| Nowadays, to cure the end-stage liver failure caused by liver diseases, orthotopic liver transplantation (OLT) remains the most effective treatment. Because of the shortage of donor, immune rejection, and economic burdens, makes a restriction in application of OLT. The field of stem cell bioengineering can potentially revolutionize cell-based therapies for functional replacement of metabolically complex tissues like that of the liver. A large number of researchers have been exploring a way to supplement than can even replace liver transplantation to treat liver disease. With the development of cytology and other related disciplines,The use of liver stem cells to treat liver disease is possible.The stem cells in the bone marrow which can differentiate into liver cells is called bone marrow derived liver stem cell (BMDLSC). The use of BMDLSC can be a new way to cure liver diseases. The bone marrow derived liver stem cell model was recently demonstrated to be a highly organotypic. Based on its successful realization of specific lineages. It is a thoroughly a new way to cure liver disease with BMDLSC transplantation. With this technology, we can repair the impaired liver which is caused by many factors. It can rebuilt the organizational structure of the damaged liver.Recently, there have been many experts trying to cure the end-stage liver failure with the help of BMDLSC and they have got some success. However, we still face many differculties ahead of use.In the existing literature, most scholars have used the technology route is to the extracted bone marrow directly to hepatic artery or portal vein infusion of transplantation, or with a simple centrifuge of the exacted bone marrow and the selected stem cell is directly infused through hepatic artery or portal vein for transplantation. although the methodology is relatively simple, but the result is not ideal, because the content of BMDLSC in bone marrow cells are very rare, even though a relatively large amount of bone marrow is exacted but the number of BMDLSC for transplant are often insufficient. We use the technology route for the expansion of bone marrow-derived liver stem cells in vitro before transplantation. This technical line in our previous animal studies have been proven safe and effective, not only can it increase the number of BMDLSC for transplantation, but also can reduce the bone marrow transplant BMDLSC requirements, and can improve the BMDLSC liver transplantation therapy.Since 2004, we have been doing research on how to repair the injuried liver with the help of bone marrow derived liver stem cells, and we have found an cffect way to isolate, purify, culture, induce and expand the BMDLSC out of the body. With this technology, we can increase the number of liver stem cells, Also this technology can induce the expression of some receptors, which can help the stem cells live in liver after the transplantation. The clinical application of this study of autologous bone marrow-derived liver stem cells in vitro amplification and it is used in liver transplantation for treatment of cirrhosis is quiet safe and effective.ObjectiveTo evaluate the therapeutic effect of autologous bone marrow derived liver stem cell transplantation after cultivation and expansion in vitro on patients with posthepatitic cirrhosis.Materials And Methods1. Case Selection:Between Jun 2008 and Mar 2009, tweleve patients with posthepatitic cirrhosis and portal hypertensive undergoing portaazygous dsvascularization in Hepatobiliary Surgery of the Nanfang Hospital affiliated by Southern Medical University. Among the 12 patients there are 3 females,9 males, with an average age of 48.8 years (39-60 years old). Patients on admission has a semi-random method were divided into six cases of trail group and control group in 6 cases. All of the patients from had history of hepatitis B, and with from mild abnormal to severe abnormal liver function obviously, positive of hepatitis B surface antigen, being from A to C grade by the Child-Pugh's grading of liver function. Their liver tissues were abnormal remarkably, with increased intrahepatic tissue fibrosis, damaged structure of hepatic lobules and formed "false lobules" and regenerative nodules.2. The bone marrow derived hepatic stem cell is grown in vitro:a week before accepting the portaazygous dsvascularization operation,We got 30-40mL by bone marrow puncture via posterior superior iliac spine bone marrow collected from the patients in the trail group.The fresh bone marrow which is collected from the patient is under gone a density gradient centrifugation to obtain cell mass full of CD117(+) cells and CD184(+) cells.The cell mass is cultured for 7. days in the culture in vivo which is consisted of high glucose DMEM with 10% self blood serum contain hepatocyte growth promoting factors(HGPF), thrombopoietin(TPO) and interleukin-3(IL-3). This jiob is done in the cell culturation room with sterilized environment The quantity changes of CD117(+) cells and CD184(+) cells are measured in flow cytometry. 