Hepatocellular transplantation for experimental ischemic acute liver failure in dogs

Recovery from acute liver failure is possible if metabolic support can be provided during the period of exogenous liver regeneration. The ability of transplanted dispersed autologous hepatocytes to alter the course of experimental ischemic acute liver failure in dogs was tested. Liver failure was induced by occlusion of blood flow in the proximal portal vein and hepatic artery(s) 48 hr after creation of a side to side portacaval shunt and immediately after a left lateral hepatic lobectomy. Dogs in Group 1 had ischemic injury with no treatment. Dogs in Group II received intrasplenic autotransplants of hepatocytes (26 ± 4x × 10⁸ intact cells) after the ischemic period. Cells for transplantation were prepared from the excised lobe during the period of liver ischemia. Dogs in Group III received intrasplenic transplants of autologous hepatocytes (26 = 3 × 10⁸ intact cells) after liver ischemia and after ligation of the main splenic artery. Serum bilirubin, serum glutamic oxalocetic transaminase, lactate dehydrogenase, and alkaline phosphatase were measured before and serially after ischemia, and showed that the degree of liver injury in all three groups was similar, although survival in Group III was better. Only 20% of nontransplanted animals (Group I) survived 10 days. Liver histology in animals that died showed hemorrhagic necrosis situation around the terminal hepatic central veins. Transplantation did not improve survival in dogs with arterialized spleens and histological examination of dogs that died showed pulmonary infarcts and additional liver injury from embolization of hepatocytes. In contrast, 70% of the animals undergoing splenic artery ligation before intrasplenic transplantation of hepatocytes were alive at 10 days. Ligation of the splenic artery reduced the tendency for hepatocytes to escape into the splenic vein and the spleen remained viable due to collateral circulation. On histological examination, hepatocytes were readily identified in the splenic parenchyma at 24 hr. 2 and 4 weeks after transplantation. In conclusion, intrasplenic hepatocytes provide sufficient metabolic support for dogs to recover from otherwise lethal ischemically induced, acute liver failure.     

Cryopreserved fetal liver cell transplants support the chronic failing liver in rats with CCl4-induced cirrhosis

Hepatocyte transplantation is a promising method for supporting hepatic function in a broad spectrum of liver diseases. The aim of this work was to test the efficacy of human fetal liver cells to support the chronic failing liver in an experimental model of carbon tetrachloride (CCl4)-induced cirrhosis in rats. Liver cirrhosis was induced by intraperitoneal administration of CCl4 at a dose of 0.2 ml (50% v/v solution)/100 g body weight, twice a week for 3 months in rats. Ten days after stopping CCl4 administration (experimental day 0), rats received intrasplenic injection of cryopreserved fetal liver cells (FLC, 1 x 10(7) cells in 0.3 ml medium). As a cirrhotic control group, CCl4-induced cirrhotic rats were used with intrasplenic injection of an equal volume of medium alone. Animals were sacrificed on experimental day 15. Human fetal liver cell transplantation almost completely prevented the death of cirrhotic animals during the 2 weeks after treatment, while high ongoing mortality was seen in the cirrhotic control group. Cell transplantation into the spleen normalized total bilirubin and TBARSs levels and increased albumin levels in blood serum, as well as restoring mitochondrial function and liver detoxification function (assessed by cytochrome P450 contents and activity) compared with the activities seen in the cirrhosis control group. In parallel with this restoration of biochemical and functional liver indices, morphological patterns of liver recovery or regeneration after liver cell transplantation were demonstrated in day 15 samples by light microscopy. These were absent in the group that had received only medium alone.     

An improved model of acute liver failure based on transient ischemia of the liver

