Safety evaluation and quality control of MSCs from hepatitis B patient in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To get mesenchymal stem cells (MSCs) from hepatitis B patient and to valuate the safety and quality after long-term culture in vitro.</p><p><b>METHODS</b>The cells obtained directly from bone marrow and cultured in Mesen Pro medium supplemented with FGF, and the morphology of MSCs was observed. Surface antigens of the MSCs were analyzed by flow-cytometry. The bacteria, virus, endotoxin and residual serum of cell suspension were detected. The MSCs and perpheral blood T lymphocytes were co-cultured in 48 well plates for 72 h and the T lymphocyte proliferation was measured by using MTT reduction method and the effect of MSCs on T lymphocyte transformation stimulated by PHA was also observed. The oncogenicity of MSCs was verified by the tumorigenesis test in sofo agar. The genetic stability of MSCs was examined by karyotype analysis.</p><p><b>RESULT</b>The MSCs from hepatitis B patient could be passaged to many generations and had strong abilities of proliferation. They expressed stem cell-surface antigens and maintained normal karyotype, prevented the pollution of bacteria and viruses, inhibited the immune response of allogenic T lymphocytes and no oncogenicity found.</p><p><b>CONCLUSION</b>The MSCs have proliferative potentials, can be passaged in long-term cultures in Mesen Pro medium without oncogenicity, can maintain normal karyotype, can inhibit the immune response of T lymphocytes and can alleviate the grafe-versus diseases. The MSCs can be served as a new type of cells in cell and gene therapy.</p>

Optimization of cultivation methods of MSCs from hepatitis B patients and investigation of their biological characteristics / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To optimize cultivation methods of bone marrow mesenchymal stem cells (MSCs) from hepatitis B patients and to investigate their biological characteristics.</p><p><b>METHODS</b>Growth curves of hepatitis B patients MSCs cultivated with five culture media and two inoculation methods were compared; the shapes, appearances, surface markers and bionomics of the cultivated MSCs were studied.</p><p><b>RESULTS</b>Inoculating the cells obtained directly from bone marrow aspirations was not as successful as using the marrow cells after their density gradient centrifugations (76% vs 88%), but the differences in the results were not statistically significant (P more than 0.05). The successful cultivation rates using five culture media were different and the differences were statistically significant (P less than 0.01). The autoserum medium was most successful, fatal bovine serum (FBS) medium was next successful and the non-patient serum medium was the least successful. The growth curves of the cultivations using the different media were parallel to this. Changing the whole culture media every 2 or 3 days was better than changing half of the media. The shapes, appearances, surface markers and the growth characteristics of MSCs from the hepatitis B patients were almost the same as MSCs from the normal adult.</p><p><b>CONCLUSION</b>The best cultivation method of MSCs from hepatitis B patients is: separating marrow cells using density gradient centrifugal separation, cultivating them using an autoserum culture medium, and completely changing the medium every 2-3 days. The biological characteristics of MSCs from the hepatitis B patients using the above methods are almost the same as those from normal adults.</p>

MxA gene-88 G/T polymorphism influences the outcomes of HBV infection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the relationship between a G/T substitution at position -88 of myxovirus resistance-1 gene (MxA) and the self-limiting or chronic infection of HBV.</p><p><b>METHODS</b>Blood samples from 100 patients with self-limiting HBV infection (positive anti-HBs and anti-HBc) and from 340 patients with chronic HBV infection were collected. MxA-88 G/T polymorphism was typed using a protocol based on competitively differentiated-polymerase chain reaction. For statistical analysis, odds ratio and chi-square test were used.</p><p><b>RESULTS</b>The detective rate of G/G genotype (low expression genotype) of MxA-88 G/T was 50.2% (221/440), those of T/T genotype (high expression genotype) and G/T heterozygous genotype were 5.5% (24/440) and 44.3% (195/440). Compared to patients with chronic infection, patients with self-limiting infection had lower frequency of G/G genotype (41.0% vs 52.9%, P < 0.05) or G allele (62.5% vs 75.9%, P < 0.01) and had higher frequency of T/T genotype (16.0% vs 2.4%, P < 0.01) or T allele (37.5% vs 24.1%, P < 0.01), but there was no significant difference in the G/T heterozygous genotype.</p><p><b>CONCLUSIONS</b>MxA gene -88 G/T polymorphism influences the natural outcomes of HBV infection to some extent. This SNP of MxA gene may be used as a clinical prognostic marker of HBV infection.</p>

Initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To probe into the initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>Forty-four CHB patients with positive HBeAg and HBV DNA were treated with interferon. Serum ALT and viral markers levels of HBsAg, HBeAg, anti-HBc and HBV DNA were examined regularly. Liver biopsy was carried out just before the treatment.</p><p><b>RESULTS</b>The rate of HBeAg seroconversion was 75% at the sixth month, and 68.2% after one year of follow up. The rate of damage sensitive stage of hepatocytes was 47.7%. The average onset time was (3.14+-1.49) weeks after the treatment, and lasted for (8.24+-3.52) weeks. The ALT level raised (1.73+-1.13) times. The occurrence of damage sensitive stage of hepatocytes was indicator for good curative effect (Fisher exact probability, P=0.028). Damage sensitive stage of hepatocytes was more often developed in patients with moderate inflammation, overexpression of HBcAg in liver and higher level of HBeAg in blood stream before treatment. HBeAg and anti-HBc levels in peripheral blood decreased in the onset period of damage sensitive stage of hepatocytes.</p><p><b>CONCLUSIONS</b>The initiative factors of the damage sensitive stage of hepatocytes may be: HBeAg decreasing in peripheral blood induced by interferon may dismiss immune lutation of HBeAg and anti-HBc to cytotoxic T lymphocyte (CTL), which recognize HBcAg as target, thus activates the cytotoxicity of HBV-infected hepatocytes mediated by CTL.</p>