3. Transplantation methods:In trail group, autologous bone marrow-hematopoietic stem cells were infused via hepatic artery after induction out-of body for 7days, while we infuse the same volume Sodium Chloride, After the operation, we give the same therapy to both of the two groups. Evaluate the therapeutic effect with the contradistinction of hepatic function before and after the operation.4. Observation target and Methods4.1. Clinical symptoms and signs Record if they had any typical symptoms and signs, such as jaundice, liver palms, spider nevus, ascites, hepatic encephalopathy. Then, we appraised the severity of above signs and symptoms.4.2. Imaging examination:The patients accept the CT and B-ultrasound examination before the transplantation and in 3 month after accepting the autologous intrahepatic transplantation of bone marrow derived liver stem cell in vitro.4.3 Preoperative and postoperative 3 months were examined (1) liver function: alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin, serum total bilirubin; (2) Coagulation:prothrombin time; (3) liver fibrosis: Hyaluronic acid (HA), human type III procollagen (HPCIII), In addition, we also recorded the laboratory test results of the patients in trail group in 2 weeks and 1 month after transplantation, but this data is affected by many factors and can only be used as a clinical reference.4.4 Pathological examination of liver tissue samples were observed by optical microscopy, after being fixed in 10% formaldehyde, paraffin-embedded, dehydrated and finally stained by hematoxylin-eosin (HE). This job is done in pathology department of Nanfang hospital.5. Statistical AnalysisThe final data are represented as X±SD, said, using SPSS 13.0 software analysis, testing levels:α=0.05. The general situation in the gender, preoperative peopleⅢ collagen before and after surgery were type III collagen changes in value because they do not obey the normal distribution and the use of non-parametric rank sum test, gender composition using x2 test, before and after surgery of AST, ALT, TBIL, ALB, PT, hyaluronic acid, after the person-Ⅲcollagen and the rest of the difference before and after the targets are subject to the normal distribution and using an independent samples t-test and compaired t test.Results1. All of the patients feel better after accepting the operation and a rank of therapy, and the sign of patients is released more or less.2. All of the patients recovered uneventfully. No side effection of the operation was found. The examination of all patients has no statistical difference before accepting operation.But there is a.statistical difference happen between the two groups,3. After the operation, ALT, AST, ALB, TBIL, PT of the trail's group significantly changed from (39±15) U/L, (64±20) U/L,(42.5±13.2)umol/L,(29.8±5.1)g/L, (15.1±1.7) s to (24±11) U/L, (35±12) U/L,(27.0±9.1)umol/L,(34.2±5.0)g/L, (13.7±1.1) s; these examinations of contol group changed from (38±8) U/L, (55±21) U/L,(31.0±9.9)umol/L,(30.0±2.6)g/L, (15.0±1.2) s to (41±13) U/L, (63±21) U/L,(29.1±9.5)umol/L,(28.2±2.7)g/L, (15.9±2.1) s. There is stantistical difference between trial group and control group.4. HA, HPCIII changed from (336.2±55.4) ng/mL, (218.8±109.4) ng/mL to (290.2±54.3) ng/mL, (186.4±88.5) ng/mL in trail group which changed from (340.5±73.7) ng/mL,(148.1±15.6) ng/mL to (342.9±67.9) ng/mL,(153.2±21.0) ng/mL. There is stantistical difference change of fiber examination of liver between trial group and control group. There is a better chang happened in trail group.5. There is no conspicuous difference of imagining examination of the patients after accepting the operation.[Conclusion]1. Autologousbone marrow-hematopoietic stem cell transplantation via hepatic artery after induction out-of-body for treatment of patient with posthepatitic cirrhosis is safe and effective. antigen characteristics of autologous bone marrow-derived liver stem cells in vitro treatment did not change with the expansion.2. Autologousbone marrow-hematopoietic stem cell transplantation via hepatic artery after induction out-of-body is a good approach for improvement of the liver functions and liver fibrosis indicators significantly.
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