转化生长因子-β_1在肝细胞移植中的变化及意义
摘要
目的：探讨同种异体肝细胞经脾脏移植对大鼠急性肝功能衰竭的治疗作用；探讨肝组织中转化生长因子-β1mRNA（TGF-β1mRNA）在肝细胞移植治疗肝功能衰竭时的变化及意义。
方法：建立大鼠急性药物性肝衰竭模型：10％D－氨基半乳糖（D-gal）溶液按1.6g/kg一次性腹腔注射；肝细胞的分离和纯化：采用肝脏原位灌注法，制备成肝细胞悬液，肝细胞浓度为4×107个/ml，于2小时内经脾脏移植；动物分组：健康成年Wistar大鼠56只制备成急性肝功能衰竭模型并随机分为两组。Ⅰ组为实验组行脾内肝细胞移植，肝细胞悬液的量为0.5 ml/只，Ⅱ组为对照组在脾内注入0.5ml生理盐水。另外取6只用作分离新鲜肝细胞。术后6小时、12小时、24小时、48小时每组分别取5只检测肝脏功能、肝脏病理改变及应用原位杂交技术检测肝脏组织中TGF-β1 mRNA的变化。
结果：肝功能变化：血清丙氨酸氨基转移酶（ALT）6小时和12小时Ⅰ组高于Ⅱ组，差别有统计学意义，P<0.01；ALT在24和48小时Ⅰ组均低于Ⅱ组，差别有统计学意义，P<0.01。总胆红素（TBil）在6小时Ⅰ组高于Ⅱ组，差别有统计学意义, P<0.01；12小时Ⅰ组高于Ⅱ组，差别无统计学意义,在24和48小时Ⅰ组均低于Ⅱ组，差别有统计学意义，P<0.01。肝组织的破坏程度在48小时Ⅰ组比Ⅱ组有较明显的减轻。在各时间点，两组间的白蛋白（ALB）差别无统计学意义。肝脏组织中TGF-β1 mRNA的变化：12小时内两组间无明显区别，24和48小时Ⅰ组均低于Ⅱ组，差别有统计学意义，P<0.01。
结论：同种异体肝细胞移植可以改善D-gal诱导的急性肝功能衰竭大鼠的肝脏功能；在24和48小时肝脏组织中的TGF-β1 mRNA Ⅰ组均低于Ⅱ组，可能有利于肝细胞的增殖。
Objective：To investigate the effect of hepatocyte transplantation on acute liver failure (ALF) of rats. To investigate the change and significance of transforming growth factor-β1 mRNA ( TGF-β1 mRNA ) in liver after hepatocyte transpl- atation.
Methods: 1.Preparation of ALF model. 10% D-gal solution was prepared and injected to rates via abdominal cavity with dosage of 1.6 g/kg respectively. 2.Cell seperation and purification. Collagenase perfusion was performed on six Wistar rats to separate liver cells and suspension was counted . 3.Hepatocyte transplantation experiments. Fifty-six of Wistar rats with ALF induced by D-gal were randomly divided into two groups: Group I and II. Group I was performed intrasplenic hepatocyte transplantation (approximately 2 ×107 cells ). Group II was performed intrasplenic injection of normal saline of same volume. 4.Post-transplantation liver function test. The blood samples of each group of experimental rates were drawn on the 6h , 12h , 24h and 48h after transplantation. ALT and TBil were assayed with automatic biochemical analyzer. Liver sample of rats was performed HE staining and in situ hybridization (ISH).
Results： The comparison between group I and II shows that signficant differences were found in ALT and TBil of 24h and 48h( p< 0.01 ). Pathological changes of group I were much mild than its control group at 48h. The comparison between group I and II shows that no signficant differences were found in TGF-β1 mRNA of 6h and 12h(p<0.01). The comparison between group I and II shows that signficant differences were found in TGF-β1 mRNA of 24h and 48h(p<0.01).
Conclusion: Intrasplenic transplantation could increase the index of hepatic function of rats with ALF induced by D-gal and hepatic pathology could be
improved within 48 hours after transplantation. The decrease of TGF-β1 mRNA in gruop I maybe facilitate to hepatocyte growth.
