Directional Differentiation Of Bone Marrow Mesenchyme Stem Cell Into Hepatocytes And Therapy Of Hepatic Fibrosis By Transplanting Hepatocyte-like Cells Through Portal Vein

Objects The research of stem cells which are of self-renewing, intrinsic versatility and transdifferentiation provides a novel method to cure various diseases. There are two types of stem cells in bone marrow,one is hematopoiesis stem cells（HSCs）,the other is mesenchymal stem cells（MSCs）.MSCs derive from the mesoblast, and obtain strong potentiality to self-renew and differentiate. Under proper conditions ,they can transdifferentiate into multilineage cells, such as osteoblasts, neurocytes, hepatocytes. MSCs have shown two advantages:firstly, it is convenient to acquire MSCs from human bodies and the procedure have been manifested harmless; Secondly, autologous mesenchymal stem cells will not be inclined to initiat immunologic rejection.Therefore,up to now, scientists have been attracted to study MSCs, which is focal issues in medical scientific research. Achievements of stem cells in biomedical fields offer new ideas and means to heal liver diseases.The fact that MSCs can differentiate to hepatocyte-like cells in vivo and vitro and show hepatocytes’ functions such as synthesizing and secreting, have been demonstrated by domestic and overseas scientists.In order to illuminate the mechanisms that MSCs transversal differentiate to hepatocytes and hepatocytes’ directional differentiateing in vivo which derived from MSCs,we transplanted these hepatocytes into homogeneitic rats’ livers which had fibrous degenerated,and evaluated the distribution and differentiation of the hepatocytes,also we assessed the therapeutic efficacy to hepatic fibrosis. Methods l.Myeloid cells were collected from the healthy male SD rats’ limbs bones, 3 to 4 weeks old, under the germfree conditions.MSCs were separated and purified with red cells clearage buffer,then were adherently culture.MSCs which passaged four times were detected surface antigen CD44,CD45 and CD90 by immunofluorescence staining.MSCs were induced with HGF,aFGF and OSM, then differentiated into hepatocyte-like cells.The changes, such as form, size,the number of nuclear and karyoplasmic ratio of the induced cells,were observed under inverted phase contrast microscope. Expressions of albumin（ALB）, alpha-fetoprotein （AFP） and cytokeratin 18（CK18） were detected in cells on the 0, 7th, 14th, 21st day though RT-PCR.2.SD male rats were divided into four subgrous:normal control group, model constructed group with CCL₄,idio-retroconversion group; anti-retroconversion group. Peritoneal injection with CCL₄ dissolved in olive oil （v/v=1:1） was applied to model constructed group, and the dose is 0.2 ml/100g rat’s weight on the fist week,after that, 0.1ml/100g twice each week until the eighth week.During this peorid,some were executed to obtain their livers and blood preparation on the 4th,6th,8th week.Normal control group were injected with olive oil only.Idio-retroconversion group were injected same as above,but stopped injiecting on the 6thweek and were executed to gain the materials on the 8th, 10th week.Anti-retroconversion group were also injected same as above,but were injiected with CCL₄ dissolved in olive oil （v/y=1:1） from the 7th week ,however the doses were 0.08ml,0.06ml,0.04ml and 0.02ml /100g rat’s weight once a week,and were put to death to acquire the materials on the 8th ,10th week. 3.SD male rats whose livers had fibrous degenerated were divided into two subgrous: transplanting control group, hepatocyte-like cells transplanted group.Six weeks before hepatocyte-like cells transplanted, SD male rats were induced to hepatic fibrosis models same as model constructed group,later same as anti-retroconversion group with 0.06ml CCL₄ group to retain fibrous degeneration.Control group was intravenous infused with physiological saline into portal vein, while transplanted group with induced cells labelled with DAPI.After cells were transplanted,rats were put to death to obtain their livers and blood preparation respectively on 1st,2nd ,3rd ,4th week. 4.We evaluatd the degree and semiquantitative statistical analysis of the hepatic fibrosis though HE dyed and Masson triple stained.The ultramicrostructures were observed under electron microscope. Frozen sections were made to detect transformation of livers which had been transplanted cells; the parameters of liver functional（ALT, AST, ALB） and hepatic fibrosis（HA, LN） were detected by the automatic biochemistry analyzer and radio-immunifaction method.5.Statistical analysis: Measurement data of variance analysis and enumeration data of ridit analysis test by the check level ofα=0.05 and the software SPSS 13.0.Results 1.When passaged four times,MSCs became fusiform shape,and had identical shape, uniform distribution, and arranged indistinct directions so that they looked like whirlpool or parallelism. The result of immunofluorescence stainting show that positive expression emerged on CD44 and CD90, and negative expression on CD45. After inducted for 21 days ,some allotypic cells became hepatocyte-like cells with the round shape or oval shape,having agminated tendency.RT-PCR demonstrated AFP positive expression emerged on the7th day and ALB and CK18 mRNA positive expression emerged on the 14th day.2.Model constructed for six weeks,we find out hepatic sinusoids expanded and congested, hepatocytes fatty degenerated,the interval of fabric tissues on portal area widen, some hepatic lobules disorganizated, inclining to form false-lobulars.The ultrastructures of hepatocytes obviously transformed. The number of organoids decreased, crista mitochondriales showed marrow-like alteration with swelling and vacuoles and lipid droplet, fibers hyperplased between hepatocytes and hepatic sinusoids but disposed regularly.Contrasted to normal group, there are statistical significances among the two groups’ fibrosis Staging and semiquantitative scoring （P<0.05） .The levels of ALT,AST,HA and LN increased obviously. Hepatic tissue would gradually recover to normal.Contrasted to normal group, each parameter of anti-retroconversing with 0.06ml CCL₄ group, which values hepatic fibrosis, showed no statistical significances （P>0.05） .3.Experimental results in vivo show that transplanted cells from the blood vessels gradually accessed into the liver, and eventually distributed between the liver cells scatteredly. But after three weeks of transplantation, fluorescence obviously decayed. In transplantation group, on the two week, liver false lobular gradually absorbed or dissipated, and fibrosis significantly lighterd than the control group, with collagen staining shallowing and distributing in the fiber interval and portal area,and liver cells presented Balloon-like degeneration.On the 4th week, liver cells arranged in normal.We could see point necrosises occasionally without fiber interval. Ultrastructure of liver cells gradually returned to normal. The differences were not significant on ALT, AST, ALB, HA, LN compared with the normal group （P>0.05） .Conclusions 1.The method that cells in the washing liquid are adherent cultured after red cells are split with RBC lysate is used to separate and purify MSCs, MSCs retains preferable activity and highly purified.2.Under certain microenvironment, HGF+aFGF+OSM, can induce and differentiate MSCs to hepatic cells expressing AFP, ALB, CK18.3.Liver fibrosis model in rats could be established by 6 weeks of twice weekly intraperitoneal injection with 50% CCl₄.But spontaneous recovery happens when stopping injection .At the 4th week after stopping injection, hepatic tissue is normal on the whole.4.Liver fibrosis model in rats could be retained for 4weeks by 6 weeks intraperitoneal injection with 50% CCl₄ firstly, then subcutaneous injection once a week. 5. Induced hepatocyte-like cells could engraft into fibrotic liver after transfusion as early as the 7th day and localized in collection tube region and liver sinus. 6. The engrafted hepatocyte-like cells could substitute liver function partly and step down the fibrosis process.