异种肝前体细胞移植治疗急性肝损伤大鼠

背景：成体肝前体细胞可在受体肝脏内定植并分化为肝细胞。不过,异种肝前体细胞移植能否促进急性肝损伤的恢复,脾脏微环境能否促进移植物的存活和向肝细胞分化,尚没有研究。 目的：评价异种肝前体细胞移植治疗急性肝损伤的作用;监测移植肝前体细胞在大鼠脾脏实质内的定植及向肝细胞的分化。 方法：体外培养雄性小鼠来源的肝前体细胞系肝上皮样前体细胞。通过CCl4腹腔注射联合2/3肝切除构建急性肝损伤大鼠模型,进行肝上皮样前体细胞脾脏移植。在肝切除后1,5,14和21 d,苏木精-伊红染色观察肝脏病理改变,全自动生化分析仪监测血清转氨酶变化,PCR反应检测脾脏组织Y染色体特异性序列Sry,脾脏CK-19和Alb免疫组织化学追踪移植肝上皮样前体细胞的植入和肝细胞分化。 结果与结论：肝上皮样前体细胞可在体外长期培养,保持增殖能力和双向分化潜能。肝上皮样前体细胞脾脏移植后,肝损伤大鼠肝细胞肿胀明显减轻,丙氨酸转氨酶和天门冬氨酸转氨酶下降更明显。移植后1,5,14和21 d,脾脏DNA中均能检测到Sry序列。在整个实验期间CK-19阳性细胞在大鼠脾脏实质内始终存在。Alb阳性细胞在移植后5 d在脾脏实质中出现,随后阳性细胞数逐渐增多。实验表明,移植肝前体细胞能在大鼠脾脏实质中植入,并分化为肝细胞,能有效促进CCl4腹腔注射联合2/3肝切除诱导的大鼠急性肝损伤的修复过程。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

L-谷氨酰胺与肝脏大部分切除后的再生

背景：L-谷氨酰胺作为DNA和谷胱甘肽等合成的氮前体,在肝组织再生,肝细胞增殖的过程中扮演着极其重要的角色。 目的：观察经饮食由来补充L-谷氨酰胺对大鼠肝脏大部切除后肝再生能力的影响。 方法：Wistar大鼠随机分组3组,L-谷氨酰胺组和L-丙氨酸组大鼠肝切除前分别灌服10% L-谷氨酰胺或10%L-丙氨酸,肝切除后继续加入饮用水中饮用,对照组肝切除前后均使用饮用水。 结果与结论：大鼠肝切除后72 h L-谷氨酰胺组肝再生率明显高于对照组及L-丙氨酸组(P < 0.05)。肝切除后24 h和72 h L-谷氨酰胺组肝细胞增殖均明显高于对照组和L-丙氨酸组(P < 0.01;P < 0.05)。肝切除后24 h和72 h总RNA水平在两种氨基酸与对照组之间差异无显著性意义。肝切除后72 h基因组DNA的含量L-谷氨酰胺组显著高于对照组和L-丙氨酸组 (P < 0.05)。提示肝损伤围手术期投用高浓度L-谷氨酰胺对大鼠肝再生有促进作用,而投用L-丙氨酸则没有此作用。     

A study of endotoxin-associated hepatotoxicity on proliferating hepatocytes.

It is thought that regeneration of the liver provides a state of preparedness for the Shwartzman reaction and contributes to the development of endotoxin-associated massive hepatic necrosis following partial hepatectomy. Therefore we examined endotoxin hepatotoxicity in rats with hepatic regeneration after 35% hepatectomy and in rats with liver cell proliferation induced by lead nitrate. Biochemical and histopathological studies showed no enhanced endotoxin hepatotoxicity in either partially hepatectomized rats or in rats with lead nitrate-induced liver cell proliferation. These results indicate that the development of endotoxin-associated hepatic damage after partial hepatectomy may not relate to regeneration and proliferation of the liver.     

