| ObjectiveTo prepare alcoholic liver fibrosis rat model according to copying and modifying continuous ethanol feeding method, select acupoints, fascia intensive area on electro-acupuncture(EA) combination with human embryonic mesenchymal stem cells (hEMSCs) transplantation, observe the changes of related molecular biological indicators, compare the influence on the targeted migration trajectory of hEMSCs in alcoholic liver fibrosis rats model by EA at acupoints and fascia intensive area, evaluate comprehensively the efficacy and their differences on alcoholic liver fibrosis model treated by EA at acupoints, fascia intensive area combination with hEMSCs transplantation; to culture hEMSCs in vitro with the serum of alcoholic liver fibrosis model rats and the rats of EA groups, further explore the possible mechanism of hEMSCs differentiation induced by EA, preliminary clarify the promotion and synergistic effect of EA for anti-liver fibrosis treatment by stem cell transplantation, so as to provide experimental and theoretical basis for guiding clinical to carry out acupuncture combined with stem cell transplantation for anti-alcoholic liver fibrosis and the effect differences between acupoints and fascia intensive area.Methods1 According to the weight of rats,102 healthy male SD rats were randomly divided into A group (normal group), B group (model group), C group (hEMSCs transplantation), D group (EA at LR14+hEMSCs transplantation), E group (EA at fascia intensive area in LR14+ hEMSCs transplantation), F group (EA at GB34+hEMSCs transplantation), G group (EA at fascia intensive area in GB34+hEMSCs transplantation), totally 7 groups. The alcoholic liver fibrosis model was copied according to using a modified liquor-lard (homemade)-pyrazole suspension gavaged continuously for 60d;2 The hEMSCs were routinely cultured, amplified and passaged to the 6th generation cells by DMEM (low sugar) medium, which contained 10%fetal bovine serum (FBS). After hEMSCs transfected by Ad5-EGFP recombinant adenovirus vector (MOI=100) for 48 h, the hEMSCs were conventionally digested and then counted; added PBS buffer according to the cell count, and diluted transfected successfully hEMSCs into single cell suspension of 2×106cells/ml; under sterile conditions, the cells were injected through the caudal vein respectively in the rats of C, D, E, F, G five groups,0.5ml/each rat;3 D, E, F, G, four groups (EA groups) were respectively treated on bilateral LR14. fascia intensive area in LR14,GB34,fascia intensive area in GB34 by EA at density wave, frequency of 2/100Hz, intensity of 0.1-0.2 mA, limbs slightly vibrating as the degree, retaining the needles for 20min, every 24h each treatment, totally 10 times. While A, B, and C three groups were respectively bounded with the cloth bags for 20min;4 The general conditions of the rats were observed including weight change, coat color, appetite and activities, etc.; the concentrations of IGF-I, C IV, MMP-2 and TIMP-1 in serum were determined by enzyme-linked immunoassay (ELLSA) method; the expression of green fluorescent were observed on frozen sections of hepatic tissue through laser scanning confocal to explore the targeted migration trajectory of hEMSCs transfected by Ad5-EGFP to the liver of alcoholic liver fibrosis model rats in vivo; the paraffin sections of hepatic tissue were stained by HE and picrosirius to observe pathologic changes of hepatic tissue and collagen deposition; the expression of TGF-β1 and TNF-alpha in hepatic tissue were determined by the immunohistochemical method;5 hEMSCs were cultured for different intervention times with the conditions of the serum from alcoholic liver fibrosis model rat and the rats of EA groups, the gene expression of AFP, ALB, and a-SMA were detected by reverse transcriptase polymerase chain reaction (RT-PCR).Results1 The rats of normal group had luster hair, weight gain, lively posture, normal diet, normal urine and stool. After modeling, the rats in model group had gradually reduced diet, weight loss, dry or unshapen stool with foul smell, yellow urine, rough, raritas and reluster fur, obvious lipsotrichia, depressed, manic irritability, and pugnacity symptoms such as biting mutually. These symptoms of the rats in each EA group were improved in some extent after treatment.2 The changes of IGF-I, C IV, MMP-2 and TIMP-1 concentrations in serum of different group rats. Compared with normal group, model group, the serum concentrations of IGF-1. IV collage and MMP-2 in model group rats significantly