同种异体胎肝组织微片移植治疗大鼠急性肝功能衰竭的实验研究

目的探讨同种异体胎肝组织微片移植对大鼠急性肝功能衰竭的治疗作用,为胎肝微组织移植的临床试验提供动物实验依据.方法选择急性肝功能衰竭SD大鼠40只,随机分为对照组(n=20)、移植组(n=20).孕14 d的SD胎鼠肝组织作为供体;移植组每只受体接受两个胎鼠肝脏组织微片移植,移植部位在受体肝组织中;对照组只行开腹手术;手术后24 h、72 h、7 d及30 d分别检测受体ALT、AST、TBIL及ALB的变化,统计存活大鼠数量.结果与对照组比较,手术后24 h、72 h、7 d移植组ALT、AST及TBIL明显下降,ALB显著升高,死亡率下降.术后30 d两组间上述指标差异无统计学意义.结论同种异体胎肝组织微片移植可以改善急性肝功能衰竭大鼠的肝功能,降低大鼠死亡率.这种作用随术后时间的延长而下降.

双相水凝胶组织固定联合脂肪酶浸泡对小鼠肝组织透明化的影响

目的:探讨双相水凝胶组织固定联合脂肪酶浸泡对小鼠肝组织透明化的影响。方法:取10只昆明小鼠的肝组织,利用双相水凝胶进行固定,切取1mm厚的肝组织10片,将其随机分为十二烷基硫酸钠(SDS)组和SDS+脂肪酶组。SDS组采用SDS清除液进行被动脂质清除9d,SDS+脂肪酶组先采用SDS清除液被动脂质清除5d,随后浸泡于脂肪酶溶液中4d。两组于清除前、清除后第1天至第9天均进行透明灰度值测定,于脂质清除完毕后予以细胞核DAPI染色检测标记深度。结果:在被动脂质清除过程中,第1天至第5天肝组织薄片的透明灰度值逐渐提升,但之后肝组织片清除速度缓慢,在第10天的清除仍然是不透明的;在第7、第9天,两组相对透明灰度值比较,差异有统计学意义(P<0.01);SDS+脂肪酶组DAPI染色深度达115μm,而SDS组DAPI染色深度达60μm,SDS+脂肪酶组DAPI渗透深度是SDS组的1.92倍。结论:双相水凝胶组织固定联合脂肪酶浸泡脂质清除技术可以快速实现小鼠肝组织透明化,为肝组织生物学研究提供技术支持。

Comparison of the liver stiffness measurement by transient elastography with the liver biopsy

AIM: To compare the liver stiffness (LS) measurement by transient elastography (TE) to the liver biopsy (LB)-considered the "gold standard" in the evaluation of patients with chronic hepatitis C. METHODS: During a period of 12 mo, we evaluated 199 consecutive patients with chronic hepatitis due to hepatitis C virus (HCV), in which LB and LS assessments (by means of TE) were performed during the same session. RESULTS: Out of 199 patients, a valid measurement of the LS could not be obtained in 8. The mean value of LS in the cohort of 191 valid measurements was 8.45 ± 4.96 kPa, ranging from 2.3 to 38 kPa. The mean value of LS in patients with signifi cant fi brosis at biopsy (161 patients with F ≥ 2 according to Metavir) was 9.02 ± 5.15 kPa, significantly higher than in patients with no or mild fi brosis (30 patients with F < 2 Metavir): 5.39 ± 1.81 kPa (P < 0.0001). For a cut- off value of 6.8 kPa, the LS had a PPV of 98%, a NPV of 30.1%, a sensitivity of 59.6% and a specificity of 93.3% for the presence of signifi cant fi brosis (at least F2 Metavir), with a diagnostic performance of 77.3% (AUROC 0.773). Using this cut-off value, we reached the best discrimination between absence of fibrosis/ mild fibrosis (F < 2 Metavir) and the presence ofmoderate to severe fi brosis (F ≥ 2 Metavir). CONCLUSION: In patients with chronic hepatitis due to HCV, a cut-off value of 6.8 kPa measured by TE can differentiate between significant fibrosis and absent or mild fi brosis, with a PPV of 98%, a NPV of 30.1%, a sensitivity of 59.6%, a specificity of 93.3%, and a diagnostic performance of 77.3%.

Liver histology in ICU patients dying from sepsis:A clinico-pathological study

AIM:To determine end-stage pathologic changes in the liver of septic patients dying in the intensive care unit. METHODS: Needle liver biopsies obtained immediately after death from 15 consecutive patients with sepsis and no underlying liver disease were subjected to routine histological examination. Liver function tests and clinical monitoring measurements were also recorded. RESULTS: Liver biochemistries were increased in the majority of patients before death. Histology of liver bi- opsy specimens showed portal inflammation in 73.3%, centrilobular necrosis in 80%, lobular inflammation in 66.7%, hepatocellular apoptosis in 66.6% and cholan- gitis/cholangiolitis in 20% of patients. Mixed hepatitic/ cholestatic type of liver injury was observed in 6/15 (40%) patients and hepatitc in 9/15 (60%). Steatosis was ob- served in 11/15 (73.3%) patients affecting 5%-80% of liver parenchyma. Among the histological features, the presence of portal inflammation in liver biopsy was as- sociated with increased hospitalization in the ICU prior death (P = 0.026). CONCLUSION: Features of hepatitis and steatosis arethe main histological findings in the liver in the majority of patients dying from sepsis.

Is liver biopsy mandatory in children with chronic hepatitis C?

Liver biopsy is considered the most accurate means to estimate the necroinflammatory activity and the extent of fibrosis. However, histology evaluation is an invasive procedure associated with risk to the patient, risk of sampling error and diagnostic inconsistencies due to inter- and intra-observer error. On the basis of histological studies performed so far, chronic hepatitis C in children appears morphologically benign in the majority of cases. At the Pediatric Liver Unit of our university, a total of 67 children with chronic hepatitis C underwent liver biopsy. Liver biopsy was repeated 5.5 years after the initial histological evaluation in 21 children. On a total number of 88 liver biopsies, micronodular cirrhosis was detected only in one genotype 1b-infected obese child. Since liver histology investigation of a child with chronic hepatitis C has few chances to highlight severe lesions, we question how liver biopsy helps in the management of children with chronic hepatitis C.

Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

AIM： To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection （LCM）. METHODS： Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS： mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer ceils derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant （2.5-fold, q value 〈 10） change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. CONCLUSION; The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.

Gene Expression Profiles in Porcine Tissues of Liver and Kidney

Microarray technologies are widely used all over the world for the gene expression analysis in various tissues, but still this technology has some limitations. The problem of eliminated reproducibility of the results, obtained in different laboratories using different platforms, is very relevant nowadays. For revelation of problems ofmicroarrays, the comparative analysis of hepatic and renal gene expression profiles （GEP） was carried out by using swine protein annotated oligonucleotide microarrays （SPAM）. Revealed differences in GEP between kidney and liver of pigs were correlated with functional and histological distinctions of these organs. It was shown that sources of errors in the comparative analysis of organ-specific GEP could be connected to the cross hybridization of one probe to transcripts （cDNA of mRNA） of different genes and to individual variability in gene expression between animals, related with the changeability of influences of exo- and endogenous regulation factors.