Stimulatory effects of small intestine mucosal factors on the proliferation and antibody formation of primarily cultured rat splenic lymphocytes

When male adult rats at the age of 8-10 weeks received 70% partial hepatectomy, a factor stimulating the proliferation and differentiation of rat splenic lymphocytes appeared in the portal blood serum taken 48 h later. This study was undertaken to investigate if this factor was present in the small-intestinal tissues which are functionally related to portal veins. Bovine small-intestine mucosal homogenate was ultracentrifuged to separate the supernatant, which was heated gently to form a precipitate. After the precipitate was removed, the supernatant was dialysed and cooled with ice. The fraction retained by the dialysing membrane was added to the medium consisting of RPMI 1640 and new-born calf serum. When rat splenic lymphocytes were cultured in medium containing the fraction, they proceeded to proliferate and their glucose metabolism increased. In the culture of lymphocytes treated with the precipitates obtained from the supernatant by addition of ammonium sulfate, the same effects on cells were observed. In addition, the numbers of antibody-forming cells increased significantly. These results indicate that a lymphocyte-activating factor was present in the fraction obtained from the extract of small-intestinal mucosa by salting-out using ammonium sulfate.     

Expression of NCAM in activated portal fibroblasts during regeneration of the rat liver after partial hepatectomy

In the portal tract of the regenerating liver after partial hepatectomy, vascular and bile ductular remodeling takes place in response to the portal hyperdynamic state and parenchymal hyperplasia. In order to reveal phenotypical changes in the portal fibroblasts, we immunohistochemically investigated neural cell adhesion molecules (NCAM) and alpha smooth muscle actin (alphaSMA) expression and the ultrastructural changes in them during liver regeneration. In the control rat liver, portal fibroblasts were negative for both NCAM and alphaSMA. They became positive for both markers two days after partial hepatectomy, increased in staining intensity, reached a maximum at three to four days, then decreased, being still clearly positive at 14 days. Under an electron microscope, portal fibroblasts from the regenerating liver had larger amounts of cytoplasm and rough endoplasmic reticulum than those from the control liver; thus they might be activated. Additionally, periportal hepatic stellate cells in the regenerating liver were activated with alphaSMA, but without NCAM. The present study has demonstrated that portal fibroblasts express NCAM and alphaSMA in the regenerating liver after partial hepatectomy via transformation into myofibroblasts following reconstruction of the portal tracts.     

IL-12 induces specific cytotoxicity against regenerating hepatocytes in vivo.

Although impaired liver regeneration is thought to be a major cause of death in patients with fulminant hepatitis, the mechanisms are not well defined. Since IL-12 synthesis has been reported to be up-regulated in murine hepatitis virus infection, we studied the influence of continuous IL-12 stimulation on murine liver regeneration using flow cytometric and functional analyses. In non-hepatectomized mice, interestingly, the number of hepatic NK cells was significantly decreased on day 7, after six IL-12 injections, and day 14, after 13 IL-12 injections. The number of hepatic NKT cells was markedly increased on day 7 and day 14 of daily IL-12 treatment. The cytotoxic activity of hepatic lymphocytes against both YAC-1 and p815 cells was enhanced on day 2, after single IL-12 injection, and day 7, after six IL-12 injections. In contrast, hepatic lymphocytes isolated 24 h after partial hepatectomy with IL-12 pretreatment did not show any cytolytic activity against either YAC-1 cells or p815 cells. However, continuous IL-12 stimulation resulted in a significantly higher serum alanine aminotransferase (sALT) level 24 h after the partial hepatectomy as compared with sALT levels in mice subjected to either partial hepatectomy or IL-12 pretreatment alone. On the other hand, the expression of hepatic TNF-alpha mRNA was markedly enhanced by continuous IL-12 stimulation even 24 h after partial hepatectomy, as compared with that in non-treated mice and hepatectomy alone. Simultaneous administration of anti-tumor necrosis factor (TNF)-alpha mAb completely inhibited IL-12-induced in vivo enhancement of liver damage after partial hepatectomy. In conclusion, IL-12 induces the specific cytolytic activity against regenerating hepatocytes in vivo mainly through the enhancement of TNF-alpha synthesis.     