方法：建立大鼠急性药物性肝衰竭模型：10％D－氨基半乳糖（D-gal）溶液按1.6g/kg一次性腹腔注射；肝细胞的分离和纯化：采用肝脏原位灌注法，制备成肝细胞悬液，肝细胞浓度为4×107个/ml，于2小时内经脾脏移植；动物分组：健康成年Wistar大鼠56只制备成急性肝功能衰竭模型并随机分为两组。Ⅰ组为实验组行脾内肝细胞移植，肝细胞悬液的量为0.5 ml/只，Ⅱ组为对照组在脾内注入0.5ml生理盐水。另外取6只用作分离新鲜肝细胞。术后6小时、12小时、24小时、48小时每组分别取5只检测肝脏功能、肝脏病理改变及应用原位杂交技术检测肝脏组织中TGF-β1 mRNA的变化。
结果：肝功能变化：血清丙氨酸氨基转移酶（ALT）6小时和12小时Ⅰ组高于Ⅱ组，差别有统计学意义，P<0.01；ALT在24和48小时Ⅰ组均低于Ⅱ组，差别有统计学意义，P<0.01。总胆红素（TBil）在6小时Ⅰ组高于Ⅱ组，差别有统计学意义, P<0.01；12小时Ⅰ组高于Ⅱ组，差别无统计学意义,在24和48小时Ⅰ组均低于Ⅱ组，差别有统计学意义，P<0.01。肝组织的破坏程度在48小时Ⅰ组比Ⅱ组有较明显的减轻。在各时间点，两组间的白蛋白（ALB）差别无统计学意义。肝脏组织中TGF-β1 mRNA的变化：12小时内两组间无明显区别，24和48小时Ⅰ组均低于Ⅱ组，差别有统计学意义，P<0.01。
结论：同种异体肝细胞移植可以改善D-gal诱导的急性肝功能衰竭大鼠的肝脏功能；在24和48小时肝脏组织中的TGF-β1 mRNA Ⅰ组均低于Ⅱ组，可能有利于肝细胞的增殖。
Objective：To investigate the effect of hepatocyte transplantation on acute liver failure (ALF) of rats. To investigate the change and significance of transforming growth factor-β1 mRNA ( TGF-β1 mRNA ) in liver after hepatocyte transpl- atation.
Methods: 1.Preparation of ALF model. 10% D-gal solution was prepared and injected to rates via abdominal cavity with dosage of 1.6 g/kg respectively. 2.Cell seperation and purification. Collagenase perfusion was performed on six Wistar rats to separate liver cells and suspension was counted . 3.Hepatocyte transplantation experiments. Fifty-six of Wistar rats with ALF induced by D-gal were randomly divided into two groups: Group I and II. Group I was performed intrasplenic hepatocyte transplantation (approximately 2 ×107 cells ). Group II was performed intrasplenic injection of normal saline of same volume. 4.Post-transplantation liver function test. The blood samples of each group of experimental rates were drawn on the 6h , 12h , 24h and 48h after transplantation. ALT and TBil were assayed with automatic biochemical analyzer. Liver sample of rats was performed HE staining and in situ hybridization (ISH).
Results： The comparison between group I and II shows that signficant differences were found in ALT and TBil of 24h and 48h( p< 0.01 ). Pathological changes of group I were much mild than its control group at 48h. The comparison between group I and II shows that no signficant differences were found in TGF-β1 mRNA of 6h and 12h(p<0.01). The comparison between group I and II shows that signficant differences were found in TGF-β1 mRNA of 24h and 48h(p<0.01).
Conclusion: Intrasplenic transplantation could increase the index of hepatic function of rats with ALF induced by D-gal and hepatic pathology could be
improved within 48 hours after transplantation. The decrease of TGF-β1 mRNA in gruop I maybe facilitate to hepatocyte growth.
