| The stem cells in the bone marrow which can differentiate into liver cells is called bone marrow derived liver stem cell (BMDLSC), which can be a new way and method to cure liver diseases. In mice, the study of BMDLSC transplantation confirmed that BMDLSC had a role of reversing CCL4 or alcohol-induced liver fibrosis. However, why the BMDLSC which settle in the bone marrow in the physiological state can migrate to the liver and play a fixed role after they were transplanted. And there are some questions still remain unclear, such as what are the factors about regulation of the post-transpanted BMDLSC migrate to the liver, and these matter is neither clear.In recent years, Hatch and his co-worker confirmed firstly that stromal cell-derived factor-la (SDF-la) in liver could recruit CXCR4-positive bone marrow-derived liver stem cells to liver and participate in the process of BMDLSC' activation and proliferation and differentiation into hepatocyte-like cells. Our past experiments also confirmed that SDF-la in the liver expression increased significantly when liver occurs fibrous hyperplasia, it suggested that SDF-1 a is likely to be play an important role in the migrating of BMDLSCs to the liver. In vitro experiment, Some papers have reported that monocyte chemoattractant protein-1 (MCP-1, the system named CCL2), regulated upon activation normal T cell expressed and secreted factor (Rantes, the system named CCL5), human monocyte chemoattractant protein-3 (MCP-3, the system named CCL7), Secondary lymphoid tissue chemokine (SLC, the system named CCL21) and human thymus expressed chemokine (TECK, the system named CCL25) can drive bone marrow-derived mesenchymal stem cells to migrate to the tissues which contain the above chemokines of high content, and the chemotaxis capacity to bone marrow-derived mesenchymal stem cells is positively correlated with the content of the above chemokines in tissues. Which suggested that these chemokines in vivo is also possible regulated of the migration of BMDLSC to the liver. However, the contents of chemokines CCL2, CCL5, CCL7, CCL21 and CCL25 in the liver is still unknown.We used the enzyme linked immunosorbent assay (ELISA) to detect the content of intrahepatic chmokines of CCL2, CCL5, CCL7, CCL21 and CCL25 of liver fibrosis and cirrhosis in patients, on the basis of the instructions of human chemokines ELISA kit. The collected liver tissue samples were weighted, then added a certain percentage of the PBS buffer to full homogenate, we assayed the contents of the above chemokines in the supernatant from the homogenate which was centrifugaled in a high-speed condition two times eventually. The results of this study showed that when liver occurs fibrous hyperplasia, the content of the chemokines of CCL2, CCL5, CCL7, CCL21, and CCL25 intrahepatic increased significantly. Comparison of cirrhosis group between liver fibrosis group,the contents of CCL2, CCL5, CCL21, and CCL25 intrahepatic are increased further, but the content of CCL7 intrahepatic reduced.[Materials And Methods]1. Liver tissues They are collected from the patients who accept the liver surgery including liver transplantation in the Hepatobiliary Surgery of the Southern Medical University affiliated Nanfang Hospital. There were 30 adult patients, from 19 to 77 years old, the average 50-year-old. And there were 17 male patients and 13female patients. The 30 cases were divided into 3 groups.①Normal control group:The donors must be healthy with normal liver function, negative of hepatitis B surface antigen and HCV antibody, being A grade by the Child-Pugh's grading of liver function. The most important thing was that their liver tissues were normal.②Liver fibrosis group:All of the patients the samples from had history of hepatitis B, and with normal or abnormal liver function, positive of hepatitis B surface antigen, HCV antibody negative, fibrotic liver structure but no "false lobule" that were confirmed by pathological examination. But their grade of liver function remained at A by the Child-Pugh's grading.③Group of cirrhosis:All of the patients the samples from had history of hepatitis B, and with from mild abnormal to severe abnormal liver function obviously, positive of hepatitis B surface antigen, HCV antibody negative, 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. Observation target and Methods2.1 Clinical symptoms and signs Record if them had some typical symptoms and signs, such as jaundice, liver palms, spider nevus, ascites, hepatic encephalopathy. Then, we appraised the severity of above signs and symptoms.2.2 CT and B-ultrasound examination.2.3 Biochemical and Immunological tests The sick person must be accepted serological testing of Hepatitis B and hepatitis C after admission. And it is necessary to test the value of ALT, AST, ALB, TBILand PT in venous blood of patients in the day before operation.2.4 Pathological examination for tissue Liver were observed by optical microscopy, after fixed in 10% formaldehyde, paraffin-embedded, dehydrated and finally stained by hematoxylin-eosin (HE).2.5 Testing of the chemokines CCL2, CCL5, CCL7, CCL21 and CCL252.5.1 Homogenate and Centrifugalization We grinded the liver tissue to homogenate, and then diluted the homogenate with a certain proportion of PBS buffer. The homogenate was centrifugal at 4℃,1000-g and 10 minutes, then the other homogenate was centrifugal at 4℃,25000rpm and 20 minutes. Supernatant was obtained to test by ELISA.2.5.2 To test the content of CCL2, CCL5, CCL7, CCL21 and CCL25. The testing was operated as the instructions of ELISA kit.We used the way of ABC-ELISA with two antibodies. The CCL2, CCL5, CCL7, CCL21 and CCL25 molecules were binding with monoclonal antibody coated on wells anticipatorily, and then binding with biotinylated the above chemokines' antibodies separately. Finally, it showed a color after adding the solution of substrates A and B that could absorb the light of 450 nm wavelength which called absorbance or OD value. There was a proportion in corresponding chemokines contents and OD values. According the proportion, we could compute the unknown chemokines' contents in sample supernatant after knew the equation of standard curve.3. Statistical AnalysisThe final measurement data are represented by x±S. The significance test of mean between multiple groups was used the One-way ANOVA and LSD-t ways by the statistical analysis software which was SPSS 13.0. But we used the Welch's approximate ANOVA and Dunnett's T3 to test the datas when the equal variances were not assumed. We adopted Pearson Correlation to analyze the relevance between the density of CCL2, CCL5, CCL7, CCL21 and CCL25 in liver and liver function and used Curve Estimation to compute standard curve equation. We usedχ2test to test the datas in R·C tables. The a's value is equal to 0.05 in all statistical analysis proceedings.