increased, the differences were statistically significant (P<0.05, P<0.05, P<0.01), the concentrations of IV collage and MMP-2 in hEMSCs transplantation group rats significantly increased, TIMP-1 significantly decreased, the differences were statistically significant (P<0.01), the TIMP-1 serum concentrations in rats of EA at fascia intensive area in GB34+hEMSCs transplantation group decreased, the difference was statistically significant (P<0.01); Compared with model group, the TIMP-1 concentrations in rats of hEMSCs transplantation group decreased significantly, the difference was statistically significant (P<0.01), the IGF-1 concentrations decreased significantly in rats of EA at fascia intensive area in LR14+hEMSCs transplantation group, the difference was statistically significant (P<0.01), the IV collage concentrations decreased significantly in rats of EA at GB34+hEMSCs transplantation group, the difference was statistically significant(P<0.05), the IGF-1, MMP-2, TIMP-1 concentrations in serum significantly decreased in rats of EA at fascia intensive area in GB34+hEMSCs transplantation group, the differences were statistically significant (P< 0.05, P<0.05, P <0.01); compared with hEMSCs transplantation group, the IV collage concentrations significantly decreased in rats of EA at LR14+hEMSCs transplantation group and TIMP-1 concentrations significantly increased, the differences were statistically significant (P<0.01), the concentrations of IGF-1, collage IV, MMP-2 in serum decreased significantly in rats of EA at fascia intensive areas in LR14+hEMSCs transplantation group, the differences were statistically significant (P<0.01, P<0.01, P<0.05), theⅣcollage, MMP-2 concentrations decreased significantly in rats of EA at GB34+hEMSCs transplantation group and fascia intensive area in GB34+hEMSCs transplantation group, the differences were statistically significant (P<0.01). Compared with EA at LR14+hEMSCs transplantation group, the serum concentrations of TIMP-1 in rats of EA at fascia intensive area in GB34+hEMSCs transplantation group significantly decreased, the difference was statistically significant (P <0.01). The comparison of IGF-1, collage IV, MMP-2 concentrations among EA groups showed no significant changes (P> 0.05).3 The targeted migration of hEMSCs with the Ad5-EGFP gene to the liver of alcoholic liver fibrosis model rats in vivo. At the same field of vision, the hepatic tissue of hEMSCs transplantation group rats could be seen very small amount of green fluorescence, and the hepatic tissue of EA groups rats also had visible green fluorescence, which slightly increased more than hEMSCs transplantation group. No distribution of green fluorescence could be seen in the kidney.4 HE staining showed:in normal group, the hepatic tissue of rats had clear and radial hepatic lobule architecture, neat hepatic sinusoid, clear cell structure, scattered lipid droplet in cytoplasm; in the hepatic tissue of model group rats, the hepatic lobule structures were disorder or incomplete, hepatocytes were swelling and severe lipoid degeneration, the proliferation of collagen fibers occurred around central veins, fiber septa widened at portal area; among the treatment groups, the degeneration and necrosis of hepatic tissue was slightly relieved compared with the model group, the proliferation of collagen fibers decreased, some hepatic lobule architecture recovered. Picrosirius staining showed that compared with normal group, the hepatic tissue of model group rats had significantly increased collagen deposition area, the difference was statistically significant (P<0.01); compared with model group, the collagen deposition significantly decreased in the hepatic tissue of rats of EA at fascia intensive area in LR14+hEMSCs transplantation group and EA at GB34+hEMSCs transplantation group, the difference was statistically significant (P<0.01), the remaining groups had no statistically significant differences (P> 0.05).5 The immunohistochemical results showed that compared with normal group, the positive expression of TGF-β1, TNF-alpha in hepatic tissue of model group, hEMSCs transplantation group and EA groups all increased (P> 0.05); compared with the model group, the positive expression of TGF-β1, TNF-alpha in hepatic tissue of hEMSCs transplantation group and EA at groups all decreased (P> 0.05); compared with hEMSCs transplantation group, the positive expression of TGF-β1, TNF-alpha in hepatic tissue of EA groups showed reducing trend (P> 0.05). among the EA groups, the