HYPOTHESIS: A reproducible and potentially reversible model of acute liver failure in the pig is feasible based on transient ischemia of the liver. DESIGN: To determine the shortest period of liver ischemia sufficient to cause 100% mortality, ischemia of the liver was induced for different lengths of time, starting with 6 hours. If the pig survived, ischemia time was prolonged for 2 hours in the next animal. In the first group, the common bile duct was not tightened. In the second group, the common bile duct was tightened. SETTING: The Laboratory for Hepatopathophysiology, Catholic University, Leuven, Belgium. PARTICIPANTS: Female stress-negative Belgian Landrace pigs weighing 18 to 22 kg. INTERVENTIONS: During preparatory surgery, all ligaments around the liver and connective tissue around the liver hilum were transected and an end-to-side portacaval shunt was made. Vessel loops were placed around the branches of the hepatic artery and bile duct. Three days later, in fully awake pigs, the loops were tightened. MAIN OUTCOME MEASURES: Mortality. Development of acute liver failure was determined based on neurologic, biochemical, and pathological variables. RESULTS: When occluded for 10 hours, all pigs in group 2 (n = 5) [corrected] died between 12 and 17 hours after the induction of ischemia. All pigs developed typical acute liver failure. Tissue specimens showed 90% necrosis of the liver parenchyma. CONCLUSION: A highly reproducible and potentially reversible model of acute liver failure in the large animal has been established.     

Major hepatic trauma: warm ischemic tolerance of the liver after hemorrhagic shock

BACKGROUND: The management of severe hepatic trauma frequently involves exposing the liver to varying periods of warm ischemia. The ischemic tolerance of the liver, in the setting of hemorrhagic shock (HS) and trauma, is presently unknown. We tested the hypothesis that warm ischemic tolerance of the porcine liver will be decreased following resuscitation from HS. MATERIALS AND METHODS: Twenty-three Yorkshire pigs were divided into three groups: 1) hepatic ischemia alone (HI, n = 9); 2) hemorrhagic shock alone (HS, n = 3); and 3) hemorrhagic shock plus hepatic ischemia combined (HSHI, n = 11). Following reperfusion, a liver biopsy was obtained and serial blood chemistries were sampled. RESULTS: Post-operative day 7 mortality was increased in the HSHI group (7/11) compared to the HI (0/9) group, P = 0.038. Notably, deaths did not result from acute liver failure, but rather from intra-operative hemodynamic collapse shortly following hepatic reperfusion. In addition, the HSHI group experienced significantly elevated lactic acid, serum creatinine and liver enzyme levels. Analysis of the liver biopsy samples is consistent with a more severe liver injury in the HSHI group. CONCLUSIONS: The warm ischemic tolerance of the liver following resuscitation from HS is significantly decreased in this porcine model compared to HS or HI alone. Mortality was associated with acute intra-operative hemodynamic collapse occurring shortly after hepatic reperfusion.     

Role of intrasplenic hepatocyte transplantation in improving survival and liver regeneration after hepatic resection in cirrhotic rats

We studied the effect of preoperative hepatocyte transplantation on the prevention of liver failure in cirrhotic rats after hepatic resection. Two groups of Lewis rats were rendered cirrhotic by i.p. injection of 1% dimethylnitrosamine and were subjected to 33% hepatectomy. Two days before the resection, 36 rats in group I received intrasplenic hepatocyte transplantation, and 25 rats in group II were given intrasplenic injection of normal saline as a control. By the end of the third postoperative day, the rats in group I had better survival and a better biochemical profile than those in group II. The liver growth rate and the labeling index of proliferating cell nuclear antigen (PCNA-LI) showed a steady rise in group I. Compared with group II, group I had a significantly lower transforming growth factor (TGF-beta1) level (p < 0.05). We conclude that preoperative intrasplenic hepatocyte transplantation improves survival and facilitates regeneration in cirrhotic rats after hepatic resection.     

Techniques for intrasplenic hepatocyte transplantation in the large animal model

Background: The preferred therapy for acute and chronic liver insufficiency and severe heritable disorders of liver metabolism is whole-organ transplantation. However, due to the shortage of organ doproposed, including transplantation of normal allogeneic hepatocytes. Recently, it has been reported that many hepatocytes transplanted into the spleen migrated to the liver. We therefore carried out a series of large-animal experiments to reexamine the intrasplenic route and to develop a method for large-scale hepatocellular transplantation in pigs. Methods: Allogeneic porcine hepatocytes were transplanted using the following routes: (1) retrograde injection of cells via the splenic vein, (2) intraarterial injection of cells, (3) direct intrasplenic injection of cells after laparotomy, (4) percutaneous intrasplenic injection of cells under laparoscopic control, (5) laparoscopic intrasplenic injection of cells. The number of cells injected varied from 2 × 10⁹ to 10 × 10⁹ cells. Results: Of all the methods tested, only direct intrasplenic injection of 2 bln of cells was found to be compatible with survival. However, even with this ``small' number of cells (2% original liver mass), there was a significant risk of spleen infarction, perisplenic adhesion formation, and portal vein thrombosis. The laparoscopic approach was found to be reliable, simple, and safe. Conclusion: Even though the spleen is considered by many authors the optimal site for hepatocellular transplantation, transplantation of cells in a number needed to support the failing liver may be associated with significant complications, morbidity, and mortality. Received: 2 March 1996/Accepted 17 May 1996     

