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Objective To optimize conditions for hypothermic preservation of rat hepatocyte spheroids without freezing in order to facilitate the application of biological artificial liver. Methods Rat hepatic cells were isolated by a two-step perfusion method, and hepatocyte spheroids formed after 48 hours of rocking culture in serum free medium (SFM). Spheroids were then maintained in rocking culture at 37℃ (control condition), or cold stored at 4℃ for 24 or 48 hours in four different cold storage solutions: SFM alone; SFM+1mmol/L deferoxamine (Def ); SFM+1μmol/L cyclosporin A (CsA); and SFM+1mmol/L Def+1μmol/L CsA. After culturing for another 4 or 5 days, survival rate, changes in ultrastructure, and the production of albumin and urea were observed. Results Cold-induced injury could be reduced significantly by the addition of the iron chelators Def and CsA. The function and structure of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def for 24 hours were similar to those in control conditions. But the function was significantly reduced after hypothermic preservation in SFM alone. After cold storage for 48 hours, the ultrastructure of hepatocyte spheroids obviously changed and the number of dead cells increased. The survival rate of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def was significantly higher than that stored in SFM or SFM+CsA(P0.05). Conclusions Hepatocyte spheroids tolerate 24 hours of cold storage with stable viability and function. Hypothermic preservation increases the availability of cell-based therapy for liver diseases. DOI: 10.11855/j.issn.0577-7402.2014.12.02

Liver failure is a life-threatened serious disease with many complications and high mortality rate. Stem cells have been applied to replacement therapy, gene therapy and tissue engineering for its capacity of self-renewal and multi-lineage differentiation. To investigate the bioactivity of the peripheral blood hematopoietic stem cells (PBHSC) in patients with acute-on-chronic liver failure, we isolated CD34+ cells from peripheral blood of patients with acute-on-chronic liver failure and healthy controls. After cultured it in serum-free medium (SFEM), we studied the bioactivity of CD34+ cells by observing the morphology, recording growth curve, detecting cell cycle and cell apoptosis. CD34+ cells and culture solution were collected at the time points of 3, 5, 7, 10, 12 and 14 days, and the levels of hepatocyte growth factor (HGF), matrix metalloproteinase-9 (MMP-9), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in culture solution were detected by ELISA. Also, the expressions of pyruvate kinase muscle isoenzyme 2 (PKM2), integrin-β1 and liver-type pyruvate kinase (LPK) were detected by RT-PCR and immunofluorescence. Our results showed the bioactivity of CD34+ cells from patients with acute-on-chronic liver failure was identified to be similar with that from healthy controls. HGF, MMP-9, TNF-α and IL-6 were found in cell culture medium. RT-PCR and immunofluorescence results indicated that PKM2, Integrin-β1 expressed on CD34+ cells from patients with acute-on-chronic liver failure. In conclusion, bioactivity of CD34+ cells of patients with acute-on-chronic liver failure was demonstrated to be normal, which could secrete HGF, MMP-9, TNF-α and IL-6, promote the growth of hepatocytes, and differentiate along a direction to hepatocyte lineage.

Objective To study the clinical characteristics of elderly patients with liver failure. Methods The clinical data of 623 elderly patients with liver failure from Jan. 2003 to Dec. 2013 in 302 Hospital of Chinese PLA were collected, and other 3791 young adult patients with liver failure during the same period were also collected as control group. The etiology and prognosis of elderly patients with liver failure were analyzed retrospectively. Results Elderly patients with liver failure accounted for 13.21% of the total number of liver failure patients in 2003, and 16.01% in 2013. The most frequent causes leading to liver failure were, in order of frequency, hepatitis B(68.06%), unknown causes (7.22%), hepatitis C (5.30%), hepatitis E (4.98%) and drug (4.65%). Acute or chronic liver failure (85.23% vs 90.56%) was the main type in the two groups. HEV infection-induced liver failure in elderly patients was the predominant cause of acute and subacute liver failure, accounting for 33.33% and 33.80% respectively in acute and subacute liver failure patients, which were significantly higher than that in the young adult group (7.77% and 12.94% respectively, P

To study the clinical features of hepatitis E virus-related liver failure.134 patients with HEV-related liver failure were analyzed retrospectively.HEV-related liver failure accounted for 8.45 percent of the total number of hepatitis E patients in the hospital. Of the 134 patients, 68 were infected with simple HEV, 66 had the superinfection with HBV. The average age of simple HEV-related liver failure patients (56.12 +/- 14.29) was higher than that of HBV superinfectiong liver failure patients (P0.05). The ratio of elderly patients (or = 60 years) in simple HEV-related liver failure patients (45.59%) was significantly higher than that of the other group (13.64%, P0.05). The ratio of direct bilirubin/total bilirubing (0.72 +/- 0.07, 0.69 +/- 0.08), and serum alanine aminotransferase [(1250.90 +/- 1593.97) U/L, (616.26 +/- 797.62) U/L] were significantly higher in simple HEV-related liver failure patients than in HBV superinfectiong liver failure patients (P0.05), but the total bilirubing had no significant difference (P0.05). The disease outcome and stage were no-significant difference in the two groups (P0.05). CONCLUSION; Simple HEV-related liver failure patients may have older age, higher aminotransferase, higher ratio of direct bilirubin/total bilirubin, but disease outcome and stage were no-significant difference in the two groups.

