| Background:Poor regeneration ability of pelvic floor muscle is the main cause of female stress urinary incontinence(SUI). In addition to traditional operation and drug therapy, stem cell transplantation is another effective way of treating SUI and shows great promise. Bone marrow mesenchymal stem cells(BMSCs) is one of the most important seed cells for tissue engineering. Afer injected around the urethra,BMSCs could replace and repair the damaged urethral sphincter, thus enhancing the tension of urethral sphincter and improving the symptom of urinary continence so that can cure SUI at root. The aggregation of stem cells to the target organs and tissues is the base of stem cell transplantation method in vivo. It is directly related to the result of the treatment, and has been studied and concerned widely in the field of tissue engineering. Nowadays, the low rate of homing cells has become a common problem that draw people’s attention. Therefore,it is urgent to find an effect way to guide stem cells gathering to the targets and improve the homing rate of stem cell.The SDF-1/CXCR4axis,which consist of stromal cell derived factor-1(SDF-1) and its receptor CXCR4, plays an important role in stem cell homing.CXCR4is widely expressing in many kinds of cells including BMSCs. SDF-1can induce the expression of CXCR4and CXCR4+cell has the speciality of migrating from low concentration to high concentration of SDF-1. Based on the chemotaxis property of BMSCs, we can control the cell chemotaxis migration indirectly through regulating the expression of SDF-1/CXCR4axis.Studies has showed that estrogen can upregulate the expression of SDF-1/CSCR4axis, suggesting that we can use estrogen to regulate the expression of SDF-1/CSCR4axis and thus regulating the migration of BMSCs.In this study, we choose female SD rats as the source of BMSCs. We use the whole marrow and adherence method to isolate and culture BMSCs, and then induced the cells with different concentrations of17β-estradiol to detect the it’s effect on the expression of CXCR4gene and protein. Furthermore, whithin the result above,we obtain the most beneficial concentration of E2, with which we gain the highest expression of BMSC s CXCR4and make a significant change of the chenmotactic ability. The main experimental results and methods are as follows:Methods:1. Isolation, culture and identification of BMSCs of rats(1) Isolation, culture and identification of BMSCs3week old female SD rats were put to death and take the bilateral tibia and fibula.Expose marrow cavity.Use DMEM-F12with10%FBS to fully rinse marrow cavity. The washing liquid was collected,and concentrated. The precipitation was added to DMEM-F12with10%FBS and cultured in incubator of37℃and5%CO2condition. The cells of third generation were treated with antibody, and then detect the expression of CD44, CD45, CD31, CD29, CD166on the surface of cells by flow cytometry method. Result:The shape of BMSCs was similar. High expression of CD44, CD29and low expression of CD166,CD45, CD31on cell surface was accorded with the expression partern of BMSCs.2. Detection of CXCR4expression of BMSCs(1) Detection CXCR4expression of BMSCs by PCRThe BMSCs of the second generation with density of106/hole were inoculated in6hole culture plates. BMSCs were cultured in DMEM-F12with10%FBS.when cells occupy60-70%of bottom, culture medium was discarded and replaced with estrogen. In this study, we set6concentrations of17β-E2, including10-10mol/L,10-9mol/L,10-8mol/L,10-7mol/L,5×10-7mol/L and10-6mol/L.2experimental groups were set as E2intervention group and ER antagonist group. Cells of ER antagonistic group were pretreated with10-8mol/L estrogen receptor antagonist named ICI182780for1h. Each group was made up of13holes and numbered from1to13.No.1was set as the control group. No.2to7and No.8to13of E2intervention group were teated with different concentrations (10-10,10-9,10-8,10-7,5×10-7,10-6mol/L) of17β-E2. Each hole of the ER antagonistic group was treat with E2as above and then treat with ICI182780of10-6mol/L. The intervent time of No.2-7was24h, and No.8-13was48h. After the intervent process, BMSCs were collected and then detect the expression of CXCR4mRNA by PCR method. Result:The expression of CXCR4mRNA induced by10-7mol/L17β-E2was the highest after24h’s intervention. The expression of CXCR4mRNA induced by5×10-7mol/L17β-E2was the highest after48h’s intervention. ICI182780of10-8mol/L can significantly inhibit the high expression of BMSCs CXCR4mRNA induced by17β-E2.(2) Detection of CXCR4protein expression of BMSCs by Western blotBMSCs of the intervention group and antagonistic group were treated by17β-E2and ICI182780as above. BMSCs were collected and then detect the expression of CXCR4protein by Western blot method. Result:The expression of CXCR4protein induced by5×10-7mol/L17β-E2was the highest both after24h and48h’s intervention.10-8mol/L ICI182780can significantly inhibit the high expression of protein of BMSCs induced by17p-E2.3. Detection of BMSCs directional migration effected by17β-E2(1)17β-E2Effect the directional migration to SDF-1of BMSCsExperiments were performed on24cell culture plate, control group and E2intervention group were set. The control group was treated without E2intervention and BMSCs of E2intervention group were precultured in DMEM-F12with10%FBS including17β-E2for24h. Each group consist of4holes and marked No.1to4. Both group were added500ul DMEM-F12including0、100、200、300ng/mL SDF-1.The suspension cell culture chambers transwell(8μm) were placed in the hole,200μl cell suspension which was pretreated was added to transwell with density of2×104per hole. The cells in the24cell culture plate was cultured for24h in37℃,5%CO2constant humidity incubator. Wipe off the cells on the inside surface by cotton ball. Fix with formaldehyde and stain by eosin for30s.5randomly selected fields were chosen to count staining cells under microscope(×100). Result:The number of migrating BMSCs reached the maximum when the concentration of SDF-1was200ng/ml in both group.The number of migrating cell in E2intervention group was larger than the control group in the same condition of SDF-1.(2) CXCR4blocking testWe set the control group and E2intervention group, both marked as No.5.cells of the2holes were treated as above and then treated with AMD3100of100ng/ml for30min. Ather procedure is same as above.Result:In the control group, the number of migrating BMSCs was no significant difference with the control No.1hole; In the E2intervention group, the number of migrating BMSCs cells was significantly more than the control No.1hole. Conclusion:Our study successfully isolated and cultured BMSCs of SD rats, and found the optimal concentration and treating time of17β-E2to obtain the highest expression of CXCR4.We also find out the optimal concentration of SDF-1for directional migration of BMSCs. Our studies demostrated that17β-E2did promote BMSCs migration towards SDF-1through up-regulating CXCR4expression. This effect of17β-E2could be blocked by CXC receptor inhibitor, ICI182780. |
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