Therapeutic Effect And Mechanism Of Umbilical Cord Mesenchymal Stem Cells With High Expression Of BFGF Combined With Collagen Scaffold In The Treatment Of Full-thickness Uterine Injury In Rat

Background:Severe uterine injury can lead to uterine scar formation and intrauterine adhesion,ultimately resulting in uterine infertility.However,the effective methods for uterine injury healing are still lacking.Our group had showed that in the treatment of the model of uterine full-thickness injury,the local transplantation of MSC(Mesenchymal stem cells)combined with collagen scaffold or the local injection of basic fibroblast growth factor(bFGF)could promote the regeneration of injured uterus.Genetic engineering modification of MSC has been shown great promise in preclinical studies.The objective of this study was to investigate the therapeutic efficiency and mechanism of the UC-MSCbFGF/scaffold on functional regeneration of the uterine full-thickness injury model.Methods:1,Construction of UC-MSCbFGF and verify transfection efficiency,stem phenotype and tumorigenicity.The plasmid of pCMV1-bFGF-hygro was constructed and transduced into the P2 UC-MSCs by electrical transduction.The transfection efficiency of bFGF in UC-MSCbFGF was identified by immunofluorescence and Western blotting(WB).Flow cytometry and three-line differentiation test were used to detect the expression of stem gene and multi-line differentiation ability of UC-MSCbFGF.1×107 UC-MSCbFGF was injected subcutaneously into nude mice and observed for 4 months to detect the tumorigenicity of UC-MSCbFGF.2,The model of uterine full-thickness injury was established in Sprague-Dawley(SD)rats by excising uterine wall of approximately 1.0 cm in length and 0.5 cm in width from each uterine horn.The rats were randomizedly classified into four groups,including the sham-operated group,the plain scaffold group,the UC-MSCs/scaffold group and the UC-MSCbFGF/scaffold group to explore the outcome of structural and functional recovery in injured uterus.3,Effect of UC-MSCbFGF on full-thickness injury model of uterine wall.At day 7,14,30 post-transplantation,we euthanized the rats and obtained the tissue sections of injured uterus containing the injured region.1)UC-MSCbFGF survive and continuously releases bFGF in vivo were observed by immunohistochemical staining.2)The degrade of inflammatory response in injured uterus was measured by immunohistochemical staining for CD45,CD68、CD206、IL-6、TNF-α和 IL-6.3)The morphological alterations of endometria were observed by hematoxylin and eosin(H&E)staining.The immunohistochemical staining for ER,Vimentin,α-SMA and CD31 were performed.4)The functional recovery of the injured uterus was assessed by fertility experiment at day 30 post-transplantation.Results:1,The plasmid of pcmv1-bFGF-hygro was successfully constructed.After transformation,bFGF was expressed stably and highly in UC-MSCs,and the gene expression and three-line differentiation ability of UC-MSCbFGF were similar to control UC-MSCs.2,The UC-MSCbFGF exhibited anti-inflammatory effect.The number of CD45+cells in the UC-MSCbFGF/scaffold group(2.01±0.89%)was significantly less than that in the UC-MSCs/scaffold group(3.88 ± 1.2%,P<0.01)and the scaffold group(8.83 ± 1.9%,P<0.01),but higher than the sham-operated group(0.38± 0.22%,P<0.01)at day 7 post-transplantation.The expression of anti-inflammatory factor IL-10 in the UC-MSCbFGF/scaffold group(1.69±0.84%)was significantly higher than UC-MSCs/scaffold group(0.91 ± 0.39%,P<0.01)and the scaffold group(0.30±0.17%,P<0.01).The pro-inflammatory factor TNF-α in UC-MSCbFGF/scaffold group(0.40 ± 0.19%)was significantly lower in UC-MSCs/scaffold group(0.68± 0.42%,P<0.01)and the scaffold group(1.03 ± 0.54%,P<0.01)3,The UC-MSCbFGF also exhibited dramatically pro-angiogenesis efficacy.The number of blood vessel in UC-MSCbFGF/scaffold group(37.5±11.78/field)was higher than the UC-MSCs/scaffold group(25.75±8.76/field,P<0.01)and the scaffold group(21.5 ± 4.11/field,P<0.01)at day 14 post-transplantation.4,UC-MSCbFGF promoted the regeneration of endometrium and myometrium of injured uterine.The thickness of endometrium and the muscle layer in each group were observed by HE and immunohistochemical staining at day 30 post-transplantation.The thickness of endometrium in UC-MSCbFGF/scaffold group was(467.6:1±115.1μm)the closed to the sham-operation group(492.1 ± 101.8μm),and significantly higher than UC-MSCs/scaffold group(210.3±22.55μm.P<0.01)and collagen scaffold group(157.6± 27.38μm,P<0.01).We observed that α-SMA positive area of UC-MSCbFGF/scaffold group(12.82 ± 3.19%)was significantly higher than that in UC-MSCs/scaffold group(8.25 ± 1.67%,P<0.05)and scaffold group(5.93%± 2.06%,P<0.01)5,The results of the fertility experiment showed that the number of uterine horn with fetus implanted into the regenerated area in the UC-MSCbFGF/scaffold group(8/16,P<0.015)was significantly higher than that in the UC-MSCs/scaffold group(1/16,P<0.015)and the scaffold group(0/16,P<0.015).Conclusion:The UC-MSCbFGF/scaffold system suppressed inflammation induced by injury,promoted angiogenesis and accelerated regeneration of the injured uterine wall,which made the time required for injured uterus healing shortened.