Roles Of CXCR4/CXCR7on Proliferation, Migration Of Mesenchymal Stem Cells Induced By SDF-1and Signal Transduction Mechanism

Objective:(1)To establish a method for isolation, purification andproliferation of bone marrow mesenchymal stem cells (MSCs）and to explore thefeasibility of MSCs differentiation into smooth muscle cells in vitro.(2)Toinvestigate roles of CXCR4/CXCR7on proliferation, migration of MSCs inducedby SDF-1and the relationship with PI3K/NF-κB signaling pathway for providingthe experimental basis for later study in vitro. Methods:(1) Using the whole bonemarrow adherent isolation and cultivation for MSCs and identified them. MSCswere cultured in basis medium containing PDGF-BB and TGF-β1for induction.Cell morphology was observed during induction. RT-PCR were used to analyzedthe expression of smooth muscle cell-specific marker α-SMA、Calponin、SM22αand SM-MHC. The expression of former three marker was detected byimmunocytochemistry.(2) RT-PCR was used to detect the expression of CXCR4and CXCR7mRNA with induction of SDF-1before and after. MTT assay andTranswell migration assay were used to detect the proliferation and migration of MSCs with Wortmanin, PDTC, anti-CXCR4and anti-CXCR7drugs as a tool.Results:(1)①MSCs mostly showed circular, polygonal, spindle after inoculationfor24-48h. At the third passage, cells formed a uniform long spindle shape. MSCsat passage3were strongly positive for CD44, weakly positive for CD106, butnegative for CD11b and CD45. After adipogenic and osteogenic induction, oil redO and alizarin red staining were positive respectively.②The cell morphology waschanged significantly-slender spindle，overlapping outgrowth，showing a tipical“peak-valley” shape like smooth muscle cells when local integration. RT-PCRshowed that expression of α-SMA、Calponin、SM22α were observed on MSCsbefore and after induction，but after induction the expression level was increasedsignificantly (P<0.01). There is no difference in results between RT-PCR andimmunocytochemistry.(2)①RT-PCR demonstrated that both CXCR4andCXCR7were expressed in MSCs. The expression of CXCR4was increased byinduced SDF-1(P<0.01)，while CXCR7was no significant change (P>0.05).②Compared with control group, the proliferation and migration of MSCs in theSDF-1group was significantly increased (P<0.01), and showed a certainconcentration-dependent.③T he proliferation of MSCs from SDF-1+Wortmaningroup(E), SDF-1+Wortmanin+PDTC group(F), SDF-1+anti-CXCR4group(G)and SDF-1+anti-CXCR7group(H) were significantly decreased than that ofSDF-1group (D)(P<0.01).There is no significant difference in the proliferation ofMSCs between the IgG group (I) and the SDF-1group (D)(P>0.05). In addition,the proliferation of MSCs from SDF-1+Wortmanin+PDTC group was significantly decreased than that of SDF-1+Wortmanin group (P<0.05).④Themigration of MSCs from SDF-1+Wortmanin group(E),SDF-1+Wortmanin+PDTC group(F) and SDF-1+anti-CXCR4group(G) weresignificantly decreased than that of SDF-1group (D)(P<0.01). There is nosignificant difference in the proliferation of MSCs between theSDF-1+anti-CXCR7group (H), IgG group (I) and the SDF-1group (D)(P>0.05).In addition, the migration of MSCs from SDF-1+Wortmanin+PDTC group wassignificantly decreased than that of SDF-1+Wortmanin group (P<0.01).Conclusions:(1)①A great many of MSCs with high purity are obtained bymethod of whole bone marrow adherence and amplification.②Smooth muscleprogenitor cells from rat bone marrow can differentiation into smooth muscle cellswith induction of PDGF-BB and TGF-β1.(2)①SDF-1/CXCR4plays a key role inpromotion of proliferation and migration of MSCs in vitro by upregulating theexpression of CXCR4, while CXCR7plays a certain role on proliferation ofMSCs，but has no significant role on migration.②PI3K/NF-κB pathway relatedsignaling moleculars may be involved in the proliferation and migration of MSCsin vitro induced by SDF-1.