液压转基因技术应用于大鼠再生肝转基因实验

目的 探讨液压转基因技术(HDT)应用于大鼠再生肝转基因的条件和方法 . 方法 以2ml/s的速度将浓度为30mg/L的含目的 基因的质粒注射入大鼠尾静脉,于注射前/后不同时间进行大鼠2/3肝切除(PH),于PH后不同恢复时间称量大鼠体重(g)和再生肝重(g),计算肝系数(Lc),并从Lc±Lc*0%、*5%、*10%、*15%、*20%、*25%、*30%、*35%等15组中找出最佳组,作为计算不同恢复时间再生肝最适注射质粒溶液量的校正系数(Trc);取大鼠肝右叶中部组织制备冷冻切片,在波长488nm的荧光显微镜下观察、计数1万个细胞中的绿色荧光蛋白阳性细胞百分率. 结果 PH后注射生理盐水和注射空质粒对肝再生的影响与对照(只进行PH)相比无显著差异.PH前液压转基因的合适时间是PH前≥12h;PH后所有时间均可进行液压转基因.PH后对肝再生大鼠进行液压转基因的转基因溶液体积为大鼠体重(g)×9%×1/3×相应的校正系数(Trc).转入基因在体内的表达时间和丰度既受载体影响,又受插入的目的 基因影响. 结论 液压转基因技术亦可有效地应用于大鼠再生肝转基因研究.     

皮质酮对大鼠早期再生肝抗酶mRNA及蛋白表达的影响

目的 研究外源皮质酮对部分肝切除(PH)诱导的大鼠早期再生肝抗酶mRNA及蛋白水平的影响.方法 PH前3d对成年雄性SD大鼠进行双侧肾上腺切除术,皮质酮悬浮于芝麻油中皮下注射去肾上腺鼠,抗酶mRNA及其蛋白水平的测定采用RT-PCR和Western blotting法. 结果 与对照组(n=6,以下相同)相比,去肾上腺组及10mg/kg、20mg/kg体重皮质酮处理组抗酶mRNA水平变化不明显,而40mg/kg体重皮质酮处理能显著刺激其转录.在PH后5、7、9h,去肾上腺术鼠抗酶蛋白水平显著低于相应时间点的对照组;皮质酮处理后,蛋白水平在所观察的整个实验过程中均显著上升,且剂量越高,蛋白含量越多,尤其在PH后5h,10、20和40mg/kg体重皮质酮处理组分别比同时间点对照组高43%、79%和93%. 结论 皮质酮对大鼠早期再生肝抗酶蛋白的合成具有剂量依赖性促进作用.     

In vivo macrophage-mediated tumor cytotoxicity in the mouse

Tumor-bearing animals injected with either LPS-activated or control macrophages were capable of suppressing tumor growth in vivo. Tumor growth rate and survival times were assessed for each group of animals. At day 12 after injection of the P815 tumor cells, no difference in tumor size could be demonstrated in any of the groups. However, by day 17 the tumors in the animals treated with 2.5 × 10⁶ or 5 × 10⁶ control as well as LPS-activated macrophages did not continue to increase in size as was seen in the case of the control animals. When examining survival times, it appeared that the animals treated with control or activated macrophages generally survived approximately 10 days longer than did the animals receiving no treatment (30-day vs 20 day survival). Thus, it appears that tumor cell growth can be slowed down in vivo when control or activated macrophages are injected into the site of the tumor mass.     

Cytokine-dependent activation of small hepatocyte-like progenitor cells in retrorsine-induced rat liver injury