Experimental models of hepatectomy and liver regeneration using newborn and weaning rats

OBJECTIVES: Liver regeneration is a complex process that has not been completely elucidated. The model most frequently used to study this phenomenon is 70% hepatectomy in adult rats; however, no papers have examined this effect in developing animals. The aims of the present study were: 1) to standardize two models of partial hepatectomy and liver regeneration in newborn suckling and weaning rats, and 2) to study the evolution of remnant liver weight and histological changes of hepatic parenchyma on the days that follow partial hepatectomy. METHODS: Fifty newborn and forty-four weaning rats underwent 70% hepatectomy. After a midline incision, compression on both sides of the upper abdomen was performed to exteriorize the right medial, left medial and left lateral hepatic lobes, which were tied inferiorly and resected en bloc. The animals were sacrificed on days 0 (just after hepatectomy), 1, 2, 3, 4 and 7 after the operation. Body and liver weight were determined, and hepatic parenchyma was submitted to histological analysis. RESULTS: Mortality rates of the newborn and weaning groups were 30% and 0%, respectively. There was a significant decrease in liver mass soon after partial hepatectomy, which completely recovered on the seventh day in both groups. Newborn rat regenerating liver showed marked steatosis on the second day. In the weaning rat liver, mitotic figures were observed earlier, and their amount was greater than in the newborn. CONCLUSIONS: Suckling and weaning rat models of partial hepatectomy are feasible and can be used for studies of liver regeneration. Although similar, the process of hepatic regeneration in developing animals is different from adults.     

Portal application of autologous CD133+ bone marrow cells to the liver: a novel concept to support hepatic regeneration

The liver has a large capacity for regeneration after resection. However, below a critical level of future liver remnant volume (FLRV), partial hepatectomy is accompanied by a significant increase of postoperative liver failure. There is accumulating evidence for the contribution of bone marrow stem cells (BMSCs) to participate in liver regeneration. Here we report on three patients subjected to intraportal administration of autologous CD133(+) BMSCs subsequent to portal venous embolization of right liver segments, used to expand left lateral hepatic segments as FLRV. Computerized tomography scan volumetry revealed 2.5-fold increased mean proliferation rates of left lateral segments compared with a group of three consecutive patients treated without application of BMSCs. This early experience with portovenous application of CD133(+) BMSCs could suggest that this novel therapeutic approach bears the potential of enhancing and accelerating hepatic regeneration in a clinical setting.     

Lateral hypothalamic lesions facilitate hepatic regeneration after partial hepatectomy in rats

It has been reported that ventromedial hypothalamic lesions facilitate hepatic regeneration through the hepatic vagal nerve after partial hepatectomy. However, whether the lateral area of the hypothalamus is involved in liver regeneration after partial hepatectomy is unknown. To determine the role of the lateral hypothalamic area in this phenomenon, we studied hepatic DNA synthesis during liver regeneration after partial hepatectomy with bilateral lesions of the area. Lesioning of the lateral hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of [methyl-³H]thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesioning were inhibited by combined hepatic vagotomy and sympathectomy. Our results demonstrate that lesioning of the lateral hypothalamus promotes hepatic regeneration through the autonomic nervous system after partial hepatectomy and suggest that the lateral hypothalamic area is involved in liver regeneration through neural mediation.     

