Empirical Study Of Fetal-Bone Mesenchymal Stem Cells Induced By Three Kinds Of Connective Tissue In Vitro

Objective: Bone marrow-derived mesenchymal stem cells (BMSCs) can be obtained easily, have the characteristic to maintain the ability of high proliferation, and the potential to differentiate into cells in three germinal layers, and is useful in the fields of tissue engineering, regenerate medicine and cellular transplant therapy in clinic. BMSCs can be induced to differentiate into epithelial cells in vitro. It was found that the BMSCs transplanted to the alkaliburn cornea of a rabbit could migrate toward corneal stroma, and differentiated into corneal epithelium. This result indicated that BMSCs could be induced by the local microenvironment in vivo and differentiated into the cells which adapted to the function of the local tissue, but the mechanism has not clear. We had found that BMSCs could be induced by fetal-intestinal connective tissue in vitro and differentiated into epithelial cells which expressed cytokeratin. Now we will compare the inducing effect of connective tissue in amnion, fetal mesentery and intestinal submucosa on the differentiation of fetal BMSCs. Try to provide the basic theory and experiment for the future research of mechanism on BMSCs differentiation induced by local body microenvironment and its application in tissue engineering.PARTⅠ1 Bone marrow cells were obtained from fetus at gestational age from 4 to 5 months. BMSCs had been isolated,cultured and amplificated in vitro by the methods of full-marrow culture and density gradient centrifugation, to compare the cellular numbers obtained by the two ways, and detect the phaenotype of these cells through analysis of flow cytometry. The results showed that the cellular numers of BMSCs isolated by full-marrow culture were more than those by density gradient centrifugation(P<0.01); The BMSCs in generation 3 (P3-BMSCs) obtained in two ways showed us a typical feature of spindle-shape and whirlpool arranged in culture. The flow cytometer-analysis showed that the P3-BMSCs expressed CD29, CD71, but unexpressed CD34, HLA-DR. The homogenicity of CD29 and CD71 was 80.73±0.17%. There was no statistically significant on the difference of fluorescence intensity between BMSCs obtained by the two ways ( P>0.05).2 The different concentrations of DAPI marked the P3-BMSCs had been studied, and the DAPI-marked rate had been compared between the P3-BMSCs cryopreservated and non-cryopreservated. After two hours, all DAPI-marked rate of BMSCs treated with four concentrations of DAPI was 100%. Although, the blue fluor of high concentration groups was much brighter than low concentration groups, the marked rate of each group decreased gradually. The statistics results showed that the DAPI- BMSCs in group of 40μg/ml was higher than those in groups of 25μg/ml or 5μg/ml(P<0.01),and there was no significant difference of DAPI-BMSCs between group of 40μg/ml and 50μg/ml. There was a significant interaction between the concentrations of DAPI and marked time, I.e., the DAPI- BMSCs decreased progressively along with the increase of time in the four concentrations of DAPI groups. The DAPI-BMSCs of cryopreserved P3- BMSCs in group of 40μg/ml was as same as that of non-cryopreserved P3-BMSCs.3 The P3-BMSCs had been cultured with different concentrations of epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1), after 7 days , most of the cells show a spindle-shape, a few of them becomed large and flat, looked like epithelial cells. The cells expressing cyokeratin (CK) and epithelial membrane antigen(EMA) had been detected, after the BMSCs had been induced 7 days. The statistics-results showed:①The positive cells of CK or EMA in the group of EGF 30 ng/ml or IGF-1 50 ng/ml were significantly higher than those of other concentration groups(P<0.05). Among the other groups, only few EMA positive cells appeared, no CK positive cells in group of 100ng/mlEGF or IGF-1.②The CK or EMA positive cells in groups induced with EGF and IGF-1 were significantly higher than those in groups induced with single factor(P<0.01); The CK positive cells in group induced with 30 ng/ml EGF and 50 ng/ml IGF-1 were higher than those in other groups induced with this two factors(P<0.01); However, there were no significant differences of EMA positive cells among the groups induced with two factors.