Complete liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the activation, expansion, and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The mechanism(s) governing activation of SHPCs after PH in retrorsine-injured rats has not been investigated. We examined the possibility that SHPCs require cytokine priming prior to becoming growth factor responsive in this model of liver injury and regeneration. Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age. Retrorsine-exposed and age-matched control rats were randomized into dexamethasone-treated and no DEX groups. DEX-treated animals were either given a single dose of DEX (2 mg/kg ip) at the time of PH or multiple DEX treatments (2 mg/kg ip each) at 24 and 1 h before PH and 1, 2, and 3 days post-PH. A subset of rats received 10 μg of recombinant IL6 protein, administered intravenously 30 min after PH. Liver tissues were harvested at 7, 14, 21, and 30 days post-PH. Treatment of retrorsine-exposed rats with the cytokine inhibitor dexamethasone (DEX) effectively blocked the emergence of SHPCs resulting in an inhibition of liver regeneration and producing significant short-term mortality. The livers of DEX-treated retrorsine-exposed rats displayed decreased numbers and smaller SHPC clusters compared to retrorsine-exposed rats in the absence of DEX treatment. Administration of recombinant IL6 to DEX-treated retrorsine-exposed rats restored the emergence of SHPCs and SHPC-mediated regenerative response. The livers of DEX-treated retrorsine-exposed rats that received IL6 displayed numbers of expanding SHPC clusters comparable to that of retrorsine-exposed rats in the absence of DEX treatment. These results combine to suggest that SHPC activation after PH in retrorsine-exposed rats is cytokine dependent and may specifically require IL6.     

外源多胺对大鼠再生肝细胞鸟氨酸脱羧酶基因转录的调节

目的 研究天然多胺(腐胺、精脒和精胺)对大鼠再生肝细胞鸟氨酸脱羧酶(ODC)基因转录的调节,探讨多胺在肝再生中的作用.方法 大鼠部分肝切除术诱导再生肝,采用原位杂交技术分析ODC mRNA的表达量,外源多胺(溶于0.9%NaCl)的处理用皮下注射法.结果 外源多胺处理后,再生肝ODC mRNA水平的变化趋势与对照组相似,但不同种类、剂量的多胺,结果差异明显.在所观察到的整个肝再生期间,高剂量腐胺(20 mg/kg体重)处理组mRNA水平始终低于对照组,特别在部分肝切除后2、4和10 h与对照组相比具有显著性差异(P＜0.05);低剂量(0.02 mg/kg体重)的腐胺处理组,只在4 h和12 h显著高于对照组.高剂量精脒(0.15 mg/kg体重)处理组的ODC mRNA水平始终低于对照组(在10 h例外);而低剂量(0.03 mg/kg体重)处理组则不同,ODC mRNA水平表现出先抑制(在2 h)后促进(在4 h和6 h)的特点.精胺的作用与精脒相似.结论 不同浓度的多胺(主要是精脒和精胺)对大鼠再生肝细胞ODC基因转录存在着反馈调节作用.     

CK19和PCNA在肝纤维化大鼠部分肝切除后再生肝中的表达

目的　检测肝纤维化大鼠部分肝切除后不同时点CK19及PCNA的表达,了解胆管再生情况。 方法　雄性SD大鼠,对照组和实验组各42只,实验组腹腔注射CCl4制备肝纤维化模型,两组均进行部分肝切除术,在不同时点取材后利用HE、免疫组化及免疫荧光双标染色等方法检测CK19和PCNA的表达情况。 结果　实验组和对照组术后随时间延长CK19表达均呈增强趋势,且实验组术后各时间点CK19的表达均高于对照组同时间点。两组PCNA的表达量都随时间推移逐步升高,但实验组大鼠明显上升缓慢,持续时间延长,表达高峰晚于对照组出现。 结论　 （1）肝纤维化大鼠部分肝切除能刺激肝卵圆细胞增殖和向胆管细胞的分化,而正常大鼠部分肝切除后再生肝中的胆管上皮细胞主要来源于原有细胞的代偿性增生。（2）术前肝纤维化大鼠的肝脏细胞就出现了增殖修复,术后由于肝纤维化大鼠本身肝脏受到伤害,因此肝脏的有效再生细胞数低于正常肝脏。     

Participation of liver progenitor cells in liver regeneration: lack of evidence in the AAF/PH rat model