Effects of partial hepatectomy on the immune responses in mice

Experiments were performed to investigate the immune responses occurring as a result of partial hepatectomy (HEP) in mice. On Day 12 mice subjected to HEP showed a twofold rise in serum levels of IgG when compared with sham-operated (ShO) controls. The effects of HEP on specific antibody production following a single immunization with sheep red blood cells (SRBCs) were investigated. An early appearance of direct (IgM) splenic plaque-forming cells (PFCs) and significantly elevated indirect (IgG) PFCs were found in HEP mice. Elevated, early-appearing mercaptoethanol-resistant (IgG) hemagglutinating antibodies were also demonstrated in the sera of HEP mice. In addition to these findings our study showed that humoral and cell-mediated responses are affected by HEP in opposing fashion. Partial hepatectomy performed immediately after skin grafting suppressed a first set allograft rejection in mice. Furthermore, inhibited delayed-type hypersensitivity (DTH) response against SRBCs, as evaluated by a footpad weight assay, was demonstrated in HEP mice compared with the ShO controls. When SRBC-primed mice were partially hepatectomized 15 days later, they responded in a manner typical of the secondary immune response, showing an increased production of indirect (IgG) splenic PFCs. A similar anamnestic response was observed in mice sensitized with leptospiral antigen 50 days prior to HEP or exposed to carbon tetrachloride (CCl4) hepatotoxin. The mice responded by elevated serum IgG-specific antibodies as measured by solid-phase enzyme-linked immunosorbent assay (ELISA). Since HEP induced in mice immunological disturbances similar to those associated with liver disease, it is suggested that partial hepatectomy creating liver deficiency followed by regeneration may be a useful experimental model to study the immune status of various forms of hepatic damage.     

Effect of toluene exposure on the liver under different experimental conditions

The effect of exposure to toluene inhalation was studied in adult mice, rats, and rabbits, and, further, in rats of different age groups and rats subjected to partial hepatectomy or ligation of the common bile duct. It was found that (1) toluene has no substantial liver damaging effect on the mouse, rat, or rabbit; (2) induction of microsomal monooxygenase enzymes by toluene is almost completely independent of physiological changes affecting the functional-anatomical state of the liver (aging), of restriction of the functional capacity of the organ (partial hepatectomy), and of irreversible hepatic damage (bile duct ligation); (3) toluene does not in any way interfere with surgically induced hepatic damage. The consequences of surgical interventions, in turn, just like the age-dependent changes, do not modify the minimal ultrastructural damaging effect of toluene.     

CK19和TGF-α在大鼠肝纤维化部分肝切除后残肝中的表达

目的　检测肝纤维化大鼠部分肝切除后不同时间点CK19及TGF-α的表达,了解其胆管再生情况。 方法　雄性SD大鼠,对照组和实验组各35只。实验组腹腔注射CCl4制备肝纤维化模型,两组均进行肝部分切除术。进行免疫组织化学、HE染色,图像分析和数据统计。 结果　验组和对照组术后随时间延长CK19表达均呈增强趋势; 实验组术后各时间点CK19的表达均高于对照组同时间点。实验组和对照组术后随时间延长TGF-α表达均呈上升趋势,但实验组上升速度明显较对照组缓慢; 实验组术后　0d、1d、3d的TGF-α表达高于对照组。 结论 (1) 肝纤维化大鼠肝部分切除后CK19呈现高表达,提示肝纤维化部分切除可以促进肝卵圆细胞的增殖和分化。(2) TGF-α对肝纤维化大鼠肝部分切除后肝卵圆细胞增殖及胆管再生的促进作用不明显。     

Increase in serum alpha foetoprotein level in hepatic regeneration of the rat. Effects of age and of magnitude of regenerative activity.

The effects of variable degrees of liver regeneration induced by two-thirds partial hepatectomy or intoxication with different doses of carbon tetrachloride (CCl4) on increased production of alpha foetoprotein (AFP) have been studied in very young (5-6 weeks) and older (15 weeks and more) rats by counter-immunoelectrophoresis (sensitivity down to 250 ng/ml). In the young animal adequate regeneration following two-thirds hepatectomy as well as 100 myl CCl4 successfully induced large increases in serum AFP levels. Smaller doses as well as a large fatal dose of the toxin, all of which resulted in inadequate regenerative activity, failed to excite AFP synthesis and secretion up to detectable levels. The adult animal did not show detectable AFP with any of these procedures. However, necrosis induced in the regenerating adult liver 24 h following partial hepatectomy did result in the detection of small amounts of protein in the serum. It is concluded that in hepatic regeneration of this species, the age of the animal as well as the magnitude of regenerating activity in the liver are critical in the occurrence of high levels of serum AFP.     

