背景和目的：严重的子宫壁损伤可导致子宫瘢痕形成和宫腔粘连，最终导致子宫性不孕。然而，目前仍缺乏有效促进损伤子宫功能性愈合的方法。本团队前期工作表明，在治疗大鼠子宫全层损伤模型中，成体干细胞复合胶原支架的局部移植，或碱性成纤维生长因子（basic fibroblast growth factor , bFGF）的局部注射，都可引导子宫组织的功能性再生，但尚不知采用基因工程编辑方法使成体干细胞高表达bFGF是否能提高治疗效果。本研究采用子宫壁全层损伤的大鼠模型，探讨负载高表达bFGF的脐带间充质干细胞（Umbilical Cord-Mesenchymal stem cells, UC-MSCs）的胶原支架（UC-MSC^bFGF/胶原支架）的对子宫损伤模型的治疗效果和治疗机制。

方法：

1、构建UC-MSC^bFGF，验证转染效率、干性表型及致瘤性。

构建pCMV1-bFGF-hygro的表达质粒，使用电转导的方式，将bFGF质粒转导到第二代UC-MSCs中。通过免疫荧光及Western blotting（WB）检测细胞中bFGF表达，鉴定转染效率。流式细胞术和三系分化试验检测UC-MSC^bFGF的干性基因的表达和多系分化能力。将1×10⁷UC-MSC^bFGF注射到裸鼠皮下，连续观察4个月，检测UC-MSC^bFGF的致瘤性。

2、大鼠子宫壁全层损伤模型的构建和实验分组。

采用成年雌性Sprague-Dawley（SD）大鼠，将大鼠的两侧子宫角分别切除长约1.0cm、宽约0.5cm的子宫壁，建立子宫壁全层损伤模型。将大鼠随机分为假手术组、胶原支架组、UC-MSCs/胶原支架组和UC-MSC^bFGF/胶原支架组，探讨不同干预方法对损伤子宫结构和功能恢复的效果。

3、UC-MSC^bFGF对大鼠子宫壁全层损伤模型的影响。

在移植后第7、14、30天，获得包含损伤区域的子宫组织切片，观察以下指标：1）UC-MSC^bFGF在大鼠体内的示踪和bFGF的缓释动力学：使用CM-Dil标记UC-MSC^bFGF和对bFGF进行免疫组化染色。2）损伤子宫的炎症反应对比：通过免疫组化和免疫荧光对大鼠损伤区域的CD45、CD68、CD206、IL-6、TNF-α和IL-6进行定量对比，分析各组表达量的差异。3）大鼠子宫内膜和肌层修复的对比：苏木精-伊红（H&E）染色观察子宫内膜形态学改变，通过免疫组化对ER、vimentin、CD31和α-SMA染色，观察各组子宫内膜结构重塑和肌层再生的情况。4）妊娠实验，在细胞移植后的第30天，进行生育力实验，评估受损子宫的功能恢复情况。

结果：

1、成功构建pCMV1-bFGF-hygro的表达质粒，经过电转之后bFGF在UC-MSCs中获得稳定高表达，并且未观察到UC-MSCs的干性基因表达和三系分化能力的改变。

2、UC-MSC^bFGF缓解大鼠子宫壁损伤模型的炎症反应。

术后第7天，在UC-MSC^bFGF/胶原支架组中（2.01 ± 0.89%），代表炎症反应的CD45+细胞数量明显少于UC-MSCs/胶原支架组（3.88 ± 1.2%，P<0.01）和单纯胶原支架组（8.83 ± 1.9%，P<0.01）。抗炎因子IL-10的表达量在UC-MSC^bFGF/胶原支架组中（1.69 ± 0.84%）明显高于UC-MSCs/胶原支架组（0.9 ± 0.39%，P<0.01）和胶原支架组（0.30 ± 0.18%，P<0.01）。而UC-MSC^bFGF/胶原支架组（0.40 ± 0.19%）的促炎因子TNF-α的表达量显著低于UC-MSCs/胶原支架组（0.68 ± 0.41%，P<0.01）和胶原支架组（1.12 ± 0.53%，P<0.01）

3、UC-MSC^bFGF促进大鼠损伤子宫壁的血管生成。

在术后14天，与UC-MSCs/胶原支架组（25.75 ± 8.76/每个视野，P<0.01）和胶原支架组（21.5 ± 4.01/每个视野，P<0.01）相比，UC-MSC^bFGF/胶原支架组（37.5 ± 11.78/每个视野）的血管内皮标志分子CD31表达数明显增加，提示微血管数量显著上升。

4、UC-MSC^bFGF促进大鼠损伤子宫壁的内膜和肌层的再生。

在术后30天，通过HE和免疫组化染色观察每组内膜的厚度及肌层的修复情况，UC-MSC^bFGF/胶原支架组内膜厚度 (467.6 ± 115.1μm)与假手术组 (492.1 ± 41.54μm)最为接近，同时显著高于UC-MSCs/胶原支架组(210.3 ± 22.55μm, P <0.01)和胶原支架组 (157.6 ± 27.38μm, P <0.01)。同时使用α-SMA评估肌层的再生情况表明，UC-MSC^bFGF/胶原支架组 (12.82% ± 3.18%)的α-SMA阳性面积占比明显高于UC-MSCs/胶原支架组 (8.25% ± 1.67%, P <0.05)和胶原支架组 (5.93% ± 2.06%, P <0.01)

5、妊娠实验结果表明，UC-MSC^bFGF/胶原支架组（8/16，P<0.015）的损伤部位着床率显著高于UC-MSCs/胶原支架组（1/16，P<0.015）和胶原支架组（0/16，P<0.015）。

结论：UC-MSC^bFGF可抑制子宫损伤引起的局部炎症反应，促进损伤区域的血管生成，加速损伤子宫壁的再生，从而缩短了损伤子宫恢复达到妊娠标准所需要的时间。

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-MSC^bFGF/scaffold on functional regeneration of the uterine full-thickness injury model.

Methods: 

1, Construction of UC-MSC^bFGF 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-MSC^bFGF 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-MSC^bFGF. 1×10⁷ UC-MSC^bFGF was injected subcutaneously into nude mice and observed for 4 months to detect the tumorigenicity of UC-MSC^bFGF.

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-MSC^bFGF/scaffold group to explore the outcome of structural and functional recovery in injured uterus. 

3, Effect of UC-MSC^bFGF 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-MSC^bFGF 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-MSC^bFGF were similar to control UC-MSCs.

2, The UC-MSC^bFGF exhibited anti-inflammatory effect.

The number of CD45+ cells in the UC-MSC^bFGF/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-MSC^bFGF/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-MSC^bFGF/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-MSC^bFGF also exhibited dramatically pro-angiogenesis efficacy.

The number of blood vessel in UC-MSC^bFGF/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-MSC^bFGF 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-MSC^bFGF/scaffold group was (467.6 ± 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-MSC^bFGF/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-MSC^bFGF/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-MSC^bFGF/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.