When hepatocyte proliferation is impaired, liver progenitor cells (LPC) are activated to participate in liver regeneration. We used the 2-acetaminofluorene/partial hepatectomy (AAF/PH) model to evaluate the contribution of LPC to liver cell replacement and function restoration. Fischer rats subjected to AAF/PH (or PH alone) were investigated 7, 10 and 14 days post-hepatectomy. Liver mass recovery (LMR) was estimated, and the liver mass to body weight ratio calculated. We used serum albumin and bilirubin levels, and liver albumin mRNA levels to assess the liver function. LPC expansion was analyzed by cytokeratin 19 (CK19), glutathione S-transferase protein (GSTp) immunohistochemistry and by CK19, CD133, transforming growth factor-β1 and hepatocyte growth factor mRNA expression in livers. Cell proliferation was evaluated by Ki67 and BrdU immunostaining. Compared with PH alone where LMR was ～100% 14 days post-PH, LMR was defective in AAF/PH rats (64.1±15.5%, P=0.0004). LPC expansion was scarce in PH livers (0.5±0.4% of CK19(+) area), but significant in AAF/PH livers (8.5±7.2% of CK19(+)), and inversely correlated to LMR (r(2)=0.63, P<0.0001). A quarter of AAF/PH animals presented liver failure (low serum albumin and high serum bilirubin) 14 days post-PH. Compared with animals with preserved function, this was associated with a lower LMR (50±6.8 vs 74.6±9.4%, P=0.0005), a decreased liver to body weight ratio (2±0.3 vs 3.5±0.6%, P=0.001), and a larger LPC expansion such as proliferating Ki67(+) LPC covered 17.4±4.2% of the liver parenchyma vs 3.1±1.5%, (P<0.0001). Amongst those, rare LPC with an intermediate hepatocyte-like phenotype were seen. Also, less than 2% of hepatocytes were engaged into the cell cycle (Ki67(+)), while more numerous (～25% of hepatocytes) in the livers with preserved function. These observations suggest that, in this model, the efficient recovery of the liver function was ensured rather by the proliferation of mature hepatocytes than by the LPC expansion and differentiation into hepatocytes.     

Protection of hepatotoxic and lethal effects of CCl4 by partial hepatectomy

CCl4 is a hepatotoxic haloalkane, capable of producing hepatocellular fatty degeneration and centrilobular necrosis. Previous reports indicate induction of liver regeneration after 36-48 hr of CCl4 treatment, which is considered as a secondary effect. The present investigation was undertaken to evaluate the primary effects of CCl4 on hepatic DNA synthesis and to correlate liver regeneration with CCl4 toxicity. These studies were conducted in normal and actively regenerating livers using male Sprague-Dawley rats undergoing sham operation (SH), or partial (70%) hepatectomy (PH). Incorporation of 3H-thymidine (3H-T) in hepatocellular nuclear DNA and autoradiographic analyses of liver sections served as indices for hepatocellular regeneration. Initial experiments established that peak regeneration occurs at 2 days post-PH (PH2) and liver regeneration phases out by 7 days post-PH (PH7). SH and PH rats were challenged with a single ip dose of either corn oil vehicle or CCl4 at either 0.1 ml/kg (to represent subtoxic dose) or 2.5 ml/kg (to represent toxic dose). The low dose of CCl4 was not toxic and did not alter 3H-T incorporation and percentage labelled cells at 6 or 24 hours after administration to SH, PH2 or PH7 groups, indicating that there was no interference with PH-stimulated hepatocellular regeneration. The high dose of CCl4 was significantly hepatotoxic and lethal in SH rats, while in PH2 rats both hepatotoxic and lethal effects were significantly decreased. 3H-T incorporation as well as percentage labelled cells, highly stimulated by PH, were significantly decreased by high dose of CCl4. However, hepatocellular regeneration in PH2 rats treated with high dose of CCl4 was still significantly higher than SH or PH7 groups by virtue of the stronger stimulatory effect of PH. In PH7 rats, where hepatocellular regeneration had returned to the SH level, the hepatotoxic and lethal effects of the large dose of CCl4 were also restored. These findings show that the progressive phase of a single high dose of CCl4 injury which normally culminates in hepatotoxic and lethal effects is significantly mitigated by previously stimulated hepatocellular regeneration. High dose of CCl4 suppresses hepatocellular regeneration at early time points after administration in contrast to the smaller subtoxic dose of CCl4. By virtue of the much stronger stimulatory effect, PH results in the protection against the hepatotoxic and lethal effects of CCl4 despite the obtunding effects of the high dose on hepatocellular regeneration.     

