人脐带间充质干细胞促进宫腔粘连大鼠子宫内膜增殖与分化

doi:10.12280/gjfckx.20201077

摘要/Abstract

摘要：

目的： 评估人脐带间充质干细胞（human umbilical cord mesenchymal stem cell,hUCMSC）对宫腔粘连大鼠损伤子宫内膜增殖分化的影响。方法： 首先以自身对照为原则构建宫腔粘连大鼠模型20只,造模14 d后将其随机分为干细胞组（n=10）和对照组（n=10）;获取人脐带标本后分离培养hUCMSC,传至第三代时将0.5 mL hUCMSC悬液多点注射于干细胞组造模侧子宫;另在对照组造模侧子宫注射等量的磷酸盐缓冲液,2组未造模侧子宫均不处理。干预7 d后获取子宫组织,使用免疫荧光染色检测人核抗体、子宫内膜上皮细胞标记物角蛋白（CK）抗体和间质细胞标记物波形蛋白抗体在大鼠子宫中的分布情况,以此评估hUCMSC在大鼠子宫中的定植和分化;使用免疫组化染色和实时荧光定量聚合酶链反应观察大鼠子宫组织增殖细胞核抗原Ki-67和CK的表达量及其mRNA表达水平。结果： 宫腔粘连大鼠模型子宫内膜组织表现为子宫腔缩窄,内膜腺体稀疏,腔上皮缺失,间质内无细胞结构区域增多;另子宫宫腔剖视可见其形态欠规则,颜色苍白,粘连带形成,宫腔欠通畅,部分闭塞。体外培养的hUCMSC在细胞形态及免疫表型方面均符合hUCMSC特征。干细胞组中,在人核与波形蛋白共同染色切片中,红色荧光与绿色荧光共同显影于同一细胞;且干细胞组的Ki-67和CK的平均光密度和mRNA表达均高于对照组,差异均有统计学意义（P<0.05）。结论： hUCMSC可在损伤部位存活并定植,并可能通过上调Ki-67和CK表达改善组织修复微环境而非经分化为子宫内膜细胞起作用。

关键词: 脐带, 间充质基质细胞, 子宫, 组织黏连, 宫腔粘连, 细胞增殖, Ki-67抗原

Abstract:

Objective: To evaluate the effect of human umbilical cord mesenchymal stem cells (hUCMSC) on the proliferation and differentiation of injured endometrium in rats with intrauterine adhesions. Methods: 20 SD rats of intrauterine adhesions model was established on the principle of self-own control. After 14 days,they were randomly divided into the stem cell group (n=10) and the control group (n=10). hUCMSC were isolated from human umbilical cord specimens, and then cultured and identified for the further study. hUCMSC in passage 3 were used to transplant into the stem cell group of uterus with intrauterine adhesions and phosphate buffer saline solution were injected into the control group of uterus with intrauterine adhesions. Immunofluorescence staining was used to detect the distribution of human nuclear antibodies, endometrial epithelial cell marker cytokeratin (CK) antibody and mesenchymal cell marker vimentin antibody in rat uterus to evaluate the location and differentiation of hUCMSC in rat uterus. Immunohistochemical staining was used to observe the expression of proliferating cell nuclear antigens Ki-67 and CK in hUCMSC and mRNA of Ki-67 and CK in rat uterus were detected by quantitative reverse transcription polymerase chain reaction. Results: The modeling side of endometrial tissue of the rat uterine showed narrow of the uterine cavity,lack of the endometrial glands and luminal epithelium,increased areas without cell structure in the stroma. Cultured cells were conformed to have the characteristics of hUCMSC in terms of cell morphology and immunophenotype. In the stem cell group, red and green fluorescence were co-developed in the same cell in the co-stained sections of human nucleus and vimentin. The average optical density and mRNA expression of Ki-67 and CK in the stem cell group were higher than those in the control group, and the differences were statistically significant (P<0.05). Conclusions: hUCMSC can survive and colonize in the injured endometrium and may improve the tissue repair microenvironment by up-regulating the expression of Ki-67 and CK, rather than by differentiation into endometrial cells.

Key words: Umbilical cord, Mesenchymal stormal cells, Uterus, Tissue adhesions, Uterine adhesion, Cell proliferation, Ki-67 antigen

汪沙, 郭正晨, 汤一群, 段华. 人脐带间充质干细胞促进宫腔粘连大鼠子宫内膜增殖与分化[J]. 国际妇产科学杂志, 2021, 48(3): 309-313.

WANG Sha, GUO Zheng-chen, TANG Yi-qun, DUAN Hua. Effects of Human Umbilical Cord Mesenchymal Stem Cells on the Proliferation and Differentiation of Endometrium in Rats with Intrauterine Adhesions[J]. Journal of International Obstetrics and Gynecology, 2021, 48(3): 309-313.

图/表 6

表1 20 μL逆转录反应体系

试剂	使用量
2×TS Reaction Mix	10 μL
Anchored Oligo（dT）18（0.5 μg/μL）	1 μL
TransStart?RT/RI Enzyme Mix	1 μL
RNA	1 μg
RNase free water	补充体系至20 μL

表2 引物序列

名称	引物	序列（5′→3′）	片段长度（bp）
GAPDH	正向	TACCCACGGCAAGTTCAACG	92
	反向	CACCAGCATCACCCCATTTG
Ki-67	正向	AAGACGAAGATCAGGCAGGA	200
	反向	CAACAATCAGGTTGGCTTCA
CK	正向	TCCTCAGCCATGTCTTCCTATG	287
	反向	CTAAAACTTCCACCGCGTCCT

图1 IUA大鼠模型的宫腔剖视图及子宫内膜组织学表现注：A、B分别为对照组未造模侧（正常子宫）、造模侧子宫（IUA模型）的HE染色（×200,标尺宽度为200 μm）;C、D分别为对照组未造模侧、造模侧子宫的Masson染色（×200,标尺宽度为200 μm）;D、E分别为对照组未造模侧、造模侧子宫的宫腔剖视图（标尺宽度为1 mm）。

图2 hUCMSC体外培养形态和鉴定结果注：A原代hUCMSC培养光镜图片（×100,标尺宽度为200 μm）;B 阴性对照;C~H流式细胞术检测hUCMSC表面抗原分子CD105、CD90、CD73和CD29阳性率>95%,HLA-DR和CD34阳性率<5%。

图3 大鼠子宫组织免疫荧光染色注：A干细胞组大鼠子宫可见少量红色荧光（HuNu）;B对照组大鼠子宫未见红色荧光表达;C干细胞组大鼠子宫绿色荧光（VIM）与红色荧光（HuNu）共表达于同一细胞;D干细胞组大鼠子宫绿色荧光（CK）与红色荧光（HuNu）并未共表达于同一细胞。标尺宽度为50 μm。

表3 2组间细胞角蛋白CK平均光密度以及mRNA对比

组别	n	Ki-67		CK
组别	n	平均光密度	mRNA	平均光密度	mRNA
干细胞组	10	0.34±0.07	0.64±0.19	0.34±0.08	0.37±0.12
对照组	10	0.16±0.06	0.22±0.17	0.17±0.07	0.17±0.08
t		6.17	5.21	5.06	4.385
P		<0.01	<0.01	<0.01	<0.01

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