Acute liver failure in rats inhibited by intrasplenic administration of OK-432

Intrasplenic administration of OK-432, an immunostimulant derived from Streptococcus, prevented hepatic failure induced in rats by D-galactosamine. When OK-432 was given 1.0 K.E. (Group I) or 0.1 K.E. (Group II) into the subcutaneously transpositioned spleen three times prior to dosing with D-galactosamine, survival rates were 100 and 87%, respectively. On the contrary, with a splenic injection of saline (Group III), the survival rate was 47 and 32% in rats given OK-432 1.0 K.E. intraperitoneally (Group IV). The poisoned rats given no pretreatment (Group V) survived at a rate of 26%. These results show that intrasplenic administration of OK-432 leads to a significant enhancement of survival. Metabolic data and histological findings were compatible with survival rates, in each group. Activation of the reticuloendothelial function by the intrasplenic administration of this immunostimulant seems to have prevented acute liver failure.     

Transplantation of hepatocytes for treatment of surgically induced acute hepatic failure in the rat

Efficacy of transplanted hepatocytes was evaluated in rats with a surgically induced acute hepatic failure. After 75% liver resection and portacaval shunt, the intrasplenic or intraperitoneal injection of 20 million isolated fresh hepatocytes was shown to significantly reduce the mortality rate. These results confirm that the transplantation of isolated hepatocytes may prevent death in rats with acute hepatic failure, and suggest that hepatocyte transplantation acts by a mechanism of hepatic support.     

Observations regarding surgical treatment in liver trauma

The high mortality rate in hepatic trauma is a concern for the surgeons on duty, who most know the physiopathological problems and the decisions needed in view of both hepatic and extrahepatic injures. The mortality rate from liver trauma has fallen from 60% at the beginning of this century, blunt trauma to the abdomen from accidents, is responsible for 80-90% of all liver injures in Europe. The severity of liver injuries in 268 patients in a prospective study (1978-1998), were treated according to a defined protocol. Non operative management was used in first day, for those who were haemodynamically stable on admission. In unstable patients who proceeded to surgery, under optimal condition the mortality rate was 34.3%. Death in patients with multiple injures should only rarely result from liver trauma. 92 patients with minor injures (grade I-II) were treated by simple suture, with mortality rate of 6%. 116 patients (43%) sustained complex hepatic injures (grade III to V); 64 patients with grade III (23%), 36 with grade IV (13%). 16 Patients (5%) grade V, injury under went finger fracture of hepatic parenchyma alone 36. The mortality rate in this group was 18% (III), 36% (IV). 16 patients with grad V injury were managed with 68% mortality rate. Juxtahepatic veins and retrohepatic V.C.I. injury continue to carry a prohibitive mortality rate (90-100%).     

Establishment of animal liver metastatic model for C-1300 murine neuroblastoma and immunotherapy for it using OK-432, streptococcus preparation.

Intrasplenic administration of 1 x 10(6) C-1300 murine neuroblastoma cells induced spleen tumor in 78.6% of mice and liver metastasis in 64.3%. When mice were pretreated with carrageenan, an antimacrophage agent, the incidence of spleen and liver tumors in this system was lower, possibly due to the rebound increase in macrophage activity following the disappearance of the carrageenan effect. Continuous daily intraabdominal injection of 1 KE of OK-432 streptococcus preparation markedly reduced the appearance of liver metastasis (P less than 0.01), whereas the spleen mass was not reduced to such an extent. However, the survival rate of the OK-432-treated group did not differ greatly from that of the nontreated group. These results demonstrate the usefulness of the model of liver metastasis induced by intrasplenic administration of tumor cells and the effectiveness of OK-432 against liver metastasis formation.     