Blockade of the renin–angiotensin system inhibits growth of colorectal cancer liver metastases in the regenerating liver

Partial hepatectomy (PH), the preferred option for selected patients with colorectal cancer liver metastases (CRCLM), is associated with 40–80 % tumor recurrence rates. Renin–angiotensin system (RAS) blockade inhibits tumor growth and has been suggested to improve liver regeneration. We documented the effect of RAS blockade on tumor growth and liver regeneration in a murine model. CRCLM induction followed by 70 % PH was performed on 78 CBA mice. Liver regeneration (days 2, 6) and CRCLM tumor load were measured by liver (and tumor) weights, percentage of CRCLM burden and tumor nodule count (days 16, 21). mRNA expression of the RAS components was characterised. Statistical analysis was performed using 2-independent sample T test or Mann–Whitney test (SPSS). Captopril did not impair liver regeneration. By day 21, Captopril decreased tumor burden (percentage of CRCLM in the liver) (48.7 ± 4.7 % control, 24.4 ± 6.2 Captopril; p = 0.008), tumor volume (1046.2 ± 200.2 mm³, 388.3 ± 150.4; p = 0.02), tumor nodule count per image field (181.1 ± 28.5, 68 ± 17.6; p = 0.005) and tumor angiogenesis (71.8 ± 6.4 vessels/mm², 43.1 ± 7.6; p = 0.015) compared to controls. Captopril enhanced tumor apoptosis (1 ± 0.2 %, 2.5 ± 0.7; p = 0.028). Liver regeneration and tumor development increased liver ACE levels. Blockade of the RAS effectively retarded CRCLM tumor growth at the late stage of tumor development within the regenerating liver without impeding liver regeneration following PH, via anti-angiogenesis and pro-tumor apoptosis. Captopril may be of therapeutic benefit in patients undergoing PH for CRCLM.     

Experimental liver metastasis: Standards for local cell implantation to study isolated tumor growth in mice

Experimental hepatic metastasis of colorectal tumors is frequently studied by local intrahepatic tumor cell implantation. However, although a variety of factors of the implantation procedure may markedly influence tumor growth characteristics, standards are not defined yet. Herein, we studied the effect of different modes of cell implantation on tumor growth and angiogenesis by in vivo fluorescence microscopy and histology seven days after grafting colorectal CT26.WT tumor cells into the left liver lobe of syngeneic BALB/c mice. We demonstrate that (i) radial growth of cells implanted within the central area of the lobe is inhibited by a regularly observed fissura which crosses at midline the surface of the lobe; (ii) cells suspended during implantation in RPMI show an uncontrolled overwhelming growth 40-fold of those suspended in PBS; (iii) cell implantation in 100 μl and 20 μl suspension medium is significantly more complicated by rupture of the liver capsule, uncontrolled intraparenchymal cell spread and recoil of the cells through the injection canal compared to cells suspended in 10 μl; (iv) the frequency of metastasis within the injection canal and at the puncture site is significantly reduced using 32G compared to 27G or 29G needles; (v) occlusion of the puncture site by acrylic glue or electric coagulation completely abolishes peritoneal tumor spread compared to no treatment or gentle compression by cotton gauze. We conclude that a standardized growth of isolated metastases is best achieved by implanting CT26.WT cells in a 10 μ l PBS blister subcapsularly into the paramedian area of the lower surface of the left liver lobe, using a 32-gauge needle and closing the puncture site with acrylic glue.     

Quantification of metastatic tumor growth in bleomycin-injured lungs

The lung is a common target organ in experimental models of tumor metastasis in which quantification usually involves counting labeled tumor cells shortly after injection, or enumeration of grossly visible pleural tumors. In this study, these approaches were used in addition to autoradiographic and morphometric methods to analyse the effect of bleomycin-mediated injury on the development, distribution and quantification of pulmonary metastases. One day after intravenous injection of 2 × 105 fibrosarcoma cells, the lungs of C57 bl/6 mice, pretreated with bleomycin (120mg/kg i.v., 5 days before) contained about nine times as many [131 I] iododeoxyuridine-labeled cells as the lungs of control animals given saline injections. At this time, autoradiographic counts of [³H] thymidine-labeled tumor cells in lung sections showed a similar increase in tumor cell localization after bleomycin, with labeled cells distributed equally between parenchymal and pleural areas. However, subsequent tumor growth was demonstrated microscopically to be predominantly in pleural and peribronchial areas, especially at sites of lung injury induced by bleomycin. Counts of grossly visible pleural tumors failed to demonstrate a difference between bleomycin groups and controls at 7 days whereas counts of nodules in lung sections, and quantification of lung area occupied by tumor both showed significantly greater tumor involvement in bleomycin-treated animals. As tumors became confluent, morphometric measurements demonstrated tumor growth in the lung more accurately than did nodule counts. We conclude that bleomycin-induced injury greatly enhances metastatic tumor growth and that morphometric methods are more sensitive than lung colony counts in their ability to quantify pulmonary metastases. Morphometry and autoradiography have also demonstrated that while there is a uniform distribution of arrested tumor cells in the lung initially, there is preferential development of metastatic tumors at sites of pulmonary damage, in particular at the pleura.     