positive expression of TGF-β1, TNF-alpha in hepatic tissue showed certain reducing or rising trend respectively (P> 0.05).6 The RT-PCR results showed that after pathological serum culturing for 24h, the cells of each induction group had no expression of AFPmRNA, ALBmRNA and a-SMA mRNA; 5d later, except the serum group from rats of EA at fascia intensive area in GB34+hEMSCs transplantation group, the cells of other groups appeared some positive expression of AFPmRNA, and very small amount of cells appeared weakly positive expression of a-SMA mRNA in serum groups from rats of alcoholic liver fibrosis model and EA at GB34+hEMSCs transplantation group, Small number of cells in serum groups from rats of EA at fascia intensive area in LR14+hEMSCs transplantation group and EA at GB34+hEMSCs transplantation group had positive expression of ALBmRNA; with prolonged incubation time. Some cells showed positive expression of AFPmRNA in serum group from rats of EA at fascia intensive area in GB34+hEMSCs transplantation group after cultured for 14d, in other induction groups, positive expression of AFPmRNA in cells decreased gradually, that of ALBmRNA increased gradually. During the whole cell culture process, the cells of control group had negative expression of AFPmRNA, ALBmRNA and a-SMA mRNA.Conclusions1 Improved method of liquor-lard (homemade)-pyrazole suspension by continuous gavage could cause rapid occurrence of severe lipoid degeneration in hepatic tissue of rats, and then lead to the occurrence of liver fibrosis, which is similar to the pathogenesis of human alcoholic liver fibrosis, and shortens the time of model preparation, has high survival rate of the models, simple and easy to operate.2 hEMSCs transplantation has certain improvement effect on alcoholic liver fibrosis. The main mechanism may be, hEMSCs were transplanted into alcoholic liver fibrosis model rats and affected by the internal environment, some of them directionally migrated to the liver and differentiated, reduced TIMP-1 concentrations in serum from liver fibrosis model rats effectively, and to certain extent reduced high expression of TGF-(31 and TNF-alpha in hepatic tissue of liver fibrosis model rats.3 EA combinations with hEMSCs transplantation have significant anti-hepatic fibrosis effect. It may be carried out by reducing the increasing serum concentrations of IGF-I, C IV, MMP-2 and TIMP-1 in alcoholic liver fibrosis model rats, improving hepatocyte degeneration and necrosis caused by liver fibrosis, inhibiting proliferation of collagen fibers and high expression of TGF-β1 and TNF-alpha in hepatic tissue, and EA promoting the targeted migration to liver and differentiation of hEMSCs to certain extent. We suppose that its mechanism may be as below, EA changes the microenvironment of local lesion under certain conditions, to make it in favor of the targeted migration of stem cells to lesions and differentiation. The certain conditions include acupoints selected in the experiment, the intensity and frequency of EA, etc.4 There are different effects among different acupoints and different fascia intensive areas:the effect of GB34 is better than that of LR14, there are no significant differences of the effect between fascia intensive areas in GB34 and LR14; the effect of fascia intensive area in LR14> LR14, the effect of GB34> that of fascia intensive area in GB34.5 The serum from rats of alcoholic liver fibrosis model group and EA groups all can induce hEMSCs to differentiate into hepatocyte-like cells, which express hepatocellular specific markers, such as AFP, ALB. And the serum from rats of EA groups can induce hEMSCs differentiation faster, and express more the specific markers. We suppose that there may be some relevant factors in the serum from rats of all groups, which may induce hEMSCs to differentiate into hepatocyte-like cells; EA may provide a more appropriate induction environment for hEMSCs differentiation to the liver cells, shorten the differentiation time, and then play an anti-hepatic fibrosis role. The induction effect of EA at fascia intensive area in LR14+hEMSCs transplantation group and EA at GB34+hEMSCs transplantation group are the most obvious.In summary, the results of this study had important guiding significances on acupuncture combined with stem cell transplantation for alcoholic liver fibrosis patients in clinic in the future, and provided some valuable experimental evidences for fascia basic research of acupuncture.
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