| In recent years, autologous bone marrow stem cell transplantation has been recognized in the treatment of cirrhosis in patients with good effect, but this treatment method has many limitations, such as the painful acquisition process, insufficient quantity, insufficient differentiation ability, etc. Experiments in vivo and in vitro suggest that umbilical cord blood stem cells and umbilical cord Mesenchymal stem cells are superior to bone marrow stem cells in self-renewal, proliferation and differentiation potential.The purpose of this study is to establish the method of separating the umbilical cord blood stem cells and the method of isolating and culturing umbilical cord mesenchymal stem cells, and then to observe the clinical effect of implantation in patients with decompensate hepatitis cirrhosis, in order to seek a safe and effective method for the treatment of liver cirrhosis after hepatitis.The first step is to separate umbilical cord blood stem cell using collection negative method under sterile conditions, at the same time, the enzyme digestion method is used to separate the umbilical cord mesenchymal stem cells. The morphology of the two types of stem cells is observed and flow cytometry is used for further identification.The second step is to select47cases of patients with decompensate hepatitis cirrhosis, on the basis of anti-viral therapy, Umbilical cord blood stem cell treatment group includes27cases, the treatment for whom is to implant umbilical cord blood stem cells through the hepatic artery intervention and intravenous pathway. As for the other20cases which form the umbilical cord mesenchymal stem cells treatment group, umbilical cord mesenchymal stem cells are implanted by the same method.The serum markers are detected at different times after implant (two weeks, one month, three months and six months), including biochemical criterion:the level of serum albumin (ALB), total bilirubin (TBIL) level and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), routine blood indicators:platelet (PLT), white blood cell (WBC) and coagulation indicators:prothrombin time (PT), tumor markers: Alpha-fetoproein (AFP). The B-ultrasound results before treatment and3months after are compared, and the improvement of clinical symptoms and signs (fatigue, anorexia, hepatic face, spider angioma etc.) and postoperative adverse reactions are observed.The volume of cord blood collected is about110ml (100-130ml), by negative collection method, single copies of stem cells are isolated by the average of2.31×10. Observed under inverted microscope, the cells are evenly distributed, of uniform size, round, translucent, with high refraction, and with the cell activity>95%. Flow cytometry instrument tests that CD34+cells account for0.23%, with the motility rate of72.71%.No cell contamination occurs.Mesenchymal stem cells are successfully obtained from the umbilical cord tissue by enzymatic digestion. Cell culture,80-90%cell fusion takes about10to14days. Passages after about3-5days. Observed under the inverted microscope, Primary cells are flat. P1, P2, P3, P4cells are more uniform, with high refraction, membrane surface has a small projection, and the nucleus is located in the center of the cell, circular or elliptical, with abundant cytoplasm, of fibroblast-like cell morphology, arranged in parallel growth or vortex-like growth. It is found in the umbilical cord blood stem cells treatment group that, preoperative and postoperative3months,6months of AST, TBIL, CHE and ALB level, the differences are statistically significant (P<0.05). White blood cell count increase significantly (P<0.05) after three months, in the six month it declines. The platelet count is no significant change, but a gradual improvement. AFP is significantly different (P<0.05) in the third month compared with that of the pre-treatment and in the sixth month it declines. Abdominal ultrasound is examination about three months after the implantation of the cells. The portal vein diameter is reduces in13cases, in12cases it increases. It remains unchanged in the other two cases. The spleen thickness in16cases decreases, and it increases in11cases. In the process of treatment cases there are no organ failure and acute or chronic GVHD. There are four fever cases and three patients suffer from Rash. Two weeks later, the appetite and diet of the patients improve significantly. Bloating, and fatigue become weaker, the decrease of ascites is found in81.48%(22/27) patients implant after six months, appetite, and fatigue improvement rate is88.89%(24/27), the mitigate rate of liver palms and spider veins is51.85%(14/27).Preoperative and postoperative ALB level of the umbilical cord mesenchymal treatment group has a significant difference (P<0.05) in the first month, continuing to the sixth month. In the third month, and6th month, compared with the preoperative comparison, the differences in AST, TBIL and PT levels are statistically significant (P<0.05). There are no significant changes in White blood cell and platelet counts, but a gradual improvement. compared with the preoperative comparison, there is a significant difference (P<0.05) in AFP levels after one month, three months. In the sixth month, there is no significant difference in the AFP level. Abdominal ultrasound is reviewed about three months after the implantation of the cells, and the portal vein diameter of seven cases reduce, it increases in nine cases, and it remains unchanged in four cases. The spleen thickness in11cases decreases, and it increase in nine cases. In the process of treatment cases there are no organ failure and acute or chronic GVHD. There are8fever cases. Two weeks after the implantation, bloating, fatigue and discomfort gradually improve and the appetite is better than before infusion.6months after implantation, the decrease rate of ascites is found in80%(16/20) patients, bloating, fatigue and discomfort improvement rate is90%(18/20), the mitigate rate of liver palms and spider veins is50%(10/20). Through the effective rate analysis of two kinds of treatments in clinical symptoms and signs, of abdominal B ultrasound results (portal vein diameter, ascites, and spleen thickness), there are no significant differents (P>0.05) between the two treatment methods.In short, the separation of the umbilical cord blood stem cells and umbilical cord Mesenchymal stem cell isolation and culture methods are safe, efficient and stable. The patient’s liver function recovery effect is significant and no significant difference between them. The separation and culture process of umbilical cord Mesenchymal stem cells are tedious, easy to be pollutioned, difficult, and the cost is higher, but the cord blood stem cells have been clinically used for a long time, the separation method is simple, highly secure, therefore more suitable for clinical application. |
PDF Full Text Request