Hepatic arterial infusion of bevacizumab in combination with oxaliplatin reduces tumor growth in a rat model of colorectal liver metastases

Unresectable colorectal liver metastases are commonly treated with systemic chemotherapy (SCT). Clinical studies on the effect of additional systemic application of bevacizumab (BE), a monoclonal antibody directed against vascular endothelial growth factor, to SCT showed a slight increase of patient survival. Herein, we studied in a rat model of colorectal liver metastasis whether a locoregional application of oxaliplatin (OX) and BE via hepatic arterial infusion (HAI) is more effective to inhibit metastatic growth compared to systemic drug application. Ten days after implantation of CC531 colorectal cancer cells into the left liver lobe of WAG/Rij rats, animals underwent either HAI or systemic intravenous application of BE (5 mg/kg body weight), OX (85 mg/m² body surface) or a combination of both. Sham-treated animals received saline and served as controls. Tumor volume was measured at days 10 and 13 using three dimensional ultrasound. At day 13 tumor tissue was analyzed histologically and immunohistochemically. Systemic application of OX, BE or their combination did not affect tumor volume when compared to controls. In contrast, HAI of BE and particularly the combination of BE and OX significantly reduced tumor volume. In the tumor tissue this was associated with a decrease of vascularization and cell proliferation as well as an increase of cell apoptosis, as indicated by a decreased number of PECAM-1- and PCNA-positive cells and an increased number of cleaved caspase-3-positive cells. Locoregional administration of BE, particularly in combination with OX, enhances the inhibitory effect on hepatic metastatic growth compared to systemic application of the drugs.     

Different expression of positive and negative regulators of hepatocyte growth in growing and shrinking hepatic lobes after portal vein branch ligation in rats

Portal vein branch embolization is often performed before hepatectomy to prevent postoperative liver failure. It is, however, still not clear how the embolized lobe shrinks and the non-embolized lobe proliferates in counterbalance. We investigated the expression of positive and negative regulators of hepatocyte growth to clarify the mechanisms of liver growth and atrophy in a rat portal vein ligation (PVL) model compared with partial hepatectomy (PH). A significant increase in DNA synthesis within the non-ligated lobe reached a peak at 36 h, a delay of 12 h as compared with PH, while no increase occurred in the ligated lobe. Expression of hepatocyte growth factor mRNA remarkably increased in the non-ligated growing lobe between 6 and 24 h, but was only slightly elevated in the ligated shrinking lobe. Contrarily, negative regulators of hepatocyte proliferation, such as TGF-beta1 and IL-1beta, were strongly expressed in the ligated shrinking lobe. Thus, the changes of portal venous flow and/or pressure caused by PVL may contribute to induction of different kinds of growth factors between the ischemic and non-ischemic lobes; these factors possibly regulate liver regeneration and atrophy after PVL.     