To study the clinical characteristics of hepatitis B virus-related acute-on-chronic liver failure patients with familial aggregation.275 patients with hepatitis B virus--related acute-on-chronic liver failure were investigated. The patients were divided into familial aggregation and non-familial aggregation group basis on their epidemiological features. Clinical data and biochemical indicators between the two groups were analyzed statistically.93 of 275 patients (33.82%) case were family aggregation. There was no significant difference compared with chronic hepatitis B patients (38.3%). The mean age of the two groups was 45.98 and 43.61 years old, respectively (P0.05). The rates of liver cirrhosis in family aggregation group were significant higher than non-familial aggregation group (73.91% vs 58.24%, p0.05). Serum total (TBil) and prothrombin activities (PTA) were no significant difference between the two groups, but ALT level in familial aggregation group was much higher (407.80 U/L vs 256.45 U/L, P 0.05).Familial aggregation were not related to acute-on-chronic liver failure in chronic HBV hepatitis patients. But the rate of liver cirrhosis were higher in patients with familial aggregation.

To construct an hybrid bioartificial liver supporting system, and observe its effectiveness and safety on patients with acute on chronic liver failure.Hybrid bioartificial liver supporting system (HBALSS) was constructed using bioreactor with HepG2 cells transfected with human augmenter of liver regeneration (hALR) gene. 12 acute on chronic liver failure patients were divided into 2 groups randomly. The treatment group was treated with the hybrid bioartificial liver support system. The group underwent plasma exchange was used as control.In the treatment group, four patients recovered, one patient died of hepatic encephalopathy, one patient died of hepatorenal syndrome, one patient recovered, but died of gastrointestnal bleeding after 1 year. In control group, two patients recovered, one patient underwent orthotropic liver transplantation, and three patients died of liver failure.The hybrid bioartificial liver supporting system with HepG2 cell line was established successfully and have certain safety and effectiveness on acute on chronic liver failure patients.

To construct an off-line hybrid bioartificial liver supporting system with human liver cell line, and study it's effect on the plasma from patients with liver failure.We established the bioreactor using Psu-2s (Fresenius) cultured with Hep G2 cell transfected with human augmenter of liver regeneration (hALR) gene, then constructed a hybrid bioartificial liver supporting system, at last using the bioartificial liver support system to purify the plasma treated 2 hours with serum bilirubin absorbent, separated from acute on chronic liver failure patients infected by hepatitis B virus.Bioreactor was successful constructed. The cell viability in perigastrum of bioreactor is 85.2% and cell propagated rapidly. Before and after treating with bilirubin absorbent, serum total bilirubin was (176.19 +/- 54.14) micromol/L and (50.1 +/- 16.85) micromol/L respectively (P = 0.0002). While there were no significance difference in the level of albumin, urea and glucose. At the begin and end of treatment with bioartificial liver, serum total bilirubin was (50.10 +/- 16.85) micromol/L and (30.27 +/- 15.02) micromol/L respectively (P = 0.000), the urea and albumin increased, urea has significantly difference, but the change of albumin hasn't.The off-line hybrid bioartificial liver supporting system with human liver cell line were builded successfully and have synthesis and metabolism functions for acute on chronic liver failure patients.

To explore the separation and culture method of adult hepatocytes.The isolated adult hepatocytes were cultivated by RPMI 1640 medium at 37 degrees C in vitro. The characteristics of the growing hepatocytes were observed. Their synthesis of urea was detected. The transformation efficiency and density's change of lidocaine were analyzed.Hepatocytes were successful separated from adult liver. And they were cultivated in common condition and hollow fiber reactor. The functional capacity of hepatocytes was for lidocaine metabolism and urea excretion.The adult hepatocytes have been successful separated from liver. And they can be cultivated in common condition and hollow fiber reactor. And it could provide a great quantity and high activity of hepatocytes for bioartificial liver.

To verify the rate of diagnostic fitting between the clinic and the indentification-aided for diagnosis and differential diagnosis system, for emerging infections diseases (EID) established.314 cases of 49 kinds of contagious diseases diagnosed and another 186 patients with fever who not diagnosed were tested by the system.Preliminary verification was made in 314 cases diagnosed which classified to 49 kinds of contagious diseases of infectious diseases and the results showed that the coincidence rate of clinical diagnosis and first diagnosis of this system was 61.9%; the suggestive rate of first three diagnoses was 78.1%, and that of first five diagnoses was 86.6%. The diagnosis of another 186 patients with fever were diagnosed by the system and the results showed that the coincidence rate of clinical diagnosis and first diagnosis was 59.7%; the suggestive rate of first three diagnoses was 77.9%, and that of first five diagnoses was 85.4%.The system can accurately suggest impossible diagnosis and differential diagnosis, and be useful for our medical work.