大部分肝切除模型大鼠肝再生组织中CD34蛋白及Delta-like-1蛋白的表达

背景：当肝脏受到严重损伤或肝脏大部分缺失时,肝干细胞迅速分裂增殖向成熟肝细胞分化修复肝组织,但肝再生是一个复杂的多信号分子调控的过程,参与调控的相关基因及蛋白质目前尚不十分清楚。 目的：观察大部分肝切除后大鼠肝再生组织中CD34蛋白、Delta-like-1蛋白的表达变化。 方法：切除2/3肝脏建立大鼠肝再生动物模型,应用苏木精-伊红染色、酶组织化学染色、免疫组织化学染色方法在不同时间点检测肝再生组织中ATP酶、增殖细胞核抗原、CD34、Delta-like-1蛋白的表达情况。 结果与结论：在肝再生的启动阶段ATP酶表达下调,CD34、Delta-like-1表达上调;在肝再生的持续增殖阶段,ATP酶表达上调到正常水平,CD34、Delta-like-1表达下调;增殖细胞核抗原表达为持续上调。说明ATP酶、CD34、Delta-like-1在不同阶段表达不同,提示在肝再生过程中各信号分子的协同作用及Notch- Delta信号通路参与其作用机制。     

肝细胞生长因子和C-met在肝纤维化肝部分切除后残余肝组织中的表达

目的 探讨肝细胞生长因子（HGF）和C-met在纤维化肝部分切除后残余肝组织中的表达变化。方法 雄性SD大鼠130只,随机分为正常组（n=7）、正常肝部分切除组（n=50）、肝纤维化组（n=7）和纤维化肝部分切除组（n=66）。正常肝部分切除组和纤维化肝部分切除组分别在术后12 h、1 d、3 d、5 d、7 d和14 d 取材,运用免疫组织化学和Western blotting方法,检测残肝组织中HGF、C-met的表达。结果 免疫组织化学显示,正常肝部分切除组,HGF表达于术后12 h达高峰,并维持高峰平台,7 d后逐渐降低,于14 d接近术前水平;C-met术后迅速上升,3 d达到高峰,而后逐步下降,14 d降回术前水平。纤维化肝部分切除组,HGF表达于术后迅速下降,12 h又迅速上升,于1 d时达高峰,然后逐步下降,14 d降至最低点;C-met术后迅速下降,12 h开始缓慢下降,于术后3 d达最低点,后缓慢上升,7 d又迅速上升,14 d达最高点。Western blotting显示,HGF、C-met蛋白条带变化规律与免疫组织化学结果吻合。结论 HGF和C-met同时高水平表达有利于肝细胞的分裂增殖,提示HGF和C-met表达不同步,可能是纤维化肝术后再生困难的重要原因。     

Facilitation of liver regeneration after partial hepatectomy by ventromedial hypothalamic lesions in rats

Whether or not the hypothalamus is involved in initiating hepatic DNA synthesis after partial hepatectomy is unclear. To determine the role of the ventromedial hypothalamic nuclei in liver regeneration after partial hepatectomy, we studied hepatic DNA synthesis during liver regeneration in rats with bilateral lesions of these nuclei. Lesions of the ventromedial hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesions were completely inhibited by hepatic vagotomy. Thus, lesions of the ventromedial hypothalamus appear to promote hepatic regeneration by increasing vagal stimulation of the liver.     