Effects of individual housing on behavior and resistance to Ehrlich tumor growth in mice

This study analyzed in Balb/C mice the effects of individual housing on behavior, serum corticosterone and resistance to Ehrlich tumor growth. Mice (60 days old) were individually (IH) or grouped housed (G) (10-12 animals/cage) for 14-21 days. The 1st day of the housing condition was considered experimental day 1 (ED1). Results showed that on ED21, IH mice, when compared to G mice, presented no differences on corticosterone serum levels when kept undisturbed; however, an increased level of this hormone was observed in IH mice after an immobilization stress challenge. An increased time spent in the plus-maze closed arms and a decreased time in the open arms were also observed in IH mice. When compared to G animals, after inoculation with 10⁵ Ehrlich tumor cells on ED1, IH mice presented an increase in volume of ascitic fluid and number of tumor cells. The survival time of IH mice was also shorter than that measured in G animals. Furthermore, IH mice injected with a different number of tumor cells on ED1 always presented increased Ehrlich tumor cells than G group. Interestingly, these effects were not observed when the tumor cells injection was done on ED4. These results suggest that individual-housing conditions induce an altered immune-endocrine response and, at the same time, decrease animals' resistance to Ehrlich tumor growth. It is proposed that the neural link between the behavioral and immunological changes observed after the stress of individual housing might involve the activation of the HPA axis.     

Heterogeneous suppression of experimentally induced colon cancer metastasis in rat liver lobes by inhibition of extracellular cathepsin B

Metastatic rat colon cancer cells but not normal rat hepatocytes showed activity of cathepsin B on their plasma membranes. Activity was visualized in living cells with a new fluorogenic substrate, [Z-Arg]²-cresyl violet, and confocal microscopy. When these cancer cells were injected into the portal vein of rats, the animals developed tumors in the liver in a heterogeneous fashion. Three- to four-fold more tumors were found in the small caudate lobe than in the other three large lobes of the liver. Oral treatment with a selective water-soluble inhibitor of extracellular cathepsin B, Mu-Phe-homo Phe-fluoromethylketone, resulted in 60% reduction of the number of tumors and 80% reduction of the volume of tumors in the three large lobes whereas tumor development was not affected in the small caudate lobe. This study supports the conclusions that (a) extra-cellular cathepsin B plays a crucial but complex role in liver colonisation by rat colon carcinoma cells in vivo,(b) its selectiv e inhibition suppresses tumor growth heterogeneously in the liver and (c) the caudate lobe of the liver is a relatively large risk factor for tumor development. © Rapid Science 1998     

Evaluation of metastatic ability at specific times during primary tumor growth: a novel spontaneous metastasis assay

A transformed NIH 3T3 fibroblast cell line, CI-e, normally does not produce spontaneous metastasis from subcutaneous or footpad tumors in nude mice. However, pulmonary tumor nodules are formed when more than 1 × 10³ cells are injected intravenously into nude mice. Co-injection of 1 × 10⁶ heavily irradiated and inactivated cells increases the clonogenic ability of the viable cells in that tumor colonies then occur with as few as 1 × 10² viable cells. Utilizing the action of these inactivated cells to enhance the lung colonizing ability of a relatively small number of viable tumor cells, we have developed a novel experimental model of spontaneous metastasis. In this model, a footpad tumor of the nude mouse metastasizes to the lungs following intravenous injection of 1 × 10⁶ inactivated cells at a specific time of tumor growth and following tumor foot amputation, whereas no spontaneous metastasis develops without injection of inactivated cells. This model enables us to detect metastatic ability which would otherwise be too low to detect using other assays. In addition, it allows us to evaluate metastatic ability at a specific time point during primary tumor growth, since no metastases can develop during the periods before inactivated cell injection and after tumor amputation. Using this model, we have determined that the metastatic ability of CI-e tumors in the footpad is constant throughout the exponential and stationary growth phases, even though cells isolated from exponentially growing tumors possess a 3.3-fold greater lung colonizing ability following intravenous injection than those from stationary tumors. This new experimental model may be applicable to other tumor cell lines and to other analyses where metastatic ability during a defined interval of tumor growth is of importance.     

