| Vital hepatitis is a common disease in our country, and gradually become a serious threat to human health, especially the viral hepatitis caused by hepatitis B virus (HBV).There are about300million patients with chronic viral hepatitis or viral carrier in the world, but in our country the number are almost attached to120million tThe virus repetitive activities caused by a variety of reasons, can destroy the liver parenchymal cells and furtherly to cause liver cell degeneration, necrosis, and regeneration with fibrous tissue and nodular liver cells, thus further accelerate the development of cirrhosis or liver cancer, which has been the main threat to the life of patients with viral hepatitis. Orthotopic liver transplantation (OLT) is the most effective treatment for the patients with various liver function decompensatation or liver failure.But because of the lack of liver source, expensive cost, and postoperation complications confine the widely application of OLT.In recent years, many new interventive therapies constantly emerged. Stem cell transplantation, extremely use of the exogenous stem cells’high proliferative activity and differentiation capacity to achieve the purpose of the damaged tissue or cell repair and regeneration. Adult stem cells from the mesenchymal source is known as mesenchymal stem cells(MSC), the concept was initially raised by Friedenstein and his colleagues in the1970s when they cultured bone marrow cells, one kind of spindle-like cells with morphology similar to fibroblast,which had the adherent growth and colony forming ability and could differentiate into bone cells, chondrocytes and stromal cells both in vivo and vitro. It was confirmed that the mesenchymal stem cells existed in various tissues of the body, including the umbilical cord, umbilical cord blood, bone marrow, fat, peripheral blood and skeletal muscle tissue. Bone marrow mesenchymal stem cells (BMSCs) which derived from bone was the most abundant and most widely used in all types of mesenchymal stem cells. A lot of studies in vitro confirmed that bone marrow mesenchymal stem cells have a high self-renewal capacity and strong potential of multi-germ layer differentiation in the induction of specific factors, which can be differentiated into bone cells, cartilage cells, skeletal muscle cells, adipocytes, endothelial cells, myocardial cells, astrocytes, neurons and liver cells.A large number of animal experiments and clinical studies have shown that the available effects of bone marrow-derived mesenchymal stem cells (BMSCs) transplantation in treating liver diseases could be listed as follows:correcting the congenital hepatic liver metabolic defect diseases, to varying degrees improving the symptoms of hepatic encephalopathy, temporarily supporting the liver function by substituting part of the the necrotic or missing cells, raising the level of serum albumin and reducing serum transaminase, ammonia and total bilirubin levels. With the development of stem cell research, BMSCs transplantation in the near future will become a conventional method of clinical treatment on liver diseases.Objective1. To explore a optimal isolation,purification and cytopreservation method of SD-rat bone marrow-derived mesenchymal stem cells (BMSCs), and to observe the tumorigenicity through implanting the BMSCs cultured in vitro into nude mices for providing an experimental basis of the BMSCs as a ideal cell type for tissue engineering and clinical rehabilitation.2. To investigate the effects of GFP gene transgenic labeled bone marrow mesenchymal stem cells transplantation on the development of fibrogenesis in rats through different injected ways and to observe the distribution of transplanted BMSCs to provide a theoretical basis for clinical applications.Methods1.6clean-grade male SD rats in1moth old were purchased. The BMSCs were obtained by flushing the bone marrow cavity of rats with low-sugar DMEM medium containing10%fetal bovine serum. Corresponding treatments were given after these cells were fully mixed. These mixed cells were divided into three groups:group A: the adherent cells were abandoned in1.5h and the supernatant along with the cell suspension were transferred to another flask culture and these cells was exchanged24hours later; group B was all exchanged after24hours for the first time; group C was exchanged after48hours for the first time. Cell adhesion and morphological changes were observed under the microscope.2. when P3cells grew to80%to85%confluence, these cells were digested and mixed with cytoprotectant solution separately containing10%.20%,30%,40%.50%serum, after gently winded and blended their gradient were adjusted to a density of1x106/mL and then were placed in-80℃refrigerator for a short-term preservation. Four weeks later all preserved cell were recovered by fast rewarming method. These cells were divided into three groups after gently mixed:group A cells were stained by trypan blue to calculate the rate of cell survival; Group B cells were seeded into96well plate and three holes of each serum concentration were digested and in which these living cells were counted from day1th to day8th to draw the cell growth curve; group C cells were cultured with adipogenic induction medium and then were stained by oil red O at D21to observe the adipogenic capacity of these recovery cells.3.6SPF-grade male mice in1moth old were purchased. The rat bone marrow mesenchymal stem cells of3th and15th generation were respectively identified by the immunohistochemical staining and adipogenic identification. In the experimental group about1×106BMSCs diluted with20uL saline of the two generation were separately injected into the unit four biceps or liver of nude mice. While20uL physiological saline was injected to corresponding parts in the control group. The survival status of nude mice along with injection site with or without new biological formation were observed, and45days later pathological tissue section from the injection site was stained by HE to observe whether there were heterotypic cells growing.4.15clean-grade SD male rats with a weight about300±50g were purchased. Phenobarbital sodium was mixed with pure water at a dose of350mg/L for rats daily drinking. After7days0.125ml carbon tetrachloride-olive oil mixture (2:3) per100g were given to each rat2times per week by gavarage for8weeks. Eight weeks later the surviving cirrhotic rats were received liver function tests. According to the test results, the successful modeled cirrhotic rats were randomly divided into the tail vein infusion group, liver local injection group, hepatic arterial infusion group, the hepatic portal vein infusion group and the saline injection group.5.2clean-grade male SD rats in1moth old were purchased. The BMSCs were obtained by flushing from the bone marrow cavity of rats and cultured with low-sugar DMEM medium containing10%fetal bovine serum. The third generation of bone marrow mesenchymal stem cells were divided into two parts, one part of these cells were used for CD34,CD44immunohistochemical staining