③The CK or EMA positive cells had not been observed in the control group.PARTⅡ1 The sample of fetal mesentery and intestinal submucosa were obtained from 20 cases of the induced-abortion fetus aged 4 to 5 months, 15 cases of the amnion were taken from normal placenta. The connective tissue of amnion,mesentery and intestinal submucosa had been prepared with mechanial avulsion and enzyme-digestive method. The epithelial cells had not been observed on the surface of prepared connective tissue with light microscope.2 The 50μl DAPI-P3-BMSCs (4.0×106/ml) had been planted on the surface of three kinds of connective tissue to induce BMSCs differentiation, and added 30ng/ml EGF and 50ng/ml IGF-1 in medium as control groups. Another control group was set up by cultured DAPI-BMSCs alone , added 30ng/ml EGF and 50ng/ml IGF-1 in medium. The DAPI-BMSCs and connective tissue were air-lifting cultured, after submerged cultivation for 7 days. The DAPI-P3-BMSCs,which had blue fluor-labeled nucleus,could be observed on the surface of three kinds of connective tissue,after 8 to 12 days cultured. There were some cells with a large volume and visible nucleus in same section stained by HE, their distribution was as same as the fluor marked cells. Observing the section stained by immunohistochemistry method, the BMSCs growed on the surface of three kinds of connective tissue could express CK or EMA. The immunohistochemical products of EGF were deposited in the three kinds of connective tissue, but the products of IGF-1 were only appeared in prepared mesentery and intestinal submucosa, but no in amnion.3 After inducing-cultured 12 days, the sections of connective tissue were stained with immunofluorescence, and observed with confocal laser scanning microscope (CLSM) . It had been showed that:①There were cells marked nucleus with blue-fluor (DAPI) on the surface of connective tissue in frozen slices and stretched preparations of amnion, mesentery and intestinal submucosa, the red-fluor (CK) also could be seen in some of these cells. A few cells with DAPI blue-fluor and scrannel green-fluor (EMA) only appeared in amnion.②Countting the cells marked with red and blue fluor and only marked with blue fluor, the percentage of CK and DAPI positive cells had been obtained. The statistics results showed that the percentage of CK and DAPI positive cells in 3 groups unite-induced by three kinds of connective tissue and two growth factors was higher than those in groups induced only by connective tissue, or two growth factors(P<0.05);but there was no significant difference of the percentage of CK and DAPI positive cells in the three unite-induced groups(P>0.05);The percentage of CK and DAPI positive cells in group alone-induced by amnion was higher than those alone-induced by mesentery and intestinal submucosa(P<0.05).4 The cellular ultrastructure had shown that, there were some short prominent on the surface of BMSCs, and the basement membrane between BMSCs amd connective tissue had not been seen.Conclusion of whole article1. The cellular numbers of BMSCs isolated and prolifereted by full-marrow culture were more than that by density gradient centrifugation.2. The best final concentration of DAPI marked BMSCs was 40ug/ml, two hours.3. BMSCs could be induced to differentiate into epithelial cells by EGF and IGF-1 in vitro. EGF and IGF-1 had synergistic effect on induced BMSCs differentiation,and the optimal synergistic effect was in 30 ng/ml EGF and 50 ng/ml IGF-1.4. BMSCs could be induced to differentiate into epithelial cells by the connective tissue of amnion, mesentery and intestinal submucosa, and the best effect was amnion. EGF was expressed in the three kinds of connective tissue, and IGF-1 was only expressed in mesentery and intestinal submucosa. The results indicated that the endogenetic growth factors in these connective tissue may be one of reasons which induced BMSCs differentiate into epithelial cells. 5. The exogenous EGF, IGF-1 had synergistic effects with three kinds of connective tissue on the induction of BMSCs to differentiate into epithelial cells in vitro.6. The basement membrane between BMSCs amd the connective tissue had not been seen with electron microscope.