Selective repopulation of mice liver after Fas-resistant hepatocyte transplantation

Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopulation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 x 10(6) cells in female MRL-+/+ (wild-type mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intravenously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The pathologic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver.     

omega-3 fatty acids decrease endothelial adhesion of human colorectal carcinoma cells

BACKGROUND: Diets rich in omega-3 fatty acids have been shown to decrease both the initiation and promotion of colon carcinogenesis although their effect on hepatic metastasis formation is less well understood. Since adhesion of human colorectal carcinoma (HCRC) cells to hepatic endothelial cells is an important step in the metastatic cascade, the effect of membrane omega-3 fatty acid alterations on endothelial cell adhesion was studied. MATERIALS AND METHODS: CX-1 cells, a moderately differentiated HCRC cell line known to produce hepatic metastases in an athymic mouse intrasplenic injection model, were used. Cells were grown in omega-3 fatty acid-enriched medium and membrane-free fatty acid modifications confirmed with gas chromatography. Both human umbilical vein and hepatic sinusoidal endothelial cells were used in the binding assays. Adhesion assays were performed in a standard fashion using (51)Cr-labeled cells to tumor necrosis factor (TNF)-stimulated endothelial cell monolayers. Immunohistochemical analysis was performed for sialyl-Lewis(x), the receptor involved in endothelial adhesion on the surface of control and fatty acid-modified cells. RESULTS: Gas chromatographic analysis confirmed membrane fatty acid modification of CX-1 cells by growth in docosahexanoic acid (omega-3) (4.761 nmol/10(6) cells vs 0.057 nmol/10(6) cells for controls). Binding of CX-1 to both human umbilical vein and hepatic sinusoidal endothelial cells decreased from 38.4 +/- 0.44 to 11.58 +/- 0.87% (P < 0.01). Immunocytochemical analysis showed a decrease in sialyl-Lewis(x) expression with omega-3 treatment. CONCLUSIONS: These data indicate that omega-3 fatty acids may also be protective against the formation of hepatic metastases. The mechanism for this may be decreased endothelial cell adhesion which in turn may be due to decreased expression of the endothelial receptor sialyl-Lewis(x).     

Aggregated colon cancer cells have a higher metastatic efficiency in the liver compared with nonaggregated cells: an experimental study

BACKGROUND: It remains unclear whether aggregated colon cancer cells have a higher tendency for metastasis formation than nonaggregated cells. Also, the absolute number of cancer cells required for hepatic metastasis remains undefined. The aim of the present study was to compare in the liver the metastatic efficiency of viable nonaggregated colon cancer cells versus cell aggregates for equivalent numbers of cancer cells. MATERIALS AND METHODS: DHD/K12/TRb colon cancer cells were administered through the portal vein in syngeneic male BD IX rats. Surgical exploration was performed 8 weeks after injection. Four groups of rats were injected with 0.25 or 0.5 x 10(6) DHD/K12/TRb viable cancer cells, either as single nonaggregated cells or as cell aggregates. RESULTS: Hepatic metastases were observed in 81% of the rats after intraportal injection of cell aggregates equivalent to 0.5 x 10(6) cancer cells. A significant lower metastatic efficiency was found after the injection of 0.5 x 10(6) non-aggregated, and 0.25 x 10(6) aggregated or nonaggregated cancer cells i.e., 16%, 32%, and 27%, respectively. CONCLUSION: Aggregated colon cancer cells have a higher metastatic efficiency in the liver compared with non-aggregated cells, although a critical number of cancer cells are necessary.     

Hepatectomy prolongs survival of mice with induced liver metastases

Resection of hepatic metastases from colorectal cancer has been shown to prolong survival in some patients. Whether this results from a reduction of tumor burden or is an indirect effect mediated by hepatectomy is questionable. Male C57BL/6Ros 8-week-old mice underwent ileocolic vein injection of a suspension of 0.3 mL of 2 x 10(5) viable liver-derived murine (MCA-38) colonic adenocarcinoma cells. This model produces hepatic metastases in all lobes of the liver. At 7, 14, or 21 days after tumor injection, mice were randomized to receive either 42% resection of the liver or laparotomy alone. Survival in the animals with hepatectomy was significantly prolonged when the hepatectomy was performed 14 or 21 days after tumor injection.     