引文
Takeshita K, et al. Hepatocellular transplantation for metabolic support in experimental acute ischemic liver failure in rats. Cell Transplant, 1993,2(4): 319-324
Khaoustov VI，Darlington GJ，Soriano HE. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell Dev Biol Anim1999,35 :501-509
Oren R, Dabeva MD，Karnezis AN, et al. Role of thyroid hormone in stimulating liver repopulation in the rat by transplanted hepatocytes .Hepatology，1999,30:903-913
Kobayashi N, Ito M，Nakamura J.Hepatocyte transplantation improves liver function and prolongs survival in rats with decompensated liver cirrhosis. . Transplant Proc,1999,31(1-2):428
Fox IJ，Roy CJ，Kaufman SS，et al. Treatment of the Crigler- Njjar syndrome type I with hepatocyte transplantation. N Engl J Med,1998,338: 1422-1426
Guha C, Chowdhury NR Jauregui H, et al. Hepatocyte-based gene therapy. J Hepatobiliary Pancreat Surg ,2001,8(l):51-57
Jayanta RC, Namita RC, Stephen C, et al. Human Hepatocyte Transplantation gene therapy and more? Pediatrics, 1998,102(3):647-648
Muraca M, Gerunda G, Neri D,et al. Hepatocyte transplantation as a treatment for glycogen storage disease type 1. Lancet ,2002 ,359 (9303 ):317318
Nada A, Terri L, Jlhn C, et al. Expansion of transplanted hepatocytes during liver regeneration. Transplantation, 1997,64(5):816
Ng VL, Alonso M, Bezerra J A. Hepatocyte transplantation:Advancing biology and treating children. Clin Liver Dis, 2000,4(4):929-945
Mahi H, Gupta S. Hepatocyte transplantationmew horizons and challenges. J Hepatobiliary Pancreat Surg ,2001,8(1):40-50
De metriou A A, Whiting J，Levenson S M，New method of hepatocyte
transplantation and extracorporeal liver support .Ann Surg,1986,204(3):259
Kobayashi N，Ito M，Nakamura J.Hepatocyte transplantation in rats with decompensated cirrhosis.Hepatology,2000,31(4):851
Strom S C，Fisher R A, Thompson M. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.Transplantation,1997，63(4):559
林言箴等.同种肝细胞移植治疗实验大白鼠药物性急性肝衰竭.中华消化杂志, 1983,4(1): 69
Matas AJ, Sutherland DE Hepatocellular transplantation for metabolic deficiencies: decrease of plasms bilirubin in Gunn rats. Science,1976, 192(4242):892-4
Baumgartner D Effects of intrasplenic injection of hepatocytes, hepatocyte fragments and hepatocyte culture supernatants on D-galactosamine-induced liver failure in rats.Eur Surg Res,1983,15(3):129-35
Onodera K Kasai S Comparative effects of hepatocellular transplantation into the spleen, portal vein, or peritoneal cavity in congenitally ascorbic acid biosynthetic enzyme-deficient rats. Transplant Proc, 1992, Dec;24(6):3006-8
Demetriou AA, Reisner A Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology,1988 Sep-Oct;8(5):1006-9
Yaron I, Namita RC, Prakash R, et al. Massive repopulation of liver by transplantation of hepatocytes into specific lobes of the liver and ligation of portal vein bronches to other lobes. Transplantation, 1997, 64: 8-13
Bojian J, et al. Enhancement of proliferation of intrasplenically transplanted hepatocytes in cirrhotic rats by hepatic stimulatory substance. Transplantation, 1997, 63: 131-135
Demetrion AA,etal.Transplanation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology, 1998, 8: 1006-1009
周京旭，杨希山，周殿元TGF-β及其受体与肿瘤关系研究进展.肿
瘤,1998,18(4):304-306
M asuhara M，Yasunaga M，Tanigawa K, et al. Expression of hepatocyte growth factor, transforming growth factor alpha, and transforming growth factor betal messenger RNA in various human liver diseases and correlation with hepatocyte proliferation. Hepatology, 1996, 24: 323-329
Masson S , Da Veall M，Hiron M ,et al. Differential regenerative response and expression of growth factors following hepatectomy of variable extent in rats . Liver ,1999 ,19 :312-317
Enami Y，Kato H，Murakami M,et al. Anti-transforming growth factor betal antibody transiently enhances DNA synthesis dueing liver regeneration after partial hepatectomy in rats. J Hepatobiliary Pancreat Surg，2001，8 :250-258
