应用单细胞RNA测序技术（scRNA-seq）探讨细胞通讯网络因子2 （CCN2，也称为CTGF）在特纳综合征（TS）胎儿颈部淋巴水囊瘤（CH）的潜在发生机制。

收集2020年1月至2020年12月于广州市妇女儿童医疗中心产前诊断中心就诊，孕11 ~ 13⁺⁶周经腹部超声检查诊断为CH而终止妊娠及相同孕周因社会因素终止妊娠病例，产前未行遗传学检测者，需同时取引产胎儿胎盘绒毛组织或骨骼肌，按照标准的操作流程进行遗传学检测。全部病例应用遗传学检测确定染色体异常类型后，CH病例中选择遗传学结果为45，X0的胎儿作为实验组，无CH病例中选择遗传学结果为46，XN的胎儿作为对照组。本研究共纳入6例样本，实验组3例，对照组3例。所有入组病例在终止妊娠后立即取颈后皮肤组织，一部分样本应用HE染色进行病理分析，一部分应用scRNA-seq分析颈后皮肤组织细胞类型并建立差异基因表达谱，并进一步应用RT-qPCR检测细胞中基因表达水平及Western blot检测相关蛋白表达情况以明确单细胞测序结果的可靠性。实验组与对照组采用独立样本t检验进行统计学分析。

病理结果提示实验组表现为淋巴管增生和扩张，淋巴结增多坏死，血管减少，间质纤维丝束增多、变长，并出现大量细胞浸润组织间隙。应用scRNA-seq分析CH病例，共捕获细胞54 488个，26个聚类，其中成纤维细胞占23.1%。同时发现基质细胞蛋白CCN2高表达。进一步定位分析显示实验组中CCN2主要高表达在成纤维细胞、内皮细胞、组织干细胞和间充质干细胞。同时Western blot验证CCN2蛋白在实验组中高表达（2.47 ± 0.49比1.00 ± 0.08，P < 0.05）。

病理结果表明淋巴管增生扩张和淋巴结增多坏死等淋巴管发育异常是TS胎儿CH的病理生理学基础。scRNA-seq结果表明基质细胞蛋白CCN2异常高表达可能导致TS胎儿CH。

Single-cell RNA sequencing (scRNA-seq) was used to investigate the potential pathogenesis of cystic hygromas (CH) in fetuses with Turner's syndrome (TS) .

The posterior cervical tissues of the terminated pregnancy fetus, which ended in termination pregnancy at 11-13⁺⁶ weeks were collected due to CH at Guangzhou Women and Children Medical Care Center from January 2020 to December 2020. The posterior cervical tissues of normal fetuses who terminated pregnancy due to social factors were collected at the same gestational age. For those who did not receive prenatal genetic testing, placental tissue or skeletal muscle of the induced fetus should be taken for genetic etiology testing according to standard operating procedures. Genetic testing was used in all cases to determine the chromosomal abnormality type. According to the genetic etiology, we selected the TS fetuses as the experimental group, and fetuses with genetic results 46, XN were the control group in cases without CH. A total of six cases were collected. The experimental group and control group, respectively had 3 cases. In all enrolled patients, posterior cervical skin tissue was taken immediately after termination of pregnancy, HE staining to some samples for pathological analysis, and some samples were analyzed by scRNA-seq to analyze the cell types of cervical skin tissue and establish differential gene expression profiles. Furthermore, RT-qPCR was used to study the gene expression level in cells, and Western blot was used to detect the expression of related proteins to confirm the reliability of the single-cell sequencing results. An independent sample test was used to compare the differences between groups.

Pathological findings showed that pathological features of CH cases were significant lymphatic hyperplasia and dilation, increased lymph nodes and necrosis, decreased blood vessels, significantly increased and lengthened interstitial fibrofilaments, and a large number of cells infiltrated into the interstitial space. In the analysis of CH cases by scRNA-seq, a total of 54 488 cells were captured and 26 clusters were identified. Among them, fibroblasts accounted for 23.1%, and stromal cell protein CCN2 was found to have high expression. Further localization analysis showed that CCN2 was highly expressed in fibroblasts, endothelial cells, muscle cells, tissue stem cells, and mesenchymal stem cells in the experimental group. Furthermore, Western blot was to verify that CCN2 protein was significantly overexpressed in the experimental group (2.47 ± 0.49 vs 1.00 ± 0.08, P < 0.05) .

Pathological analysis confirmed that lymphatic abnormalities such as lymphatic hyperplasia and dilation, and increased lymph nodes and necrosis were the common pathophysiological basis of fetuses with TS. ScRNA-seq results suggested that abnormal overexpression of CCN2 may be related to CH in fetuses with TS.