The Experimental Study Of Therapeutic Effect Of Bone Marrow Mesenchymal Stem Cells Transplantation On Thin Endometrium

Part I:Isolation, culture and identification of rat bone marrow mesenchymal stem cells in vitroObjective:To establish a simple and reliable method for isolation, culture and identification of the SD rat bone marrow mesenchymal stem cells (BMSCs) in vitro.Methods:The whole bone marrow adherent culture method was used to isolate of BMSCs from male SD rat. The proliferation ability of cells was analyzed by MTT examination; Identified cultivated mesenchymal stem cells by detecting the surface antigens of BMSCs with flow cytometry, inducing BMSCs differentiate into adipocytes and osteoblasts in conditioned medium, and observing the morphological characteristics. And last, BMSCs were cryopreserved and thawed in order to observe the influence of cryopreservation on cellular proliferation.Results:The primary cultured BMSCs were adherent, round or elliptical in shape at beginning of in vitro culture with big obvious nucleus, and then become colony-like. All adherent cells took on typical morphologically resembled fibroblast and maintained similar morphology with passages. The growth curve was similar between P1, P3and P5BMSCs. Flow cytometric analysis of BMSCs at passage3showed that these cells were negative for CD34, CD45and were positive for CD90. BMSCs can be induced to differentiate into osteoblasts and adipocytes under appropriate conditions. The thawed BMSCs were round, suspension, and turned to spindle-shaped and adherent. The proliferation became slower, but was similar with that of uncryopreserved after passage.Conclusions:The whole bone marrow adherent culture method could obtain bone marrow mesenchymal stem cells with high purity, which had great potential ability of proliferation and multi-lineage differentiation, and was a simple and feasible method. Part II:Identification and establishment of the rat model of thin endometriumObjective:To establish and identify a rat model of thin endometrium.Methods:1. Twelve SD rats, weighted about220-280g, were randomly divided into two groups:model group (perfusing alcohol into uterine cavities for about5min) and control group (perfusing saline into uterine cavities for about5min).2. Identify whether the model was established successfully by observing the endometrium morphological change with Hematein Eosin (HE) staining, measuring the endometrium thickness, examinating the expression of cytokeratin and vimentin with immunohistochemical (IHC), which were the markers of endometrial cells.Results:1. Compared with control group, the endometrium lining was thinner and flat. The glands were sparse and inactive, and the endometrial interstitial edema.2. The expression of cytokeratin and vimentin was significantly weaker than that of control group. Furthermore, the expression of Integinβ3was also weaker obviously in model group (P<0.05).Conclusions:Successful rat model of thin endometrium could be established by perfusing alcohol into uterine cavities, and the success rate was100%. Part III:The therapeutic effect and the possible mechanisms of bone marrow mesenchymal stem cells transplantation on thin endometriumObjective:To explore the therapeutic effection of rat bone marrow mesenchymal stem cells transplantation on thin endometrium in vivo and its possible mechanisms.Methods:1. BrdU was used to label the BMSCs in vitro, and then the BMSCs were observed by IHC. The Polymerase Chain Reaction (PCR) technique was used to trace the transplanted cells by detecting the sex-determining gene (SRY) of rats. Seventy-two adult female rats were selected and divided randomly into six groups:the normal control group (normal rats without any treatment), the PBS control group (thin endometrium model following PBS administration), intravenous transplantation earlier group (intravenous transplantation of1×107BMSCs six to eight hours after establishment of model), intrauterine transplantation earlier group (intrauterine transplantation of1×107BMSCs six to eight hours after establishment of model), intravenous transplantation later group (intravenous transplantation of1×107BMSCs twelve days after establishment of model), intrauterine transplantation later group (intrauterine transplantation of1×107BMSCs twelve days after establishment of model). The uterine were dissected three estrous cycles after transplanting of BMSCs.2. HE staining was used to observe the morphology of endometrium, and measure the thickness of endometrium; IHC and Western Blot (WB) were used to examine the expression of cytokeratin, vimentin, CD34, as well as the endometrial receptivity markers-Integrinavβ3and Leukemia Inhibitory Factor (LIF).3. The expression of BrdU in endometrium was detected by IHC and Western WB. The Polymerase Chain Reaction (PCR) technique was used to trance the transplanted cells by detecting the sex-determining gene (SRY) of rats.4. The expression of VEGFmRNA、bFGFmRNA、TNF-amRNA、 IL-1βmRNA、IL-6mRNA was evaluated by Real-Time PCR.Results:1. HE