and adipogenic identification, another part was infected with lentiviral vectors carrying the EGFP gene. 6. These GFP-marked BMSCs were transplanted into the rats with liver cirrhosis through the tail vein, local liver, hepatic arterial and the hepatic portal vein pathway. Liver injury maintained for1month by carbon tetrachloride after transplantation Four weeks later these rats were killed to finish the liver fuction test containing serum albumin, alanine aminotransferase and aspartate aminotransferase; Liver tissue was taken to make section to stain with HE and Sirius red-picric acid to observe the degree of liver cirrhosis; Liver tissue sections were made to observe fluorescence distribution under the fluorescence microscopy.Results1. A large number of bone marrow mesenchymal stem cells adherent can be seen, mostly with a shape of short spindle and irregular triangle and a oval, large nuclear located in the central of the cell and some with a visible dual-core in the group A. Despite visible short spindle cells, other large round or long spindle cells adherent also could be seen in the group B and group C, which had a more complexed composition shape compared to group A. Bone marrow mesenchymal stem cell obtained from group A could have highest activity and purity.2. Cryopreservation and recovery have a certain impact on survival and proliferation of bone marrow mesenchymal stem cells. The cell growth incubation period had been extended, this effect has become even more obvious with the decreasing of serum concentrations; high inoculation density is conducive to the growth and proliferation of cell recovery, inoculation density of30%serum group was too low and the cell growth rate is slower than other groups, but as cell proliferation attained to a certain number, the cell proliferation became significantly faster;3. BMSCs passaged to15generations maintained the characteristics of mesenchymal stem cells. BMSCs transplanted into the liver of nude mice can survive in the local liver site for45days, with a similar growth state as in vitro, without atypia and infiltrative growth, suggesting that BMSCs after long term cultured in vitro,when transplanted into nude mice, can survive in the local infusion site and with no tumorigenicity.4. Round adherent cells can been observed at4h after the bone marrow mesenchymal stem cells obtained from the bone. These cells were all exchanged after48h to remove unattached cells, after that scattered adherent cells were seen with a relatively uniform appearance as a shape of short spindle or star. With the build-up culture days, some colonies gradually emerged in a radial array. After cultured for7-12days, these cells proliferate exuberantly and the colonies gradually integrated and tightly packed into a spiral shape. BMSCs of P3-generation expressed CD34negative, CD44positive by imunohistochemical staining and oil red0staining positive after adipogenic inducation, which were the biological characteristics of bone marrow mesenchymal stem cells.5. Liver function tests suggest that the Alb level of rats with chronic liver fibrosis induced by phenobarbital combined with carbon tetrachloride for eight weeks (before transplantation) was lower than that of normal rats, meanwhile the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were higher.The Alb level of all groups gradually decreased after transplantation and the level of ALT and AST continued to decline along with the persistent liver damage and the magnitude declined of each group was considerable.6. Liver tissue biopsy stained by HE of the rats after bone marrow mesenchymal stem cell transplantation through different infusion ways showed that normal lobular architecture was destroyed, the liver cells arranged in disorder, a large number of liver cells changed into vacuolar degenaration, there was a wide range of collagen fibers proliferated in the hepatic portal area and strentched into parenchyma to form pseudolobule. Picrosirius-picric acid staining showed that a large number of red-stained collagen fiber sedimented in the liver parenchyma and hepatic portal vein area which could be dyed red and formed the pseudolobule both in the bone marrow mesenchymal stem cells transplantation group and the control saline injection group.7. The bone marrow mesenchymal stem cells after GFP-labeled were similar with the bone marrow mesenchymal stem cells of P3generation. The green fluorescence of the cells gradually diminished into a yellow-green and even yellow color along with the extension of the culture time in vitro. Liver tissue biopsies after transplantation were observed under the immunofluorescence microscopy, these results showed that a lot of expression of yellow fluorescent signal cells distributed in the fiber interval of the portal area in the tail vein transplant group, and other yellow fluorescent proteins in the local injection group, the hepatic artery group and the hepatic port vain group were expressed in the local injection site, around the hepatic artery and hepatic port vain respectively after the four weeks’transplatation, and were consistent with the distribution of the collagen fiber.Conclusion1. Our laboratory was the first one to use1.5h differential adherent binding24h first exchange method, by which BMSCs with a uniform shape, rapid proliferation rate, stable trait could be obtained in the original generation, with advantages of simple, convenient and practical operation and smaller damage, that provided an experimental base could be used as seed cells of tissue engineering.2. Bone marrow mesenchymal stem cells cryopreserved with freezing medium containing30%concentration of serum, can guarantee to maximize their activity and proliferation, but also ensure the genetic characteristics and differentiation ability.3. BMSCs within15generations cultured in vitro transplanted into nude mice could be survived and localized to the liver of injection site and with no tumor formation, but with the increase in the number of cultured in vitro has increased risk of tumor formation.4. Cells expressed fluorescent signal were observed in the different distribution of hepatic lobule under the immunofluorescence microscopy, considered possibly related to cell transplantation pathways, but were all distributed in the liver lobule periportal area or lobular perivascular site of more collagen deposition.5. There were no significant improvements in the liver function test and fibrosis degree of those rats of liver fibrosis through BMSCs transplantation with different inject ways. BMSCs distributed in different site of liver along with the different ways of transplanted BMSCs and may be involved in the formation of liver fibrosis.6. Transplanted BMSCs via the hepatic artery had become a common treatment in clinical, which could be conducted as hepatic arteriography,differential diagnosis, zoning embolism chemotherapy and so on, that could be used as the preferred method for cells transplantation in vivo in animal experiments. |
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