Establishment of a syngeneic model of hepatic colorectal oligometastases

BACKGROUND: Regional and systemic therapies aimed at improving the outcome for patients with colorectal hepatic metastases have met with modest yet tangible success. Currently, liver resection remains the only curative treatment, but only a minority of patients are candidates for surgery. Animal models are an ideal way to study new treatments for patients with metastatic colorectal cancer. We propose a syngeneic animal model of hepatic colorectal metastases that simulates oligometastases, which is a clinical state considered amenable to regional therapeutic strategies. MATERIALS AND METHODS: BDIX (BD-9) rats underwent intrasplenic injection of DHD/K12/TRb (Prob/K12) cells to create hepatic metastases via the portal system. After injection of 5 x 10(6) cells, rats underwent laparotomy to determine metastatic burden. Histological analysis confirmed the presence of metastases from resected tumors. RESULTS: Fifty-three animals were prospectively treated and observed for the development of oligometastases defined as between 1 and 10 hepatic lesions. Thirty-six (68%) of the animals developed detectable metastases while 32 (60%) developed oligometastases (average = 4.40 +/- 2.67). Four animals had overwhelming metastatic liver and peritoneal disease. All animals underwent peritoneal examination and thoracotomy to ensure localized disease. Histological analysis of five hepatectomy specimens confirmed the presence of metastatic cancer. Animals with oligometastases were healthy as evidenced by normal feeding and grooming behavior. CONCLUSIONS: An animal model of oligometastatic colorectal cancer to the liver can reproducibly mimic the stage IV state in humans conducive to regional therapy and can be used reliably to test novel treatments and mechanisms of metastatic colorectal cancer.     

Characterization of human liver dendritic cells in liver grafts and perfusates.

It is generally accepted that donor myeloid dendritic cells (MDC) are the main instigators of acute rejection after organ transplantation. The aim of the present study was to characterize MDC in human donor livers using liver grafts and perfusates as a source. Perfusates were collected during ex vivo vascular perfusion of liver grafts pretransplantation. MDC, visualized in wedge biopsies by immunohistochemistry with anti-BDCA-1 monoclonal antibody (mAb), were predominantly observed in the portal fields. Liver MDC, isolated from liver wedge biopsies, had an immature phenotype with a low expression of CD80 and CD83. Perfusates were collected from 20 grafts; perfusate mononuclear cells (MNC) contained 1.5% (range, 0.3-6.6%) MDC with a viability of 97 +/- 2%. Perfusates were a rich source of hepatic MDC since 0.9 x 10(6) (range, 0.11-4.5 x 10(6)) MDC detached from donor livers during vascular perfusion pretransplantation. Perfusate MDC were used to further characterize hepatic MDC. Perfusate MDC expressed less DC-LAMP (P = 0.000), CD80 (P = 0.000), CD86 (P = 0.003), and CCR7 (P = 0.014) than mature hepatic lymph node (LN) MDC, and similar CD86 (P = 0.140) and CCR7 (P = 0.262) as and more DC-LAMP (P = 0.007) and CD80 (P = 0.002) than immature blood MDC. Perfusate MDC differed from blood MDC in producing significantly higher amounts of interleukin (IL)-10 in response to lipopolysaccharide (LPS), and in being able to stimulate allogeneic T-cell proliferation. In conclusion, human donor livers contain exclusively immature MDC that detach in high numbers from the liver graft during pretransplantation perfusion. These viable MDC have the capacity to stimulate allogeneic T-cells, and thus may represent a major player in the induction of acute rejection.     

Evaluation of Pringle maneuver during liver resection in a rat model of surgical obstructive jaundice.

Temporary portal triad clamping (Pringle maneuver) during liver resection reduces intraoperative blood loss. A normal liver can safely tolerate normothermic ischemia for up to 60 min. However, its safety in patients with surgical obstructive jaundice (SOJ) is not known. Therefore, we investigated the effect of hepatic ischemia in an experimental rat model of SOJ created by ligating the bile duct. Four groups of rats were created: Group I (sham operation, 10 days later, liver resection); Group II (sham operation, 10 days later, liver resection with 5 min of hepatic ischemia); Group III (bile duct ligation, 10 days later, liver resection); and Group IV (bile duct ligation, 10 days later, liver resection with 5 min of hepatic ischemia). The ischemic injury was assessed by the survival of rats, liver tissue malondialdehyde and total glutathione (markers of free radical injury), serum alanine aminotransferase, aspartate aminotransferase, and liver histology. The results showed decreased survival (47.6% vs. 90% [p = .046]), increased liver tissue malondialdehyde (161 +/- 35 vs. 129 +/- 33 microg/gm liver tissue [p = .05]), and decreased liver tissue total glutathione (565 +/- 169 vs. 1075 +/- 276 nmol/gm liver tissue [p = .05]) in rats with SOJ subjected to hepatic ischemia when compared to nonjaundiced rats. The changes in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase showed an increasing trend in the SOJ group but were not statistically significant. Ischemic changes in liver histology were seen more often in the SOJ group but were not statistically significant. These data suggest that temporary portal triad clamping in an experimental model of SOJ is detrimental to the outcome of liver resection.     

