrane fraction and supernatant by centrifugation. To investigate their proliferative effects, we added each fraction to cultured hepatocytes and DNA synthesis of hepatocytes was measured by 3H-thymidine. Results: Study A: In the platelet(+) group, significant increment of 3H-thymidine uptake was recognized in comparison to the platelet( ) group (p <0.001). This phenomenon was not observed in the platelet separated group (Figure 1). Effect of platelet increased in proportion to platelet concentration in medium (Figure 2). Study B: Significant increment of 3H-thymidine uptake was observed in cultures with whole disrupted platelets and supernatant groups (p < 0.0001), but not with membrane fraction group (Figure 3).
3 ~.,25
:1:
~'
:1:
~- 1.5
~
0.~
0 contlol Figme 3
disnlpted
supematant
lnembl ane
Conclusions: Our results suggest that direct contact of platelets induces hepatocyte regeneration and platelets contain a substance which strongly promotes hepatocyte regeneration.
•
PLATELETS PROMOTE LIVER REGENERATION EXTENSIVE HEPATECTOMY
AFTER
S. Murata, R. Matsuo, O. Ikeda, N. Ohkohchi. Institute of Clinical Medicine, Gm'troenterological Surgery, Uniuersity of Tsukuba, Tsukuba, Ibaraki, Japan
Background and Aims: Platelets contain many growth factors in alpha granules, such as, platelet derived growth factor, hepatocyte growth factor, and epidermal growth factor. In previous in vitro study we reported