Khaoustov VI，Darlington GJ，Soriano HE. Induction of three-dimensional assembly of human liver cells by simulated microgravity. In Vitro Cell Dev Biol Anim1999,35 :501-509
Oren R, Dabeva MD，Karnezis AN, et al. Role of thyroid hormone in stimulating liver repopulation in the rat by transplanted hepatocytes .Hepatology，1999,30:903-913
Kobayashi N, Ito M，Nakamura J.Hepatocyte transplantation improves liver function and prolongs survival in rats with decompensated liver cirrhosis. . Transplant Proc,1999,31(1-2):428
Fox IJ，Roy CJ，Kaufman SS，et al. Treatment of the Crigler- Njjar syndrome type I with hepatocyte transplantation. N Engl J Med,1998,338: 1422-1426
Guha C, Chowdhury NR Jauregui H, et al. Hepatocyte-based gene therapy. J Hepatobiliary Pancreat Surg ,2001,8(l):51-57
Jayanta RC, Namita RC, Stephen C, et al. Human Hepatocyte Transplantation gene therapy and more? Pediatrics, 1998,102(3):647-648
Muraca M, Gerunda G, Neri D,et al. Hepatocyte transplantation as a treatment for glycogen storage disease type 1. Lancet ,2002 ,359 (9303 ):317318
Nada A, Terri L, Jlhn C, et al. Expansion of transplanted hepatocytes during liver regeneration. Transplantation, 1997,64(5):816
Ng VL, Alonso M, Bezerra J A. Hepatocyte transplantation:Advancing biology and treating children. Clin Liver Dis, 2000,4(4):929-945
Mahi H, Gupta S. Hepatocyte transplantationmew horizons and challenges. J Hepatobiliary Pancreat Surg ,2001,8(1):40-50
De metriou A A, Whiting J，Levenson S M，New method of hepatocyte
transplantation and extracorporeal liver support .Ann Surg,1986,204(3):259
Kobayashi N，Ito M，Nakamura J.Hepatocyte transplantation in rats with decompensated cirrhosis.Hepatology,2000,31(4):851
Strom S C，Fisher R A, Thompson M. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.Transplantation,1997，63(4):559
林言箴等.同种肝细胞移植治疗实验大白鼠药物性急性肝衰竭.中华消化杂志, 1983,4(1): 69
Matas AJ, Sutherland DE Hepatocellular transplantation for metabolic deficiencies: decrease of plasms bilirubin in Gunn rats. Science,1976, 192(4242):892-4
Baumgartner D Effects of intrasplenic injection of hepatocytes, hepatocyte fragments and hepatocyte culture supernatants on D-galactosamine-induced liver failure in rats.Eur Surg Res,1983,15(3):129-35
Onodera K Kasai S Comparative effects of hepatocellular transplantation into the spleen, portal vein, or peritoneal cavity in congenitally ascorbic acid biosynthetic enzyme-deficient rats. Transplant Proc, 1992, Dec;24(6):3006-8
Demetriou AA, Reisner A Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology,1988 Sep-Oct;8(5):1006-9
Yaron I, Namita RC, Prakash R, et al. Massive repopulation of liver by transplantation of hepatocytes into specific lobes of the liver and ligation of portal vein bronches to other lobes. Transplantation, 1997, 64: 8-13
Bojian J, et al. Enhancement of proliferation of intrasplenically transplanted hepatocytes in cirrhotic rats by hepatic stimulatory substance. Transplantation, 1997, 63: 131-135
Demetrion AA,etal.Transplanation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology, 1998, 8: 1006-1009
周京旭，杨希山，周殿元TGF-β及其受体与肿瘤关系研究进展.肿
瘤,1998,18(4):304-306
M asuhara M，Yasunaga M，Tanigawa K, et al. Expression of hepatocyte growth factor, transforming growth factor alpha, and transforming growth factor betal messenger RNA in various human liver diseases and correlation with hepatocyte proliferation. Hepatology, 1996, 24: 323-329
Masson S , Da Veall M，Hiron M ,et al. Differential regenerative response and expression of growth factors following hepatectomy of variable extent in rats . Liver ,1999 ,19 :312-317
Enami Y，Kato H，Murakami M,et al. Anti-transforming growth factor betal antibody transiently enhances DNA synthesis dueing liver regeneration after partial hepatectomy in rats. J Hepatobiliary Pancreat Surg，2001，8 :250-258