| Interventions as cesarean sections(CS)and myomectomy operation cause severe damage to the uterus.This damage may lead to uterine myometrium and endometrium repair defects and result in future infertility,miscarriages,preterm deliveries,even uterine rupture during subsequent pregnancy,which affects the women’s reproductive health.And the rupture of the uterus is a major cause of maternal and infant mortality.Therefore,exploring the uterine wound healing mechanism is very important for maintaining female reproductive health.However,the limited access of human uterine tissue during the repair period not only impairs the research on the mechanism of uterine damage repair and remodeling but also hinders the progress of clinical diagnosis and treatment of uterine damage diseases.Thus,animal model is a valuable tool to explore the unknown field.Tissue engineering is a new subject that combines cell biology with material science to construct tissues and organs in vitro or in vivo.It provides an ideal treatment for injured or defective tissue.In recent year,more and more tissue engineering materials were used to enhance tissue healing.Collagen has been widely used in tissue engineering because of its good histocompatibility and biodegradability.The clinical efficacy of using collagen scaffolds loaded with cells or bioactive factors to promote tissue repair is satisfactory.Leukemia inhibitor factor(LIF)is a kind of cytokine with multiple biological functions.Recently,it has been found that LIF had important biological functions in repairing various tissues such as skeletal muscles,nerves and myocardium,and it effectively promoted the regeneration and repair of these tissuesand organs.LIF is abundantly expressed in uterine tissues,including endometrial glands,stroma and myometrium,which suggesting that LIF may also play a biological role in promoting the repair of uterine injury.This study has carried on the preliminary exploration to this question.In the first part of this study,the rat model of full-thickness uterine wall injury was developed.The collagen scaffold loaded with different doses of LIF was transplanted into the defect area of uterine wall.The feasibility and optimal dosage of LIF to promote uterine repair were explored by comparing the effect of LIF and PBS at 2 weeks after operation.And our results showed that 0.5μg LIF had better therapeutic effect.Inflammatory cells and factors in the collagen scaffold were analyzed during the inflammatory phase after injury.It was found that LIF regulated the local inflammatory response during the repair of uterine wall in rats.And the possible mechanism of LIF promoting the repair of uterine injury was explored.In the second part of the study,the collagen scaffold loaded with 0.5μg LIF or PBS were used to repair the uterine wall injury in rats.In order to explicit the effect of LIF,we have compared and analyzed the changes of uterine tissue structure and remodeling from 2 to 12 weeks after operation,and analyzed the difference of uterine function through pregnancy experiment.It was determined that LIF can effectively promote the repair of uterine tissue structure and function.In this study,we found and clarified the biological function of LIF in promoting the repair of uterine injury in rats,and accumulated experimental data for the clinical treatment of uterine injury diseases by LIF.Objective: 1.To explore the feasibility of LIF in the repair of uterine injury and investigate the possible mechanism.Method:1.The experimental rat model of uterine wall injury developed previously in our laboratory was applied,namely a 1.5 cm × 0.5 cm uterine wall was excision opposite to the mesangial side.2.Twenty four adult female Sprague-Dawley(SD)rats were randomly divided into 4 groups(6 rats in each group): PBS/collagen group,0.25μg LIF/collagen group,0.5μg LIF/collagen group and 1.0μg LIF/collagen group,After uterine wall injury,1.5 cm × 0.5 cm collagen scaffold was sutured to the uterine defect.The collagen scaffolds were loaded with 0.25μg,0.5μg,1.0μg LIF or PBS respectively.Two weeks after operation the injured uterus was removed for examination.Histological observations included Hematoxylin-eosin staining(HE)and immunohistochemical(IHC)staining were made to evaluate the shape of uterine wall,the number of infiltrating cells and neovascularization in the collagen scaffold.The expression of von-Willebrand-factor(v WF)were detected to analyse the neovascularization.3.Ten female SD rats were randomly divided into two groups: the LIF/ collagen scaffold group(5 rats were sutured with collagen scaffold loaded with 0.5 μg LIF after uterine injury)and the PBS/ collagen scaffold group(5 rats were sutured with PBS infiltrated collagen scaffold)(LIF: number of uterus =10;PBS: number of uterus = 10).One week after operation,the the damaged uterus were prepared for HE,IHC and immunofluorescence staining of CD45 to compare the changes of inflammatory cells between two groups.Total RNA was extracted and the m RNA levels of IL-10,IL-12,IL-6,IL-1β,TNF-α and TGF-β were detected by reverse transcription PCR and real-time quantitative PCR analysis.4.Fourty eight female SD rats were randomly divided into two groups: the