Immunologisch induzierte Hemmung und Förderung des Tumorwachstums (Ehrlich-Ascites-Tumor): Bedeutung verschiedener antigenhaltiger PrÄparationen

Growth of solid Ehrlich-Ascites-Tumor (EAT) is inhibited, when animals are immunized by four s.c. injections of the soluble moiety of EAT-material. However, enhancement of tumor growth was observed when the unsoluble part of EAT-material was used for immunization.—Immunizing the animals in the same way with antigenic material from normal connective tissue, the same effect was observed as with EAT-material, though to a lesser extent. A single i.v. injection of soluble EAT-material—obtained by freezing and thawing and injected a few days before tumor transplantation—resulted in facilitating tumor development. In contrast, soluble material obtained by ultrasonic treatment showed no effect. Intravenous injection of unsoluble EAT-material, obtained by ultrasonic treatment, resulted in facilitating tumor growth when injected three days before tumor transplantation. This effect is abolished by heating the material for 30 min at 56? C. Principly the same results were obtained using unsoluble material of spleen tissue. Intravenous injection of unsoluble EAT-material 19 days before tumor transplantation resulted in an inhibition of tumor growth. This effect is not abolished by heating the material. Unsoluble material from spleen tissue resulted equally in an inhibition of tumor growth. In contrast to the EAT-assay, no inhibition could be observed when heated unsoluble material was used. These results are interpreted in the sense of the existence of special antigenic factors characteristic for EAT cells.     

Reconstitution Properties of Thymus Stem Cells in Murine Fetal Liver

Injection of day-12 murine fetal liver cells into thymus lobes of Thy-1 congenic adult recipients results in a wave of thymocyte development. The kinetics of repopulation by donor cells reaches a peak after 20–25 days. The frequency of thymic stem cells (TSC) in day-12 fetal liver was estimated, by limit dilution, as 1 in 4x10⁴ cells. Within 8 hr of injection into a thymus lobe, fetal liver TSC commit to T-cell development, losing stem-cell activity. When fetal liver cells are maintained in culture for 7 days, with no exogenous cytokines added, and then injected intra-thymically (I.T.), thymus recolonization is not observed. However, TSC can be maintained in culture for 7 days with IL-1β, IL-3, IL-6, or LIF added, alone or in combination, with steel factor (SLF). Poisson analysis of fetal liver cells cultured with SLF and IL-3 together revealed a precursor frequency of 1 in 1.8x 10⁵ cells. In contrast, the frequency of TSC in adult bone marrow was estimated by limit dilution as 1 in 12,000 cells.     

Murine Kupffer cells and hepatic natural killer cells regulate tumor growth in a quantitative model of colorectal liver metastases

This investigation aimed to develop a biologically relevant murine model of colorectal liver metastases and determine if Kupffer cells (KC) and hepatic natural killer cells (hNKC) regulate tumor growth. The model involves the injection of murine colon adenocarcinoma 26 (MCA 26) tumor cells into the portal vein of female-specific pathogen-free BALB/c mice. Metastases developed in all animals, and the growth was limited entirely to the liver. To determine if KC and hNKC control the development of liver metastases, the in vivo function of these hepatic effector cells was modulated. Tumor growth was quantitated by the uptake of ¹²⁵I into tumor DNA. Stimulation of the KC and hNKC produced a significant (P < 0.01) dose-dependent decrease in ¹²⁵I uptake in the liver in both treatment groups, which was associated with a significant improvement in survival (P < 0.05). The in vivo cytotoxic function of the liver was inhibited with an intravenous injection of gadolinium chloride (for KC) or asialo GM₁ antiserum (for hNKC). Inhibition of KC and hNKC cytotoxic function led to a significant (P < 0.01) increase in 1251 uptake in the liver and a significant decrease in survival (P < 0.05).     

Effect of retinoic acid-induced transglutaminase on cell growth in regenerating liver

Transglutaminase 2 (TG2) is implicated in the inhibitory regulation of the hepatocyte growth in vitro. In vivo, however, the role of TG2 in liver regeneration after partial hepatectomy (PH) is almost unknown. A dramatic increase of TG2 expression and activation is induced by retinoic acid (RA). Here we show the effect of the RA-induced overexpression of TG2 on liver regeneration after PH. Regenerating rat liver was prepared by 70% PH. RA was intraperitoneally injected immediately after PH. TG2 activity was determined by incorporation of 14C-putrescine into dimethylcasein. Cell cycle was evaluated for incorporation of BrdU into hepatocytes and detected by a flow cytometric analysis. The treatment of RA greatly increased TG2 activity at 1 day after PH. At that time, DNA synthesis was significantly reduced by the treatment of RA. The recovery of liver weight after PH was significantly delayed by the treatment of RA. These results suggested that TG2 was involved in growth capacity in regenerating rat liver after PH.