A Study On The Effect Of C-kit~+ Cardiac Stem Cells Differentiation By CGRP In Vitro

Object: Nowadays, the incidence rate of AMI is rising and the age of onset is also younger than before, which becomes an important public health problem that we should not ignore. The limited effect of AMI’s existing clinical treatment makes people put the hope on the cells treatment of stem cells. The cells treatment of stem cells is mainly in two directions: one is through its differentiation into target cells to repair damaged heart, the other is through the paracrine of various bioactive factors to promote internal / exogenous stem cells survival and differentiation. c-kit+CSCs are valued for its specificity of cardiac tissue. The feasibility and effectiveness of c-kit+CSCs in the treatment of AMI are confirmed by a number of rodent animal experiments and preliminary clinical studies, but the poor local microenvironment after MI makes a low survival rate of transplanted cells. To enhance the ability of cells to withstand harsh environments and improve the viability of cells, people used a variety of interventions, but the effect is not ideal. How to improve the ability of differentiation of c-kit+CSCs after transplantation has become an urgent problem to be solved. Our preliminary studies show that CGRP treated c-kit+CSCs can inhibit the NF-κB signaling pathway, reduce the inflammatory reaction, increase the survival of transplanted cells. But it is still not clear whether CGRP has the effect on differentiation of c-kit+CSCs. CGRP, as a neuropeptide with strong cardiovascular protective effect, not only have the effect of anti-inflammatory, anti-apoptosis and promoting bioactive factors release, it also induced the differentiation of ADMSCs, MSCs and other cells. In order to further study the effect of CGRP on the differentiation of c-kit+CSCs and its possible mechanism, we use CGRP to deal with c-kit+CSCs in vitro, and observe the change of myocardial cells, smooth muscle cells, endothelial cells’ specific markers, to investigate whether the change of c-kit+CSCs is related to the induction of CGRP. To provide a new theoretical basis and experimental data for the treatment of AMI with c-kit+CSCs. Methods:1. Sorting out the c-kit+CSCs with enzyme digestion combined with MACS from rat auricle and identify cells purity with FACS, and drawing the growth curve of c-kit+CSCs by CCK-8.2. Make the newly naive selected c-kit+CSCs as the control group, using different concentrations of CGRP(0、10-7、10-8、10-9M) treated c-kit+CSCs to see the expression of cardiac cells’ specific marker by PCR and to obtain the optimal concentration of CGRP.3. The expression of the differentiation markers of c-kit+CSCs treated CGRP and CGRP+CGRP8-37 was observed by immunofluorescence.4. The cells were divided into four groups with dexamethasone, CGRP, CGRP+ its antagonist CGRP8-37 treatment, the normal c-kit+CSCs as the control, the group above as dexamethasone, CGRP, CGRP + CGRP 8-37.5. Detect myocardial cells’, smooth muscle cells’ and endothelial cells’ specific markers with PCR and Western Blot, and observe changes in the morphology of cells in each group by inverted phase contrast microscope to study CGRP induced differentiation.6. In order to further study the mechanism of CGRP induced differentiation of c-kit+CSCs, use c-kit+CSCs without any treatment as the control and use ELISA to assess the situation of c-kit+CSCs secreting growth factor. Results:1. Culture and sort the rats c-kit+CSCs combined enzyme digestion with MACS, cells are in spindle shape or branched shape under microscope observation. The identification of FACS cells phenotype shows c-kit 87.9%, CD34 2.3%, CD45 2.2%.2. When using 10-8M CGRP to deal with c-kit+CSCs cells, we find that cardiomyocyte differentiation marker Nkx2.5 expression is the highest(P < 0.05), as the best CGRP concentration, and use the concentration in the follow-up experiment.3. Immunofluorescence observation after CGRP induced c-kit+CSCs, Nkx2.5, cTnT, cells expression of red fluorescence and green fluorescence of α-SMA and CD31 expression increases compared with the control.4. PCR detection shows that CGRP can be induced at the mRNA level increased specificity of c-kit+CSCs of the early and late myocardial cells, smooth muscle cells and endothelial cells marker expression, but its expression level is lower than dexamethasone group compared with the blank group, all these specific markers expressions are inhibited and the expression tendency of Nkx2.5, cTnT, α-SMA and CD31 tends to be consistent with PCR detected by WB after adding CGRP’s antagonists. The results are of statistical significance; the morphology of the cells among the groups has no change.5. We find that secretion growth factors of c-kit+CSCs have been improved after treated CGRP detected by ELISA compared with the control, and the difference has statistical significance. Conclusion:1. The experiment develops a high purity and good growth state of c-kit+CSCs by enzyme digestion and immunomagnetic beads sorting method;2. CGRP can promote the differentiation of c-kit+CSCs to cardiac muscle cells, smooth muscle cells and endothelial cells, and the best concentration of promoting cardiac muscle cells differentiation is 10-8M;3.CGRP can promote the secretion of growth factors in c-kit+CSCs, which may be related to the differentiation of c-kit+CSCs.