To investigate the characteristics of the hepatic pathological and clinical features of patients with hepatitis B virus (HBV) in immune tolerant stage and find the better measure of diagnosing patients chronic infected by HBV in immune tolerant phase.135 patients with HBV chronic infection and persistently normal serum alanine aminotransferase (ALT) levels were involved in this study, whose serum HBeAg and HBV DNA were positive. Statistical analysis included the ages, sex, serum levels of HBVDNA, ALT and histological grade. The grades of inflammation and fibrosis were obtained through hepatic biopsy performed on all the patients.Mean age in those patients was 22.61 +/- 8.95 years old. All those patients were divided into two groups according to histological grade: low- histological grade group, Gor = 1 and Sor = 1; and high-histological grade group, Gor = 2, Sor = to 2. Levels of histological grade were low in most of patients (99/135). Patients of low-histological grade had no difference in age, sex and family history of HBsAg carriers. Compared with low-normal ALT (ALT less than 30U/L), those with high-normal ALT (ALTor = 30U/L) had a greater frequencies of high-histological grade. Compared with high HBVDNA (HBVDNAor = 10(7) copies/ml), those with low HBVDNA (HBVDNA less than 10(7) copies/ml) had a greater frequencies of high-histological grade.Levels of histological grade were low in most of patients with HBV chronic infection, serum HBeAg and HBVDNA positive, persistently normal serum ALT levels, but some of them were high-histological grade. It showed those patients were not all in immune tolerant stage of chronic HBV infection. Examination of ALT and HBVDNA are helpful to evaluate hepatic pathological damage for them.

CD4(+)CD25(high) regulatory T cells (Treg) have been shown to play an important role in maintaining peripheral tolerance against self and foreign antigens, and in suppressing T cell immune response. Our aim was to characterize circulating Treg in HBV-infected patients.Treg in peripheral blood from 72 chronic hepatitis B (CHB) patients, 16 acute hepatitis B (AHB) patients and 32 healthy subjects were quantitatively analyzed using flow cytometry. Serum HBV markers were evaluated for each subject. HBV-DNA levels were measured using real-time RT-PCR.CHB patients presented a higher fraction of circulating Treg (3.9% +/- 1.4%) than those in AHB patients (3.1% +/- 0.9%) (P0.05), but were similar to healthy controls (3.5% +/- 0.7%). CHB patients with greater than 10(7) copies/ml of serum HBV DNA loads had a higher frequency (4.5% +/- 1.9%) of circulating Treg than health controls (P0.01) and the patients with less than 10(7) copies/ml of serum viral loads (3.4% +/- 0.7%). A correlation was found between circulating Treg and HBV DNA level (r = 0.32, P0.01). Furthermore, Treg was more frequent in convalescent phase (6.0% +/- 1.7%) than in early acute phase (3.0% +/- 0.6%).Increased peripheral Treg is found to be associated with HBV replication in chronic hepatitis B. In acute HBV infection, Treg is less frequent in early phase. The related mechanisms is under further investigation.

To investigate the prognostic significance and role of coagulation factor V (CFV) levels in clinical diagnostic criteria for severe hepatitis.The CFV level and prothrombin activity (PTA) were tested by turbidimetry for 129 times in 58 patients with severe hepatitis. Comparative studies and clinical significance of CFV and PTA were analyzed by SPSS and SDAS softwares.1. The levels of CFV and PTA were 15.3%+/-9.7% and 23.5%+/-10.0%, respectively, at the onset of severe hepatitis. 2. The mortality of severe hepatitis gradually increased with the gradual decrease of CFV or PTA during the most severe stage of the illness (P=0.000). 3. The levels of CFV and PTA decreased continually and rapidly in patients who died but gradually increased in survivors. The decrease or increase of PTA preceded that of CFV on the exacerbation or convalescent stage. 4. Hepatic encephalopathy occurred in 14 cases (24.14%). In 10 cases, it occurred in the terminal stage of the illness, far later than the time of the decrease of CFV. 5. The level of CFV was closely related to PTA (the correlation coefficient was 0.812), the level of CFV was almost consistent with that of PTA.1. The level of CFV is an important prognostic indicator in severe hepatitis and is more specific than PTA. 2. Simultaneous determination of CFV and PTA may be helpful in earlier and more accurate diagnosis of severe hepatitis. 3. Possible use of CFV as one of the criteria for liver transplantation in patients with severe hepatitis should be studied.