LIF/ collagen group(the defect was sutured with collagen scaffold loaded with LIF0.5μg)and the PBS/collagen group(collagen stent wetted with PBS).Animals were sacrificed at 2,4,8 and 12 weeks(6 rats in each group at every time point)aftersurgery.The uterine horns were prepared for the patency test and gross examination,histological sections of the damaged uterus were prepared for IHC.The area of uterine smooth muscle and gland regeneration were evaluated by IHC staining of alpha smooth muscle actin(a-SMA)and Cytokeratin 7(CK7).5.Twenty five female SD rats were divided into two groups,including LIF/collagen scaffold group(15 rats)and PBS/collagen scaffold group(10 rats)(LIF: number of uterus = 30;PBS: number of uterus = 20),two different methods were used to repair the uterine wall for 8 weeks after operation and then two groups of animals were mated with male SD rats,the number of pregnant uterus and embryos were counted at 15-19 days of gestation to evaluate the effect of LIF on pregnancy function of the uterus.Result:1.Short-term comparative observation after operation:(1)Local cellularization and angiogenesis of uterine damage: Two weeks after operation,cell infiltration was observed in the undegraded collagen scaffold,no obvious inflammatory reaction was found in the local area.The number of cells infiltrated in the LIF /collagen group was significantly higher than that in the PBS group(P < 0.001).IHC staining of vascular density in 0.25 μg LIF and 0.5 μg LIF group was significantly higher than that in PBS group(P < 0.001).(2)Changes in inflammatory cells and factors in collagen scaffolds: One week after surgery,HE staining showed that collagen had no obviously degradation both in LIF/collagen and PBS/collagen group.A large number of inflammatory cell infiltration was found in collagen scaffolds in the injured area,anti-CD45 IHC and immunofluorescence staining showed that the number of CD45 positive cells(inflammatory cell)infiltration in LIF group was less than that in PBS group,The AOD value of CD45 in LIF group was significantly lower than that in the PBS group(P<0.01).The expression of IL-10,IL-12,IL-6,IL-1β,TNF-α and TGF-β in injured uterine tissues were detected.The expression level of IL-10,a crucial anti-inflammatory cytokine for wound healing,was upregulated 2.74 folds in LIF/collagen group(P<0.01);while the expression level of IL-12,a proinflammatory cytokine,was downregulated 2.09 folds in LIF/collagen group(P < 0.05).There was no significant difference in the expression of IL-6,IL-1β,TNF-α and TGF-β between the two groups(P > 0.05).2.Long-term comparative observation after operation:(1)The appearance of uterine healing: At 2 and 4 weeks after uterine healing,there were hyperemia and edema on the surface of the two groups,and granulation tissue was found in both groups.The appearance of the operation area in the LIF group was similar to that of the surrounding tissue at 8 and 12 weeks after operation.While scar and local collapse could be seen in the repaired area in PBS/collagen group,the uterine patency test showed that there was no obstruction in both groups.(2)Regeneration of uterine smooth muscle: The percentage of α-SMA positive area in the LIF/collagen group was significantly higher than that in the PBS/collagen group at 8 and 12 weeks after operation(P < 0.001).the percentage of α-SMA positive area at 8 and 12 weeks was significantly higher than that in the LIF/collagen group 2 weeks after operation(P < 0.01).(3)Regeneration of endometrium: There was no significant change in endometrial thickness from 4 to 12 weeks after operation in each group.The endometrial thickness of LIF/collagen group was higher than that of the PBS/collagen group without significantly difference(P > 0.05).The number of regenerated endometrialglands in the LIF/collagen group was significantly incresead than that in the PBS group at 8 and 12 weeks respectively(P < 0.001).3.Pregnancy test results: the average pregnancy rate and the average number of embryos per uterine were significantly higher in the LIF/collagen group than those in the PBS/collagen group(86.67% vs 60.00%)(P < 0.05)and(3.63 ± 0.34 vs 2.35 ± 0.51)(P<0.05).Furthermore,embryos in the LIF/collagen group were found both in grafted and normal tissue in 10 uterine horns(10/30).Whereas in the PBS/collagen group,only 3 uterus(3/20)had embryo implantation in the repair area.No uterine rupture occurred in both groups during the entire gestation period.Conclusion: Collagen scaffold loaded with LIF could promote the healing of uterine wall injury in rats.In the early stage of uterine damage,LIF mainly promoted the cellulization of the injured area and promoted the vascularization in a dose-dependent manner.LIF had a significant effect on the regeneration of myometrium and endometrial glands.The uterus repaired promoted by LIF could receive embryo and was competent for pregnancy.During the inflammatory phase of uterine injury repair,LIF inhibits the infiltration of inflammatory cells and the release of pro-inflammatory factors,and promotes the expression of anti-inflammatory factors in the injured tissues,which may be the potential mechanism for LIF promoting regeneration of uterus. |
