| Stroke is a leading cause of death and no pharmacological treatment is presently available to protect brain tissue from the multiple neurochemical cascades that are triggered by ischemia and reperfusion. Mounting experimental data obtained from animal models suggest that bone marrow mesenchymal stem cells (MSCs), administered locally or systemically, can improve functional outcome after stroke. In rat models for stroke and trauma, intravenously and transcerebrally injected MSCs have been demonstrated to go through the blood-brain barrier and accumulate in the parenchyma of the injured brain, and thereby induce neurological and functional recovery. It therefore seems that the injured tissues can specifically attract MSCs and mediate their migration behavior. However, the mechanisms regulating MSCs migration and accumulation in the injured brain remain to be revealed.Stromal cell-derived factor-1 (SDF-1α) and its cellular receptor CXCR4 have been demonstrated to direct the migration of stem cells associated with injury repair in many species and tissue types. In rat myocardial infarction model, SDF-1 plays an essential role in promoting the homing in of cells toward the ischemic myocardium by recruiting MSCs that express CXCR4. Expression of both SDF-1α/CXCR4 is predominantly promoted in hypoxic conditions, such as acute renal failure, ischemic cardiomyopathy, and ischemic brain. This raises the possibility that the SDF-1α/CXCR4 axis also plays essential roles in directing MSCs migration in ischemic brain.RNA interference (RNAi) technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. In an effort to investigate the migration of rat MSCs (rMSCs) within the injured brain and the regulating mechanisms, we first constructed CXCR4 RNAi lentiviral vector and established CXCR4 gene silencing rMSCs. Then we focused on characterizing the effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivo. This study consisted of three parts. Part1:Construction and identification of the lentiviral RNAi vector of CXCR4 gene Materials and Methods1) Three specific target sequences were selected according to rat CXCR4 mRNA. The complementary DNA which contained both sense and antisense oligonucleotides were synthesized.2) After phosphorylation and annealing, these double strands DNA were cloned to BglⅡand HindⅢsites of pSUPER.3) Then the product pRiCXCR4a, b, c was confirmed by electrophoresis and sequencing.4) CXCR4 shRNA was cloned to XbaI site of pNL, a transfer vector of lentivirus. Then the product pNL-RiCXCR4a, b, c were obtained.Results and Conclusions1) It is confirmed by restriction enzyme digestion and sequencing that CXCR4 shRNA expression structure is correctly cloned to pSUPER and pNL respectively.2) RNAi lentivirus vector of rat CXCR4 gene has been constructed, which is the essential building block required for the CXCR4 functional research. Part2:Establishment, culture and identification of CXCR4 gene silenced MSCsMaterials and Methods1) pNL-RiCXCR4 was cotransfected along with pHELPER and pVSVG into 293T to package lentivirus particles.2) According to the enhanced green fluorescent protein (EGFP) expression,the functional titer was determined by flow cytometry (FCM) after transduction into 293T cells.3) The rMSCs were transduced with lentiviral vectors at 2MOI and selected for stable integrants by using EGFP reporter gene to establish stable silenced cell line.4) Real-time RT-PCR analysis of CXCR4 mRNA, Western Blot analysis of CXCR4 protein and FCM analysis of CXCR4 on the cell membrane were conducted to compare the RNAi effect.5) The growth curve was obtained and the population doubling time was calculated to compare the growth of CXCR4-downregulated rMSCs and their parental cells.Results and Conclusions1) After cotransfection, lentiviral vector can be packaged in 293T cells. 2) The functional titer of unconcentrated virus and concentrated virus are 6.4×104TU/mL and 6.9×106TU/mL respectively.3) CXCR4 silenced cell line MSC-CXCR4- was successfully established.4) Down-regulation of CXCR4 expression in rMSCs was confirmed by Real-time RT-PCR, Western blot and FCM.Part3:Effect of CXCR4 gene silencing and SDF-1αon the migration of rMSCs in vitro and vivoMaterials and Methods1) In vitro migration of rMSCs in response to SDF-1αwas assessed in a modified 48-well microchemotaxis chamber using polycarbonate membranes with 8μm pore size. The cells were added to the upper chambers. SDF-1αwas added to the lower wells in different concentrations. The migrated cells were counted and the migration index was calculated to show the difference of migration.2) The healthy adult male SD rats were subjected to transient middle cerebral artery occlusion (MCAO).The rMSCs expressed EGFP (EGFP+) were infused into the femoral vein of rats at 24 hours before or after MCAO. The rMSCs with CXCR4 gene downregulation were also transplanted at 24 hours after MCAO.3) Immediately following femoral vein injection of EGFP-labeled rMSCs, the SD rats received an intracerebral injection of SDF-1αinto the left cerebral cortex. As control, the opposite side was injected with PBS alone.4) The tissue samples of brain, heart, lung, liver, spleen, kidney, skeletal muscle, small intestine and bone marrow of rats were collected. Then the frozen sections and bone marrow smear were prepared. The distribution of EGFP+ cells was observed under the fluorescent microscope.5) To clarify the relation and location of SDF-1αand EGFP-labeled rMSCs in the brain, SDF-1αimmunofluorescence staining was performed in the brain frozen sections.Results and Conclusions1) In vitro chemotaxis assay, the number of migrating cells significantly increased in the concentration of 200ng/mL SDF-1α, and significantly decreased while CXCR4 was knockdown with siRNA.2) In the groups of rats transplanted before or without MCAO, EGFP+ cells were found in bone marrow sample, lung, spleen and intestine samples, but not in the other tissues. 3) In the group of rats transplanted after MCAO, EGFP+ cells were localized in the ischemic penumbra, few marked cells were observed in the contralateral cerebral hemisphere or other tissues except a few in lung. CXCR4 downregulation in rMSCs exhibit strong inhibitory effects on rMSCs migration to the ischemic region of brain.4) Immunofluorescence staining showed that the source of SDF-1αis mainly from neurons and astrocytes in the ischemic penumbra, and the location and density of EGFP+ cells appears to be roughly associated with the level of SDF-1αimmunoreactivity in the brain frozen sections.5) The cerebral injury before transplantation is very important and necessary for intravenously grafted rMSCs selectively migrating to ischemic focus, otherwise, the grafted rMSCs will settle down in bone marrow, spleen, and never migrate to other tissues again.6) The interaction of locally produced SDF-1αand CXCR4 expressed on the rMSCs surface plays an important role in the migration of transplanted cells, suggesting that it might be a potential approach to modulate the expression of the two molecules in order to further facilitate the therapeutic effects using MSCs.
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