staining of endometrium12days after BMSCs transplantationCompared with PBS control group, the endometrium was thickened with variant degree after BMSCs transplantation with increased number of glands and lightened tissue edema, but still not to their normal morphology.2. Measurement of endometrium thickness12days after BMSCs transplantationCompared with PBS control group, the endometrium thickness was thicker in the other five groups with significantly difference (P<0.05); intrauterine transplantation earlier group and intravenous transplantation later group has slightly thinner endometrium compared with normal group without significant difference (P>0.05); the endometrium in intravenous transplantation earlier group and intrauterine transplantation later group was thinner than that of normal group with significant difference (P<0.05); there was no any difference between the four group with BMSCs transplantation (P>0.05).3. The expression of cytokeratin12days after BMSCs transplantationThe results of IHC:The mean optical density was significantly higher in the other five groups compared with the PBS group (P<0.01), and was weaker than that of normal group with significant difference (P<0.05); there was no any significant difference between each group with BMSCs transplantation (P>0.05).The results of WB:The expreesion of cytokeratin in the other groups was significant stronger compared with the PBS control group (P<0.05); the expression of cytokeratin in the normal control group was stronger than that of the other five groups with significant difference (P<0.05); the expression of cytokeratin increased in the order of intravenous transplantation earlier, intravenous transplantation later, intrauterine transplantation earlier, intrauterine transplantation later, there were no significant differences among them (P>0.05).4. The expression of vimentin12days after BMSCs transplantationThe results of IHC:The mean optical density was significantly higher in the other five groups compared with the PBS group (P<0.01); the intravenous transplantation later had a lower mean optical density than that of the other three groups with BMSCs transplantation (P<0.05), and there were no any differences in the three groups (P>0.05).The results of WB:The expression of vimentin in the four groups with BMSCs transplantation was stronger than that of the PBS control group, and weaker than that of normal control group with significant difference (P<0.05); the expression of vimentin increased in the order of intrauterine transplantation earlier, intravenous transplantation earlier, intrauterine transplantation later, intravenous transplantation later, there were no significant differences among them (P>0.05).5. The expression of CD3412days after BMSCs transplantationThe results of IHC:The mean optical density was significant higher in the groups with BMSCs transplantation except for the intravenous transplantation later group (P<0.05); compared with the PBS control group, the mean optical density was significant higher in the intrauterine transplantation later group and the intravenous transplantation earlier group (P<0.05), and was slight decreased in the intravenous transplantation later group and the intrauterine transplantation earlier group with no significant difference (P>0.05).The results of WB:The expression of CD34was weakest in the normal control group; the expression of CD34in the intrauterine transplantation later group and the intravenous transplantation earlier group was significant stronger than that of the PBS control group and the normal control group, respectively (P<0.05), and meanwhile slightly stronger than that of the intravenous transplantation later group and the intrauterine transplantation earlier group (P>0.05).6. The expression of integrinavβ312days after BMSCs transplantationThe results of IHC:The intrauterine transplantation later group has slightly higher mean optical density compared with the PBS control group (P>0.05), and the other groups have significant higher mean optical density than that of the PBS control group (P<0.05); the normal control group had a significant higher mean optical density than that of the other groups (P<0.05), and was slightly decreased in the intravenous transplantation later group and the intrauterine transplantation earlier group (P>0.05); the mean optical density increased in the order of intrauterine transplantation later group, intrauterine transplantation earlier group, intravenous transplantation earlier group, and there had significant differences among them (P<0.05).The results of WB:Compared with the PBS control group, the expression of integrinav+β3in the groups with BMSCs transplantation was significant stronger than that of the PBS control group, and weaker than that of the normal control group (P<0.05); the expression of integrinavdecreased in the order of intravenous transplantation later, intravenous transplantation earlier, intrauterine transplantation later, intrauterine transplantation earlier, and there had significant