Liver damage using suicide genes. A model for oval cell activation

Liver regeneration from the facultative hepatic stem cells, the oval cells, takes place in situations in which liver regeneration from pre-existing hepatocytes is prevented. Different models have been used to stimulate oval cell response. Many of them involve the use of carcinogenic agents with or without partial hepatectomy. In this study we show that adenovirus-mediated gene transfer of the suicide gene thymidine kinase followed by ganciclovir administration caused hepatotoxicity of variable intensity. Rats with moderate elevation in serum transaminases recovered normal liver architecture few weeks after adenovirus injection. In contrast, rats with severe liver damage exhibited a marked and persisting activation of oval cells accompanied by ductular hyperplasia. In some rats, such lesion eventually evolved to cholangiofibrosis and in one rat to cholangiocarcinoma. Deposition of fibronectin and increased number of hepatic stellate cells were found in association with oval cells and cholangiofibrotic lesions. Hepatocyte growth factor was hyperexpressed in the livers with intense oval cell response or ductular proliferation, suggesting a participation of this factor in those lesions. In summary, our data demonstrate activation of oval cell response after gene transfer of thymidine kinase followed by ganciclovir administration. These findings indicate that high doses of this therapy causes liver damage together with an impairment in hepatocellular regeneration.     

The mitogenic activity of hepatocyte growth factor on rat hepatocytes is dependent upon endogenous transforming growth factor-alpha

Both transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) induce DNA synthesis in hepatocytes in vitro and in vivo. Hepatic and circulating levels of HGF have been reported to increase before an increase in TGF-alpha levels in several rat models of liver regeneration. In addition, serum TGF-alpha levels increase after an increase in serum HGF levels in patients with either partial hepatectomy or acute hepatitis. In this study, we investigate the significance of TGF-alpha in hepatocyte proliferation. TGF-alpha contents and DNA synthesis in cultured rat hepatocytes increased in response to HGF addition to the culture medium in a dose-related manner. These increases were suppressed by the addition of anti-sense TGF-alpha mRNA oligonucleotide. Furthermore, the addition of anti-TGF-alpha rabbit IgG suppressed the increase in DNA synthesis. When the anti-TGF-alpha antibody was administered to rats after partial hepatectomy, the number of mitotic hepatocytes was reduced in comparison to rats treated with normal rabbit IgG. These results were observed even though hepatic HGF levels were increased equally in rats given either anti-TGF-alpha antibody or normal rabbit IgG. Our results suggest that HGF stimulates TGF-alpha production in rat hepatocytes, and that the mitogenic activity of HGF depends on endogenous TGF-alpha activity.     

Temporal relation between a hepatic erythropoietic factor and the site of rat erythropoietin production

Serum borne hepatic erythropoietic factor (HEF), which can stimulate hepatic erythropoietin (Ep) production in the adult rat, is found at elevated levels in the serum of partially hepatectomized rats and of rats subjected to hepatotoxic injury. It is also detected in sera of patients with liver disease. The purpose of the present study was to determine whether or not HEF activity is increased in the serum of the normal neonatal rat at a time when the liver is the primary site of Ep production. Our results show significantly increased HEF activity in the serum of young rats during the second to fifth weeks of life. Negligible activity was detected in rats over five weeks of age. In the rat, the kidney is reported to begin producing Ep by the third week of life and by the eighth week the kidney is the major site of synthesis with liver production at this age significantly diminished. Thus, our findings show a temporal relation between HEF activity in the serum and the reported transition from liver to kidney production of Ep.     

Further studies on the tumor-initiating activity of the beta-blocker DL-ZAMI 1305

The purpose of this study was to evaluate the initiating activity of the hepatocarcinogen beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylamino-propan-2-ol (DL-ZAMI 1305) by the initiation-promotion protocol of Pereira. Female Wistar rats were given a single dose 150 mg/kg of body weight of DL-ZAMI 1305 by gavage 24 hours before or 24 hours after partial hepatectomy. One week later rats were given phenobarbital (0.05%) in the diet for a period of 7 weeks. DL-ZAMI 1305-treatment resulted in the appearance of gamma-glutamyltranspeptidase foci and of other preneoplastic lesions in all animals. Preneoplastic lesions were also present in a fraction of DL-ZAMI 1305-treated animals not subjected to partial hepatectomy, whether given or not phenobarbital. Results obtained in a separate experiment demonstrated that DL-ZAMI 1305-treatment inhibits cell proliferation and induces DNA damage in the regenerating rat liver. The results of this study clearly demonstrated that the beta-blocker DL-ZAMI 1305 is an initiating carcinogen for the liver of female Wistar rats.