[Results]1. The contents of CCL2, CCL5, CCL7, CCL21, and CCL25 intrahepatic in three group.1.1 The content of CCL2 was (0.46±0.34) pg/mg in the control group, (19.30±4.26) pg/mg in fibrosis group and (81.23±11.47) pg/mg in cirrhosis group. The level of CCL2 expression in cirrhosis group was remarkably higher than that in the control group (P=0.000) and fibrosis group (P=0.000), and the level of CCL2 in fibrosis group was remarikably higher than that in the control group (P=0.000).1.2 The content of CCL5 was (0.30±0.37) pg/mg in the control group, (0.88±0.52) pg/mg in fibrosis group and (3.75±1.45) pg/mg in cirrhosis group. The level of CCL5 expression in cirrhosis group was remarkably higher than that in the control group (P=0.000) and fibrosis group (P=0.000), and the level of CCL5 in fibrosis group was remarikably higher than that in normal liver (P=0.200).1.3 The content of CCL7 was (2.03±1.14) pg/mg in the control group, (5.09±1.51)pg/mg in fibrosis group and (4.57±1.42)pg/mg in cirrhosis group. The level of CCL7 expression in the control group was remarkably lower than that in the fibrosis group (P=0.000) and cirrhosis group (P=0.000), but there was no significant difference between that in the fibrosis group and cirrhosis group (P=0.390)。1.4 The content of CCL21 was (0.14±0.21) pg/mg in the control group, (0.92±0.69) pg/mg in fibrosis group and (3.22±1.45) pg/mg in cirrhosis group. The level of CCL21 expression in cirrhosis group was remarkably higher than that in the control group (P=0.000) and fibrosis group (P=0.000), but there was no significant difference between that in the fibrosis group and the control group (P=0.092).1.5 The content of CCL25 was (4.06±2.23) pg/mg in the control group, (14.46±4.10) pg/mg in fibrosis group and (21.97±2.98) pg/mg in cirrhosis group. The level of CCL25 expression in cirrhosis group was remarkably higher than that in the control group (P=0.000) and fibrosis group (P=0.000), and the level of CCL25 in the fibrosis group was remarkably higher than that in the control group (P=0.000).2. Correlation analysis chemokines of CCL2, CCL5, CCL7, CCL21, and CCL25 levels and values of liver function2.1 The correlation analysis between CCL2, CCL5, CCL21, and CCL25 and ALT, AST was statistically significant (P<0.05), the correlation analysis between CCL2, CCL5,CCL21and CCL25 and PT was also statistically significant, but the correlation coefficients were relatively small.2.2 The correlation analysis between CCL7 and AST was statistically significant, but the correlation coefficient is small; and there has no significant correlation with other values of liver function.3. The correlation analysis between CCL2, CCL5, CCL7, CCL21 and CCL25 separately showed that, the correlation coefficient between CCL2 and CCL5, CCL21, CCL25 were more than 0.812 (P<0.01). The correlation coefficient between CCL5 and CCL21, CCL25 were 0.584 (P<0.01),0.788 (P<0.01), separately. The correlation coefficient between CCL21 and CCL25 was 0.728 (P<0.01). But the correlation coefficient between CCL7 and other chemokines had no statistical significance except it with the CCL25 (r=0.550).[Conclusion]1. The results of this study observed that the liver not only expressed the chemokines of CCL2, CCL5, CCL7, CCL21 and CCL25, when the liver occurs fibrous hyperplasia, the expression of these five kinds chemokines have a significant increase. As the severity of liver injury, intrahepatic chemokines'levels increased gradually, when the liver occur fibrosis, the levels of CCL7 reached a peak. Then the cirrhosis of the liver occurs, the levels of CCL2, CCL5, CCL21 and CCL25 intrahepatic peaked. However, the significance of these chemokines expressed increasing when the liver occur fibrous hyperplasia and the role of these five kinds chemokines participated in migrating to the liver should be further explored.2. The correlation analysis between the contents of CCL2, CCL5, CCL21 and CCL25 intrahepatic and the liver function showed that:the contents of chemokines CCL2, CCL5, CCL21 and CCL25 expression in the liver was positively correlated with the ALT and AST in the serum. Which suggested that these four chemokines' expression levels in the liver can be used as indicators to judge whether the liver was damaged. When liver cells are damaged, these four kinds of chemokines expressed up-regulated. Although the levels of CCL7 in the liver were also positively correlated with the ALT and AST, the correlation coefficient is only 0.337 and 0.380.3. The correlation analysis of CCL2, CCL5, CCL7, CCL21 and CCL25 showed that the correlation coefficients between CCL2, CCL5, CCL21 and CCL25 were above 0.728 significantly, it suggested that these four kinds of chemokines' expression mechanism may has similarities. The levels of CCL7 only correlated with CCL25, and the correlation coefficient is 0.550, and there is no statistically significant of the correlation coefficients between CCL7 and the CCL2, CCL5, CCL21, it may suggested that the expression mechanism of CCL7 is different from the other four kinds of chemokines.
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