differences among them (P<0.05). the expression of integrinβ3decreased in the order of intravenous transplantation later, intrauterine transplantation earlier, intrauterine transplantation later, intravenous transplantation earlier, and there had significant differences among them except for the later two groups (P<0.05).7. The expression of LIF12days after BMSCs transplantationThe results of IHC:The mean optical density was significant higher in the groups with BMSCs transplantation compared with the PBS control group (P<0.05), and slightly lower than that of the normal control group (P>0.05), and there was no significant difference among the groups with BMSCs transplantation (P>0.05).The results of WB:The expression of LIF was strongest in the normal control group and weakest in the PBS control group; the expression of LIF decreased in the order of intrauterine transplantation later group, intravenous transplantation later, intrauterine transplantation earlier, intravenous transplantation earlier, and there had significant differences among them (P<0.05).8. Tracing and location of BMSCs12days after BMSCs transplantationThe expression of Sry:We did not detect the expression of Sry mRNA in any groups with PCR technology.The expression of BrdU:The expression of BrdU increased in the order of intrauterine transplantation earlier group, intravenous transplantation earlier group, intrauterine transplantation later group, intravenous transplantation later group, and the intravenous transplantation later group expressed stronger than that of the intrauterine transplantation earlier group (P<0.05), and similar expression was seen in the intravenous transplantation earlier group and the intrauterine transplantation later group (P>0.05); there was no expression of BrdU in the PBS control group and the normal control group.9. The expression of VEGFmRNA、bFGFmRNA、IL-1βmRNA、 TNF-αmRNA、IL-6mRNA12days after BMSCs transplantationThe result of VEGFmRNA expression:The expression of VEGFmRNA decreased in the order of the intravenous transplantation later group, PBS control group, intravenous transplantation earlier group, normal control group, intrauterine transplantation earlier group, and there was no significant difference among them (P>0.05).The result of bFGFmRNA expression:The expression of bFGFmRNA decreased in the order of the intravenous transplantation later group, PBS control group, intrauterine transplantation later group, intravenous transplantation earlier group, intrauterine transplantation earlier group, normal control group, and there were significant differences among them (P<0.05); but there was no significant difference when compared between the intravenous transplantation earlier group and the intrauterine transplantation earlier group (P>0.05).The result of IL-1βmRNA expression:The expression of IL-1βmRNA was strongest in the PBS control group, and weakest in the normal control group; the expression of IL-1βmRNA was significant weaker in the intravenous transplantation earlier group and the intrauterine transplantation earlier group compared with the transplantation later groups (P<0.05); with the same transplantation time, the expression of IL-1βmRNA had no difference between the two different transplantation ways (P>0.05).The result of TNF-αmRNA expression:The expression of TNF-αmRNA was increased in the order of the intrauterine transplantation later group, normal control group, intravenous transplantation later group, PBS control group, intravenous transplantation earlier group, intrauterine transplantation earlier group, and there was no significant difference among them (P>0.05).The result of IL-6mRNA expression:The expression of IL-6mRNA was weakest in the normal control group and the PBS control group; the expression of IL-6mRNA was significant stronger in the four groups with BMSCs transplantation compared with the normal control group and the PBS control group (P<0.05); The intravenous transplantation later group expressed slightly higher than that of the other three groups with BMSCs transplantation (P>0.05).Conclusions:1. Regeneration of the endometrial cells, reparation of endometrial tissue, and improvement of endometrial receptivity could be seen after the BMSCs transplantation, suggesting that BMSCs transplantation was feasible as a treatment of thin endometrium;2. No matter which way and no matter when the BMSCs transplantation, the treatment effect in various degrees on the thin endometrium could be seen and further research was needed to chose the optical way and time for transplantation;3. BrdU was detected in the endometrium after BMSCs transplantation, suggesting that BMSCs could migrate and home to sites of damage, which may be one of the mechanisms of treatment;4. The VEGFmRNA, bFGFmRNA, and IL-6mRNA were up-regulated after BMSCs transplantation and the expression of pro-inflammatory factors TNF-amRNA, IL-1βmRNA were down-regulated after BMSCs transplantation, suggesting that differentiation, paracrine, and immunomodulatory may be mechanisms of treatment.