Protective effect of steroids on liver ischemia.

Occlusion of the afferent liver circulation for variable periods of time would be advantageous to temporarily control bleeding from profound lacerations or during extensive resections. Because of its low tolerance to ischemia we attempted to protect the liver with steroids during inflow occlusion. Total hepatic ischemia was produced in rabbits by ligating the portal triad and gastrohepatic ligament for 30 minutes. A 10 per cent survival was obtained in untreated controls whereas pre-treatment with methylprednisolone improved survival to 100 per cent. Methylprednisolone injection after occlusion improved survival only to 57 per cent. There were profound pathohistologic and electron microscopic changes in untreated controls. In animals treated with methylprednisolone either before or after occlusion changes were minimal or absent. This treatment was used in four trauma patients in whom occlusion of the liver inflow was carried out for various periods of time. Even though no significant statement can be made from such small group, the early postoperative course was remarkably smooth and stable. Methylprednisoline protects the liver during warm ischemia, especially if given before occlusion, and decreases the mortality from this maneuver in experimental animals.     

Hepatic metastasis alters the immune function of murine liver nonparenchymal cells.

To examine the effect of a single hepatic focus of metastatic colon tumor on the immune function of liver non-parenchymal cells (NPCs) from C57Bl/6 mice, we injected 2.5 x 10(5) liver-derived murine colon adenocarcinoma (LD-MCA-38) cells beneath the liver capsule. Three weeks following injection of the tumor cells, the immune function of the NPCs was studied. The NPCs from tumor-bearing mice exhibited increased cytotoxic and proliferative activity. The NPCs from tumor-bearing mice also contained a greater percentage of CD8+ and T-cell receptor gamma/delta+ liver-associated T lymphocytes. Levels of interleukin 6 and tumor necrosis factor were increased in the NPC supernatant, and interleukin 6 levels were increased in serum from tumor-bearing mice. We conclude that the presence of a single hepatic focus of metastatic tumor results in augmented immune function of murine liver NPCs.     

缺血预处理减轻大鼠肝缺血再灌注损伤的免疫机制研究

目的探讨大鼠肝缺血再灌注损伤(HIRI)的免疫机制和缺血预处理(IPC)的保护作用。 方法80只大鼠被随机分为假手术组(A组)、肝门阻断20 min组(B组)、30 min组(C组)、40 min组(D组)以及肝门阻断30 min前预处理组(E组),每组再分为再灌注2 h亚组和24 h亚组,各8只。检测再灌注后2 h、24 h的血丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、白细胞介素10、12(IL-10、IL-12)以及外周血T淋巴细胞亚群的水平,观察再灌注后2 h、24 h时的存活率及肝脏病理情况。 结果随着肝门阻断时间的延长,ALT、AST显著升高,肝内炎症细胞浸润增加,24 h存活率逐渐降低。D组再灌注2 h时,CD8+ T淋巴细胞显著升高,CD4+/CD8+比值下降,调节性T淋巴细胞显著减少,血清IL-10显著降低,而IL-12水平显著升高。再灌注后24 h,B组大鼠各项指标逐步恢复至假手术组水平,而D组大鼠CD4+T淋巴细胞、CD4+/CD8+比值尤其是Treg显著升高,且IL-10水平显著升高,IL-12水平明显降低。与C组相比,E组阻断30 min后无一例死亡,再灌注2 h的ALT、AST水平显著降低,Treg和IL-10水平显著升高,IL-12水平明显降低,再灌注24 h后各项指标恢复并接近假手术组水平,肝脏病理损伤较轻。 结论肝缺血再灌注引起肝脏损伤甚至死亡,可能与诱导T淋巴细胞尤其是Treg和细胞因子的紊乱有关。缺血预处理可以增加再灌注早期的Treg细胞,有效纠正免疫紊乱,减轻损伤。